JPH0956389A - Purification of avermectin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain the subject highly pure compound useful as a raw material for parasite medicines, etc., by extracting insolubles obtained from the fermentation culture solution of the avermectin with the water- containing solution of a solvent miscible with water and subsequently concentrating the extract or adding water to the extract to generate precipitates. SOLUTION: A method for purifying avermectin comprises culturing Streptomyces avermitilis K2038 (FERM BP-2775) in a spore-forming flat culture medium at 28 deg.C for 7 days, transplanting the formed spores in a culture medium, subjecting the spores to a shaking culture treatment at 28 deg.C for 168hr, filtering the obtained culture solution of the avermectin, washing the avermectin mixture insolubilized together with the cells with water, bringing the obtained aqueous solution into contact with the water-containing solution of a solvent (e.g. methanol) miscible with water to extract the avermectin, concentrating the extract or adding water to the extract, separating the produced precipitates, and subsequently drying the separated product. Thus, the objective purified avermectin is simply and inexpensively obtained in a high purification efficiency without using chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for simply purifying high-purity avermectin from an avermectin fermentation culture solution without using industrially complicated chromatography.

[0002]

BACKGROUND ART Avermectin is widely used as a synthetic raw material for synthesizing avermectin derivatives such as ivermectin, a parasite drug, and as a parasite drug for domestic animals. When avermectin is obtained, the fermentation broth of avermectin contains many analogs and other impurities in addition to avermectin B1a having high antiparasitic activity. As a general method for purifying avermectin, fermentation culture liquid of avermectin, or after extracting avermectin by adding an organic solvent to a cell cake obtained by filtering the fermentation culture liquid, silica gel, alumina oxide,
A method is known in which a fraction containing avermectin is concentrated to dryness by subjecting it to chromatography using a carrier such as dextran gel. There is also known a method in which a mixed solvent of methanol-ethylene glycol is added to the residue obtained by concentration to dryness, and methanol is concentrated and distilled off to obtain crystals after cooling (Japanese Patent Publication No. 17558/1990).

However, in these methods using chromatography, a large amount of a simple substance of chromatography and an organic solvent must be used because the load on the carrier is low.
The productivity is low, the obtained avermectin purified product is expensive, and the purification method is not always satisfactory industrially. Further, a method for purifying avermectin is known, in which a solvent that mixes with water is added to a fermentation culture solution of avermectin to extract avermectin, and then water is added to the extract to obtain a precipitated precipitate (WO95 / 0341
9). However, with this method, the precipitate is obtained in the form of an oil, paste or wax. There is also a description that it is necessary to add an electrolyte, a surfactant and the like together with a solvent that is miscible with water in order to improve crystallinity, but in the examples, even if these are added, the precipitate is oily, paste-like or Only available in waxy form.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for purifying avermectin, which is simple, inexpensive and has high purification efficiency without using industrially complicated chromatography.

[0005]

Means for Solving the Problems The present invention comprises the steps of contacting an insoluble matter in a fermentation culture solution of avermectin with an aqueous solution of a solvent mixed with water to extract avermectin, and then concentrating the extract or extracting the solution. By adding water to
The present invention relates to a method for purifying avermectin, which comprises precipitating avermectin as a solid. Further, the avermectin fermentation culture liquid is contacted with an organic solvent that is immiscible with water to extract avermectin, and the organic solvent is distilled off,
Avermectin is characterized by precipitating avermectin as a solid by adding an aqueous solution of a solvent miscible with water to the residue to dissolve avermectin, and then filtering and concentrating the filtrate or adding water to the filtrate. Related to the purification method. In these cases, avermectin B1a can be selectively precipitated as a solid depending on the concentration of avermectin B1a in the fermentation broth of avermectin. Further, the avermectin B1a obtained above is dissolved in a solvent mixed with water, filtered if necessary, and then water is added to the solution or the solution is concentrated to selectively crystallize the avermectin B1a. be able to.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The avermectin fermentation culture medium used in the present invention is a culture medium in which a microorganism having an avermectin-producing ability is cultured in a nutrient medium. Examples of microorganisms capable of producing avermectin include Streptomyces avermitilis K2038 (FERM
BP-2775; JP-A-3-254678) and the like.
The nutrient medium may be any medium that is commonly used for culturing microorganisms having avermectin-producing ability, and may be, for example, the medium described in Japanese Patent Publication No. 17558/1990. The cultivation is performed under aerobic conditions such as shaking culture and aeration / agitation culture. Cultivation temperature is 20 to 40 ° C, pH
Is adjusted to 6.0 to 8.0, preferably 7.0 to 7.5. The avermectin concentration in the fermentation broth is usually preferably 0.4 g / l or higher, particularly preferably 0.5 g / l or higher. When avermectin B1a is selectively precipitated, the concentration of avermectin B1a in the fermentation broth is preferably 0.2 g / l or more,
The amount of avermectin B1a in the total avermectin is preferably 30% or more. Above all, the avermectin B1a concentration in the fermentation broth is particularly preferably 0.3 g / l or more, and the amount of avermectin B1a in the total avermectin is particularly preferably 35% or more.

As the insoluble matter in the fermentation culture liquid of avermectin, a precipitate obtained by centrifuging the fermentation culture liquid of avermectin obtained by the above-mentioned production method, a residue obtained by filtering the fermentation culture liquid of avermectin, etc. can give. Examples of the solvent that can be mixed with water include methanol, acetone, and ethanol having a boiling point lower than that of water. As a water-containing aqueous solution that mixes with water, water-containing methanol having a water content of 15 to 50%, water-containing acetone having a water content of 20 to 60%, water-containing ethanol having a water content of 20 to 60%, and the like are preferably used. Water-containing methanol with a water content of less than 15%,
When water-containing acetone having a water content of less than 20% or water-containing ethanol having a water content of less than 20% is used, avermectin is colloidally dispersed in the liquid when water is added or the solution is concentrated, and solid-liquid separation is performed. It will be impossible. On the other hand, when hydrated methanol having a water content of more than 50%, hydrated acetone having a water content of more than 60%, or hydrated ethanol having a water content of more than 60% is used, avermectin cannot be sufficiently extracted from the insoluble matter in the fermentation broth. I have a problem. When extracting the avermectin by contacting the insoluble matter in the fermentation broth of avermectin with the aqueous solution of the solvent mixed with water, add 3 to 6 times the amount of the aqueous solution to the insoluble matter,
It is preferable to carry out the extraction at 20 to 40 ° C. for 15 minutes to 2 hours. When the extract is concentrated or water is added to the extract, for example, when water-containing methanol is used, the water content becomes 50% or more, and when water-containing ethanol is used, the water content becomes 60%. Until the above, when water-containing acetone is used, it is preferable to concentrate or add water until the water content becomes 60% or more. In addition, when avermectin is extracted with a solvent that mixes directly with water from an avermectin fermentation broth or an aqueous solution thereof, avermectin is dispersed colloidally in the liquid when water is added or the solution is concentrated to give a solid-liquid solution. Separation becomes impossible.

Examples of the organic solvent which is immiscible with water include ethyl acetate, chloroform and methyl isobutyl ketone. When the avermectin is extracted by bringing the avermectin fermentation culture solution into contact with an organic solvent that is immiscible with water, add 0.3 to 1.0 times the volume of the organic solvent to the fermentation culture solution at 20 to 40 ° C. It is preferable to carry out the extraction for 15 minutes to 2 hours. When the avermectin is dissolved by distilling off the organic solvent after extraction and adding an aqueous solution of a solvent that mixes with water to the residue, the avermectin concentration is 1 to 3 g.
It is preferable to add an aqueous solution so that the amount becomes 1 / l. When the filtrate is concentrated after the solution is filtered or water is added to the filtrate, for example, when water-containing methanol is used, the water content becomes 50% or more, and when water-containing ethanol is used, the water content becomes Is preferably 60% or more, and when water-containing acetone is used, it is preferable to concentrate or add water until the water content becomes 60% or more. The water content is 1
When less than 5% water-containing methanol, water content less than 20% water-containing acetone, water content less than 20% water-containing ethanol, etc. is used, avermectin is colloidally dispersed in the liquid when water is added or the filtrate is concentrated. Then, solid-liquid separation becomes impossible. On the other hand, when water-containing methanol with a water content of more than 50%, water-containing acetone with a water content of more than 60%, or water-containing ethanol with a water content of more than 60% is used, the residue after distilling off the solvent immiscible with water However, there is a problem that avermectins of the above can not be sufficiently extracted.

As the avermectin B1a to be crystallized, a solution containing avermectin B1a, for example, as it is, which is precipitated by adding a solvent, a solution containing avermectin B1a, or a solution thereof is dried. Any of the ones used. When a solvent that is miscible with water is added to the avermectin B1a to be crystallized to dissolve the avermectin B1a, it is preferable to add a solvent that is miscible with water so that the avermectin B1a concentration is 1 to 10 g / l. In particular, 1 to 5 g of the avermectin B1a-containing solution as-prepared by adding a solvent to the solution or a concentrated avermectin B1a-containing solution is used.
4 when the dried product is used so that
It is particularly preferable to add a solvent that is miscible with water so that the amount becomes 10 to 10 g / l. When the filtrate is concentrated after the solution is filtered or water is added to the filtrate, for example, when water-containing methanol is used, the water content becomes 50% or more, and when water-containing ethanol is used. It is preferable to concentrate or add water until the water content becomes 60% or more, and when water-containing acetone is used, the water content becomes 60% or more. When the solution of the dried product is concentrated, the avermectin B1a concentration is 50 to 150 g /
It is preferable to concentrate so that the concentration becomes 1.

The obtained precipitate or crystal can be isolated by filtration, centrifugation or the like. Hereinafter, embodiments of the present invention will be specifically described with reference to examples.

[0011]

EXAMPLES As a sporulation plate medium and a production medium,
The following composition was used. Spore-forming plate medium Malt extract (Difco) 10 g Yeast extract (Difco) 4 g Soluble starch (Difco) 4 g Agar 20 g Distilled water 1 L Adjusted to pH 7.2 with 2 L aqueous 2N potassium hydroxide solution, and then added at 121 ° C to 15
Autoclaved for minutes. After the sterilization was completed, magnesium chloride and potassium nitrate were added so as to be 10 mM and 8 mM, respectively. Production medium Glucose 45 g Peptone milk 24 g Yeast autolysate 2.5 ml Polypropylene glycol (average molecular weight 2000) 20 g Distilled water 1 L Adjusted to pH 7.2 with 2N potassium hydroxide aqueous solution
Autoclaved for minutes.

The analysis of the total amount of avermectin and the amount of avermectin B1a in each purification step was performed by high performance liquid chromatography under the following conditions using a pure product as a standard. Guard column; Inertsil ODS-2 [GL Science Co., Ltd.] (5 μm, 4 × 10 mm) Separation column; Inertsil ODS-2 [GL Science Co., Ltd.]
(5 μm, 4 × 250 mm) Column temperature; 55 ° C. Mobile phase; Acetonitrile: methanol: water = 5: 3: 2 Flow rate: 0.6 ml / min Detection; UV 243 nm Sample injection volume; 10 μl at 0.2 g / l

Example 1 Streptomyces avermitilis cultured on a spore-forming plate medium at 28 ° C. for 7 days
30 spores of K2038 (FERM BP-2775) strain containing 30 ml of production medium
Transfer to 20 0 ml flasks, 210 rpm at 28 ° C, 2.5 cm amplitude
And cultured for 168 hours. 500 ml of the avermectin fermentation culture solution obtained above was filtered, and the avermectin mixture insolubilized with the cells was washed with water. 1000 ml of methanol containing 20% of water was added to the obtained solid matter.
After mixing for 60 minutes, filtration was performed to obtain an extract. To this extract was added 500 ml of water to obtain crude avermectin B1a as a solid precipitate. 200 ml of methanol was added to the obtained crude avermectin B1a to dissolve it, the insoluble matter was filtered off, and 150 ml of water was added to the obtained filtrate to obtain crystals of avermectin B1a. The crystals were separated and dried to obtain 112 mg of avermectin B1a. The avermectin B1a purity at each stage is shown in Table 1.

[0014]

[Table 1]

Comparative Example 1 Streptomyces avermitilis cultured in a sporulation plate medium at 28 ° C. for 7 days
30 spores of K2038 (FERM BP-2775) strain containing 30 ml of production medium
Transfer to 20 0 ml flasks, 210 rpm at 28 ° C, 2.5 cm amplitude
And cultured for 168 hours. 500 ml of the avermectin fermentation culture solution obtained above was filtered, and the avermectin mixture insolubilized with the cells was washed with water. 1000 ml of methanol was added to the obtained solid and mixed for 60 minutes, followed by filtration to obtain an extract. 300 ml of water in this extract
Was added, avermectin formed a colloid and solid-liquid separation was impossible. No change in avermectin was observed even when water was added.

Example 2 Streptomyces avermitilis cultured in a sporulation plate medium at 28 ° C. for 7 days
Spores of K2038 (FERM BP-2775) strain contained in 300 ml of production medium
The cells were transferred to 28 2 L flasks and cultured at 28 ° C under conditions of 210 rpm and an amplitude of 2.5 cm for 192 hours. 8000 ml of the avermectin fermentation culture solution obtained above was filtered, and the avermectin mixture insolubilized with the bacterial cells was washed with water. To the solid obtained, add 20000 ml of methanol containing 40% of water.
After mixing for 60 minutes, filtration was performed to obtain an extract. 3000 ml of water was added to this extract to obtain crude avermectin B1a as a solid precipitate. The obtained precipitate was vacuum dried at 30 ° C. to remove water. After adding 500 ml of methanol to the dried product to dissolve it, the solution was concentrated to 20 ml and avermectin B1a was added.
Was obtained. The crystals were separated and dried to obtain 1.85 g of avermectin B1a. The avermectin B1a purity at each stage is shown in Table 2.

[0017]

[Table 2]

Example 3 Streptomyces avermitilis cultured in a sporulation plate medium at 28 ° C. for 7 days
30 spores of K2038 (FERM BP-2775) strain containing 30 ml of production medium
Transfer to 20 0 ml flasks, 210 rpm at 28 ° C, 2.5 cm amplitude
And cultured for 168 hours. 20 ml of methyl isobutyl ketone was added to 500 ml of the avermectin fermentation broth obtained above.
After adding 0 ml and mixing for 30 minutes, the mixture was allowed to stand for 60 minutes. The methyl isobutyl ketone solution containing avermectin in the upper layer was separated and concentrated to dryness. To this, 200 ml of methanol containing 30% of water was added, and the dried product was dissolved with stirring, and then filtered to obtain a solution. 80 ml of water was added to this solution to obtain crude avermectin B1a as a solid precipitate. 200 ml of methanol was added to the obtained crude avermectin B1a to dissolve it, the insoluble matter was filtered off, and 150 ml of water was added to the obtained filtrate to obtain crystals of avermectin B1a. The crystals were separated and dried to obtain 112 mg of avermectin B1a. The avermectin B1a purity at each stage is shown in Table 3.

[0019]

[Table 3]

Comparative Example 2 Streptomyces avermitilis cultured in a sporulation plate medium at 28 ° C. for 7 days
30 spores of K2038 (FERM BP-2775) strain containing 30 ml of production medium
Transfer to 20 0 ml flasks, 210 rpm at 28 ° C, 2.5 cm amplitude
And cultured for 168 hours. 20 ml of methyl isobutyl ketone was added to 500 ml of the avermectin fermentation broth obtained above.
After adding 0 ml and mixing for 30 minutes, the mixture was allowed to stand for 60 minutes. The methyl isobutyl ketone solution containing avermectin in the upper layer was separated and concentrated to dryness. To this, 200 ml of methanol was added, and the dried product was dissolved with stirring, and then filtered to obtain a solution.
When 200 ml of water was added to this solution, avermectin formed a colloid and solid-liquid separation was impossible. No change in avermectin was observed even when water was added.

[0021]

INDUSTRIAL APPLICABILITY The present invention provides a simple, inexpensive and highly efficient purification method for avermectin that does not require chromatography.

Claims (10)

[Claims] 1. Avermectin is extracted by contacting an insoluble matter in a fermentation broth of avermectin with an aqueous solution of a solvent that mixes with water, and then concentrating the extract or adding water to the extract to obtain avermectin. A method for purifying avermectin, which comprises precipitation. 2. Avermectin is prepared by contacting an avermectin fermentation culture solution with an organic solvent immiscible with water to extract avermectin, distilling off the organic solvent, and adding an aqueous solution of a solvent miscible with water to the residue. Is dissolved and then filtered, and the filtrate is concentrated or water is added to the filtrate to precipitate avermectin, which is a method for purifying avermectin. 3. A water-containing aqueous solution of a solvent miscible with water, the water-containing methanol having a water content of 15 to 50%, and the water content of 20 to 60%.
The method for purifying avermectin according to claim 1 or 2, which is water-containing acetone or water-containing ethanol having a water content of 20 to 60%. 4. The method for purifying avermectin according to claim 2, wherein the organic solvent immiscible with water is ethyl acetate, chloroform or methyl isobutyl ketone. 5. Avermectin B1a is prepared by contacting an insoluble matter in a fermentation culture solution of avermectin with an aqueous solution of a solvent mixed with water to extract avermectin, and then concentrating the extract or adding water to the extract to obtain avermectin B1a. A method for purifying avermectin, which comprises selectively precipitating a. 6. Avermectin is extracted by contacting an avermectin fermentation culture solution with an organic solvent immiscible with water to extract avermectin, distilling off the organic solvent, and adding an aqueous solution of a solvent miscible with water to the residue. Is dissolved and then filtered, and the filtrate is concentrated or water is added to the filtrate to selectively precipitate avermectin B1a, thereby purifying avermectin. 7. A water-containing aqueous solution of a solvent miscible with water, the water-containing methanol having a water content of 15 to 50%, and the water content of 20 to 60%.
7. The method for purifying avermectin according to claim 5, which is water-containing acetone or water-containing ethanol having a water content of 20 to 60%. 8. The method for purifying avermectin according to claim 6, wherein the organic solvent immiscible with water is ethyl acetate, chloroform or methyl isobutyl ketone. 9. The avermectin B1a obtained in claim 5, 6, 7 or 8 is dissolved in a solvent mixed with water, filtered if necessary, and water is added to crystallize the avermectin B1a. A method for crystallizing avermectin B1a. 10. The avermectin B1a obtained in any one of claims 5, 6, 7 or 8 is dissolved in a solvent 8 which mixes with water, filtered if necessary, and the solution is concentrated to crystallize the avermectin B1a. Avermectin B characterized by
Crystallization method of 1a.