CN105232457A - Method for preparing doramectin sterile solution - Google Patents
Method for preparing doramectin sterile solution Download PDFInfo
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- CN105232457A CN105232457A CN201510787886.3A CN201510787886A CN105232457A CN 105232457 A CN105232457 A CN 105232457A CN 201510787886 A CN201510787886 A CN 201510787886A CN 105232457 A CN105232457 A CN 105232457A
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- Prior art keywords
- doractin
- organic solvent
- sterile solution
- preparation
- doramectin
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- 238000000034 method Methods 0.000 title claims abstract description 32
- 239000008174 sterile solution Substances 0.000 title claims abstract description 20
- QLFZZSKTJWDQOS-YDBLARSUSA-N doramectin Chemical compound O1[C@@H](C)[C@H](O)[C@@H](OC)C[C@@H]1O[C@@H]1[C@@H](OC)C[C@H](O[C@@H]2C(=C/C[C@@H]3C[C@@H](C[C@@]4(O3)C=C[C@H](C)[C@@H](C3CCCCC3)O4)OC(=O)[C@@H]3C=C(C)[C@@H](O)[C@H]4OC\C([C@@]34O)=C/C=C/[C@@H]2C)/C)O[C@H]1C QLFZZSKTJWDQOS-YDBLARSUSA-N 0.000 title abstract description 9
- 229960003997 doramectin Drugs 0.000 title abstract description 9
- 238000000855 fermentation Methods 0.000 claims abstract description 42
- 230000004151 fermentation Effects 0.000 claims abstract description 42
- 239000002893 slag Substances 0.000 claims abstract description 41
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 35
- 239000003960 organic solvent Substances 0.000 claims abstract description 33
- 239000008213 purified water Substances 0.000 claims abstract description 31
- 238000001914 filtration Methods 0.000 claims abstract description 30
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 22
- 239000012043 crude product Substances 0.000 claims abstract description 18
- 239000007787 solid Substances 0.000 claims abstract description 17
- 238000001035 drying Methods 0.000 claims abstract description 15
- 238000001471 micro-filtration Methods 0.000 claims abstract description 13
- 238000001816 cooling Methods 0.000 claims abstract description 11
- 238000002425 crystallisation Methods 0.000 claims abstract description 10
- 230000008025 crystallization Effects 0.000 claims abstract description 10
- 238000000605 extraction Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 41
- 241000894006 Bacteria Species 0.000 claims description 40
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 36
- 238000003756 stirring Methods 0.000 claims description 34
- 239000007788 liquid Substances 0.000 claims description 28
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 15
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000000706 filtrate Substances 0.000 claims description 12
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- 239000013078 crystal Substances 0.000 claims description 8
- 235000011187 glycerol Nutrition 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 229920002565 Polyethylene Glycol 400 Polymers 0.000 claims description 6
- 229910021529 ammonia Inorganic materials 0.000 claims description 6
- JLFNLZLINWHATN-UHFFFAOYSA-N pentaethylene glycol Chemical compound OCCOCCOCCOCCOCCO JLFNLZLINWHATN-UHFFFAOYSA-N 0.000 claims description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 239000012670 alkaline solution Substances 0.000 claims description 4
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 claims description 4
- 238000007781 pre-processing Methods 0.000 claims description 4
- 125000002573 ethenylidene group Chemical group [*]=C=C([H])[H] 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 239000000047 product Substances 0.000 abstract description 3
- 238000000746 purification Methods 0.000 abstract description 2
- 241001468227 Streptomyces avermitilis Species 0.000 abstract 1
- 230000000869 mutational effect Effects 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- 238000011084 recovery Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- NZNMSOFKMUBTKW-UHFFFAOYSA-N cyclohexanecarboxylic acid Chemical compound OC(=O)C1CCCCC1 NZNMSOFKMUBTKW-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 239000005660 Abamectin Substances 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 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 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- -1 ammonia carboxylic acid Chemical class 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- VZFUCHSFHOYXIS-UHFFFAOYSA-N cycloheptane carboxylic acid Natural products OC(=O)C1CCCCCC1 VZFUCHSFHOYXIS-UHFFFAOYSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Extraction Or Liquid Replacement (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention relates to a method for preparing a doramectin sterile solution. The method comprises the following steps: firstly, pre-treating a doramectin fermentation broth prepared by fermenting mutational streptomyces avermitilis which serves as a strain; performing flash drying to obtain dried strain slag, and extracting with an organic solvent a; cooling and adding purified water to separate; filtering to obtain a doramectin crude product, and dissolving the crude product with an organic solvent; performing ultrafiltration and crystallization; and dissolving a solid doramectin obtained by one more filtration with an organic solvent B; and performing microfiltration to obtain the doramectin sterile solution. The method can be used for realizing effective separation and purification on the doramectin in the fermentation broth, the total extraction yield is over 85 percent, the product quality accords with the requirement, the production cost is reduced, and the international market competition of doramectin can be improved.
Description
Technical field
The invention belongs to antibiotic extractive technique field, particularly relate to a kind of preparation method of doractin sterile solution.
Background technology
Doractin be by the Avid kyowamycin new strains of gene recombinaton by add ring ammonia carboxylic acid be precursor substance ferment a kind of Avermectins antibiotic, belong to third generation avermitilis strain class medicine.
In prior art, the extraction of Duola's mycin adopts Organic Alcohol lixiviate coupled ion switching technology or Organic Alcohol lixiviate in conjunction with chromatographic technique extraction purification doractin fermentation liquid, obtains its finished product, and its subject matter existed is:
1 extract yield is low.Average extract yield is only about 70%.
2 do not carry out pretreatment to doractin fermentation liquid, and in wet bacteria slag, moisture has exceeded 60%, directly with an organic solvent carry out lixiviate, cause liquor capacity to increase, and add the intractability of lower procedure.
3 production technologies are comparatively complicated, and the production cycle is long.
4 production costs are higher, and contrast like product, the market competitiveness is more weak.
Summary of the invention
The object of the invention is to overcome above-mentioned defect of the prior art, provide one effectively to improve extract yield, technique is simple, with short production cycle, and reduces the preparation method of the doractin sterile solution of subsequent technique intractability.
Technical scheme taked for achieving the above object is:
A kind of preparation method of doractin sterile solution, it is characterized in that its technical process is: first the doractin fermentation liquid produced for strain fermentation with the Avid kyowamycin that suddenlys change is carried out pretreatment, the dry bacterium slag organic solvent A lixiviate obtained after expansion drying, lower the temperature and add purified water and separate out, after filtering, the doractin crude product organic solvent B of gained is dissolved, ultrafiltration, crystallization, after again filtering, the solid doractin organic solvent B that obtains of gained is dissolved, and microfiltration can obtain doractin sterile solution;
Described preprocessing process is: first, doractin fermentation liquid acid solution is adjusted pH2 ~ 3, filter after stirring 10 ~ 20min, gained wet bacteria slag dilute with water, continues stirring 10 ~ 20min, again filters, pH9-10 is adjusted with alkaline solution after gained wet bacteria slag dilute with water, again stir 20-40min, leave standstill 80-100min, plate-and-frame filtration;
Described organic solvent A is methanol or ethanol or butanols or isobutanol or acetone;
Described organic solvent B is medicinal glycerin or PEG400.
Hydrochloric acid or the sulfuric acid solution of described acid solution to be mass concentration be 20-30%, ammonia or the sodium hydroxide solution of alkaline solution to be mass concentration be 30-40%.
Described expansion drying uses flash dryer to bacterium slag moisture lower than 5%, and control inlet temperature 100 ~ 120 DEG C, leaving air temp 70 ~ 90 DEG C, reinforced rotating speed is 50 ~ 400r/min.
Described dry bacterium slag organic solvent A lixiviate twice, first time lixiviate adds 4-5L organic solvent A according to the dry bacterium slag of every 1kg, and second time adds 2-2.5L organic solvent A according to the dry bacterium slag of every 1kg, each extraction time 100-150min.
Described cooling also adds purified water precipitation process and is: organic solvent lixiviating solution is cooled to 0-5 DEG C, adds 4.5-5 times of purified water of its volume, leaves standstill 150-190min, filters, obtains doractin crude product.
Described doractin crude product or solid doractin are dissolved in organic solvent B, control its mass concentration 20-30%.
In described ultra-filtration process, Stress control is at 0.2-0.3MPa, and the material of ultrafilter membrane is vinylidene, and aperture is 100nm.
Described crystallization process is: under stirring, the doractin ultrafiltrate of ultrafiltration gained is cooled to-10-0 DEG C, cooling rate controls at 2-3 DEG C/h, when there is crystalline polamer, add crystal seed doractin, consumption is the 0.1-0.3% of doractin ultrafiltrate volume, continues to stir 60-100min, then growing the grain 180-200min, filtration can obtain solid doractin.
Described microfiltration adopts membrane filter system, and this equipment is separately installed with the filter element that specification is 0.45um and 0.22um, and in filter process, Stress control, at 0.2-0.3MPa, filters and terminates, and collects filtrate, obtains aseptic doractin solution.
Described preprocessing process is: it is the 20-30% of fermentating liquid volume that first time dilutes purified water consumption used, and it is the 40-50% of fermentating liquid volume that second time dilutes purified water consumption used.
Technical advantage of the present invention is embodied in:
1 extract yield is higher, reaches more than 85%.
2 the present invention adopt filtering technique to replace ion exchange and chromatographic technique, reduce the difficulty of production technology, shorten the production cycle.
First 3 the present invention have carried out pretreatment to fermentation liquid, thus reduce the intractability (reason: doractin fermentation liquid after pretreatment, can remove most of insoluble granule, and insoluble granule accounts for more than 80% of total impurities) of subsequent technique.
The preparation mainly injection of 4 doractin clinical practices.According to external veterinary drug Doramectin injection fluid quality standard, the invention provides a kind of method preparing doractin sterile solution, the doractin solution obtained by the method carries out compatibility, and its solution quality meets quality standard.
specific implementation method
Be explained the present invention with example below, it should be understood that example is for illustration of the present invention instead of limitation of the present invention.Scope of the present invention and core content are determined according to claims.
Fermentation liquid source in following embodiment:
Adopt second order fermentation mode of manufacture doractin, it produces strain for sudden change Avid kyowamycin, and doractin fermentation medium seed formula is corn starch, soybean cake powder, glucose, cottonseed meal, fermentative medium formula is corn starch, soybean cake powder, yeast powder, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, calcium carbonate, amylase, precursor cyclohexanecarboxylic acid.Doractin fermentation temperature is 28 DEG C, and fermentation period is 11 days, and fermentation unit is generally at 1200mg/L.
The material of ultrafilter membrane is vinylidene, and aperture is 100nm.
The filter element used in microfiltration provides membrane filter system by Co., Ltd in Mi Libo, and this equipment is separately installed with the filter element that specification is 0.45um and 0.22um, and in filter process, Stress control is at 0.2-0.3MPa.
Described crystal seed is sterile bulk drug doractin.
embodiment 1
Doractin fermentation liquid 10m
3, tire as 1324mg/L.
Fermentation liquor pretreatment: first, is adjusted to 2 with 20% hydrochloric acid solution by fermentation liquid pH, stirs 10min, obtains wet bacteria slag after filtration.Then, in this wet bacteria slag, purified water 2m is added
3, stir 10min, again filter.Finally, in the wet bacteria slag again filtering gained, purified water 4m is added
3, the ammonia spirit with 30% regulates pH to 9, stirs 20min, then leaves standstill 80min.Leave standstill after terminating and fermentation liquid is carried out plate-and-frame filtration, obtain doractin wet bacteria slag.
Expansion drying: use flash dryer, control inlet temperature 100 ~ 120 DEG C, leaving air temp 70 ~ 90 DEG C, reinforced rotating speed is 50 ~ 400r/min.Expansion drying terminates, and obtain doractin dry bacterium slag 756kg, wherein moisture is 4.1%.
Organic solvent A lixiviate: first time lixiviate, adds methanol 3050L; Second time lixiviate, adds methanol 1550L, merges twice lixiviating solution.
Lower the temperature and add purified water and separate out: lixiviating solution cooling temperature is down to 0 DEG C, adds purified water 20700L, leaves standstill 150-180min.Leave standstill after terminating and filter, obtain doractin crude product 12.5kg.
Dissolve: by doractin dissolving crude product in medicinal glycerin, concentration controls 20%.
Ultrafiltration: in ultra-filtration process, Stress control is at 0.2-0.3MPa.Ultrafiltration terminates, and collects filtrate 62L.
Crystallization: under stirring, is cooled to-10 DEG C by doractin ultrafiltrate, and cooling rate controls at 2-3 DEG C/h.When there is crystalline polamer, add crystal seed 62g.Continue to stir 60min, then growing the grain 180min.Growing the grain terminates rear filtration, obtains solid doractin 11.4kg.
Dissolve: be dissolved in medicinal glycerin by solid doractin, concentration controls 20%.
Microfiltration: adopt membrane filter system to cross microfiltration doractin solution, this equipment is separately installed with the filter element that specification is 0.45um and 0.22um.In filter process, Stress control is at 0.2-0.3MPa.Filter and terminate, collect filtrate, obtain 20% aseptic doractin solution.
Extract total recovery: 85.6%.
embodiment 2
Doractin fermentation 10m
3, tire as 1296mg/L.
Fermentation liquor pretreatment: first, is adjusted to 2.3 with 23% sulfuric acid solution by fermentation liquid pH, stirs 13min, obtains wet bacteria slag after filtration.Secondly, in this wet bacteria slag, purified water 2.3m is added
3, stir 13min, again filter; Finally, purified water 4.2m is added again filtering in gained wet bacteria slag
3, the sodium hydroxide solution with 33% regulates doractin fermentation liquid pH to 9.3, stirs 25min, then leaves standstill 85min.Leave standstill after terminating and fermentation liquid is carried out plate-and-frame filtration, obtain doractin wet bacteria slag.
Expansion drying: use flash dryer, control inlet temperature 100 ~ 120 DEG C, leaving air temp 70 ~ 90 DEG C, reinforced rotating speed is 50 ~ 400r/min.Expansion drying terminates, and obtain doractin dry bacterium slag 731kg, wherein moisture is 4.0%.
Organic solvent lixiviate: first time lixiviate, adds methanol 3080L; Second time lixiviate, adds methanol 1535L, merges twice lixiviating solution.
Lower the temperature and add purified water and separate out: organic solvent lixiviating solution temperature is down to 1 DEG C, adds purified water 21300L, leave standstill 160min.Leave standstill after terminating and filter, obtain doractin crude product 12.2kg.
Dissolve: by doractin dissolving crude product in PEG400, concentration controls 22%.
Ultrafiltration: in ultra-filtration process, Stress control is at 0.2-0.3MPa.Ultrafiltration terminates, and collects filtrate 55.3L.
Crystallization: under stirring, is cooled to-8 DEG C by doractin ultrafiltrate, and cooling rate controls at 2-3 DEG C/h.When there is crystalline polamer, add crystal seed 83g.Continue to stir 70min, then growing the grain 185min.Growing the grain terminates rear filtration, obtains solid doractin 11.29kg.
Dissolve: be dissolved in PEG400 by solid doractin, concentration controls 23%.
Microfiltration: by doractin solution membrane filter system microfiltration, this equipment is separately installed with the filter element that specification is 0.45um and 0.22um.In filter process, Stress control is at 0.2-0.3MPa.Filter and terminate, collect filtrate, obtain 23% aseptic doractin solution.
Extract total recovery: 86.5%.
embodiment 3
Doractin fermentation 10m
3, tire as 1247mg/L.
Fermentation liquor pretreatment: first, is adjusted to 2.5 with 25% hydrochloric acid solution by fermentation liquid pH, stirs 15min, obtains wet bacteria slag after filtration.Secondly, in this wet bacteria slag, purified water 2.5m is added
3, stir 15min, again filter; Finally, purified water 4.5m is added again filtering in gained wet bacteria slag
3, the ammonia spirit with 35% regulates doractin fermentation liquid pH to 9.5, stirs 30min, then leaves standstill 90min.Leave standstill after terminating and fermentation liquid is carried out plate-and-frame filtration, obtain doractin wet bacteria slag.
Expansion drying: use flash dryer, control inlet temperature 100 ~ 120 DEG C, leaving air temp 70 ~ 90 DEG C, reinforced rotating speed is 50 ~ 400r/min.Expansion drying terminates, and obtain doractin dry bacterium slag 747kg, wherein moisture is 3.9%.
Organic solvent lixiviate: first time lixiviate, adds methanol 3365L; Second time lixiviate, adds methanol 1718L, merges twice lixiviating solution.
Lower the temperature and add purified water and separate out: organic solvent lixiviating solution is down to 3 DEG C, adds purified water 23890L, leave standstill 170min.Leave standstill after terminating and filter, obtain doractin crude product 12kg.
Dissolve: by doractin dissolving crude product in medicinal glycerin, concentration controls 25%.
Ultrafiltration: in ultra-filtration process, Stress control is at 0.2-0.3MPa.Ultrafiltration terminates, and collects filtrate 48L.
Crystallization: in whipping process, is cooled to-5 DEG C by doractin ultrafiltrate, and cooling rate controls at 2-3 DEG C/h.When there is crystalline polamer, add crystal seed 96g.Continue to stir 80min, then growing the grain 190min.Growing the grain terminates rear filtration, obtains solid doractin 10.86kg.
Dissolve: doractin dissolution of solid is in medicinal glycerin, and concentration controls 25%.
Microfiltration: by doractin solution film filter plant, this equipment is separately installed with the filter element that specification is 0.45um and 0.22um.In filter process, Stress control is at 0.2-0.3MPa.Filter and terminate, collect filtrate, obtain 25% aseptic doractin solution.
Extract total recovery: 87.1%.
embodiment 4
Doractin fermentation 10m
3, tire as 1301mg/L.
Fermentation liquor pretreatment: first, is adjusted to 2.7 with 28% hydrochloric acid solution by fermentation liquid pH, stirs 18min, obtains wet bacteria slag after filtration.Secondly, in this wet bacteria slag, purified water 2.7m is added
3, stir 18min, again filter; Finally, purified water 4.7m is added again filtering in gained wet bacteria slag
3, the sodium hydroxide solution with 38% regulates doractin fermentation liquid pH to 9.7, stirs 35min, then leaves standstill 95min.Leave standstill after terminating and fermentation liquid is carried out plate-and-frame filtration, obtain doractin wet bacteria slag.
Expansion drying: use flash dryer, control inlet temperature 100 ~ 120 DEG C, leaving air temp 70 ~ 90 DEG C, reinforced rotating speed is 50 ~ 400r/min.Expansion drying terminates, and obtain doractin dry bacterium slag 750kg, wherein moisture is 3.9%.
Organic solvent lixiviate: first time lixiviate, adds methanol 3525L; Second time lixiviate, adds methanol 1800L, merges twice lixiviating solution.
Lower the temperature and add purified water and separate out: organic solvent lixiviating solution temperature is down to 4 DEG C, adds purified water 23890L, leave standstill 170min.Leave standstill after terminating and filter, obtain doractin crude product 12.21kg.
Dissolve: doractin dissolving crude product is in PEG400, and concentration controls 28%.
Ultrafiltration: in ultra-filtration process, Stress control is at 0.2-0.3MPa.Ultrafiltration terminates, and collects filtrate 43.6L.
Crystallization: under stirring, is cooled to-3 DEG C by doractin ultrafiltrate, and cooling rate controls at 2-3 DEG C/h.When there is crystalline polamer, add crystal seed 110g.Continue to stir 90min, then growing the grain 195min.Growing the grain terminates rear filtration, obtains solid doractin 11.28kg.
Dissolve: doractin dissolution of solid is in PEG400, and concentration controls 28%.
Microfiltration: by doractin solution film filter plant, this equipment is separately installed with the filter element that specification is 0.45um and 0.22um.In filter process, Stress control is at 0.2-0.3MPa.Filter and terminate, collect filtrate, obtain 28% aseptic doractin solution.
Extract total recovery: 86.7%.
embodiment 5
Doractin fermentation 10m
3, tire as 1290mg/L.
Fermentation liquor pretreatment: first, is adjusted to 3 with 30% sulfuric acid solution by fermentation liquid pH, stirs 20min, obtains wet bacteria slag after filtration.Secondly, purified water 3m is added in wet bacteria slag
3, stir 18min, again filter; Finally, purified water 5m is added in wet bacteria slag
3, the ammonia spirit with 40% regulates doractin fermentation liquid pH to 10, stirs 40min, then leaves standstill 100min.Leave standstill after terminating and fermentation liquid is carried out plate-and-frame filtration, obtain doractin wet bacteria slag.
Expansion drying: use flash dryer, control inlet temperature 100 ~ 120 DEG C, leaving air temp 70 ~ 90 DEG C, reinforced rotating speed is 50 ~ 400r/min.Expansion drying terminates, and obtain doractin dry bacterium slag 753kg, wherein moisture is 4.0%.
Organic solvent lixiviate: first time lixiviate, adds methanol 3765L; Second time lixiviate, adds methanol 1882L, merges twice lixiviating solution.
Lower the temperature and add purified water and separate out: organic solvent lixiviating solution temperature is down to 5 DEG C, adds purified water 28237L, leave standstill 180min.Leave standstill after terminating and filter, obtain doractin crude product 12.12kg.
Dissolve: doractin dissolving crude product is in medicinal glycerin, and concentration controls 30%.
Ultrafiltration: in ultra-filtration process, Stress control is at 0.2-0.3MPa.Ultrafiltration terminates, and collects filtrate 40.4L.
Crystallization: under stirring, is cooled to 0 DEG C by doractin ultrafiltrate, and cooling rate controls at 2-3 DEG C/h.When there is crystalline polamer, add crystal seed 120g.Continue to stir 100min, then growing the grain 200min.Growing the grain terminates rear filtration, obtains solid doractin 11.13kg.
Dissolve: doractin dissolution of solid is in medicinal glycerin, and concentration controls 30%.
Microfiltration: by doractin solution film filter plant, this equipment is separately installed with the filter element that specification is 0.45um and 0.22um.In filter process, Stress control is at 0.2-0.3MPa.Filter and terminate, collect filtrate, obtain 30% aseptic doractin solution.
Extract total recovery: 86.2%.
Claims (10)
1. the preparation method of a doractin sterile solution, it is characterized in that its technical process is: first the doractin fermentation liquid produced for strain fermentation with the Avid kyowamycin that suddenlys change is carried out pretreatment, the dry bacterium slag organic solvent A lixiviate obtained after expansion drying, lower the temperature and add purified water and separate out, after filtering, the doractin crude product organic solvent B of gained is dissolved, ultrafiltration, crystallization, after again filtering, the solid doractin organic solvent B that obtains of gained is dissolved, and microfiltration can obtain doractin sterile solution;
Described preprocessing process is: first, doractin fermentation liquid acid solution is adjusted pH2 ~ 3, filter after stirring 10 ~ 20min, gained wet bacteria slag dilute with water, continues stirring 10 ~ 20min, again filters, pH9-10 is adjusted with alkaline solution after gained wet bacteria slag dilute with water, again stir 20-40min, leave standstill 80-100min, plate-and-frame filtration;
Described organic solvent A is methanol or ethanol or butanols or isobutanol or acetone;
Described organic solvent B is medicinal glycerin or PEG400.
2. according to the preparation method of doractin sterile solution according to claim 1, it is characterized in that described acid solution to be mass concentration be hydrochloric acid or the sulfuric acid solution of 20-30%, ammonia or the sodium hydroxide solution of alkaline solution to be mass concentration be 30-40%.
3. according to the preparation method of doractin sterile solution according to claim 1, it is characterized in that described expansion drying uses flash dryer to bacterium slag moisture lower than 5%, control inlet temperature 100 ~ 120 DEG C, leaving air temp 70 ~ 90 DEG C, reinforced rotating speed is 50 ~ 400r/min.
4. according to the preparation method of doractin sterile solution according to claim 1, it is characterized in that described dry bacterium slag organic solvent A lixiviate twice, first time lixiviate adds 4-5L organic solvent A according to the dry bacterium slag of every 1kg, second time adds 2-2.5L organic solvent A according to the dry bacterium slag of every 1kg, each extraction time 100-150min.
5. according to the preparation method of doractin sterile solution according to claim 1, it is characterized in that described cooling and add purified water precipitation process be: organic solvent lixiviating solution is cooled to 0-5 DEG C, add 4.5-5 times of purified water of its volume, leave standstill 150-190min, filter, obtain doractin crude product.
6., according to the preparation method of doractin sterile solution according to claim 1, it is characterized in that described doractin crude product or solid doractin are dissolved in organic solvent B, control its mass concentration 20-30%.
7. according to the preparation method of doractin sterile solution according to claim 1, it is characterized in that in described ultra-filtration process, Stress control is at 0.2-0.3MPa, and the material of ultrafilter membrane is vinylidene, and aperture is 100nm.
8. according to the preparation method of doractin sterile solution according to claim 1, it is characterized in that described crystallization process is: under stirring, the doractin ultrafiltrate of ultrafiltration gained is cooled to-10-0 DEG C, cooling rate controls at 2-3 DEG C/h, when there is crystalline polamer, adds crystal seed doractin, consumption is the 0.1-0.3% of doractin ultrafiltrate volume, continue to stir 60-100min, then growing the grain 180-200min, filtration can obtain solid doractin.
9. according to the preparation method of doractin sterile solution according to claim 1, it is characterized in that described microfiltration adopts membrane filter system, this equipment is separately installed with the filter element that specification is 0.45um and 0.22um, in filter process, Stress control is at 0.2-0.3MPa, filter and terminate, collect filtrate, obtain aseptic doractin solution.
10. according to the preparation method of doractin sterile solution according to claim 1, it is characterized in that described preprocessing process is: it is the 20-30% of fermentating liquid volume that first time dilutes purified water consumption used, it is the 40-50% of fermentating liquid volume that second time dilutes purified water consumption used.
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| CN108976270A (en) * | 2017-12-08 | 2018-12-11 | 北大方正集团有限公司 | A kind of preparation method of high-purity doractin |
| CN109234194A (en) * | 2018-09-26 | 2019-01-18 | 武汉回盛生物科技股份有限公司 | A kind of doractin producing strains and its application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105754921A (en) * | 2016-04-27 | 2016-07-13 | 东北农业大学 | Genetically engineered bacterium producing 5-keto-22,23-dihydro doramectin and preparation method and application of bacterium |
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| CN108976270A (en) * | 2017-12-08 | 2018-12-11 | 北大方正集团有限公司 | A kind of preparation method of high-purity doractin |
| CN109234194A (en) * | 2018-09-26 | 2019-01-18 | 武汉回盛生物科技股份有限公司 | A kind of doractin producing strains and its application |
| CN109234194B (en) * | 2018-09-26 | 2021-08-24 | 武汉回盛生物科技股份有限公司 | Doramectin producing strain and application thereof |
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