MXPA97002761A - Procedure for recovery of clavulan acid without use of ami - Google Patents
Procedure for recovery of clavulan acid without use of amiInfo
- Publication number
- MXPA97002761A MXPA97002761A MXPA/A/1997/002761A MX9702761A MXPA97002761A MX PA97002761 A MXPA97002761 A MX PA97002761A MX 9702761 A MX9702761 A MX 9702761A MX PA97002761 A MXPA97002761 A MX PA97002761A
- Authority
- MX
- Mexico
- Prior art keywords
- clavulanic acid
- salts
- fermentation
- salt
- purification
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000011084 recovery Methods 0.000 title claims abstract description 6
- 239000002253 acid Substances 0.000 title description 6
- HZZVJAQRINQKSD-PBFISZAISA-N Clavulanic acid Chemical compound OC(=O)[C@H]1C(=C/CO)/O[C@@H]2CC(=O)N21 HZZVJAQRINQKSD-PBFISZAISA-N 0.000 claims abstract description 61
- 229960003324 Clavulanic Acid Drugs 0.000 claims abstract description 58
- 238000001556 precipitation Methods 0.000 claims abstract description 20
- 238000007792 addition Methods 0.000 claims abstract description 19
- 239000007864 aqueous solution Substances 0.000 claims abstract description 18
- 239000002904 solvent Substances 0.000 claims abstract description 15
- 229910052783 alkali metal Inorganic materials 0.000 claims abstract description 14
- 150000001447 alkali salts Chemical class 0.000 claims abstract description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims abstract description 11
- KFZMGEQAYNKOFK-UHFFFAOYSA-N iso-propanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 9
- 150000001340 alkali metals Chemical class 0.000 claims abstract description 7
- 239000008346 aqueous phase Substances 0.000 claims abstract description 4
- 150000001412 amines Chemical class 0.000 claims abstract description 3
- 239000011780 sodium chloride Substances 0.000 claims description 36
- 238000000855 fermentation Methods 0.000 claims description 34
- 230000004151 fermentation Effects 0.000 claims description 34
- 150000003839 salts Chemical class 0.000 claims description 26
- 239000002609 media Substances 0.000 claims description 18
- CSCPPACGZOOCGX-UHFFFAOYSA-N acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 16
- 238000000746 purification Methods 0.000 claims description 16
- 229910052799 carbon Inorganic materials 0.000 claims description 15
- 102000004169 proteins and genes Human genes 0.000 claims description 15
- 108090000623 proteins and genes Proteins 0.000 claims description 15
- 239000008121 dextrose Substances 0.000 claims description 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 12
- 235000013312 flour Nutrition 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 10
- 235000013619 trace mineral Nutrition 0.000 claims description 10
- 239000011573 trace mineral Substances 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 9
- -1 alkali metal salt Chemical class 0.000 claims description 8
- 229910052803 cobalt Chemical class 0.000 claims description 8
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 8
- 235000010755 mineral Nutrition 0.000 claims description 8
- 239000011707 mineral Substances 0.000 claims description 8
- 235000010469 Glycine max Nutrition 0.000 claims description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N acetic acid ethyl ester Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 7
- 229910052742 iron Chemical class 0.000 claims description 7
- 239000012071 phase Substances 0.000 claims description 7
- 210000004215 spores Anatomy 0.000 claims description 7
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 235000017060 Arachis glabrata Nutrition 0.000 claims description 5
- 240000005781 Arachis hypogaea Species 0.000 claims description 5
- 235000010777 Arachis hypogaea Nutrition 0.000 claims description 5
- 235000018262 Arachis monticola Nutrition 0.000 claims description 5
- 229920001353 Dextrin Polymers 0.000 claims description 5
- 239000004375 Dextrin Substances 0.000 claims description 5
- 241000187433 Streptomyces clavuligerus Species 0.000 claims description 5
- 241000209149 Zea Species 0.000 claims description 5
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 5
- 238000010521 absorption reaction Methods 0.000 claims description 5
- 150000001720 carbohydrates Chemical class 0.000 claims description 5
- 235000014633 carbohydrates Nutrition 0.000 claims description 5
- 235000005822 corn Nutrition 0.000 claims description 5
- 235000005824 corn Nutrition 0.000 claims description 5
- 235000012343 cottonseed oil Nutrition 0.000 claims description 5
- 235000019425 dextrin Nutrition 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 5
- 239000003456 ion exchange resin Substances 0.000 claims description 5
- 229920003303 ion-exchange polymer Polymers 0.000 claims description 5
- 235000012054 meals Nutrition 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 5
- 235000020232 peanut Nutrition 0.000 claims description 5
- 159000000001 potassium salts Chemical class 0.000 claims description 5
- 150000003626 triacylglycerols Chemical class 0.000 claims description 5
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 claims description 4
- 229920002261 Corn starch Polymers 0.000 claims description 4
- 229910052802 copper Inorganic materials 0.000 claims description 4
- 239000008120 corn starch Substances 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 4
- 159000000011 group IA salts Chemical class 0.000 claims description 4
- 239000001963 growth media Substances 0.000 claims description 4
- WHXSMMKQMYFTQS-UHFFFAOYSA-N lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 claims description 4
- 229910052744 lithium Inorganic materials 0.000 claims description 4
- 229910052749 magnesium Inorganic materials 0.000 claims description 4
- 229910052759 nickel Inorganic materials 0.000 claims description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 159000000000 sodium salts Chemical class 0.000 claims description 4
- 229910052725 zinc Inorganic materials 0.000 claims description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N β-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- NTIZESTWPVYFNL-UHFFFAOYSA-N Methyl isobutyl ketone Chemical compound CC(C)CC(C)=O NTIZESTWPVYFNL-UHFFFAOYSA-N 0.000 claims description 3
- 235000019764 Soybean Meal Nutrition 0.000 claims description 3
- 150000004649 carbonic acid derivatives Chemical group 0.000 claims description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 3
- 229910052700 potassium Inorganic materials 0.000 claims description 3
- 239000011591 potassium Substances 0.000 claims description 3
- 230000000644 propagated Effects 0.000 claims description 3
- KEAYESYHFKHZAL-UHFFFAOYSA-N sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 239000004455 soybean meal Substances 0.000 claims description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical class OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims description 2
- 229920000742 Cotton Polymers 0.000 claims description 2
- 150000001242 acetic acid derivatives Chemical class 0.000 claims description 2
- 239000008272 agar Substances 0.000 claims description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 2
- 239000011575 calcium Substances 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- 159000000007 calcium salts Chemical class 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 150000007522 mineralic acids Chemical class 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- 230000002378 acidificating Effects 0.000 claims 2
- 240000007842 Glycine max Species 0.000 claims 1
- 238000005352 clarification Methods 0.000 claims 1
- 239000003480 eluent Substances 0.000 claims 1
- 235000010633 broth Nutrition 0.000 description 11
- 239000000203 mixture Substances 0.000 description 5
- LWIHDJKSTIGBAC-UHFFFAOYSA-K Tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 230000003115 biocidal Effects 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241000187747 Streptomyces Species 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 229910003002 lithium salt Inorganic materials 0.000 description 3
- 159000000002 lithium salts Chemical class 0.000 description 3
- 229940064005 Antibiotic throat preparations Drugs 0.000 description 2
- 229940083879 Antibiotics FOR TREATMENT OF HEMORRHOIDS AND ANAL FISSURES FOR TOPICAL USE Drugs 0.000 description 2
- 229940042052 Antibiotics for systemic use Drugs 0.000 description 2
- 229940042786 Antitubercular Antibiotics Drugs 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- 229940093922 Gynecological Antibiotics Drugs 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M Potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- YBRBMKDOPFTVDT-UHFFFAOYSA-N Tert-Butylamine Chemical class CC(C)(C)N YBRBMKDOPFTVDT-UHFFFAOYSA-N 0.000 description 2
- 229940024982 Topical Antifungal Antibiotics Drugs 0.000 description 2
- 235000010419 agar Nutrition 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 239000002518 antifoaming agent Substances 0.000 description 2
- 102000006635 beta-Lactamases Human genes 0.000 description 2
- 108020004256 beta-Lactamases Proteins 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 229940079866 intestinal antibiotics Drugs 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229940005935 ophthalmologic Antibiotics Drugs 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 235000019798 tripotassium phosphate Nutrition 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 102000005922 Amidases Human genes 0.000 description 1
- 108020003076 Amidases Proteins 0.000 description 1
- 229960005069 Calcium Drugs 0.000 description 1
- 229960003563 Calcium Carbonate Drugs 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate dianion Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 229940090805 Clavulanate Drugs 0.000 description 1
- 241001517488 Clavus Species 0.000 description 1
- 241000206672 Gelidium Species 0.000 description 1
- 241000152160 Ira Species 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L Iron(II) sulfate Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 241000424942 Streptomyces clavuligerus ATCC 27064 Species 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 230000000845 anti-microbial Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 1
- 239000003781 beta lactamase inhibitor Substances 0.000 description 1
- 230000037348 biosynthesis Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking Effects 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 230000002759 chromosomal Effects 0.000 description 1
- 239000000701 coagulant Substances 0.000 description 1
- 230000001112 coagulant Effects 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000001066 destructive Effects 0.000 description 1
- 201000009910 diseases by infectious agent Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drugs Drugs 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000008394 flocculating agent Substances 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 230000000813 microbial Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 229940040975 systemic penicillins Beta-lactamase inhibitors Drugs 0.000 description 1
- 230000001131 transforming Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000002132 β-lactam antibiotic Substances 0.000 description 1
Abstract
The present invention relates to a process for the recovery of clavulanic acid without the use of amines, in which the alkali salts of clavulanic acid are precipitated in extremely pure form by extracting an organic solution of clavulanic acid with an aqueous solution of an alkali metal at a pH between 6.0 and 6.5 adding a lower alcohol to the aqueous phase and causing precipitation at low temperature by the addition of a solvent such as isopropanol or aceto
Description
PROCEDURE OF RECOVERY OF CLAVULAN ACID WITHOUT THE USE OF AMINES
The present invention describes a process for recovering alkali salts of clavulanic acid obtained by fermentation.
Clavulanic acid is described as (2R, 5R, Z) -3- (2-idroxietilidene) -7-oxo-4-oxa-1-azabicyclo-2,3,0-heptan-2-carboxylic acid and its structural formula is:
Since the discovery of beta-lactamic antibiotics, a phenomenon of microbial resistance to these products has been observed.
The magnitude of this phenomenon has grown over time, reducing the effectiveness of these drugs and causing an increase in dosage to control infections.
It is well known that resistant microorganisms express enzymes (such as beta lactamase or acylases) that modify the structure of the beta-lactam antibiotic, reducing its antimicrobial activity.
One of the strategies used by pharmacologists to avoid this problem has been the introduction into the original molecule obtained by fermenting functional groups that reduce the possibility of attack of the molecule by the destructive enzymes. An even more recent strategy has been to associate the beta-lactamic antibiotic with substances that have the property of blocking the action of said enzymes.
These substances therefore protect the antibiotic and allow a considerable increase in its effectiveness.
Among the beta lactamase inhibitors, the best known is clavulanic acid, which has a very high activity against chromosomal beta-lactamase and Gram-negative microorganism plasmids.
Clavulanic acid has been initially isolated from fermentation broths of strains of the genus Streptomyces.
In particular, patent GB 1508977 reports on the biosynthesis and isolation of clavulanic acid from fermentation broths using the Streptomyces clavuligerus strain ATCC 27064.
Undesirable components of the fermentation broth as proteins and in particular other beta-lactamic antibiotics are eliminated with the use of fractionation techniques.
In any case, this purification is difficult using conventional techniques, particularly due to the similar behavior of the different beta-lactamic carboxylic acids.
Several efforts have been made to purify clavulanic acid through different derivatives such as lithium salt (GB 1543563) and t-butylamine salt (US 4647659-Derwent abstract). The known methods of purification have several disadvantages, insofar as they are often problematic or result in low yields and / or quality.
Furthermore, the aforementioned methods do not allow the direct preparation of pure salts other than those of lithium and of tert-butylamine, additional steps being required for the transformation to pharmaceutically useful compounds such as the potassium salt which is a common component of many specialties.
Now a new process for the purification of clavulanic acid has been found in surprise form that eliminates the disadvantages of the previous methods.
From the fermentation broths the alkaline salts of clavulanic acid (in particular the potassium and lithium salts) can be obtained with a novel method of recovery that eliminates the isolation of intermediates and directly precipitates the alkali salts of clavulanic acid in high yield and purity .
In more detail, according to the present method, the alkali salts of clavulanic acid are precipitated in extremely pure form by extracting from organic solution of clavulanic acid with an aqueous solution of an alkali metal at a pH between 6.0 and 6.5, adding an alcohol lower than the aqueous phase and causing precipitation at low temperature by the addition of a solvent such as isopropanol or acetone
In particular, the preparation of high purity salts of clavulanic acid in direct form, according to the present invention, comprises the following steps:
a) Purification of the solid-free fermentation broth by absorption on a column containing an ion exchange resin; elution of the absorbed activity by means of an aqueous solution of a sodium salt at temperatures preferably between 0/5 C.
b) Extraction of the eluate obtained from step a) by means of a solvent immiscible with water at an acid pH
c) Addition to the purified organic solution of an aqueous solution of an alkali metal base at a pH between 6 and 6.5
d) Precipitation of the alkali metal salts of clavulanic acid by adding a lower alcohol to the aqueous solution obtained previously and followed by the addition to the hydroalcoholic phase of a solvent such as isopropanol or acetone at low temperature.
The precipitation method mentioned above and the purification sequence mentioned above represent a novation under the operating conditions that has not been described above.
The fermentation broth is clarified according to well-known methods after the addition of quaternary ammonium salts as protein flocculants.
In more detailed form, the resin used for the ion exchange of the clarified broth is of the polystyrene type and the sodium salt is the sodium chloride.
The water-immiscible solvent used for the extraction is preferentially ethyl or butyl acetate or methyl isobutyl ketone.
The acid pH varies between 1.0 and 1.5 and is preferentially 1.1 and is controlled using sulfuric acid or other inorganic acids
The alkali metal salts used are preferentially carbonates, bicarbonates or acetates of lithium, potassium and sodium and the pH of step c) is 6.2 The low molecular weight alcohol added to the aqueous solution of the alkaline salt of clavulanic acid it is preferably methanol.
During the course of the precipitation phase, the temperature is preferably maintained between +5 and +10 C and in particular at + 10 C.
The production of clavulanic acid by means of the Streptomyces Clavuligerus strain is carried out in the presence of assimilable carbon sources, nitrogen sources, mineral salts and triglycerides.
The fermentation is carried out under aerobic conditions at temperatures from +20 to +37 C and preferably at + 25 / + 30C under agitation
The preferred protein sources are corn flour, peanut flour, soybean meal and in particular cottonseed meal.
The sources of carbon used for growth are in particular dextrin, dextrose or corn starch
The amounts of protein sources, carbohydrate sources and triglycerides in the culture medium vary in general between 1% and 10% and more particularly between 2% and 5%
Calcium and sodium potassium salts, such as carbonates and phosphates, are usually added to the culture medium.
Trace elements can also be added to the culture medium in the form of inorganic salts of Co, Fe, Mg, Ni, Zn and Cu. An antifoaming agent can also be added to prevent an excess of foam during the course of the fermentation.
In a preferential way, the fermentation is carried out under aerobic conditions under agitation at a temperature of + 24 / + 27 C and at a pH of 6.8 / 7.0 in a medium with the components and in the concentrations mentioned above.
During the fermentation the pH is controlled by the addition of alkali.
The fermentation process is usually preceded by steps in which the microorganism grows and more specifically:
a) Preparing a suspension of spores of Streptomyces Clavuligerus.
b) Inoculating said suspension in a seed tank.
c) Transferring the cultivation developed from the seed tank to the fermenting tank in which the fermentation takes place as indicated above.
The spore suspension is prepared preferentially by growing the microorganism on a solid agar medium containing a carbon source (dextrin or dextrose), calcium salts, mineral salts and trace elements such as Co and Fe.
The best producing colonies are selected according to their production capacity at the flask level in fermentative media containing soy or peanut flour as a protein source, dextrose as a carbon source, potassium salts and trace elements such as Co and Fe; the colonies that are better producers are then propagated in solid medium.
The seed tank contains a medium containing soy flour, cotton or corn as a protein source, dextrose or dextrose as a carbon source, and sodium salts
All the aforementioned cultivation steps are carried out at temperatures between + 20 / + 37C and preferentially between + 25 / + 30C under aerobic conditions and under agitation
Clavulanic acid is obtained from the fermentation broth in the form of its alkaline salts according to the extraction-precipitation method mentioned above
The present invention is illustrated in a non-limiting manner with the following examples and according to the following procedure: production process of purified clavulanic acid or its salts by means of a fermentation of a strain selected from Streptomyces Clavu gerus var FAC comprising, a) Preparation of a suspension of spores of Streptomyces Clavuhgerus var FAC, b) Inoculation of said suspension in a seed tank, c) Fermentation of the strain d) Purification of fermentation broth free of solids and clarified of clavulanic acid by absorption on a column containing a ream of ion exchange and elution with an aqueous solution of a sadistic salt at low temperature, e) Extraction of the eluate obtained in step d) by means of a solvent immiscible with water at an acid pH, f) Addition to the organic solution purified from an aqueous solution of an alkali metal salt at a pH between 6 and 6, g) Precipitation of the salt of a alkali metal of clavulanic acid by the addition of a low molecular weight alcohol to the aqueous solution obtained in the previous step, followed by the addition to the hydroalcohoxide phase of a solvent selected from isopropanol and / or acetone at low temperature
EXAMPLE 1 Starting from an ophthalmic vial of Streptomyces Clavuligerus var Fac, we proceed to the selection of colonies grown in a solid medium with the following composition Source of carbohydrates (dextrin or dextrose) 10 gr / lt calcium carbonate 2 gr / lt trace elements (chloride of cobalt, ferrous sulfate) 1 ml / lt agar agar 20 gr / lt
After incubation at constant temperature (25 C) the colonies are selected. The best producers are round, elevated and with a green sporification at the top.
The selected colonies are transferred to inclined agarose tubes containing the same solid medium mentioned above and incubated at temperatures of 25 c for 7-10 days until complete sporification.
The mature tubes are tested to check their productivity using a vegetative medium with the following composition:
protein source 25 gr / lt carbohydrate source 10 gr / lt potassium phosphate 2 gr / lt soybean oil 2 gr / lt
The inoculated medium is incubated under agitation for 45/60 hrs and aseptically transferred to fermentative medium with the following composition:
Soy flour 35 gr / l dextrose 8 gr / lt potassium phosphate 1 gr / lt trace elements (Ni, Co as sulfates, 1% sol) 1 ml The flasks are incubated under agitation for 3/5 days at constant temperature (25C ) and are titled to check productivity as clavulanic acid.
The best producing colonies are propagated in solid medium to obtain sufficient biomass for industrial inoculum.
The suspension of spores of the colonies with better productivity is inoculated in a seed tank containing a sterile medium of the following composition
Soy flour 50 gr / lt sodium phosphate 1 gr / lt dextrose (or other carbohydrate source) 40 gr / lt antifoam 0.5 gr / lt
Other protein sources that give good results may be cottonseed meal or corn flour
The seed tank usually develops under aeration and at a temperature between 25/30 C in 40/50 hrs and once the biomass has developed it is transferred to a fermentation tank with the following composition:
Soy flour 35 gr / l dextrin 35 gr / ltr triglycerides 25 gr / lt trace elements (1% Mg sol, Fe, Zn, Cu) 1 ml
The fermentation takes place in 5/7 days at a constant temperature of 24/27 C and the pH is controlled between 6.5 and 7 with the addition of alkali.
In the course of fermentation, different nutrients are added in order to improve production.
The most convenient containers to carry out this production are typical agitated and gritty containers.
The fermentation broth rich in clavulanic acid is already treated in the recovery phase with coagulants of the proteins and then the solids are eliminated with conventional separation methods (vacuum rotary filter, ultrafiltration or centrifugation systems).
The solids-free broth is absorbed at low temperature (+ 3 / + 10C and preferably + 5C) on ion exchange resins (as IRA, registered trademark of Rohm and Haas Corp).
The activity absorbed on the resin is eluted by means of a sodium chloride solution at low temperature and the rich eluate is further processed.
Example 2 3000 Its rich eluate containing 6/8 active kg is extracted countercurrent in a liquid / liquid extractor with 6/8 volumes of ethyl acetate or another water immiscible solvent such as methyl isobutyl ketone.
The pH is automatically controlled to values around 1 (1 to 1.5) using mineral acids such as sulfuric acid or preferably hydrochloric acid
The rich organic solution is anhydrified and decolorized and is concentrated at half volume in vacuum; A solution of potassium acetate in water is added immediately to obtain a pH between 6 and 6.5.
The rich aqueous phase is separated from the organic phase and diluted with a volume of methanol or other low molecular weight alcohol.
The rich aqueous solution is added at low temperature in 40/50 volumes of acetone or isopropanol.
The white crystalline precipitate (potassium clavulanate with a purity above 800 mcgr / mg) is separated by filtration and dried in vacuo at room temperature.
In this way 40 kg of pure potassium clavulanate are obtained.
Example 3
Using the same amounts of raw materials according to the previous example and adding a lithium salt (such as carbonate or acetate), pure lithium clavulanate is obtained.
Claims (16)
1) A process of purification of clavulanic acid by direct precipitation of the alkaline salts of clavulanic acid in very pure form characterized the process because it comprises the addition to a purified organic solution of clavulanic acid from an aqueous solution of an alkali metal salt to a pH between 6 and 6 and precipitation of the alkali salt of clavulanic acid by the addition of a low molecular weight alcohol followed by the addition to the hydroalcoholic phase of a solvent selected from isopropanol and / or acetone at low temperature
2) A process of purification of clavulanic acid by direct precipitation of the alkali salts of clavulanic acid in very pure form according to clause 1 characterized the process because it comprises the following steps, a) Pur? F? Cac? On of the free fermentation broth of solids and clarification of clavulanic acid by absorption on a column containing an ion exchange resin and elution with an aqueous solution of a sadistic salt at low temperature, b) Extraction of the eluate obtained in step a) by means of a solvent immiscible with water at an acidic pH, c) Addition to the purified organic solution of an aqueous solution of an alkali metal salt at a pH between 6 and 6 5, d) Prec? p? tac? on of the salt of an alkali metal of clavulanic acid by adding a low molecular weight alcohol to the aqueous solution obtained in the previous step followed by the addition to the hydroalcoholic phase of a solvent selected from isopropanol and / or acetone at low temperature
3) A process of purification of clavulanic acid by direct precipitation of the alkali salts of clavulanic acid in very pure form according to clauses 1 and 2 characterized in that in step c) the alkali metal salt is selected from carbonates, bicarbonates or acetates of lithium, potassium and sodium 4).
A process of purification of clavulanic acid by direct precipitation of the alkali salts of clavulanic acid in very pure form according to clauses 1 and 2 characterized in that in step c) the pH is 6.2.
5). A process of purification of clavulanic acid by direct precipitation of the alkali salts of clavulanic acid in very pure form according to clauses 1 and 2 characterized in that in step d) the low molecular weight alcohol is methanol.
6). A process of purification of clavulanic acid by direct precipitation of the alkali salts of clavulanic acid in very pure form according to clauses 1 and 2, characterized in that in step d) the temperature of precipitation is kept between 5 C to 15 C.
7). A process of purification of clavulanic acid by direct precipitation of the alkali salts of clavulanic acid in very pure form according to clauses 1 and 2 characterized in that in step a) the ion exchange resin is of the polystyrenic type, the sadistic salt used as eluent is the sodium chloride and the temperature at which the absorption is carried out is selected between 0 C and 5 C.
8). A process of purification of clavulanic acid by direct precipitation of the alkali salts of clavulanic acid in very pure form according to clauses 1 and 2, characterized in that in step b) the water-immiscible solvent is selected from ethyl or butyl acetate and / or methyl isobutyl ketone.
9). A process of purification of clavulanic acid by direct precipitation of the alkali salts of clavulanic acid in very pure form according to clauses 1 and 2 characterized in that in step b) the pH varies between 1.0 to 1.5 preferably 1.1 and is controlled using sulfuric acid and other inorganic acids and the temperature varies between 5 C up to 10 C preferably 10 C.
10). A process for the production of purified clavulanic acid or its salts by means of a fermentation of a strain selected from Streptomyces Clavuligerus var FAC comprising; a) Preparation of a spore suspension of Streptomyces Clavuligerus var FAC; b) Inoculation of said suspension in a seed tank; c) Fermentation of the strain characterized by the following steps d) Purification of fermentation broth free of solids and clarified of clavulanic acid by absorption on a column containing an ion exchange resin and elution with an aqueous solution of a sadistic salt at low temperature; e) Extraction of the eluate obtained in step d) by means of a solvent immiscible with water at an acidic pH; f) Addition to the purified organic solution of an aqueous solution of an alkali metal salt at a pH between 6 and 6.5; g) Precipitation of the salt of an alkali metal of clavulanic acid by the addition of a low molecular weight alcohol to the aqueous solution obtained in the previous step, followed by the addition to the hydroalcoholic phase of a solvent selected from isopropanol and / or acetone at low temperature.
eleven ). A process for producing purified clavulanic acid or its salts by means of fermentation according to clause 10 characterized in that in step a) the spore suspension is preferably prepared by growing the microorganism on a solid agar medium containing a carbon source such as dextrin or dextrose, a calcium salt, mineral salts and Co and Fe salts as trace elements, selecting the best producing colonies by first growing them in a solid vegetative medium containing a protein source, corn starch as a carbon source, a potassium salt as a source of minerals and then in a fermentative medium containing soybean or peanut flour as a protein source, dextrose as a carbon source, a potassium salt as a source of minerals and trace elements Ni and Co, the colonies that are better producers are propagated then in a solid medium.
12). A process for the production of purified clavulanic acid or its salts by means of fermentation according to clause 10, characterized in that in step b) the suspension of spores of the colonies with the best productivity is inoculated in a seed tank containing a sterile medium having flour of soy, cotton or corn as a protein source, dextrose or dextrose as a source of carbon and sodium salts
13) A process for producing purified clavulanic acid or its salts by means of fermentation according to clause 10, characterized in that in step a) and b) the temperature is maintained between 20 C to 37 C and under agitation
14) A process for producing purified clavulanic acid or its salts by means of fermentation according to clause 10, characterized in that in step c) the fermentation is carried out under aerobic conditions at temperatures from 20 C to 37 C, preferably 25 C to 30 C low agitation and selected protein sources of corn flour, peanut flour, soybean meal, cottonseed meal and degreased cottonseed, the carbon source is selected from dextnna, dextrose or corn starch, the mineral salts are selected potassium, calcium, sodium and as trace elements are used inorganic salts of Co, Fe, Mg, Ni, Zn and Cu
15) A process for the production of purified clavulanic acid or its salts by means of fermentation according to clause 10, characterized in that in step c) the fermentation is carried out under aerobic conditions at a temperature of between 24 C to 7 C and at a pH selected from 6 8 to 7 0 in a medium consisting of a selected protein source of soybean meal, peanut meal and / or cottonseed meal, a carbon source selected from dextpna, dextrose or corn starch, selected tgglicepdos and trace elements of inorganic salts of Fe, Mg, Zn and Cu
16) A process for the production of purified clavulanic acid or its salts by means of fermentation according to clause 10 characterized in that in step c) the fermentation is carried out in the presence of assimilable carbon sources, nitrogen sources, mineral salts and triglycerides where the amounts of protein sources, carbohydrate sources and triglycerides in the culture medium vary from 1% to 10%, preferably from 2% to 5%.
SUMMARY
The present invention relates to a process for the recovery of clavulanic acid without the use of amines, in which the alkali salts of clavulanic acid are precipitated in extremely pure form by extracting an organic solution of clavulanic acid with an aqueous solution of an alkali metal at a pH between 6.0 and 6.5, adding a lower alcohol to the aqueous phase and causing precipitation at low temperature by the addition of a solvent such as isopropanol or acetone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| MXPA/A/1997/002761A MXPA97002761A (en) | 1997-04-16 | Procedure for recovery of clavulan acid without use of ami |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| MXPA/A/1997/002761A MXPA97002761A (en) | 1997-04-16 | Procedure for recovery of clavulan acid without use of ami |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| MX9702761A MX9702761A (en) | 1998-10-31 |
| MXPA97002761A true MXPA97002761A (en) | 1999-01-11 |
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