CN115385970A - Extraction method of kitasamycin - Google Patents
Extraction method of kitasamycin Download PDFInfo
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- CN115385970A CN115385970A CN202210296238.8A CN202210296238A CN115385970A CN 115385970 A CN115385970 A CN 115385970A CN 202210296238 A CN202210296238 A CN 202210296238A CN 115385970 A CN115385970 A CN 115385970A
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- 229950007634 kitasamycin Drugs 0.000 title claims abstract description 56
- XYJOGTQLTFNMQG-KJHBSLKPSA-N leucomycin V Chemical compound CO[C@H]1[C@H](O)CC(=O)O[C@H](C)C\C=C\C=C\[C@H](O)[C@H](C)C[C@H](CC=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](N(C)C)[C@H](O[C@@H]2O[C@@H](C)[C@H](O)[C@](C)(O)C2)[C@@H](C)O1 XYJOGTQLTFNMQG-KJHBSLKPSA-N 0.000 title claims abstract description 56
- 238000000605 extraction Methods 0.000 title abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 30
- 239000000919 ceramic Substances 0.000 claims abstract description 27
- 239000011347 resin Substances 0.000 claims abstract description 25
- 229920005989 resin Polymers 0.000 claims abstract description 25
- 238000000855 fermentation Methods 0.000 claims abstract description 24
- 230000004151 fermentation Effects 0.000 claims abstract description 24
- 239000012535 impurity Substances 0.000 claims abstract description 15
- 238000002425 crystallisation Methods 0.000 claims abstract description 5
- 230000008025 crystallization Effects 0.000 claims abstract description 5
- 238000001035 drying Methods 0.000 claims abstract description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical group [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 45
- 238000001914 filtration Methods 0.000 claims description 37
- 239000012528 membrane Substances 0.000 claims description 37
- 239000007788 liquid Substances 0.000 claims description 32
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 26
- 239000000706 filtrate Substances 0.000 claims description 26
- 238000000108 ultra-filtration Methods 0.000 claims description 22
- 238000004042 decolorization Methods 0.000 claims description 15
- 239000000047 product Substances 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 9
- 239000003957 anion exchange resin Substances 0.000 claims description 9
- 238000005189 flocculation Methods 0.000 claims description 9
- 230000016615 flocculation Effects 0.000 claims description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 239000002585 base Substances 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- 239000003513 alkali Substances 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 claims description 2
- 239000008394 flocculating agent Substances 0.000 claims description 2
- 230000020477 pH reduction Effects 0.000 claims description 2
- 238000003825 pressing Methods 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 abstract description 15
- 238000000926 separation method Methods 0.000 abstract description 15
- 238000004519 manufacturing process Methods 0.000 abstract description 12
- 239000002904 solvent Substances 0.000 abstract description 8
- 238000004945 emulsification Methods 0.000 abstract description 7
- 238000011084 recovery Methods 0.000 abstract description 5
- 238000003912 environmental pollution Methods 0.000 abstract description 3
- 238000005374 membrane filtration Methods 0.000 abstract description 3
- 238000001879 gelation Methods 0.000 abstract description 2
- 238000001179 sorption measurement Methods 0.000 abstract description 2
- 238000001728 nano-filtration Methods 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 45
- 239000000243 solution Substances 0.000 description 35
- 239000008367 deionised water Substances 0.000 description 22
- 229910021641 deionized water Inorganic materials 0.000 description 22
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 18
- 238000005406 washing Methods 0.000 description 17
- 239000012065 filter cake Substances 0.000 description 15
- 238000003756 stirring Methods 0.000 description 9
- 239000013078 crystal Substances 0.000 description 8
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 6
- 235000006408 oxalic acid Nutrition 0.000 description 6
- 238000001291 vacuum drying Methods 0.000 description 6
- 241001052560 Thallis Species 0.000 description 5
- 238000000638 solvent extraction Methods 0.000 description 5
- 238000003828 vacuum filtration Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 239000012467 final product Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000002351 wastewater Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000588832 Bordetella pertussis Species 0.000 description 1
- 241000193449 Clostridium tetani Species 0.000 description 1
- 241000186227 Corynebacterium diphtheriae Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 241000193996 Streptococcus pyogenes Species 0.000 description 1
- 241001312524 Streptococcus viridans Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000005276 aerator Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000006286 aqueous extract Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- WGFMTHGYKYEDHF-UHFFFAOYSA-L disodium 2-hydroxy-2-oxoacetate Chemical compound [Na+].[Na+].OC(=O)C(O)=O.[O-]C(=O)C([O-])=O WGFMTHGYKYEDHF-UHFFFAOYSA-L 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- ZOOODBUHSVUZEM-UHFFFAOYSA-N ethoxymethanedithioic acid Chemical compound CCOC(S)=S ZOOODBUHSVUZEM-UHFFFAOYSA-N 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 235000012247 sodium ferrocyanide Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000012991 xanthate Substances 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/13—Crystalline forms, e.g. polymorphs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a method for extracting kitasamycin from fermentation liquor, which comprises the following steps: the kitasamycin fermentation liquor is subjected to pretreatment, ceramic membrane filtration, resin adsorption impurity removal, shaking screen separation, nanofiltration concentration, crystallization and drying to obtain a finished product of kitasamycin. The kitasamycin extraction method provided by the invention can avoid the use of organic solvents in production, thereby solving series problems brought by the use of solvents in production, such as environmental pollution, solvent recovery, extraction emulsification, product gelation and the like.
Description
Technical Field
The invention relates to an extraction method of antibiotics, in particular to an extraction method of kitasamycin, belonging to the field of pharmaceutical chemicals.
Background
Kitasamycin, also known as cylindromycin, is a sixteen-membered macrolide antibiotic. Has strong inhibitory effect on gram-positive bacteria such as Staphylococcus, streptococcus pyogenes, streptococcus viridans, streptococcus pneumoniae, bacillus tetani, and Bacillus diphtheriae. It also has good inhibitory effect on gram-negative bacteria such as gonococcus and Bordetella pertussis.
At present, the method generally adopted for extracting kitasamycin is as follows: pre-treating kitasamycin fermentation liquor and then filtering to obtain kitasamycin filtrate; then adding hydrophobic organic solvent (such as ethyl acetate, butyl acetate, etc.) into the kitasamycin filtrate, and extracting kitasamycin into organic solvent phase; then, carrying out back extraction on the organic solvent phase by using an acidic buffer solution to obtain a kitasamycin phosphate buffer solution; and adjusting the pH of the solution to crystallize to obtain kitasamycin crystals, and drying to obtain finished products.
The method needs to use a large amount of organic solvents in the production process, and brings a series of problems to the production: (1) The production wastewater contains a large amount of organic solvents, and the recovery of the solvents needs higher investment and cost. (2) The volatile organic solvent also pollutes the environment seriously in the production process. (3) The solvent extraction method is easy to have serious emulsification phenomenon in the production process, and even if the demulsifier and the centrifuge are added for demulsification, the problem can not be effectively solved. Therefore, the whole demulsification process only depends on the use of the fermentation liquor flocculant of the salt of the xanthate, which can cause cyanide pollution in the wastewater. (4) The back extraction liquid contains a large amount of organic solvents, so that a serious glue formation phenomenon is generated in a subsequent crystallization process, and the quality of a finished product has a large risk. Therefore, the stripping solution must be treated by blowing solvent, but the air after solvent blowing must be discharged after special environmental protection treatment because of serious odor.
At present, research reports on extraction of kitasamycin bulk drugs mainly focus on a solvent extraction system, for example, patent CN201010158957.0 discloses application of a buffer liquid system in kitasamycin extraction, and patent CN201610610224.3 researches a method for reducing the cyclic pressure retaining force by recycling fermentation liquor raffinate to a production system. However, all these cannot be removed from the solvent extraction system, and the above-mentioned disadvantages cannot be avoided. Therefore, it is necessary to develop a non-solvent extraction method.
Disclosure of Invention
The invention provides an extraction method of kitasamycin, which can avoid the use of organic solvents in the extraction process, thereby avoiding the problems of organic solvent recovery and extraction emulsification and solving the problem of environmental pollution caused by production from a process source.
The technical scheme of the method is as follows:
a method for extracting kitasamycin from fermentation liquor comprises the following process steps:
(1) Adjusting the pH of the pretreated fermentation liquor to 5.0-6.9 by using alkali, and then filtering by using a tubular porous ceramic membrane to obtain a ceramic membrane filtrate;
(2) Adding anion exchange resin into the ceramic membrane filtrate to remove impurities, and then separating the resin by using an oscillating screen to obtain feed liquid after impurity removal;
(3) Adjusting the pH of the feed liquid after impurity removal to 5.0-6.9 by using acid, adding activated carbon for decolorization, and filtering to obtain an activated carbon decolorized liquid;
(4) And (3) performing ultrafiltration concentration on the decolorized solution of the active carbon by 5-10 times by using an ultrafiltration membrane with the molecular weight of 1000-3000 to obtain a concentrated solution, and then adding alkali to crystallize to obtain the finished product of the kitasamycin.
In the invention, the traditional extraction-back extraction operation is replaced by adopting the technologies of ceramic membrane filtration, anion exchange resin impurity removal, ultrafiltration membrane concentration and the like, organic solvents are not used, the recovery, extraction and emulsification of the organic solvents are fundamentally avoided, and the method is more environment-friendly.
The pretreatment method comprises the steps of acidification, flocculation and filtration. Preferably, the flocculating agent used for flocculation is polyaluminum chloride, polyferric chloride or polyaluminum ferric silicate. Because the subsequent process does not involve solvent extraction and does not have the condition of extraction and emulsification, the flocculant which has larger harm to the environment such as yellow bloody salt, zinc sulfate and the like can be avoided. Preferably, the filtration is plate and frame filter pressing.
The separation and purification method comprises a ceramic membrane separation technology. The aperture of the ceramic membrane is generally 30-200 nm and is used for removing macromolecular organic impurities, and the preferred aperture of the ceramic membrane is 30-50 nm. The inventors have also found that the pH of the broth prior to ceramic membrane separation is also a critical factor, and preferably, prior to ceramic membrane separation, the pH is adjusted with a base to a pH of 6.0 to 6.5, more preferably 6.1 to 6.5, in which case impurities are more efficiently removed and filtration efficiency is improved.
The separation and purification method comprises a resin impurity removal technology. Researches show that pigments in kitasamycin fermentation liquor are impurities with anions, and the pigment can be well separated by using anion exchange resin. The resin used will greatly affect the yield and quality of the product, and preferably, the resin includes strong-base or weak-base anion exchange resins such as D201, D301, LSA700A, LSA700B, etc. The amount of resin used is generally 2 to 10%, preferably 4 to 6%.
The separation and purification method of the invention also comprises a vibrating screen separation technology. After the kitasamycin feed liquid is subjected to resin impurity removal, the pH value can rise to more than 10, and at the moment, part of kitasamycin can be suspended in the feed liquid in a crystal form. Therefore, the feed liquid and the resin can not be separated in a filtering mode, and the invention skillfully utilizes the vibrating screen separation technology to solve the problem. Further, the oscillating screen is a standard screen No. 3.
The separation and purification method comprises an ultrafiltration membrane separation technology. The molecular weight of the ultrafiltration membrane used is generally 1000-3000, and the preferred molecular weight of the ultrafiltration membrane is 3000 for removing small-molecule impurities. Although kitasamycin molecular weight is less than 1000, we have surprisingly found that kitasamycin can be perfectly retained by an ultrafiltration membrane of 3000 molecular weight.
Preferably, when the ultrafiltration membrane separation is adopted for separation, the pH value of the feed liquid is also a key factor, the pH value of the feed liquid rises to more than 10 after the resin is subjected to impurity removal, the pH value of the feed liquid needs to be adjusted to weak acidity, and further, the pH value is adjusted to 6.0-6.5 and further adjusted to 6.1-6.5.
Specifically, the extraction method comprises the following steps:
(1) Fermentation liquor pretreatment and filtration: acidifying the fermentation liquor to pH3.0-4.0 with acid, adding flocculant to flocculate, and press filtering to obtain filtrate. And adjusting the pH value back to 6.0-6.5 by using alkali.
(2) Ceramic membrane filtration: and filtering the obtained filtrate by using a tubular porous ceramic membrane to obtain a ceramic membrane filtrate.
(3) Resin decoloring: adding 2-10% (W/V) anion exchange resin into the ceramic membrane filtrate for decolorization for 1h, and separating the resin by using an oscillating screen to obtain decolorized feed liquid.
(4) Activated carbon decolorization: adjusting the pH of the feed liquid after resin separation to 5.5-6.5 by using dilute hydrochloric acid, adding 0.3-0.5% of activated carbon for decoloring for 1h, and filtering to obtain filtrate.
(5) And (3) ultrafiltration concentration: and (3) performing ultrafiltration concentration on the active carbon decolorized solution by using an ultrafiltration membrane with the molecular weight of 3000 for 5-10 times to obtain a concentrated solution.
(6) And (3) crystallization: slowly adding 10% sodium hydroxide solution into the ultrafiltration concentrated solution under stirring, adjusting the pH value to 9.0-10.0, and controlling the crystallization temperature to 30-35 ℃. The crystals were obtained by centrifugation, and washed with a small amount of purified water.
(7) And (3) drying: vacuum drying at-0.090-0.100 MPa and 70-80 ℃ for 5h to obtain the finished product.
Compared with the prior art, the invention has the beneficial effects that:
the kitasamycin extraction method provided by the invention can avoid the use of organic solvents in production, thereby solving series problems brought by the use of solvents in production, such as environmental pollution, solvent recovery, extraction emulsification, product gelation and the like.
Detailed Description
The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
Guitar mold in the examplesThe fermentation broth is prepared from fermentation filtrate of large-scale production tank-release of Pylo Biotech Limited company in Zhejiang, and the preparation process comprises the following steps: preparing a culture medium in a 60-ton fermentation tank, sterilizing and inoculating. At pH6.0-8.0, 28-32 deg.C and air flow of 36m 3 Culturing for 50-100 hours under the conditions of min and stirring speed of 120-135 rpm, and placing in a tank.
Example 1
Taking the kitasamycin in a workshop, placing the kitasamycin in a tank 10L (the titer is 11268 u/ml), adjusting the pH value to be 3.2 by oxalic acid, adding 50g of polyaluminum chloride for flocculation, centrifuging the thalli by a centrifugal machine, washing a filter cake by about 4kg of deionized water, and combining the filtrate and the top water. The pH of the solution was adjusted to 6.3 with 10% sodium hydroxide solution, and the solution was filtered through a 50nm tubular porous ceramic membrane. 5kg of deionized water was added to the final stage of filtration to keep the top water rate and filtration rate consistent. 15.6kg of filtrate is collected together, the titer is 7412u/ml, and the filtration yield is 102.6%.
And (3) adding 0.8L LSA700A resin into the ceramic membrane filtrate for decoloring for 1h. After decolorization, the resin and feed liquid are separated by using a 3# standard sieve, and the resin is top-washed by using a small amount of deionized water. The separated feed liquid and the top water are combined, and the pH value of the feed liquid is adjusted back to 6.3 by using 2mol/L dilute hydrochloric acid. Adding 90g of activated carbon, stirring and decoloring for 1h, carrying out vacuum filtration, and carrying out top washing on a filter cake with a small amount of water. The obtained decolorized solution is 18.9kg in total, the titer is 5642u/ml, and the decolorization yield is 92.2%.
The decolorized solution is concentrated to about 2kg by ultrafiltration with a 3000 molecular weight membrane, and then is top-washed with 5kg deionized water while maintaining the same top water rate and filtration rate. 1.97kg of ultrafiltration concentrate was obtained.
Adjusting pH of the concentrated solution to 9.5 with 10% sodium hydroxide solution, maintaining the temperature at 35 deg.C for 30min, vacuum filtering, and washing the filter cake with a small amount of deionized water to obtain kitasamycin wet crystal. Vacuum drying at 80 deg.C and-0.095 MPa for 5 hr to obtain final product 52.6g of kitasamycin with potency of 1695u/mg and total yield of 79.12%. Through detection, all quality indexes (92.6 percent of the total components, 0.3 percent of residues on ignition, 1.6 percent of dry loss and pH 8.7) of the product meet the requirements of pharmacopoeia.
The method does not use an organic solvent, does not relate to the problem of extraction and emulsification, does not have the problems of organic solvent residue and post-treatment, and is more environment-friendly.
Example 2
Taking a workshop kitasamycin, putting into a tank 10kg (the titer is 13108 u/ml), adjusting the pH value to 3.0 by oxalic acid, adding 50g of polyaluminum chloride for flocculation, centrifuging the thalli by a centrifugal machine, washing a filter cake by about 4kg of deionized water, and combining the filtrate and the top water. The pH was adjusted to 6.3 with 10% sodium hydroxide solution and then filtered through a 35nm format tubular porous ceramic membrane. In the final stage of filtration, 5kg of deionized water was added to maintain the top water rate and filtration rate consistent. The filtrate was collected together 16.1kg, titer was 8126u/ml, and filtration yield was 99.8%.
And adding 0.7L LSA700B resin into the ceramic membrane filtrate for decolorization for 1h. After decolorization, the resin and feed liquid are separated by using a 3# standard sieve, and the resin is top-washed by using a small amount of deionized water. The separated feed liquid and the top water are combined, and the pH value of the feed liquid is adjusted back to 6.0 by using 2mol/L dilute hydrochloric acid. Adding 90g of active carbon, stirring and decoloring for 1h, carrying out vacuum filtration, and washing a filter cake by using a small amount of water. The obtained decolorized solution is 19.3kg in total, the titer is 6412u/ml, and the decolorization yield is 94.6%.
The decolorized solution is concentrated to about 2kg by ultrafiltration with a 1000 molecular weight membrane, and then is washed by flowing and top washing with 5kg deionized water, so that the top water rate and the filtration rate are kept consistent. Finally, 2.23kg of ultrafiltration concentrated solution is obtained.
Adjusting pH of the concentrated solution to 9.2 with 10% sodium hydroxide solution, maintaining the temperature at 35 deg.C for 30min, vacuum filtering, and washing the filter cake with a small amount of deionized water to obtain kitasamycin wet crystal. Vacuum drying at 80 deg.C and-0.095 MPa for 5 hr to obtain final product of kitasamycin 62.4g, titer 1706u/mg, and total yield 81.21%. Through detection, all quality indexes (93.1 percent of the total components, 0.3 percent of residues on ignition, 1.5 percent of dry loss and pH 8.8) of the product meet the requirements of pharmacopoeia.
Example 3
Taking the kitasamycin in a workshop, putting the kitasamycin in a tank of 10kg (the titer is 12008 u/ml), adjusting the pH value to 3.5 by oxalic acid, adding 50g of polyaluminum chloride for flocculation, centrifuging the thalli by a centrifugal machine, washing a filter cake by about 4kg of deionized water, and combining the filtrate and the top water. The pH was adjusted to 6.5 with 10% sodium hydroxide solution and then filtered through a 50nm format tubular porous ceramic membrane. In the final stage of filtration, 5kg of deionized water was added to maintain the top water rate and filtration rate consistent. 15.9kg of filtrate is collected together, the titer is 7869u/ml, and the filtration yield is 104.2%.
The ceramic membrane filtrate was decolorized for 1 hour by adding 1.1L of D201 resin. After decolorization, the resin and feed liquid are separated by a 3# standard sieve, and the resin is top-washed by a small amount of deionized water. The separated feed liquid and the top water are combined, and the pH value of the feed liquid is adjusted back to 6.2 by using 2mol/L dilute hydrochloric acid. Adding 90g of active carbon, stirring and decoloring for 1h, carrying out vacuum filtration, and washing a filter cake by using a small amount of water. The obtained decolorized solution is 19.1kg in total, the titer is 5958u/ml, and the decolorization yield is 90.95%.
The decolorized solution is concentrated to about 2kg by ultrafiltration with a 3000 molecular weight membrane, and then is top-washed with 5kg deionized water while maintaining the same top water rate and filtration rate. 2.11kg of ultrafiltration concentrate was obtained.
Adjusting pH of the concentrated solution to 9.7 with 10% sodium hydroxide solution, maintaining the temperature at 35 deg.C for 30min, vacuum filtering, and washing the filter cake with a small amount of deionized water to obtain kitasamycin wet crystal. After vacuum drying is carried out for 5 hours at 80 ℃ and-0.095 MPa, 54.3g of finished kitasamycin product is obtained, the titer is 1682u/mg, and the total yield is 76.06%. Through detection, all quality indexes (91.8 percent of the total components, 0.4 percent of residues on ignition, 1.2 percent of dry loss and 8.5 percent of pH) of the product meet the requirements of pharmacopoeia.
Example 4
Taking a workshop kitasamycin, putting into a tank 10kg (the titer is 11358 u/ml), adjusting the pH value to 3.0 by oxalic acid, adding 50g of polyaluminum chloride for flocculation, centrifuging the thalli by a centrifugal machine, washing a filter cake by about 4kg of deionized water, and combining the filtrate and the top water. The pH was adjusted to 6.1 with sodium hydroxide solution and then filtered through a 35nm tubular porous ceramic membrane. In the final stage of filtration, 5kg of deionized water was added to maintain the top water rate and filtration rate consistent. 15.3kg of filtrate is collected together, the titer is 7378u/ml, and the filtration yield is 99.4%.
0.9L of D301 resin is added into the ceramic membrane filtrate for decolorization for 1 hour. After decolorization, the resin and feed liquid are separated by using a 3# standard sieve, and the resin is top-washed by using a small amount of deionized water. The separated feed liquid and the top water are combined, and the pH value of the feed liquid is adjusted back to 6.1 by using 2mol/L dilute hydrochloric acid. Adding 90g of activated carbon, stirring and decoloring for 1h, carrying out vacuum filtration, and carrying out top washing on a filter cake with a small amount of water. The obtained decolored liquid is 18.5kg in total, the titer is 5536u/ml, and the decolored yield is 90.7%.
The decolorized solution is concentrated to about 2kg by ultrafiltration with a 1000 molecular weight membrane, and then is washed by flowing and top washing with 5kg deionized water, so that the top water rate and the filtration rate are kept consistent. Finally, 2.36kg of ultrafiltration concentrated solution is obtained.
Adjusting pH of the concentrated solution to 9.6 with 10% sodium hydroxide solution, maintaining the temperature at 35 deg.C for 30min, vacuum filtering, and washing the filter cake with a small amount of deionized water to obtain kitasamycin wet crystal. Vacuum drying at 80 deg.C and-0.095 MPa for 5 hr to obtain final product 54.8g of kitasamycin with titer 1659u/mg and total yield 80.04%. Through detection, all quality indexes (92.5 percent of the total components, 0.4 percent of residues on ignition, 1.9 percent of dry loss and pH 8.5) of the product meet the requirements of pharmacopoeia.
Comparative example 1
Taking and placing the kitasamycin in a workshop into a tank 10kg (the potency is 11358 u/ml), adjusting the pH value to 3.0 by oxalic acid, adding 50g of polyaluminium chloride and 20g of yellow blood salt for flocculation, centrifuging the thalli by a centrifugal machine, washing a filter cake by about 5kg of deionized water, combining the filtrate and the top water solution to obtain 13.3kg of the kitasamycin, wherein the potency is 8853u/ml, and the yield is 103.7%.
The pH of the mixture was adjusted to 9.5 with 10% sodium hydroxide solution, and 5.0L of butyl acetate was added thereto and extracted with stirring for 2min. The feed liquid is put into 4 separating funnels of 5L for standing and layering for 30min, and the lower layer raffinate is separated. And combining the emulsion layer and the butyl acetate extract, adding 50g of activated carbon, stirring, adsorbing and filtering to obtain clear butyl acetate extract. The butyl acetate extract was back-extracted with 2500ml of oxalic acid-sodium oxalate buffer (2.0% oxalic acid, 0.4% sodium hydroxide) having a pH of 2.0 to obtain kitasamycin aqueous extract. Adjusting pH to 4.5 with 10% sodium hydroxide solution, adding 25g active carbon for adsorption and decolorization for 1h, vacuum filtering, and top washing the filter cake with a small amount of water. 2.58kg of decolored liquid is obtained, the titer is 34308u/ml, and the extraction yield is 75.2 percent.
Blowing the solvent for 2h by using an air aerator while stirring, then adjusting the pH value to 9.5 by using a 10% sodium hydroxide solution, preserving the heat at 35 ℃ for 30min, carrying out vacuum filtration, and washing a filter cake by using a small amount of deionized water to obtain the kitasamycin wet crystal. Vacuum drying at 80 deg.C and-0.095 MPa for 5 hr to obtain final product of kitasamycin 48.3g, with potency 1636u/mg and total yield 69.6%.
In the practical production of the scheme, the separation of the extraction liquid needs a three-phase centrifuge to break emulsion; the raffinate contains about 1 percent of butyl acetate and a small amount of cyanide, so the environment-friendly treatment is difficult.
Claims (10)
1. A method for extracting kitasamycin from fermentation liquor is characterized by comprising the following process steps:
(1) Adjusting the pH of the pretreated fermentation liquor to 5.0-6.9 by using alkali, and then filtering by using a tubular porous ceramic membrane to obtain a ceramic membrane filtrate;
(2) Adding anion exchange resin into the ceramic membrane filtrate to remove impurities, and then separating the resin by using an oscillating screen to obtain feed liquid after impurity removal;
(3) Adjusting the pH of the feed liquid after impurity removal to 5.0-6.9 by using acid, adding activated carbon for decolorization, and filtering to obtain an activated carbon decolorized liquid;
(4) And (3) performing ultrafiltration concentration on the decolorized solution of the active carbon by 5-10 times by using an ultrafiltration membrane with the molecular weight of 1000-3000 to obtain a concentrated solution, and then adding alkali to crystallize to obtain the finished product of the kitasamycin.
2. The method for extracting kitasamycin from fermentation broth according to claim 1, wherein in step (1), said pretreatment comprises acidification, flocculation and filtration; flocculating agent used for flocculation is polyaluminium chloride, polyferric chloride or polyaluminium ferric silicate;
the filtration is plate and frame filter pressing.
3. The method for extracting kitasamycin from fermentation broth according to claim 1, wherein in step (1), said base is sodium hydroxide solution, adjusting pH to 6.0-6.5;
the aperture of the tubular porous ceramic membrane is 30-50 nm.
4. The method for extracting kitasamycin from fermentation broth according to claim 1, wherein in step (2) the anion exchange resin used is either strongly basic or weakly basic.
5. The method for extracting kitasamycin from fermentation broth according to claim 1, wherein in step (2), the anion exchange resin is used in an amount of 2 to 10% based on the ceramic membrane filtrate.
6. The method for extracting kitasamycin from fermentation broth according to claim 1 or 4, wherein in step (2), the anion exchange resin is one or more of D201, D301, LSA700A and LSA 700B.
7. The method for extracting kitasamycin from fermentation broth according to claim 1, wherein in step (3), the acid is diluted hydrochloric acid and the pH is adjusted to 6.0-6.5.
8. The method for extracting kitasamycin from fermentation liquor according to claim 1, wherein in the step (3), the dosage of the activated carbon is 0.3-0.5% of the mass of the feed liquor, and the decolorization time is 1-3 h.
9. The method for extracting kitasamycin from fermentation broth according to claim 1, wherein in step (4), said base is sodium hydroxide solution with a concentration of 10-20% by mass and the pH value after adding base is 9.0-10.0.
10. The method for extracting kitasamycin from fermentation broth according to claim 1, wherein in step (4), said crystallization temperature is between 30 ℃ and 35 ℃;
the drying is carried out under the conditions of-0.090 to-0.100 MPa and 70 to 80 ℃, and the drying time is 5 to 10 hours.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845068A (en) * | 2010-04-29 | 2010-09-29 | 浙江普洛康裕生物制药有限公司 | Buffer liquid system and application thereof to extraction of kitasamycin |
| CN103304612A (en) * | 2013-06-25 | 2013-09-18 | 伊犁川宁生物技术有限公司 | Concentration method of erythrocin fermentation liquor |
| CN106188186A (en) * | 2016-07-29 | 2016-12-07 | 天方药业有限公司 | A kind of kitasamycin cleaning extraction process |
| CN109553650A (en) * | 2017-09-25 | 2019-04-02 | 联邦制药(内蒙古)有限公司 | The aqueous extraction method of erythromycin fermentation liquid |
| CN110066843A (en) * | 2019-04-16 | 2019-07-30 | 天方药业有限公司 | Kitasamycin fermentation medium, kitasamycin and its fermentation process |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845068A (en) * | 2010-04-29 | 2010-09-29 | 浙江普洛康裕生物制药有限公司 | Buffer liquid system and application thereof to extraction of kitasamycin |
| CN103304612A (en) * | 2013-06-25 | 2013-09-18 | 伊犁川宁生物技术有限公司 | Concentration method of erythrocin fermentation liquor |
| CN106188186A (en) * | 2016-07-29 | 2016-12-07 | 天方药业有限公司 | A kind of kitasamycin cleaning extraction process |
| CN109553650A (en) * | 2017-09-25 | 2019-04-02 | 联邦制药(内蒙古)有限公司 | The aqueous extraction method of erythromycin fermentation liquid |
| CN110066843A (en) * | 2019-04-16 | 2019-07-30 | 天方药业有限公司 | Kitasamycin fermentation medium, kitasamycin and its fermentation process |
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