CN109652358A - The breeding method of sirolimus producing strains - Google Patents
The breeding method of sirolimus producing strains Download PDFInfo
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- CN109652358A CN109652358A CN201910123881.9A CN201910123881A CN109652358A CN 109652358 A CN109652358 A CN 109652358A CN 201910123881 A CN201910123881 A CN 201910123881A CN 109652358 A CN109652358 A CN 109652358A
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- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 title claims abstract description 47
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 title claims abstract description 47
- 229960002930 sirolimus Drugs 0.000 title claims abstract description 47
- 238000009395 breeding Methods 0.000 title claims abstract description 20
- 239000011521 glass Substances 0.000 claims abstract description 28
- 241000894006 Bacteria Species 0.000 claims abstract description 25
- 239000000243 solution Substances 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 16
- 238000010008 shearing Methods 0.000 claims abstract description 16
- 239000012895 dilution Substances 0.000 claims abstract description 14
- 238000010790 dilution Methods 0.000 claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 claims abstract description 14
- 230000001580 bacterial effect Effects 0.000 claims abstract description 12
- 239000002504 physiological saline solution Substances 0.000 claims description 12
- 235000015097 nutrients Nutrition 0.000 claims description 10
- 241000187391 Streptomyces hygroscopicus Species 0.000 claims description 9
- 241000233866 Fungi Species 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 239000012530 fluid Substances 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 7
- 238000011218 seed culture Methods 0.000 claims description 6
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 5
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- 238000012549 training Methods 0.000 claims description 4
- 241000187747 Streptomyces Species 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 claims description 2
- 239000001257 hydrogen Substances 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 2
- -1 phosphoric acid hydrogen Chemical class 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 abstract description 24
- 230000004151 fermentation Effects 0.000 abstract description 24
- 230000001488 breeding effect Effects 0.000 abstract description 6
- 231100000225 lethality Toxicity 0.000 description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000012092 media component Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- BVHLGVCQOALMSV-JEDNCBNOSA-N L-lysine hydrochloride Chemical compound Cl.NCCCC[C@H](N)C(O)=O BVHLGVCQOALMSV-JEDNCBNOSA-N 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000001857 anti-mycotic effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000002543 antimycotic Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 229940041033 macrolides Drugs 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000004112 neuroprotection Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The present invention provides the breeding method of sirolimus producing strains, and by the access of sirolimus producing strains equipped in the culture bottle of glass dregs, culture bottle is placed in shaking table shaking, after shaking, plate, the bacterial strain that picking single colonie, as anti-shearing force and sirolimus production capacity improve are applied after bacterium solution dilution.Breeding is carried out to sirolimus producing strains using the method for the present invention, the anti-shearing force and sirolimus production capacity of obtained strains increase, and finally improve the yield of fermentation liquid sirolimus.
Description
Technical field
The present invention relates to Microbial Breeding technologies, specifically, being related to the breeding method of sirolimus producing strains.
Background technique
Sirolimus (Rapamycin) is a kind of macrolides compound by streptomyces hygroscopicus fermenting and producing, it has
There are antimycotic, immunosupress, antitumor, neuroprotection and anti-aging isoreactivity, is clinically applied to the anti-row of organ transplant
The treatment of reprimand reaction and autoimmune disease.
Sirolimus is a kind of with multiple biological activities, valuable natural products, but wild type sirolimus is raw
Producing the lower sirolimus yield of strain is to limit a key factor of its industrialized production and application.Therefore, west is improved
Luo Mosi fermentation level is imperative.
The fermentability of microbial strains depends not only on the performance of thallus itself, and growing environment is suitable for and thallus is efficient
The necessary factor of expression product.In fermentation industry, deep layer leads to oxygen stirring fermentation and plays an important role, and the purpose is to make system gas-liquid
Two-phase comes into full contact with, the mixing of strengthening process and mass transfer, nutrient media components in promotion system, and temperature, pH value and dissolved oxygen etc. are equal
Even distribution, guarantee thallus can in good environment normal growth and metabolism etc..But it also has unfavorable aspect, high-speed rotating to stir
Device shearing production mycelia is mixed, cellular damage is easily caused；After speed of agitator is slack-off, dissolved oxygen is insufficient, and fermentation unit increasess slowly.
It stirs finally closely bound up with fermenter productivity and product quality.
Summary of the invention
The object of the present invention is to provide the breeding methods of sirolimus producing strains, to improve the anti-shearing force and Xi Luo of bacterial strain
Do not take charge of production capacity.
Present inventive concept is as follows: carrying out anti-shearing force with glass dregs by the mycelia to sirolimus producing strains in seed bottle
Screening, the bacterial strain that acquisition hyphal cell can be resistant to strong pressure are subtracted with improving bacterial strain in the fermenter to the adaptability of shearing force
The damage of few cell, the final production capacity for improving sirolimus.
In order to achieve the object of the present invention, the present invention provides the breeding method of sirolimus producing strains, and sirolimus is generated
Culture bottle is placed in shaking table shaking equipped in the culture bottle of glass dregs by bacterium access, after shaking, applies plate after bacterium solution dilution,
The bacterial strain that picking single colonie, as anti-shearing force and sirolimus production capacity improve.
Further, the inclined-plane of sirolimus producing strains is dug into fungus block and accesses the culture of seed bottle, it will culture after culture
Then culture bottle is placed in shaking table shaking, after shaking, bacterium solution is dilute equipped in the culture bottle of glass dregs by mature mycelia access
Plate, the bacterial strain that picking single colonie, as anti-shearing force and sirolimus production capacity improve are applied after releasing.
In the present invention, the sirolimus producing strains include but is not limited to streptomyces hygroscopicus (Streptomyces
Hygroscpicus), such as bacterial strain FC904-25.
Method above-mentioned, shaking table culture condition are as follows: 26-30 DEG C, 260-300rpm cultivate 30-60min, preferably 28 DEG C,
300rpm cultivates 60min.
Preferably, the shape of the glass dregs is triangle, area 0.1-0.2cm2。
Sirolimus producing strains breeding method of the invention, specifically includes the following steps:
A, inclined-plane and seed culture: sirolimus producing strains are passed into inclined-plane, 26-30 DEG C of cultivation temperature, are cultivated 5-6 days；
B, fungus block access in inclined-plane is equipped in the seed bottle of fluid nutrient medium, 26-30 DEG C, shaking speed 250-280rpm,
The bottled liquid measure 100ml/750ml of seed cultivates 45-55h；
C, take the bacterium solution of 1ml step B in the triangular flask equipped with 9ml physiological saline and 12-15g glass dregs, 26-30 DEG C,
260-300rpm shaking table shakes 30-60min；
D, after shaking, gained bacterium solution applies plate after gradient dilution, separates to obtain single colonie.
In the specific embodiment of the present invention, the method is as follows:
A, streptomyces hygroscopicus is passed into inclined-plane, 28 DEG C of cultivation temperature, cultivated 5 days；
B, by the access of inclined-plane fungus block equipped in the seed bottle of fluid nutrient medium, 28 DEG C, shaking speed 260rpm, seed is bottled
Liquid measure 100ml/750ml cultivates 48h；
C, take the bacterium solution of 1ml step b in the triangular flask equipped with 9ml physiological saline and 15g glass dregs, 28 DEG C, 300rpm
Shaking table shakes 60min；
D, after shaking, gained bacterium solution applies plate after gradient dilution, separates to obtain single colonie.
Fluid nutrient medium described in step B are as follows: glucose 18-22g/L, dipotassium hydrogen phosphate 05-1.5g/L, bean cake powder 19-
23g/L and ammonium sulfate 1.5-2.5g/L (being prepared with water).Preferably, the fluid nutrient medium are as follows: glucose 20g/L, phosphoric acid hydrogen
Dipotassium 1g/L, bean cake powder 21g/L and ammonium sulfate 2g/L.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
Breeding is carried out to sirolimus producing strains using the method for the present invention, the anti-shearing force and sirolimus of obtained strains are raw
Production capacity power increases, and finally improves the yield of fermentation liquid sirolimus.
Sirolimus producing strains need higher speed of agitator in fermenting and producing to meet in production process to dissolved oxygen etc.
Demand, but since it is der Pilz fermentation, too strong shearing force easily causes to produce that bacterium mycelium is mechanically damaged, physiological status
Abnormal, inhibition thalli growth, and the sirolimus producing strains Jing Guo anti-shearing force breeding are resistant to certain cut in production
The mixing of nutriment and mass transfer in speed of agitator strengthening process, culture medium group in promotion system thus can be improved in shear force
Point, temperature, pH value and dissolved oxygen etc. are uniformly distributed, guarantee thallus can normal growth and metabolism in good environment, finally mention
High fermentation unit.68.7% left side can be improved in the upper average fermentation unit of production in sirolimus producing strains by anti-shearing force breeding
It is right.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
Streptomyces hygroscopicus used in following example 1-3 is bacterial strain FC904-25.Absorb water chain used in embodiment 4-6
Mould is bacterial strain WY93.
Glass dregs used are self-control: breaking teat glass (thickness of pipe wall 0.15cm) into pieces selection area in 0.1-0.2cm2Three
It is angular.
The breeding method of 1 sirolimus producing strains of embodiment
1, inclined-plane and seed culture: inclined-plane is passed with streptomyces hygroscopicus, 28 DEG C of cultivation temperature, is cultivated 5 days.By mature inclined-plane bacterium
Block accesses the culture of seed bottle (containing seed culture medium), and 28 DEG C, shaking speed 260rpm, shaking flask liquid amount 100ml/750ml, training
Bacterium dense 38% (volumetric method measurement) after feeding 48h draws 1ml bacterium solution in the triangular flask equipped with 9ml physiological saline and 15g glass dregs
(while glass dregs not to be added, the triangular flask of 9ml physiological saline is only housed as a control group), triangular flask is placed on shaking table,
28 DEG C, 300rpm shakes 30min, and gradient dilution separates to obtain single colonie, and compared with the control group, experimental group lethality is only 37%.
Lethality=(every milliliter of single colonie number of control group-every milliliter of experimental group single colonie number) every milliliter of single bacterium of/control group
Fall number × 100
Experimental group, control group are picked them separately into single colonie after culture is mature, biography connects inclined-plane, is inoculated with sub- bottle.Seed training used
Support base are as follows: glucose 20g/L, dipotassium hydrogen phosphate 1g/L, bean cake powder 21g/L and ammonium sulfate 2g/L (preparing with water).
Fermented and cultured: mature seed liquid is accessed into fermentation flask culture.1,2,3 are separately added into fermentation flask culture medium
The bead of diameter 3mm.The inoculum concentration 2ml/20ml of seed liquor, 28 DEG C, shaking speed 260rpm, shaking flask liquid amount 20ml/
With the potency of HPLC measurement fermentation liquid sirolimus after 250ml, the 192h that ferments.
Fermentation medium used are as follows: glucose 60g/L, dipotassium hydrogen phosphate 1g/L, cottonseed meal 21g/L, sodium chloride 5g/L,
L lysine HCL 15g/L (is prepared) with water.After fermentation, experimental group (shaking 30min with shaking table on glass dregs) is measured
Compared with the control group, potency generally improves 16% or so to isolated single colonie.
The breeding method of 2 sirolimus producing strains of embodiment
Seed bottle draws 1ml bacterium solution in the triangular flask equipped with 9ml physiological saline and 15g glass dregs after cultivating, above shake
Bed shaking 45min, with embodiment 1, gradient dilution separates to obtain single colonie for remaining operation, compared with the control group, experimental group lethality
49%.
After fermentation, measure experimental group (shaking 45min with shaking table on glass dregs) isolated single colonie with compare
Group is compared, and potency generally improves 38% or so.
The breeding method of 3 sirolimus producing strains of embodiment
Seed bottle draws 1ml bacterium solution in the triangular flask equipped with 9ml physiological saline and 15g glass dregs after cultivating, above shake
Bed shaking 60min, remaining operation is the same as embodiment 1.Gradient dilution separates to obtain single colonie, compared with the control group, experimental group lethality
61%.
After fermentation, measure experimental group (shaking 60min with shaking table on glass dregs) isolated single colonie with compare
Group is compared, and potency generally improves 51% or so.
Previous fermentor revolving speed maximum can only achieve 150rpm, if further increasing revolving speed, will lead to feed liquid and turns dilute.It adopts
250rpm can be improved with revolving speed after the strain of anti-shearing force, mixing and the mass transfer of nutriment can be promoted in this way, promote
Nutrient media components in system, temperature, pH value and dissolved oxygen etc. are uniformly distributed, guarantee thallus can in good environment normal growth
And metabolism, it is final to improve strain unit.Sirolimus producing strains by anti-shearing force breeding are producing upper average fermentation unit
It can be improved 68.7% or so.
The breeding method of 4 sirolimus producing strains of embodiment
1, inclined-plane and seed culture: inclined-plane WY93 is passed with streptomyces hygroscopicus, 25 DEG C of cultivation temperature, is cultivated 15 days.It will be mature
Inclined-plane fungus block accesses the culture of seed bottle (containing seed culture medium), and 28 DEG C, shaking speed 260rpm, shaking flask liquid amount 50ml/
250ml cultivates bacterium dense 32% (volumetric method measurement) after 48h, draws 1ml bacterium solution in being equipped with 9ml physiological saline and 15g glass dregs
(while glass dregs not to be added, the triangular flask of 9ml physiological saline is only housed as a control group) in triangular flask, triangular flask is placed in
On shaking table, 28 DEG C, 300rpm shakes 30min, and gradient dilution separates to obtain single colonie, and compared with the control group, experimental group lethality is only
It is 29%.
Experimental group, control group are picked them separately into single colonie after culture is mature, biography connects inclined-plane, is inoculated with sub- bottle.Seed training used
Support base are as follows: glucose 15g/L, starch 3g/L, soybean cake powder 18g/L (are prepared) with water.
Fermented and cultured: mature seed liquid is accessed into fermentation flask culture.1,2,3 are separately added into fermentation flask culture medium
The bead of diameter 3mm.The inoculum concentration 5ml/50ml of seed liquor, 25 DEG C, shaking speed 260rpm, shaking flask liquid amount 50ml/
With the potency of HPLC measurement fermentation liquid sirolimus after 500ml, the 96h that ferments.
Fermentation medium used are as follows: glucose 50g/L, dipotassium hydrogen phosphate 1g/L, soybean cake powder 21g/L, sodium chloride 5g/L,
(being prepared with water).After fermentation, measure experimental group (shaking 30min with shaking table on glass dregs) isolated single colonie with it is right
It is compared according to group, potency generally improves 20% or so.
The breeding method of 5 sirolimus producing strains of embodiment
Seed bottle draws 1ml bacterium solution in the triangular flask equipped with 9ml physiological saline and 15g glass dregs after cultivating, above shake
Bed shaking 45min, with embodiment 4, gradient dilution separates to obtain single colonie for remaining operation, compared with the control group, experimental group lethality
37%.
After fermentation, measure experimental group (shaking 45min with shaking table on glass dregs) isolated single colonie with compare
Group is compared, and potency generally improves 43% or so.
The breeding method of 6 sirolimus producing strains of embodiment
Seed bottle draws 1ml bacterium solution in the triangular flask equipped with 9ml physiological saline and 15g glass dregs after cultivating, above shake
Bed shaking 60min, remaining operation is the same as embodiment 4.Gradient dilution separates to obtain single colonie, compared with the control group, experimental group lethality
55%.
After fermentation, measure experimental group (shaking 60min with shaking table on glass dregs) isolated single colonie with compare
Group is compared, and potency generally improves 60% or so.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. the breeding method of sirolimus producing strains, which is characterized in that the access of sirolimus producing strains is equipped with to the training of glass dregs
It supports in bottle, culture bottle is placed in shaking table shaking, after shaking, apply plate after bacterium solution dilution, picking single colonie is as anti-shearing
The bacterial strain that power and sirolimus production capacity improve.
2. the method according to claim 1, wherein the inclined-plane of sirolimus producing strains, which is dug fungus block, accesses seed
Then culture bottle is placed in and is shaken equipped in the culture bottle of glass dregs after culture by bottle culture by the mature mycelia access of culture
Bed shaking after shaking, applies plate, picking single colonie, as anti-shearing force and sirolimus production capacity after bacterium solution dilution
The bacterial strain of raising.
3. the method according to claim 1, wherein the sirolimus producing strains include streptomyces hygroscopicus
(Streptomyces hygroscpicus)。
4. the method according to claim 1, wherein shaking table shakes condition are as follows: 26-30 DEG C, 260-300rpm vibration
30-60min, preferably 28 DEG C are shaken, 300rpm shakes 60min.
5. the method according to claim 1, wherein the shape of the glass dregs is triangle, area 0.1-
0.2cm2。
6. method according to claim 1-5, which comprises the following steps:
A, inclined-plane and seed culture: sirolimus producing strains are passed into inclined-plane, 26-30 DEG C of cultivation temperature, are cultivated 5-6 days；
B, fungus block access in inclined-plane is equipped in the seed bottle of fluid nutrient medium, 26-30 DEG C, shaking speed 250-280rpm, seed
Bottled liquid measure 100ml/750ml cultivates 45-55h；
C, take the bacterium solution of 1ml step B in the triangular flask equipped with 9ml physiological saline and 12-15g glass dregs, 26-30 DEG C,
260-300rpm shaking table shakes 30-60min；
D, after shaking, gained bacterium solution applies plate after gradient dilution, separates to obtain single colonie.
7. according to the method described in claim 6, characterized by comprising the following steps:
A, streptomyces hygroscopicus is passed into inclined-plane, 28 DEG C of cultivation temperature, cultivated 5 days；
B, fungus block access in inclined-plane is equipped in the seed bottle of fluid nutrient medium, 28 DEG C, shaking speed 260rpm, the bottled liquid measure of seed
100ml/750ml cultivates 48h；
C, take the bacterium solution of 1ml step b in the triangular flask equipped with 9ml physiological saline and 15g glass dregs, 28 DEG C, 300rpm shaking table
Shake 60min；
D, after shaking, gained bacterium solution applies plate after gradient dilution, separates to obtain single colonie.
8. according to the method described in claim 6, it is characterized in that, fluid nutrient medium described in step B are as follows: glucose 18-22g/
L, dipotassium hydrogen phosphate 05-1.5g/L, bean cake powder 19-23g/L and ammonium sulfate 1.5-2.5g/L.
9. according to the method described in claim 8, it is characterized in that, the fluid nutrient medium are as follows: glucose 20g/L, phosphoric acid hydrogen
Dipotassium 1g/L, bean cake powder 21g/L and ammonium sulfate 2g/L.
10. according to the described in any item methods of claim 7-9, which is characterized in that the streptomyces hygroscopicus is bacterial strain FC904-
25。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101486976A (en) * | 2008-01-16 | 2009-07-22 | 杭州华东医药集团生物工程研究所有限公司 | Streptomyces hygroscopicus and use thereof |
| CN102199638A (en) * | 2011-02-21 | 2011-09-28 | 西南大学 | Amino acid adding method for raising sirolimus fermentation yield |
| CN102229968A (en) * | 2011-06-07 | 2011-11-02 | 华东理工大学 | Method for cumulatively producing Sirolimus by using streptomyces hygroscopicus |
| CN107557403A (en) * | 2017-10-31 | 2018-01-09 | 无锡福祈制药有限公司 | A kind of method for improving sirolimus fermentation yield |
| CN108624524A (en) * | 2018-04-02 | 2018-10-09 | 华东师范大学 | The bacterial strain and its separating screening method of one plant of production bacteria cellulose |
-
2019
- 2019-02-19 CN CN201910123881.9A patent/CN109652358A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101486976A (en) * | 2008-01-16 | 2009-07-22 | 杭州华东医药集团生物工程研究所有限公司 | Streptomyces hygroscopicus and use thereof |
| CN102199638A (en) * | 2011-02-21 | 2011-09-28 | 西南大学 | Amino acid adding method for raising sirolimus fermentation yield |
| CN102229968A (en) * | 2011-06-07 | 2011-11-02 | 华东理工大学 | Method for cumulatively producing Sirolimus by using streptomyces hygroscopicus |
| CN107557403A (en) * | 2017-10-31 | 2018-01-09 | 无锡福祈制药有限公司 | A kind of method for improving sirolimus fermentation yield |
| CN108624524A (en) * | 2018-04-02 | 2018-10-09 | 华东师范大学 | The bacterial strain and its separating screening method of one plant of production bacteria cellulose |
Non-Patent Citations (6)
| Title |
|---|
| 吴晓良: "顶头孢霉的遗传育种方法初步研究和头孢菌素C发酵行为突变筛选", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 张莉等: "用庆大霉素抗性筛选法选育西罗莫司高产菌株", 《中国抗生素杂志》 * |
| 杨雪霞等: "剪切力对木葡糖醋杆菌及细菌纤维素合成的影响", 《纤维素科学与技术》 * |
| 芦安娜等: "He-Ne激光与紫外线复合诱变选育西罗莫司高产菌株", 《河北化工》 * |
| 董超: "剪切力及抑制物对木葡糖醋杆菌和纤维素合成的影响及耐受菌株选育", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 陈希杨: "高产雷帕霉素吸水链霉菌的遗传改造和发酵过程优化", 《中国博士学位论文全文数据库 工程科技I辑》 * |
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