CN102311486B - Method for separating and extracting enramycin by using macroporous weakly-acidic cationic resin - Google Patents
Method for separating and extracting enramycin by using macroporous weakly-acidic cationic resin Download PDFInfo
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Abstract
The invention provides an effective method for separation and purification of enramycin, and particularly provides a method for separating and extracting enramycin by using macroporous weakly-acidic cationic resin. The method comprises the following steps: completely activating the macroporous weakly-acidic cationic resin by using methanol, and then adding sufficient water in the activated resin for stirring and mixing; carrying out column packing, and then passing a weak-alkaline methanol solution through the column for balancing; heating enramycin mycelium in a water bath by using ethanol, and then refluxing so as to obtain a crude product; mixing the obtained crude product with a weakly-acidic methanol water solution; filtering, and adjusting the pH of the obtained solution to alkalescence; loading the column, adsorbing, and then washing with methanol water to remove impurities; and eluting with a proper amount of methanol water, collecting eluent, concentrating, and drying by distillation so as to obtain the product enramycin. The method provided by the invention is simple in process, strong in maneuverability, and high in recovery rate and purity.
Description
Technical field
The invention belongs to separation technology field, relate to a kind of method of using macropore acidulous cation resin separation and Extraction enramycin.
Background technology
Since microbiotic invention, not only effect is remarkable aspect the treatment of human diseases and prevention, is also bringing into play huge effect aspect the livestock feed additive simultaneously.Microbiotic is to be found by people such as Stocktadt in the later stage forties 20th century to the growth promoting function of animal, find in succession that later on tetracyclines, Macrolide, β-microbiotic such as Nei phenol amine add in feed, the growth of promotion, weightening finish, volume increase are arranged, improve price of deed effect.Nineteen fifty U.S. FDA official approval allows to add microbiotic in feed.Microbiotic is sure for the growth of livestock and poultry and the effect of raising production performance, and very stable, and the poultry industry of many countries is the prolonged application Antibiotic Additive all.Facts have proved, reasonably use Antibiotic Additive can promote growth of animals or poultry, improve food conversion ratio.Also have simultaneously report to show, do not allow to add the country of microbiotic or other antivirus somatotropic agents in feed, can cause the livestock industry production cost to increase, profit reduces, and also might cause such as the problems such as trade war between ecotope and country simultaneously.But for fear of causing the Resistant strain amount reproduction due to antibiotic abuse or the residual quantity of medicine in livestock product being increased, growth of animals or poultry and HUMAN HEALTH are caused direct harm, and the antibiotic feed additive of using the livestock and poultry special use is extremely urgent.Because enramycin has good growth promotion and improves the effect of efficiency of feed utilization, therefore recommended as the microbiotic growth promoter by many countries in the world.
Enramycin (Enramycin), have another name called Enramycin, enramycin, enramycin, enramycin, it is a kind of polypeptide antibiotics that is produced by the actinomycetes Streptomyces fungicidious NO.B5477 fermentation of separating in soil, be soluble in dilute hydrochloric acid, be slightly soluble in water, methyl alcohol, ethanol.On market, main source is the mycelium pre-mixture of Schering Plough company.
Have the following advantages as the fodder additives enramycin:
1. the clinical application research aspect, compare with other Antibiotic Additive, and in the few situation of consumption, feed coefficient is lower, and the speed of growth is faster.2. pharmacodynamic study aspect, enramycin all has powerful germicidal action to main gram-positive microorganism.Enramycin has stronger restraining effect to hepatitis B virus (HBV) antigen, comprises that hepatitis B virus e antigen (HBeAg) is also had inhibition preferably.Enramycin and do not have cross resistance between existing microbiotic or antimicrobial drug clinically.Sensitive organism produces resistance to enramycin hardly, and under experiment condition, the generation of resistance is also very slow, even occur, resistance is also unstable, and very easily loses.3. drug residue research aspect, enramycin is for oral administration extremely to be difficult for being absorbed, and drug main will excrete by ight soil.4. toxicologic study aspect, enramycin toxicity is very low.Acute toxicity test shows, enramycin reality when oral, subcutaneous or administered intramuscular is nontoxic.5. the hydrochloride of enramycin has fabulous stability to heat, illumination and humidity.Enramycin has very high stability in feed, standing storage is seldom degraded at ambient temperature, and is also highly stable in granulation material process, with after feed mixes at room temperature standing storage tire descend very little.Enramycin is not degraded in enteron aisle, can keep original anti-microbial activity.
Antibiotic extraction mainly contains following several approach:
The precipitator method, solvent extraction, absorption method, ion exchange chromatography, high performance liquid chromatography, membrane separation technique, capillary electrophoresis, Two-phase system, reverse micelle, supercritical extraction etc. are arranged.The method for newly beginning in recent years to use such as membrane separation technique, capillary electrophoresis, Two-phase system, reverse micelle, supercritical extraction wherein, cost is high, there is no comparatively ripe industrialization pattern, mostly in the laboratory small-scale, indivedual kinds is explored.
The traditional methods such as the precipitator method, solvent extraction, absorption method, because gac can not well separate impurity with most of solvents, the material purity that obtains is inadequate, thereby do not reach the effect that separation and Extraction requires, mostly be applied at present slightly carry, the crude product pretreatment stage coordinates additive method jointly to use.In high performance liquid chromatography, by come the exclusive segment related impurities with pre-column, pre-column is often because can not regeneration causing the consume cost compare high mostly.Most microbiotic kind separate, extract, the aspect widespread uses such as concentrated, purifying, decolouring be chromatography.
In Chinese invention patent application " a kind of enramycin producing strain and utilize the method for macroporous resin extraction " (200910032340.1), record comes the separation and Extraction Enramycin with macroporous adsorbent resin Amberlite XAD-4, Amberlite XAD-16 etc.The absorption essence of macroporous adsorbent resin is that a kind of object height is disperseed or surface molecular is subjected to the unequal surface adsorption phenomenon that produces of reactive force, and this absorption property is the result due to Van der Waals force or generation hydrogen bond.Vesicular structure due to macroporous adsorbent resin makes it have the screening effect to the different material of molecular size simultaneously.By above-mentioned this absorption and screening principle, organic compound is according to the difference of adsorptive power and the size of molecular weight, reaches separation, purifying, removal of impurities, the different purposes such as concentrated on macroporous adsorbent resin through certain solvent elution.Its physico-chemical property is stable, is insoluble to acid, alkali and organic solvent, and is good to the organism selectivity, is not subjected to the impact of inorganic salts and strong ion, low molecular compound existence, also is widely used in recent years the separation and Extraction of Chinese medicine, microorganism.But this inventive method can only be controlled at 60%～71% for the productive rate of enramycin in mycelium, and the rate of recovery is lower, and is too many for large-scale industrial production consume, and content detection aspect also difficulty satisfies accuracy requirement.
The ion exchange resin that ion exchange chromatography is applied to is divided into resin cation (R.C.) and the large class of resin anion(R.A) two, they can be respectively with solution in positively charged ion and negatively charged ion carry out ion-exchange.Divide two kinds of gel-type and macroporous types, all title macroporous ion-exchange resins with physical holes structure according to pore texture.Resin cation (R.C.) is divided into again slightly acidic and strongly-acid, and wherein acidulous cation resin contains the slightly acidic group, as carboxyl-COOH, can go out H at dissociation in water
+And be acid.Resin can be combined by other cation-adsorption in solution from separating rear remaining negative electricity group, thereby produces the cationic exchange effect.The acidity of this resin be dissociative a little less than, but more easily regenerate than highly acidic resin.But for enramycin, it is generally acknowledged and to use ion-exchange resin purification, mainly use polymeric adsorbent (" the microbiology content assaying method of enramycin research in feed " Chinese veterinary drug magazine 2008,42 (9): 17～21 Gu Xin etc.).This area need to be sought better method and come the separation and Extraction enramycin, improves the rate of recovery and purity.
Summary of the invention
Technical problem to be solved by this invention is that the separation and purification for enramycin provides a kind of effective means.The technical scheme that addresses this problem has been to provide a kind of method of using macropore acidulous cation resin separation and Extraction enramycin.The technical scheme of the method for macropore acidulous cation resin separation and Extraction enramycin of the present invention comprises the following steps:
A, extract to such an extent that to contain the enramycin extracting solution standby by the enramycin crude product;
B, the macropore acidulous cation resin is soaked with capacity methyl alcohol after, remove the upper strata methanol liquid, then add enough water, standing in order to the dress post;
C, with pretreated macropore acidulous cation resin dress chromatography column, use 1: 1 methanol-water of pH 8.0 to cross post, stand-by after balance;
D, the described enramycin extracting solution that contains of step a is transferred to pH 8.0, refilter, use the supernatant liquor upper prop; Cross post with 1: 1 methanol aqueous solution, impurity is removed in washing; Use 7: 3 methanol aqueous solutions to cross post again, collect elutriant, concentrated, evaporate to dryness obtains the enramycin product.
Wherein, the acidulous cation resin of macropore described in aforesaid method is the HD-2 type.
Wherein, the method that the described enramycin crude product of aforesaid method step a extracts is for to add 1: 1 methanol-water of pH3.0 appropriate the enramycin crude product, fully mixes rear standingly, and filtration is got supernatant liquid standby as extracting solution.
Wherein, the temperature of the described evaporate to dryness of aforesaid method steps d is no more than 45 ℃.
Separately, described 7: 3 methanol-waters refer to that volume ratio is the mixing solutions of 70% methyl alcohol and 30% water, and methanol-water in like manner can be prepared in 1: 1.
Wherein, the material of the pH of adjusting described in aforesaid method value is the aqueous solution of hydrochloric acid and sodium hydroxide.
Wherein in aforesaid method, described enramycin crude product can be the mycelium that produces the enramycin bacterium, also can be the commercially available enramycin mycelium pre-mixture of Schering Plough company.
The inventive method concrete operations are as follows:
The enramycin crude product extracts: sample adds with the methanol-water (1: 1) of transferring pH 3.0 appropriate, fully mixes rear standingly, filters, and gets supernatant liquid standby as extracting solution;
The resin pre-treatment: after acidulous cation resin HD-2 is soaked with methyl alcohol, remove upper strata methyl alcohol, then add enough water, standing in order to the dress post;
Chromatography column dress post and processing: with pretreated resin dress chromatography column, use the methanol-water (1: 1) of pH 8.0 to cross post, stand-by after balance.In whole dress post process, the pillar of dress post can not dry up;
Separation and purification: aforementioned extracting solution is transferred to pH 8.0 with NaOH, and the supernatant liquor upper prop after refiltering is crossed post with methanol-water (1: 1), and impurity is removed in washing; Use methanol-water (7: 3) to cross post again, collect elutriant, concentrated, evaporate to dryness obtains the enramycin product.
Separation and Extraction enramycin of the present invention be mainly to have broken routine; introduce the purifying that acidulous cation resin HD-2 type carries out enramycin; and the method is optimized; extracting product purity all reaches more than 98%; the rate of recovery is up to more than 85%; be applicable to large-scale industrial production, for this area purifying enramycin provides a kind of new selection.
Below by embodiment, the present invention is specifically described.
Embodiment
The inventive method immerses the macropore acidulous cation resin in the methyl alcohol of capacity, makes abundant activation, after placement, removes the upper strata methanol liquid, adds the water of capacity, mixes rear dress post, crosses column equilibration with weakly alkaline methanol solution.The enramycin mycelium refluxes through the ethanol heating in water bath and obtains crude product, and crude product mixes with weakly acidic methanol aqueous solution, stirs, filter, filtrate transfers to weakly alkaline, again filters, get this filtrate flow through chromatography column, after adsorbing, then wash away impurity with common methanol-water, then wash with water and be neutrality, use at last appropriate methanol-water wash-out, collect whole elutriants, concentrated, get the product enramycin after evaporate to dryness.
In aforesaid method, the preferred HD-2 type resin cation (R.C.) that uses, preferably use methanol-water (1: 1) and methanol-water (7: 3) to wash and wash-out.
Particularly, the inventive method is:
The enramycin crude product extracts: sample adds with the methanol-water (1: 1) of transferring pH 3.0 appropriate, fully mixes rear standingly, filters, and gets supernatant liquid standby as extracting solution.Transfer pH generally to use concentrated hydrochloric acid and sodium hydroxide.
Resin pre-treatment: after acidulous cation resin HD-2 is soaked with methyl alcohol, remove upper strata methyl alcohol, then with adding the same water gaging of methyl alcohol, standing 20 minutes in order to the dress post.
Chromatography column dress post and processing: with pretreated resin dress chromatography column, the methanol-water (1: 1) of the pH 8.0 of use is crossed post, and flow velocity 0.4ml/min is stand-by after balance.In whole dress post process, the pillar of dress post can not dry up.
Separation and purification: aforementioned extracting solution is transferred to pH 8.0 with NaOH, supernatant liquor upper prop after refiltering, upper column flow rate 0.5ml/min crosses post, flow velocity 0.4ml/min with methanol-water (1: 1), impurity is removed in washing, use methanol-water (7: 3) to cross post, flow velocity 0.3ml/min collects elutriant again, concentrated, evaporate to dryness obtains the enramycin product.
Implementation process reaches and the existing methodical embodiment that relatively asks for an interview in detail.
The enramycin crude product that uses in following instance is the commercially available enramycin 4% mycelium pre-mixture of Schering Plough company.The enramycin standard substance are that Schering Plough company is commercially available, and purity detecting uses conventional H PLC to carry out.Macropore acidulous cation resin HD-2, Hebei deep blue grace Chemical Manufacture.
Embodiment one use HD-2 type resin cation (R.C.) separation and purification enramycin
The present embodiment is the condition of using on the macroporous adsorbent resin of diverting from one use to another.
1,50% methanol-water of enramycin crude product 10g+50ml (pH 3.0) mixes, and stirs, and filters, and gets supernatant liquid standby as extracting solution.
2, separation and purification, the present embodiment are the conditions of using on the macroporous adsorbent resin of diverting from one use to another.
(1) resin pre-treatment:
HD-2 type macropore acidulous cation resin immerses in the methyl alcohol of capacity, slowly stirs, and makes abundant mixing, after placement, carefully removes the upper strata methanol liquid, adds the water of capacity, after mixing, places, and is standby.
(2) chromatography column is processed:
Select the chromatography column of 1cmX20cm specification, the resin dress post that pre-treatment is good in whole dress post process, can not dry up the pillar of dress post.Use the methanol-water (1: 1) of pH 8.0 alkalescence of 25ml to cross post with the 0.4ml/min flow velocity, after balance, stand-by.
(3) separation and purification:
It is that alkalescence (pH=8.0) is filtered that extracting solution is transferred to PH with NaOH liquid, get filtrate with 0.5ml/min speed stream process chromatography column, cross post with appropriate pH value 8.0 methanol solutions (concentration is 1: 1) flow velocity with 0.4ml/min again, impurity is removed in washing, then use the pH3.0 methanol solution (concentration 7: 3) of 50ml to cross post with the 0.3ml/min flow velocity, collect whole eluting liquids, concentrated, get the product enramycin after evaporate to dryness.The gained enramycin rate of recovery is 73.1% after testing, and purity is 97.4%.
Embodiment two uses the inventive method macropore acidulous cation resin HD-2 separation and purification enramycin
1, sample is slightly carried
Enramycin crude product 130g+200ml (methyl alcohol)+200mlH
2O
With standing after the abundant stirring 120min of concentrated hydrochloric acid HCl accent pH 3.0, filter, get supernatant liquid standby as extracting solution.
2, separation and purification, this example are to use the inventive method to carry out middle amount purifying.
(1) resin pre-treatment:
Acidulous cation resin HD-2 removes methyl alcohol after soaking 60min with methyl alcohol, then the 20min dress post that is soaked in water.
(2) chromatography column is processed:
Select the chromatography column of 1cmX20cm specification, the resin dress post that pre-treatment is good in whole dress post process, can not dry up the pillar of dress post.Use the methanol-water (1: 1) of the pH 8.0 of 25ml to cross post with the 0.5ml/min flow velocity, after balance, stand-by.
(3) separation and purification:
The extracting solution of step 1 gained is transferred to pH 8.0 with NaOH, and the supernatant after filtration is crossed post, 0.4ml/min with the 0.5ml/min upper prop with methanol-water (1: 1); Impurity is removed in washing, and washing shares 650ml.Use 650ml methanol-water (7: 3) 0.3ml/min to cross post again, collect elutriant, concentrated, evaporate to dryness obtains product.The gained enramycin rate of recovery 86.3% after testing, purity is 98.5%.
Claims (3)
1. use the method for macropore acidulous cation resin separation and Extraction enramycin, it is characterized in that comprising the following steps:
A, extract to such an extent that to contain the enramycin extracting solution standby by the enramycin crude product;
B, the macropore acidulous cation resin is soaked with capacity methyl alcohol after, remove the upper strata methanol liquid, then add enough water, standing in order to the dress post; Described macropore acidulous cation resin is the HD-2 type;
C, with pretreated macropore acidulous cation resin dress chromatography column, use the 1:1 methanol-water of pH8.0 to cross post, stand-by after balance;
D, the described enramycin extracting solution that contains of step a is transferred to pH8.0, refilter, use the supernatant liquor upper prop; Cross post with the 1:1 methanol aqueous solution, impurity is removed in washing; Cross post with the 7:3 methanol aqueous solution again, collect elutriant, concentrated, evaporate to dryness obtains the enramycin product.
2. the method for use macropore acidulous cation resin separation and Extraction enramycin according to claim 1, it is characterized in that: the method that the described enramycin crude product of step a extracts is for to add the 1:1 methanol-water of pH3.0 appropriate the enramycin crude product, fully mix rear standing, filter, get supernatant liquid standby as extracting solution.
3. the method for use macropore acidulous cation resin separation and Extraction enramycin according to claim 1, it is characterized in that: the temperature of the described evaporate to dryness of steps d is no more than 45 ℃.
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| CN102898509B (en) * | 2012-11-04 | 2014-05-21 | 乐占线 | Method for preparing enramycin crude product |
| CN103232530B (en) * | 2013-04-17 | 2014-10-29 | 南京工业大学 | Enramycin A and B separation method |
| CN104402976B (en) * | 2014-12-29 | 2016-06-01 | 江西兴鼎科技有限公司 | A kind of method preparing enramycin fine powder |
| CN105037497B (en) * | 2015-03-27 | 2018-06-26 | 中牧实业股份有限公司 | A kind of method of purification of enramycin |
| CN108864256B (en) * | 2018-07-17 | 2022-02-01 | 浙江工商大学 | Method for separating and purifying enramycin A and enramycin B |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101597578A (en) * | 2009-07-01 | 2009-12-09 | 南京工业大学 | A kind of enramycin producing strain and utilize the method for macroporous resin extraction |
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Non-Patent Citations (6)
| Title |
|---|
| 周昕 等.大孔弱酸性树脂( HD-2) 提取尿激酶性能的研究.《衡阳医学院学报》.1998,第26卷(第3期),274－277. |
| 大孔弱酸性树脂( HD-2) 提取尿激酶性能的研究;周昕 等;《衡阳医学院学报》;19980930;第26卷(第3期);274－277 * |
| 大孔树脂分离纯化持久霉素*;王丹丹 等;《食品与发酵工业》;20100430;第36卷(第4期);194－197 * |
| 王丹丹 等.大孔树脂分离纯化持久霉素*.《食品与发酵工业》.2010,第36卷(第4期),194－197. |
| 顾欣 等.饲料中恩拉霉素的微生物学含量测定方法研究.《中国兽药杂志》.2008,第42卷(第9期),17－21. |
| 饲料中恩拉霉素的微生物学含量测定方法研究;顾欣 等;《中国兽药杂志》;20081231;第42卷(第9期);17－21 * |
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