CN102453737A - Method for producing rapamycin fermentation liquid by culturing streptomyces hygroscopicus - Google Patents
Method for producing rapamycin fermentation liquid by culturing streptomyces hygroscopicus Download PDFInfo
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Abstract
The invention discloses a new method for producing rapamycin fermentation liquid by culturing streptomyces hygroscopicus. According to the invention, the fermentation liquid containing rapamycin is prepared by fermentation culture of a microbial strain, namely streptomyces hygroscopicus ACCC No.40417. The rapamycin fermentation liquid prepared by the method disclosed by the invention has high titer, thus being more favorable to extraction and industrialized production.
Description
Technical field
The invention belongs to the biological medicine technology field, be specifically related to the method that a kind of microbial fermentation is produced rapamycin.
Background technology
Rapamycin (Rapamycin; RPM; RAPA), having another name called sirolimus (Sirolimus), is the triolefin macrolide antibiotics of being separated from streptomyces hygroscopicus (Strepomyces hygroscopicus) by Canadian Ayerst institute in 1975; It has caused widely as a kind of novel efficient immunosuppressor pays close attention to, and it is again a kind of effective anti-mycotic agent and has antineoplastic action simultaneously.
Rapamycin is 31 yuan of triolefin Macrocyclic lactone compounds that ring is formed, and contains the half carboxylic ketone that special α, beta-diketon second nipecotic acid amine molecule cover, and its molecular formula is C
51H
79O
13, molecular weight is 991.Rapamycin is one type of close ester class microbiotic, and outward appearance is generally the white crystals body.Be soluble in organic solvents such as methyl alcohol, ethanol, acetone, chloroform, ETHYLE ACETATE, be dissolved in ether, hexane, sherwood oil hardly, atomic water-soluble.PH is low neutral in blood plasma, and it is fast especially to degrade in the time of 37 ℃, and the transformation period is 1～5h in blood plasma, and is unstable in methyl alcohol.Its fusing point is 183～185 ℃, D25=153 ° of specific rotatory power [α] (methyl alcohol).Vezinia equals to find that it had antifungic action in 1975 that later Tanaka etc. find that again it is a kind of extremely promising immunosuppressor and has antitumor action.Sirolimus has very strong resisting transplant rejection reaction simultaneously, and can work in coordination with the prolongation graft survival time with ciclosporin, and sirolimus can also reverse ongoing graft-rejection.In addition, sirolimus prevents and treats the effect of experimental oneself soup-dissolving courage and uprightness anemia in addition.
Rapamycin is better than Ciclosporin A (the cyclosporin A of existing wide clinical application greatly in the effect that prevents the effect of animal organ's transplant rejection; CsA); Also being better than the similar medicine FK506 of Macrolide (tacrolimus), is very potential clinically immunosuppressor.Because the chemical structure of RPM and CsA is different, both similar with FK506, variant again, couplings such as RPM and CsA, FK506 all have good synergy, and the benefit of coupling is: reduced the consumption of various immunosuppressor in the regimen; Reduced the spinoff of immunosuppressor; Strengthened the immunosuppressor effect.The mechanism of action of rapamycin is all different with CsA and FK506.CsA and FK506 respectively with cytoplasm in ring spore avidin and FKBP12 (FK506 is conjugated protein) combine, suppress Calcineurin (calcineurin), the expression of prevention cytokine such as interleukin-2 (IL-2) and transcribe suppressor T cell activatory commitment.RPM has similar molecular structure with FK506, also can combine RAPA-FKBP12 mixture and mammals rapamycin target protein (mammalian target of rapamycin with FKBP12; MTOR) combine; But it is the generation of blockage of T cells growth factor receptor not, but blocks the hyperplasia signal that these factors provide, and suppresses the T that multiple stimulation causes and the hyperplasia of bone-marrow-derived lymphocyte; Be the late period that cell rests on the cell cycle G1 phase, stop these cells to get into the S phase.In sum, rapamycin is as a kind of immunosuppressor of new high-efficiency, and it has great importance in clinical organ transplantation and treatment autoimmune disease.
1993, U.S. Merck company was converted into rapamycin through the method for microbial transformation, and after early stage, seed liquor was cultivated, fermented liquid had finally produced the rapamycin of 300mg/L.The India scientist has screened high yield rapamycin streptomycete from soil, and by product is few, in the dextrin substratum of high pass tolerance, has produced the rapamycin of 300～310mg/L through fed-batch fermentation.2004, India scholar Vaid etc. carried out mutagenesis to a streptomycete, and vegetables oil is fermented as only carbon source, and the result shows the 144～192h that continuously ferments, and the productive rate of rapamycin can reach 180～330mg/L.On October 20th, 1999, rapamycin went on the market in the U.S., was oral liquid formulation.After this adopt the 1mg tablet of the nanocrystal technology preparation of Yi Lan (Elan) company also to go on the market in the U.S..In March, 2000, rapamycin went on the market in states such as Britain, Germany, Denmark, and rapamycin is also sold with the mode of oral suspensions in Britain in addition.
Because the rapamycin of produced in usa fails to obtain administrative protection in China, enterprise can produce rapamycin in the native land.It is reported; From calendar year 2001 so far; State Food and Drug Administration has successively ratified 7 tame enterprise production rapamycins, and wherein Xinchang, Zhejiang pharmaceutical factory, the auspicious medicine company of Fujian section, the East China pharmacy of Hangzhou Sino-U.S., 4 families of North China pharmacy scientific research development company are rapamycin raw material pharmaceutical factory.At present, have the fermentation level of reported in literature all not high, production peak is at 600mg/L-700mg/L.
Hangzhou Sino-U.S. Huadong Medicine Co., Ltd. has dropped into suitability for industrialized production (Chinese patent CN200810019207.8), on 10 tons, 50 tons fermentor tanks, cultivates the streptomyces hygroscopicus fermentation capacity about 600mg/L.Zhang Wei etc. disclose strain improvement and culture condition optimization in " rational selection of thunder handkerchief rhzomorph superior strain and new process for fermenting research " (Zhejiang University's master thesis in 2009) literary composition, be 700mg/L 20 tons of fermentation cylinder for fermentation abilities.
Hence one can see that, and the deficiency that rapamycin biological fermentation production at present exists is that rapamycin biological fermentation unit is not high, is unfavorable for extracting purifying, can not fully satisfy industrial production requirement.
Summary of the invention
The objective of the invention is to overcome the deficiency of existing rapamycin production technology, thereby a kind of fermentation process that can carry out suitability for industrialized production better is provided, specifically comprise the control method of a cover culture medium prescription and culture condition.
For achieving the above object, the present invention has carried out dull and stereotyped cultivation, shake-flask seed cultivation, seeding tank seed culture and ferment tank to streptomyces hygroscopicus (Streptomyces hygroscopicus) ACCC No.40417 and has cultivated.The practical implementation process is following:
A. dull and stereotyped the cultivation: in the petridish that contains solid medium, constant temperature culture is 5-7 days after the inoculation with the streptomyces hygroscopicus bacterial classification inoculation;
B. shake-flask seed is cultivated: the seed culture medium of in triangular flask, packing into, and the dull and stereotyped streptomyces hygroscopicus bacterial classification of cultivating gained is inserted in the back of sterilizing, and is placed on the vertical shaking table, cultivates 50-70h under the 220-230rpm condition, gets the shake-flask seed nutrient solution;
C. seeding tank seed culture: the shake-flask seed nutrient solution is inoculated in the seed tank culture base according to the long-pending 11%-15% inoculum size of seed tank culture matrix, cultivates 35-46h under the 200-210rpm condition, the seeding tank seed liquor;
D. ferment tank is cultivated: the seeding tank seed liquor is inoculated in fermentation tank culture medium according to the 11%-15% inoculum size of fermentation tank culture medium volume; Initial mixing speed is 180-220rpm; Cultivate 72-96h; During thalline weight in wet base 0.22-0.23g/ml, the adjusting rotating speed is 255-300rpm, cultivates 105-150h; Fermentor cultivation total time 170-230h.
The method of preferred above-mentioned cultivation streptomyces hygroscopicus (Strepomyces hygroscopicus ACCC No.40417) fermentative prodn rapamycin fermented liquid specifically may further comprise the steps:
A. dull and stereotyped the cultivation: behind the petridish that contains solid medium, the inoculation back was in 28 ℃ of constant temperature culture 5-7 days with the streptomyces hygroscopicus bacterial classification inoculation, and taking-up is put in 4 ℃ of refrigerators and preserves;
B. shake-flask seed is cultivated: the 50mL seed culture medium of in the 250mL triangular flask, packing into, the streptomyces hygroscopicus bacterial classification that 4 ℃ of refrigerators are preserved is inserted in the sterilization back, is placed on the vertical shaking table 29 ℃, cultivates 50-70h under the 220-230rpm condition, the shake-flask seed nutrient solution;
C. seeding tank seed culture: the shake-flask seed nutrient solution is inoculated in the seed tank culture base according to the long-pending 11%-15% inoculum size of seed tank culture matrix,, cultivates 35-46h under the 200-210rpm condition in 29 ℃, the seeding tank seed liquor;
D. ferment tank is cultivated: the seeding tank seed liquor is inoculated in fermentation tank culture medium according to the 11%-15% inoculum size of fermentation tank culture medium volume; In 29 ℃; Initial mixing speed is 180-220rpm, cultivates 72-96h, when treating that the thalline weight in wet base is 0.22-0.23g/ml; Regulate rotating speed to 255-300rpm, cultivate 105-150h; Fermentor cultivation total time 170-230h, wherein, when cultivating 24h and 48h, disposablely respectively add two kinds of materials: concentration is that 1.1% L-Ala and concentration are 0.5% Methionin.
The used substratum of above-mentioned steps contains following component respectively:
(1) solid medium contains rolled oats, glucose, yeast extract, lime carbonate and agar.
(2) seed culture medium contains Zulkovsky starch, peptone, yeast extract, peanut meal and glycerine.
(3) fermention medium contains W-Gum, soybean cake powder, peptone, glucose, K
2HPO
4, KH
2PO
4And NaCL.
The component content of the used substratum of above-mentioned steps is:
(1) solid medium contains rolled oats 30-48g, glucose 15-25g, yeast extract 5-18g, lime carbonate 1-4g and agar 10-20g, and pH is 6.8-7.4.
(2) seed culture medium contains Zulkovsky starch 30-48g, peptone 2-8g, yeast extract 5-16g, peanut meal 4-15g, glycerine 4-15g and lime carbonate 1-4g, and pH is 6.7-7.3.
(3) fermention medium contains W-Gum 9-18g, soybean cake powder 4-15g, peptone 3-10g, glucose 28-42g, K
2HPO
40-8g, KH
2PO
41-10g and NaCL1-4g, lime carbonate 1-5g and bubble enemy 1-6g, pH is 6.5-7.3.
The optimization formula of the used substratum of above-mentioned steps is:
(1) pH of solid medium is 7.2, and every liter of solid medium contains rolled oats 40g, glucose 20g, yeast extract 10g, lime carbonate 2g and agar 16g.
(2) pH of seed culture medium is 7.0, and every liter of seed culture medium contains Zulkovsky starch 40g, peptone 5g, yeast extract 10g, peanut meal 10g, glycerine 10g and lime carbonate 2g.
(3) pH of fermention medium is 6.9, and every liter of fermention medium contains W-Gum 13g, soybean cake powder 10g, peptone 6g, glucose 35g, K
2HPO
43g, KH
2PO
43g and NaCL1g, lime carbonate 3g and bubble enemy 3g.
The shake-flask seed substratum is identical with seeding tank seed culture based formulas, and this paper unifies the called after seed culture medium.
Through the selection of substratum and the control of culture condition; Especially the control of rotating speed; The present invention can produce the fermented liquid that contains the higher concentration rapamycin in cultivating the streptomyces hygroscopicus process; The rapamycin mean concns of the fermented liquid that in the fermenting experiment of 100L fermentor tank, obtains has reached 908.69mg/L; The rapamycin mean concns of the fermented liquid that in the fermenting experiment of 1000L fermentor tank, obtains has reached 895.28mg/L, more helps the extraction work of rapamycin, can satisfy need of industrial production.
Embodiment
For better explanation the present invention, further specify through embodiment below, but the present invention never only limits to following examples.
The fermentation culture of embodiment 1100L fermentor tank
(1) culture medium preparation
The solid culture based formulas is: every liter of solid medium contains rolled oats 40g, glucose 20g, yeast extract 10g, lime carbonate 2g and agar 16g, and pH is 7.2.Preparation 0.6L.
The seed culture based formulas is: every liter of seed culture medium contains Zulkovsky starch 40g, peptone 5g, yeast extract 10g, peanut meal 10g, glycerine 10g and lime carbonate 2g, and pH is 7.0.Preparation 7L.
Fermentative medium formula is: every liter of fermention medium contains W-Gum 13g, soybean cake powder 10g, peptone 6g, glucose 35g, K
2HPO
43g, KH
2PO
43g and NaCL1g, lime carbonate 3g and bubble enemy 3g, pH is 6.9.Preparation 60L.
(2) flat board of streptomyces hygroscopicus is cultivated
Pack into after solid medium configures (1x750mL) in the triangular flask, 121 ℃ of sterilizations 21 minutes simultaneously will clean petridish be wrapped 121 ℃ and were sterilized 21 minutes.After waiting that then the solid medium of being sterilized cools off slightly, the petridish after each sterilization is poured the about 50mL of solid medium into, and is subsequent use.
The streptomyces hygroscopicus bacterial classification inoculation in the petridish that contains solid medium, after postvaccinal petridish is placed in 28 ℃ of constant incubators or cultivates 5-7 days in the biochemical incubator, is taken out and is put in 4 ℃ of refrigerators and preserves subsequent use.
(3) the 250mL shake-flask seed is cultivated
In the 250mL triangular flask, pack into the seed culture medium of 50mL, the streptomyces hygroscopicus bacterial classification that 4 ℃ of refrigerators are preserved is inserted in the sterilization back, is placed on the vertical shaking table 29 ℃, 230rpm and cultivates 58h, gets the shake-flask seed nutrient solution, and is subsequent use.
(4) seed culture of 10L seeding tank
The seed culture medium 7L that in the 10L seeding tank, packs into, shake-flask seed nutrient solution 800mL is inserted in the sterilization back, 29 ℃ of temperature, air flow 1: 0.5, tank pressure 0.05MPa cultivates 46h under the condition of mixing speed 210rpm, obtains 10L seeding tank seed culture fluid.
(5) fermentation culture of 100L fermentor tank
The fermention medium 70L that in the fermentor tank of 100L, packs into, real jar of sterilization back moved into the seed culture fluid of 8L by the preparation of 10L seeding tank, air flow 1: 0.5; Tank pressure 0.05MPa, 29 ℃ of temperature, initial mixing speed is under the 200rpm condition; Fermentation culture 72h; During thalline weight in wet base 0.22-0.23g/ml, the adjusting rotating speed is 260rpm, cultivates 108h.Fermentor cultivation total time 180h.
During the fermentation, vegetative stage, disposablely respectively when selecting fermentation culture 24h and 48h add two kinds of materials: concentration is that 1.1% L-Ala and concentration are 0.5% Methionin.
Produce five batches of products as stated above respectively, fermentation culture is collected five batch fermentation liquid after finishing; Respectively get fermented liquid 2ml respectively; After centrifugal, use the 3ml80% acetone extract, extraction liquid is used efficient liquid phase chromatographic analysis; Obtain the concentration of the rapamycin of every batch fermentation liquid in the five batch fermentation liquid, mean concns is 908.69mg/L.Data are seen table 1.
Table 1,100L ferment tank result
The fermentation culture of embodiment 2100L fermentor tank
(1) culture medium preparation
The solid culture based formulas is: every liter of solid medium contains rolled oats 30g, glucose 25g, yeast extract 15g, lime carbonate 2g and agar 16g, and pH is 7.1.Preparation 0.6L.
The seed culture based formulas is: every liter of seed culture medium contains Zulkovsky starch 30g, peptone 5g, yeast extract 7g, peanut meal 10g, glycerine 10g, lime carbonate 2g, and pH is 6.9.Preparation 7L.
Fermentative medium formula is: every liter of fermention medium contains W-Gum 18g, soybean cake powder 13g, peptone 4g, glucose 35g, KH
2PO
44g and NaCL1g, lime carbonate 3g, bubble enemy 4g, pH is 6.5.Preparation 60L.
(2) flat board of streptomyces hygroscopicus is cultivated
Pack into after solid medium configures (1x750mL) in the triangular flask, 121 ℃ of sterilizations 21 minutes simultaneously will clean petridish be wrapped 121 ℃ and were sterilized 21 minutes.After waiting that then the solid medium of being sterilized cools off slightly, the petridish after each sterilization is poured the about 50mL of solid medium into, and is subsequent use.
The streptomyces hygroscopicus bacterial classification inoculation in the petridish that contains solid medium, after postvaccinal petridish is placed in 28 ℃ of constant incubators or cultivates 5-7 days in the biochemical incubator, is taken out and is put in 4 ℃ of refrigerators and preserves subsequent use.
(3) the 250mL shake-flask seed is cultivated
In the 250mL triangular flask, pack into the seed culture medium of 50mL, the streptomyces hygroscopicus bacterial classification that 4 ℃ of refrigerators are preserved is inserted in the sterilization back, is placed on the vertical shaking table 29 ℃, 222rpm and cultivates 62h, gets the shake-flask seed nutrient solution, and is subsequent use.
(4) seed culture of 10L seeding tank
The seed culture medium 7L that in the 10L seeding tank, packs into, shake-flask seed nutrient solution 850mL is inserted in the sterilization back, 29 ℃ of temperature, air flow 1: 0.5, tank pressure 0.05MPa cultivates 44h under the condition of mixing speed 205rpm, obtains 10L seeding tank seed culture fluid.
(5) fermentation culture of 100L fermentor tank
The fermention medium 70L that in the fermentor tank of 100L, packs into, real jar of sterilization back moved into the seed culture fluid of 8.5L by the preparation of 10L seeding tank, air flow 1: 0.5; Tank pressure 0.05MPa, 29 ℃ of temperature, initial mixing speed is under the 208rpm condition; Fermentation culture 80h; During thalline weight in wet base 0.22-0.23g/ml, the adjusting rotating speed is 270rpm, cultivates 106h.Fermentor cultivation total time 174h.
During the fermentation, vegetative stage, disposablely respectively when selecting fermentation culture 24h and 48h add two kinds of materials: concentration is that 1.1% L-Ala and concentration are 0.5% Methionin.
Produce five batches of products as stated above respectively, fermentation culture is collected five batch fermentation liquid after finishing; Respectively get fermented liquid 2ml respectively, centrifugal after, use the 3ml80% acetone extract; Extraction liquid is used efficient liquid phase chromatographic analysis; Obtain the concentration of the rapamycin of every batch fermentation liquid in the five batch fermentation liquid, mean concns is 896.32mg/L, and data are seen table 2.
Table 2,100L ferment tank result
The fermentation culture of embodiment 31000L fermentor tank
(1) culture medium preparation
The solid culture based formulas is: every liter of solid medium contains rolled oats 40g, glucose 20g, yeast extract 10g, lime carbonate 2g and agar 16g, and pH is 7.2.Preparation 1L
The seed culture based formulas is: every liter of seed culture medium contains Zulkovsky starch 40g, peptone 5g, yeast extract 10g, peanut meal 10g, glycerine 10g and lime carbonate 2g, and pH is 7.0.Preparation 70L
Fermentative medium formula is: every liter of fermention medium contains W-Gum 13g, soybean cake powder 10g, peptone 6g, glucose 35g, K
2HPO
43g, KH
2PO
43g and NaCL1g, lime carbonate 3g and bubble enemy 3g, pH is 6.9.Preparation 700L
(2) flat board of streptomyces hygroscopicus is cultivated
Pack into after solid medium configures (1x750mL) in the triangular flask, 121 ℃ of sterilizations 21 minutes simultaneously will clean petridish be wrapped 121 ℃ and were sterilized 21 minutes.After waiting that then the solid medium of being sterilized cools off slightly, the petridish after each sterilization is poured the about 50mL of solid medium into, and is subsequent use.
The streptomyces hygroscopicus bacterial classification inoculation in the petridish that contains solid medium, after postvaccinal petridish is placed in 28 ℃ of constant incubators or cultivates 5-7 days in the biochemical incubator, is taken out and is put in 4 ℃ of refrigerators and preserves subsequent use.
(3) the 250mL shake-flask seed is cultivated
In the 250mL triangular flask, pack into the seed culture medium of 50mL, the streptomyces hygroscopicus bacterial classification that 4 ℃ of refrigerators are preserved is inserted in the sterilization back, is placed on the vertical shaking table 29 ℃, 230rpm and cultivates 60h, gets the shake-flask seed nutrient solution, and is subsequent use.
(4) seed culture of 10L seeding tank
The seed culture medium 7L that in the 10L first class seed pot, packs into, shake-flask seed nutrient solution 900mL is inserted in the sterilization back, 29 ℃ of temperature, air flow 1: 0.5, tank pressure 0.05MPa cultivates 40h under the condition of mixing speed 210rpm, obtains the first class seed pot nutrient solution.
(5) seed culture of 100L seeding tank
The seed culture medium 70L that in 100L secondary seed jar, packs into, first class seed pot nutrient solution 9L is inserted in the sterilization back, 29 ℃ of temperature, air flow 1: 0.5, tank pressure 0.05MPa cultivates 36h under the condition of mixing speed 206rpm, obtains secondary seed jar nutrient solution.
(6) fermentation culture of 1000L fermentor tank
The fermention medium 700L that in the fermentor tank of 1000L, packs into, real jar of sterilization back moved into the secondary seed jar nutrient solution of preparation in the 90L seeding tank, air flow 1: 0.5; Tank pressure 0.05MPa, 29 ℃ of temperature, initial mixing speed is under the 185rpm condition; Fermentation culture 78h; During thalline weight in wet base 0.22-0.23g/ml, the adjusting rotating speed is 255rpm, cultivates 140h.Fermentation culture total time 218h.
During the fermentation, vegetative stage, disposablely respectively when selecting fermentation culture 24h and 48h add two kinds of materials: concentration is that 1.1% L-Ala and concentration are 0.5% Methionin.
Produce five batches of products as stated above respectively, fermentation culture is collected five batch fermentation liquid after finishing; Respectively get fermented liquid 2ml respectively, centrifugal after, use the 3ml80% acetone extract; Extraction liquid is used efficient liquid phase chromatographic analysis; Obtain the concentration of the rapamycin of every batch fermentation liquid in the five batch fermentation liquid, mean concns is 895.28mg/L, and data are seen table 3.
Table 3,1000L ferment tank result
The fermentation culture of embodiment 41000L fermentor tank
(1) culture medium preparation
The solid culture based formulas is: every liter of solid medium contains rolled oats 30g, glucose 25g, yeast extract 15g, lime carbonate 2g and agar 16g, and pH is 7.1.Preparation 1L.
The seed culture based formulas is: every liter of seed culture medium contains Zulkovsky starch 30g, peptone 5g, yeast extract 7g, peanut meal 10g, glycerine 10g, lime carbonate 2g, and pH is 6.9.Preparation 70L.
Fermentative medium formula is: every liter of fermention medium contains W-Gum 18g, soybean cake powder 13g, peptone 4g, glucose 35g, KH
2PO
44g and NaCL1g, lime carbonate 3g, bubble enemy 4g, pH is 6.5.Preparation 700L.
(2) flat board of streptomyces hygroscopicus is cultivated
Pack into after solid medium configures (1x750mL) in the triangular flask, 121 ℃ of sterilizations 21 minutes simultaneously will clean petridish be wrapped 121 ℃ and were sterilized 21 minutes.After waiting that then the solid medium of being sterilized cools off slightly, the petridish after each sterilization is poured the about 50mL of solid medium into, and is subsequent use.
The streptomyces hygroscopicus bacterial classification inoculation in the petridish that contains solid medium, after postvaccinal petridish is placed in 28 ℃ of constant incubators or cultivates 5-7 days in the biochemical incubator, is taken out and is put in 4 ℃ of refrigerators and preserves subsequent use.
(3) the 250mL shake-flask seed is cultivated
In the 250mL triangular flask, pack into the seed culture medium of 50mL, the streptomyces hygroscopicus bacterial classification that 4 ℃ of refrigerators are preserved is inserted in the sterilization back, is placed on the vertical shaking table 29 ℃, 225rpm and cultivates 54h, gets the shake-flask seed nutrient solution, and is subsequent use.
(4) seed culture of 10L seeding tank
The seed culture medium 7L that in the 10L first class seed pot, packs into, shake-flask seed nutrient solution 780mL is inserted in the sterilization back, 29 ℃ of temperature, air flow 1: 0.5, tank pressure 0.05MPa cultivates 45h under the condition of mixing speed 208rpm, obtains the first class seed pot nutrient solution.
(5) seed culture of 100L seeding tank
The seed culture medium 70L that in 100L secondary seed jar, packs into, first class seed pot nutrient solution 7.8L is inserted in the sterilization back, 29 ℃ of temperature, air flow 1: 0.5, tank pressure 0.05MPa cultivates 39h under the condition of mixing speed 204rpm, obtains secondary seed jar nutrient solution.
(6) fermentation culture of 1000L fermentor tank
The fermention medium 700L that in the fermentor tank of 1000L, packs into, real jar of sterilization back moved into the secondary seed jar nutrient solution of preparation in the 78L seeding tank, air flow 1: 0.5; Tank pressure 0.05MPa, 29 ℃ of temperature, initial mixing speed is under the 195rpm condition; Fermentation culture 89h; During thalline weight in wet base 0.22-0.23g/ml, the adjusting rotating speed is 265rpm, cultivates 125h.Fermentation culture total time 201h.
During the fermentation, vegetative stage, disposablely respectively when selecting fermentation culture 24h and 48h add two kinds of materials: concentration is that 1.1% L-Ala and concentration are 0.5% Methionin.
Produce five batches of products as stated above respectively, fermentation culture is collected five batch fermentation liquid after finishing; Respectively get fermented liquid 2ml respectively, centrifugal after, use the 3ml80% acetone extract; Extraction liquid is used efficient liquid phase chromatographic analysis; Obtain the concentration of the rapamycin of every batch fermentation liquid in the five batch fermentation liquid, mean concns is 877.92mg/L, and data are seen table 4.
Table 4,1000L ferment tank result
Claims (11)
1. cultivate the method for streptomyces hygroscopicus (Strepomyces hygroscopicus ACCC No.40417) fermentative prodn rapamycin fermented liquid, it is characterized in that this method may further comprise the steps:
A. dull and stereotyped the cultivation: in the petridish that contains solid medium, constant temperature culture is 5-7 days after the inoculation with the streptomyces hygroscopicus bacterial classification inoculation;
B. shake-flask seed is cultivated: the seed culture medium of in triangular flask, packing into, and the dull and stereotyped streptomyces hygroscopicus bacterial classification of cultivating gained is inserted in the back of sterilizing, and is placed on the vertical shaking table, cultivates 50-70h under the 220-230rpm condition, gets the shake-flask seed nutrient solution;
C. seeding tank seed culture: the shake-flask seed nutrient solution is inoculated in the seed tank culture base according to the long-pending 11%-15% inoculum size of seed tank culture matrix, cultivates 35-46h under the 200-210rpm condition, the seeding tank seed liquor;
D. ferment tank is cultivated: the seeding tank seed liquor is inoculated in fermentation tank culture medium according to the 11%-15% inoculum size of fermentation tank culture medium volume; Initial mixing speed is 180-220rpm; Cultivate 72-96h; During thalline weight in wet base 0.22-0.23g/ml, the adjusting rotating speed is 255-300rpm, cultivates 105-150h; Fermentor cultivation total time 170-230h.
2. the method for cultivation streptomyces hygroscopicus (Strepomyces hygroscopicus ACCC No.40417) fermentative prodn rapamycin fermented liquid as claimed in claim 1 is characterized in that this method may further comprise the steps:
A. dull and stereotyped the cultivation: in the petridish that contains solid medium, the inoculation back is after 28 ℃ of constant temperature culture 5-7 days with the streptomyces hygroscopicus bacterial classification inoculation, and taking-up is put in 4 ℃ of refrigerators and preserves;
B. shake-flask seed cultivation: the 50mL seed culture medium of in the 250mL triangular flask, packing into, the streptomyces hygroscopicus bacterial classification that 4 ℃ of refrigerators are preserved is inserted in the sterilization back, is placed on the vertical shaking table, in 29 ℃, cultivates 50-70h under the 220-230rpm condition, gets the shake-flask seed nutrient solution;
C. seeding tank seed culture: the shake-flask seed nutrient solution is inoculated in the seed tank culture base according to the long-pending 11%-15% inoculum size of seed tank culture matrix,, cultivates 35-46h under the 200-210rpm condition in 29 ℃, the seeding tank seed liquor;
D. ferment tank is cultivated: the seeding tank seed liquor is inoculated in fermentation tank culture medium according to the 11%-15% inoculum size of fermentation tank culture medium volume; In 29 ℃; Initial mixing speed is 180-220rpm, cultivates 72-96h, during thalline weight in wet base 0.22-0.23g/ml; The adjusting rotating speed is 255-300rpm, cultivates 105-150h; Fermentor cultivation total time 170-230h, wherein, the disposable respectively concentration of adding is that 1.1% L-Ala and concentration are 0.5% Methionin when cultivating 24h and 48h.
3. the method for claim 1 is characterized in that said solid medium contains rolled oats, glucose, yeast extract, lime carbonate and agar.
4. method as claimed in claim 3 is characterized in that said solid medium contains rolled oats 30-48g, glucose 15-25g, yeast extract 5-18g, lime carbonate 1-4g and agar 10-20g, pH6.8-7.4.
5. method as claimed in claim 4, the pH that it is characterized in that said solid medium is 7.2, and every liter of solid medium contains rolled oats 40g, glucose 20g, yeast extract 10g, lime carbonate 2g and agar 16g.
6. the method for claim 1 is characterized in that the seed culture medium that is adopted contains Zulkovsky starch, peptone, yeast extract, peanut meal and glycerine.
7. method as claimed in claim 6 is characterized in that the seed culture medium that is adopted contains Zulkovsky starch 30-48g, peptone 2-8g, yeast extract 5-16g, peanut meal 4-15g, glycerine 4-15g and lime carbonate 1-4g, and pH is 6.7-7.3.
8. method as claimed in claim 7, the pH of the seed culture medium that it is characterized in that being adopted is 7.0, and every liter of seed culture medium contains Zulkovsky starch 40g, peptone 5g, yeast extract 10g, peanut meal 10g, glycerine 10g and lime carbonate 2g.
9. the method for claim 1 is characterized in that said fermention medium contains W-Gum, soybean cake powder, peptone, glucose, K
2HPO
4, KH
2PO
4And NaCL.
10. method as claimed in claim 9 is characterized in that said fermention medium contains W-Gum 9-18g, soybean cake powder 4-15g, peptone 3-10g, glucose 28-42g, K
2HPO
40-8g, KH
2PO
41-10g and NaCL1-4g, lime carbonate 1-5g and bubble enemy 1-6g, pH is 6.5-7.3.
11. method as claimed in claim 10, the pH that it is characterized in that said fermention medium is 6.9, and every liter of fermention medium contains W-Gum 13g, soybean cake powder 10g, peptone 6g, glucose 35g, K
2HPO
43g, KH
2PO
43g and NaCL1g, lime carbonate 3g and bubble enemy 3g.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104388492A (en) * | 2014-11-24 | 2015-03-04 | 苏州嘉禧萝生物科技有限公司 | Method for producing rapamycin by using streptomyces hygroscopicus |
| CN104604946A (en) * | 2015-01-12 | 2015-05-13 | 邱德文 | Biological protein preparation for preventing and controlling citrus huanglongbing and preparation method for biological protein preparation |
| CN107365811A (en) * | 2017-09-15 | 2017-11-21 | 常州兰陵制药有限公司 | Utilize the technique of actinoplanes fermenting and producing rapamycin |
Citations (1)
| 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 |
-
2010
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Patent Citations (1)
| 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 |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388492A (en) * | 2014-11-24 | 2015-03-04 | 苏州嘉禧萝生物科技有限公司 | Method for producing rapamycin by using streptomyces hygroscopicus |
| CN104604946A (en) * | 2015-01-12 | 2015-05-13 | 邱德文 | Biological protein preparation for preventing and controlling citrus huanglongbing and preparation method for biological protein preparation |
| CN104604946B (en) * | 2015-01-12 | 2017-10-13 | 邱德文 | It is a kind of for biologic protein agents of prevention and control Citrus Huanglongbing pathogen and preparation method thereof |
| CN107365811A (en) * | 2017-09-15 | 2017-11-21 | 常州兰陵制药有限公司 | Utilize the technique of actinoplanes fermenting and producing rapamycin |
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Effective date of registration: 20151218 Address after: 273400 Shandong province Feixian County North Ring Road No. 1 Patentee after: Lunan New Era Biological Technology Co., Ltd. Address before: 276005 Shandong province Feixian County North Ring Road No. 1 Patentee before: Shandong Xinshidai Pharmaceutical Industry Co., Ltd. |