CN103255189A - Preparation method for teicoplanin - Google Patents
Preparation method for teicoplanin Download PDFInfo
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- CN103255189A CN103255189A CN2013101710709A CN201310171070A CN103255189A CN 103255189 A CN103255189 A CN 103255189A CN 2013101710709 A CN2013101710709 A CN 2013101710709A CN 201310171070 A CN201310171070 A CN 201310171070A CN 103255189 A CN103255189 A CN 103255189A
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Abstract
The invention relates to the field of fermentation for antibiotics, and discloses a preparation method for teicoplanin. The preparation method comprises the steps of: fermenting by teicoplanin producing bacteria to prepare teicoplanin, and continuously removing the generated teicoplanin out of a fermentation system during the fermentation process. After the generated teicoplanin is removed out of the fermentation system, the concentration of antibiotics in fermentation liquor can be reduced, and the adverse effects of the teicoplanin producing bacteria can be avoided, thus greatly raising the fermentation level of teicoplanin, and achieving an output of 3.9 g/L.
Description
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Technical field
The present invention relates to antibiotic fermentation field, more specifically, relate to a kind of preparation method of teicoplanin.
Background technology
Microbiotic is a kind of secondary metabolite of microorganism, building-up process at it is subjected to the control that metabolism is regulated, it is produced bacterium have certain influence, the research antibiotics generated bacterium is to the tolerance mechanism of himself product, to improving the production of antibiotics ability, the microbiotic of developing new kind has important directive significance.
Teicoplanin (Teicoplanin) claims teicoplanin (Teicomycin A2) again, and it is the important microbiotic that the another present clinical treatment multi-drug resistant bacteria after vancomycin infects.Vancomycin and teicoplanin be present only can useful effect in the active drug of streptococcus aureus (MRSA), the worldwide problem of MRAS has caused the demand of vancomycin and teicoplanin to increase.Compare with vancomycin, teicoplanin is lower because of its toxic side effect, and the transformation period is longer and have more advantage in vivo.
For many years, the researchist is endeavouring to improve the output of teicoplanin always, be mostly from the screening mutant strain with improve fermentation condition and improve, and obtained certain effect.The teicoplanin production peak is 2.8 g/L (Lee et al., Optimization of culture conditions and scale-up to plant scales for teicoplanin production by Actinoplanes teichomyceticus. Appl Microbiol Biotechnol (2008) 80:21 – 27) at present.But want to utilize fermentation method further to improve the output of teicoplanin, comparatively difficult technically.
Conventional teicoplanin purification process is that fermentation is finished back adding sodium hydroxide in the fermented liquid and made pH be upgraded to 11 to carry out alkalinisation treatment, the dissolving mycelium is in order to discharge teicoplanin, stir and be neutralized to pH8.0 with hydrochloric acid after one hour, if 5000 L ferment tank teicoplanins, well imagining needs a large amount of sodium hydroxide and hydrochloric acid, and environment is caused great pressure.
Summary of the invention
Technical problem to be solved by this invention is, can't further improve the deficiency of teicoplanin output in order to overcome the fermentation method that utilizes that exists in the prior art, and a kind of preparation method of teicoplanin is provided.
Technical problem to be solved by this invention is achieved by the following technical programs:
A kind of preparation method of teicoplanin, this preparation method is: utilize teicoplanin to produce bacterium fermentative preparation teicoplanin, constantly the teicoplanin that produces is shifted out fermentation system during the fermentation.
The contriver finds that the microbiotic teicoplanin is to the deleterious effect of self thalline.At this phenomenon, the present invention during the fermentation, constantly shifts out fermentation system with the teicoplanin that produces, and reduces the concentration of microbiotic in fermented liquid, is avoided it teicoplanin is produced the deleterious effect of bacterium, thereby improve the output of teicoplanin.
A kind of preparation method of teicoplanin, this preparation method is: utilize teicoplanin to produce bacterium fermentative preparation teicoplanin, constantly the teicoplanin that produces is shifted out fermentation system during the fermentation, make the teicoplanin content in the fermentation system be lower than 15 mg/L.
As a kind of preferred version, make the teicoplanin content in the fermentation system be lower than 5 mg/L.
As a kind of preferred version, this preparation method is: utilize teicoplanin to produce bacterium fermentative preparation teicoplanin, produce in the fermenting process of bacterium at teicoplanin, add the resin of adsorbable teicoplanin.
The resin that adds the adsorbable microbiotic teicoplanin of significant quantity in the fermented liquid effectively reduces accumulation owing to teicoplanin and causes deleterious effect to self thalline, has improved the output of teicoplanin greatly.And use this method can simplify product and extract purge process, avoid ordinary method to extract in the purifying teicoplanin process and used bronsted lowry acids and bases bronsted lowry, saved production cost, be conducive to environment protection.
As a kind of preferred version, described resin is low-pole macroporous resin, non-polar macroporous resin and/or affine resin.
As a kind of most preferably scheme, described non-polar resin is polystyrene resin; Described low-pole resin is poly-alkane ester resin; Described affine resin is the D-Ala-DAla-AGA resin.
Above-mentioned resin can both be bought from the market and obtain.For example, the polystyrene resin commodity have Amberlite XAD 16 HP resins.
As a kind of preferred version, the adding volume of resin is 1 ~ 10% of culture volume or fermentating liquid volume.
As a kind of most preferably scheme, the adding volume of resin is 5 ~ 7% of culture volume or fermentating liquid volume.
As a kind of preferred version, the time of adding resin is for directly adding resin in the preceding substratum of sterilization when fermentation is initial or after for some time is carried out in fermentation resin being added in the fermented liquid.For some time is carried out in described fermentation, refers to ferment carry out adding after 12 ~ 72 hours.Such as can when fermentation is carried out 12,24,36,48,60 or 72 hours, adding.
As a kind of most preferably scheme, the time that adds resin is directly resin to be added in the preceding substratum of sterilization when fermentation is initial.
As a kind of preferred version, it is actinoplanes A. that described teicoplanin produces bacterium
Teichomyceticus
As a kind of preferred version, after fermentation is finished, be that 4 ~ 9.5 desorbed solution is resolved with the resin that is adsorbed with teicoplanin with the pH value, namely get teicoplanin; Described desorbed solution is organic solvent.
As a kind of most preferably scheme, described pH value is 4 ~ 7; Described organic solvent is that volume fraction is 50 ~ 100% methyl alcohol, 50 ~ 100% ethanol and/or 50 ~ 100% acetone.
As a kind of preferred version, also comprise inorganic salt in the described desorbed solution.Add inorganic salt and can accelerate resolution speed.
As a kind of most preferably scheme, described inorganic salt are ammonium salt or phosphoric acid salt.
As a kind of preferred version, described desorbed solution volume is 3 ~ 10 times of resin volume.
As a kind of preferred version, described resolution temperature is 5 ~ 30 ℃, stirs the mixing of desorbed solution and resin
Liquid 30 ~ 60 minutes.
As a kind of preferred version, before carrying out the desorbed solution parsing, the resin that is adsorbed with teicoplanin is washed.The adsorbed impurity of resin is removed in acting as of washing.
Compared with prior art, the present invention has following beneficial effect:
(1) preparation method of teicoplanin of the present invention has improved the teicoplanin fermentation level greatly, and output reaches 3.9 g/L, surpasses the output of the highest 2.8g/L of report at present;
(2) simplified the downstream extraction process, more friendly to environment.Because of the present invention in the teicoplanin fermenting process, utilize the resin absorption teicoplanin, the content of remaining teicoplanin seldom in the fermented supernatant fluid, comparing with traditional method does not need with sodium hydroxide fermented liquid to be carried out alkalinisation treatment and sour neutralization procedure, in the leaching process of teicoplanin, saved a large amount of bronsted lowry acids and bases bronsted lowries, reduce the managing risk in the production process, be conducive to environmental protection and reduce cost.
Description of drawings
Fig. 1 adds teicoplanin to the feedback inhibition of end product in the fermented liquid.
Fig. 2 adds teicoplanin to the influence of thalline weight in wet base in the fermented liquid.
Embodiment
Further explain the present invention below in conjunction with specific embodiment, but embodiment does not do any type of restriction to inventing itself.
The titration method of embodiment of the invention teicoplanin
The agar plate diffusion process is adopted in titration, and the indicator of measuring the teicoplanin biological value is subtilis
Bacillus subtilisCMCC 63501.With known teicoplanin standardized solution of tiring and product to be tested solution, carry out contrast culture at the substratum of bacillus subtilis bacteria growing, produce transparent inhibition zone.The size of standard of comparison product and product to be tested inhibition zone utilizes two dosimetries to calculate, and obtains the antibacterial potency of teicoplanin.
Embodiment 1 teicoplanin is to the influence of self thalli growth
Teicoplanin solid plate culture medium prescription consists of (g/L): glucose 1.0, yeast powder 2.0, K
2HPO
41.0 KCl 6.0, MgSO
4.7H
2O 0.6, FeSO
4.7H
2O 0.04, and pH 7.0.The teicoplanin finished product is mixed with certain density solution, is applied to teicoplanin after the filtration sterilization and produces on the bacterium solid medium, again with the coating of teicoplanin spore suspension thereon.28 ℃ of incubators were cultivated 7 ~ 10 days, observed the spore growing state.The result is as shown in table 1.
Show teicoplanin to the influence of self thalline from table 1, along with the teicoplanin concentration that is coated on planar surface increases, the time that spore begins to sprout is more long, and the speed of growth is slow, and when being coated with 0.012 g teicoplanin, himself spore is grown hardly.
Embodiment 2 teicoplanins are to the influence of fermentation
The teicoplanin fermentative medium formula consists of (g/L): dextrin 12, Zulkovsky starch 20, analysis for soybean powder 24, corn steep liquor 16, CaCO
32.0 pH 6.8.Fermentation volume 150 mL, (24 hours) add the teicoplanin product solution of the degerming after filtration of different concns (0 to 30 mg/L) in the fermented liquid in fermentation way.28 ℃, survey them behind 150 rpm shaking table oscillation and fermentation cultivation, 8 d and tire and thalline weight in wet base (thalline weight/fermented liquid weight * %), observe production productive rate and the thalli growth of teicoplanin whether be affected.The result as depicted in figs. 1 and 2.
Can find out that from Fig. 1, Fig. 2 not adding tiring of teicoplanin is 521.32 U/mL, the thalline weight in wet base is 20.7%, and when the interpolation concentration of teicoplanin was 5 ~ 15 mg/L, tiring of teicoplanin was slow increase trend, and weight in wet base slowly descends; When the concentration of teicoplanin was 20 mg/L, final tiring of teicoplanin was 306.70 U/mL, descended 41%, and weight in wet base is 13.63%, has descended 35%; When teicoplanin concentration was 30 mg/L, finally tiring only had 116.62 U/mL, and its output has nearly descended 80%, and the thalline weight in wet base has descended 79%.Its formation is subjected to the severe inhibition effect.
The accumulation that above result clearly states secondary metabolite teicoplanin in the fermenting process is to himself thalline and produce and all have stronger restraining effect.
Embodiment 3
The teicoplanin fermention medium shown in embodiment 2,28 ℃ of leavening temperatures, fermentation volume 1L.In fermentation 0,24,48,72 hour the time, add respectively 0% ~ 10% the poly-alkane ester resin of low-pole (%, M/V) or nonpolar polystyrene resin (%, M/V), all fermentation times are 8 days.The result that tires of the poly-alkane ester resin of low-pole of (during the fermentation beginning) adding 0% to 10% when table 2 is depicted as 0 hour.
Described resin is before adding, need through pre-treatment, treatment process can be as follows: 12 h soak with 100% methyl alcohol in elder generation, to remove impurity wherein, after being washed till neutrality with purified water, resin is put into the ultrasonic cleaner ultrasonic degas, and the resin after the processing adds in the unpasteurized fermention medium or independent 121 ℃, 30 min sterilization.
The result shows, in different resin add-ons, and the best results of 5% ~ 7% (resin volume/fermentating liquid volume) wherein.As shown in table 2,0 hour the time, add 7% the poly-alkane ester resin of low-pole and nonpolar polystyrene resin in fermentation, the output of teicoplanin is respectively 1892.42 U/mL and 3173.20U/mL, is respectively not add 3.36 times and 5.63 times of resin.Amounting to output is 3.9 g/L, and the output of teicoplanin has surpassed present teicoplanin production peak 2.8 g/L among the present invention.And the content of remaining teicoplanin is few in the fermented liquid, only is 1% of ultimate production.
The purifying of embodiment 4 teicoplanins
After finishing to the fermentation of the fermented liquid of embodiment 3, fermented liquid is isolated polymeric adsorbent after filtration, and polymeric adsorbent is dipped in 80% methanol solution and resolves.Resolve and at room temperature carry out the mixed solution of agitating resin and 80% methanol solution 30 to 60 minutes.
The routine fermentation of Comparative Examples 1 teicoplanin
The teicoplanin fermentative medium formula consists of (g/L): dextrin 12, Zulkovsky starch 20, analysis for soybean powder 24, corn steep liquor 16, CaCO
32.0 pH 6.8.28 ℃ of leavening temperatures, fermentation volume 1L.Fermentation time 8 days.
The result shows: among the embodiment 3, the poly-alkane ester resin of the low-pole of adding 7% (%, rate ratio Comparative Examples 1 W/V) is high 3.36 times, and (%, W/V) rate ratio Comparative Examples 1 is high 5.63 times for the nonpolar polystyrene resin of adding 7%.
The conventional purifying of Comparative Examples 2 teicoplanins
After Comparative Examples 1 fermentation finishes, fermentation is finished back adding sodium hydroxide in the fermented liquid and is made pH be upgraded to 11 to carry out alkalinisation treatment, the dissolving mycelium is in order to discharge teicoplanin, stir and be neutralized to pH8.0 with hydrochloric acid after one hour, filter back upper prop chromatographic column, with ealkaline buffer such as pH 9.5,4mol/ L NaHCO
3(containing acetonitrile) washes post, collects significant part.The result is low more about 70% ~ 80% than the fermentation level of the embodiment of the invention 4, and needs a large amount of bronsted lowry acids and bases bronsted lowries.
Claims (10)
1. the preparation method of a teicoplanin, this preparation method is: utilize teicoplanin to produce bacterium fermentative preparation teicoplanin, it is characterized in that, constantly the teicoplanin that produces is shifted out fermentation system during the fermentation.
2. the preparation method of a teicoplanin, this preparation method is: utilize teicoplanin to produce bacterium fermentative preparation teicoplanin, it is characterized in that, constantly the teicoplanin that produces is shifted out fermentation system during the fermentation, make the teicoplanin content in the fermentation system be lower than 15 mg/L.
3. preparation method according to claim 1 and 2, this preparation method is: utilize teicoplanin to produce bacterium fermentative preparation teicoplanin, it is characterized in that, produce in the fermenting process of bacterium at teicoplanin, add the resin of adsorbable teicoplanin.
4. preparation method according to claim 3 is characterized in that, described resin is low-pole macroporous resin, non-polar macroporous resin and/or affine resin.
5. preparation method according to claim 4 is characterized in that, described non-polar resin is polystyrene resin; The low-pole resin is poly-alkane ester resin; Described affine resin is the D-Ala-DAla-AGA resin.
6. preparation method according to claim 3 is characterized in that, the adding volume of resin is 1 ~ 10% of culture volume or fermentating liquid volume.
7. preparation method according to claim 6 is characterized in that, the adding volume of resin is 5 ~ 7% of culture volume or fermentating liquid volume.
8. preparation method according to claim 1 and 2 is characterized in that, it is actinoplanes A. that described teicoplanin produces bacterium
Teichomyceticus
9. preparation method according to claim 1 and 2 is characterized in that, after fermentation is finished, is that 4 ~ 9.5 desorbed solution is resolved with the resin that is adsorbed with teicoplanin with the pH value, namely gets teicoplanin; Described desorbed solution is organic solvent.
10. preparation method according to claim 9 is characterized in that, the pH value of described desorbed solution is 4 ~ 7; Described organic solvent is that volume fraction is 50 ~ 100% methyl alcohol, 50 ~ 100% ethanol and/or 50 ~ 100% acetone.
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| CN2013101710709A CN103255189A (en) | 2013-05-10 | 2013-05-10 | Preparation method for teicoplanin |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1671640A1 (en) * | 2004-12-17 | 2006-06-21 | Biongene Co. Ltd. | Mutant strain of Actinoplanes teichomyceticus for the production of teicoplanin |
| CN102417919A (en) * | 2011-09-02 | 2012-04-18 | 山东鲁抗医药股份有限公司 | Method for producing high-purity teicoplanin by way of fermentation |
| CN102718843A (en) * | 2012-06-30 | 2012-10-10 | 华北制药集团新药研究开发有限责任公司 | Preparation method of single teicoplanin components |
-
2013
- 2013-05-10 CN CN2013101710709A patent/CN103255189A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1671640A1 (en) * | 2004-12-17 | 2006-06-21 | Biongene Co. Ltd. | Mutant strain of Actinoplanes teichomyceticus for the production of teicoplanin |
| CN102417919A (en) * | 2011-09-02 | 2012-04-18 | 山东鲁抗医药股份有限公司 | Method for producing high-purity teicoplanin by way of fermentation |
| CN102718843A (en) * | 2012-06-30 | 2012-10-10 | 华北制药集团新药研究开发有限责任公司 | Preparation method of single teicoplanin components |
Non-Patent Citations (2)
| Title |
|---|
| J.C. LEE，ET AL.: "Improved production of teicoplanin using adsorbent resin in fermentation", 《LETTERS IN APPLIED MICROBIOLOGY》 * |
| 邓小宽等: "糖肽类抗生素提取分离的研究概况", 《中国医药工业杂志》 * |
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Application publication date: 20130821 |