JPH101496A - Avermectin purification method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple, inexpensive and highly purified purification method of avermectin B1a without using chromatography. SOLUTION: After extracting avermectin by bringing an avermectin-containing substance into contact with a solvent miscible with water, the extract is concentrated, and then a solvent and a salt miscible with water are added again to obtain an insoluble substance. A method for purifying avermectin B1a is provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for easily and simply purifying high-purity avermectin B1a from a fermentation broth of avermectin without using industrially complicated chromatography.

[0002]

BACKGROUND OF THE INVENTION Avermectin is widely used as a raw material for synthesizing avermectin derivatives such as ivermectin, a parasite drug, and itself as a parasite drug for livestock 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.

[0003] As a general method for purifying avermectin, avermectin is extracted by adding an organic solvent to a fermentation broth of avermectin or an insoluble substance containing avermectin obtained by subjecting the fermentation broth to centrifugation, filtration or the like. After that, a method is known in which a fraction containing avermectin is subjected to chromatography using a carrier such as silica gel, alumina oxide, or dextran gel to concentrate and dry the fraction containing avermectin. Further, a methanol-ethylene glycol mixed solvent was added to the residue obtained by concentration to dryness, and methanol was concentrated and distilled off.
A method of obtaining crystals after cooling is also known (Japanese Patent Publication No. 2-17)
558). However, in these methods using chromatography, since the load on the carrier is low, a large amount of chromatographic simple substance and organic solvent must be used, the productivity is low, and the purified avermectin purified product is expensive. However, it is not always an industrially satisfactory purification method.

Avermectin fermentation broth is subjected to centrifugation, filtration, and the like, and a solvent mixed with water is added to an insoluble material containing avermectin to extract avermectin. Then, water and sodium chloride are added to the extract. Avermectin purification method of obtaining a precipitate precipitated by adding an inorganic salt or the like such as is known (WO95 / 0341).
9). However, according to this method, the volume at the time of solid-liquid separation of the precipitate increases, and it is difficult to obtain an industrial production method.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for purifying avermectin B1a which is simple, inexpensive and has high purification efficiency without using industrially complicated chromatography.

[0006]

According to the present invention, an avermectin-containing substance is brought into contact with a solvent miscible with water to extract avermectin, the extract is concentrated, and then a solvent and a salt miscible with water are added again. And a method for purifying avermectin B1a, wherein insoluble matter is obtained.
After contacting the obtained insolubles with a solvent that mixes with water,
Avermectin B1a having higher purity can be obtained by concentrating the solution from which residual insolubles have been removed and obtaining a precipitate. The salt may be added before, after, or simultaneously with the addition of a solvent that is miscible with water.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION As avermectin-containing substances,
Examples include insolubles in a fermentation broth of avermectin, and synthetic avermectin. The fermentation culture solution of avermectin used in the present invention is a culture solution obtained by culturing a microorganism capable of producing avermectin in a nutrient medium. As the microorganism having an avermectin-producing ability, a microorganism that mainly produces avermectin B is preferable. For example, Streptomyces avermitilis ( Streptomyces avermitilis )
K2038 (FERM BP-2775; JP-A-3-254678). 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. Culture temperature is 20 to 40 ° C
And the pH is 6.0 to 8.0, preferably 7.0 to 7.0.
Adjust it to 5. The avermectin concentration in the fermentation broth is usually preferably 0.4 g / l or more, more preferably 0.5 g / l.
/ L or more is particularly preferred. The avermectin B1a concentration in the fermentation broth is preferably 0.2 g / l or more, and the amount of avermectin B1a in the total avermectin is 30%.
% Is preferable. 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.

The insoluble matter in the avermectin fermentation broth includes a precipitate obtained by centrifuging the avermectin fermentation broth obtained by the above-mentioned production method, a residue obtained by filtering the avermectin fermentation broth, and the like. 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. When avermectin is extracted by contacting the insoluble matter in the fermentation broth of avermectin with a solvent that is miscible with water, the concentration of the solvent that is miscible with water is set to 75% or more, and at 20 to 40 ° C.
It is preferred to perform the extraction over a period of minutes to 2 hours. When the extract is concentrated, it is preferable that the concentration of the solvent miscible with water is reduced to 30% or less. When the solvent miscible with water is added again, the concentration of the solvent miscible with water is reduced. 5
It is preferable to add until it becomes 0 to 70%.

Examples of the salt include sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, sodium hydrogencarbonate and the like. Of these, sodium chloride is preferred. The salt concentration is preferably at least 10% after the addition of the solvent and salt miscible with water. When the obtained insoluble matter is brought into contact with a solvent that is mixed with water, it is preferable that the avermectin B1a concentration be 1 to 10 g / l, and particularly preferably 4 to 10 g / l. Particularly preferred. When concentrating the solution from which residual insolubles had been removed, the avermectin B1a concentration was 50 to 150 g.
/ L is preferred. In addition, removal of the residual insoluble matter can be performed by centrifugation, filtration, or the like.

The isolation of the resulting insoluble matter or precipitate is
It can be performed by centrifugation, filtration 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 fermentation broth of avermectin obtained above (containing 0.6 g of B1a body and 0.62 g of B2a body) was filtered, and the avermectin mixture which had been insolubilized together with the cells was washed with water. To the obtained solid (135 ml), 470 ml of methanol was added and mixed for 60 minutes, followed by filtration to obtain an extract (containing 0.58 g of the B1a form and 0.6 g of the B2a form; the ratio of the B1a form to the total avermectin 45%). Was. After concentrating this extract to 100 ml, sodium chloride 15g
Was added. At this time, the methanol concentration is 15%,
The avermectin B1a concentration and the avermectin B2a concentration in the supernatant were both 0.5 g / l or less. Subsequently, 150 ml of methanol was added, and a mixture of crude avermectin B1a and sodium chloride (avermectin B1a) was centrifuged.
Content 100mg / g; ratio of B1a body to total avermectin
90%). After 100 ml of methanol was added to the obtained mixture to dissolve it, the insoluble matter was filtered and concentrated to 20 ml to obtain avermectin B1a, 4 having a purity of 98% or more.
50 mg were obtained.

Example 2 Streptomyces avermitilis cultured at 28 ° C. for 7 days on a spore-forming plate medium
Spores of K2038 (FERM BP-2775) strain contained in 300 ml of production medium
The cells were transferred to 28 2L flasks and cultured at 28 ° C under the conditions of 210 rpm and an amplitude of 2.5 cm for 192 hours. 15 L of the fermentation broth of avermectin obtained above (containing 24 g of B1a body and 26 g of B2a body) was filtered, and the avermectin mixture which had been insolubilized together with the cells was washed with water. Methanol 15 is added to the obtained solid (5 L).
After adding L and stirring for 2 hours, the mixture was filtered and the extract (B
(1g body 21g, B2a body 23g; ratio of B1a body to total avermectin 48%). After adding 400 g of sodium chloride to this extract, it was concentrated to 1.5 L. After adding 2 L of methanol and stirring for 1 hour, a mixture of crude avermectin B1a and sodium chloride (avermectin B1a content 120 mg / g; ratio of B1a form to total avermectin 91%) was obtained by centrifugation. 3 L of methanol was added to the obtained mixture, and the mixture was stirred for 1 hour. The insoluble matter was filtered and concentrated to 600 ml to obtain 18 g of avermectin B1a having a purity of 97% or more (recovery rate from fermentation broth of 75%). ) Got.

[0015]

According to the present invention, avermectin B which is simple, inexpensive and has high purification efficiency without using chromatography
A method for purification of 1a is provided.

Claims (8)

[Claims] (1) extracting avermectin by bringing the avermectin-containing substance into contact with a solvent miscible with water, concentrating the extract, and then adding a solvent and salt miscible with water again to obtain an insoluble substance; A method for purifying avermectin B1a, comprising: 2. After extracting avermectin by bringing the avermectin-containing substance into contact with a solvent miscible with water, concentrating the extract, and then adding a solvent and salt miscible with water again to obtain an insoluble substance, A method for purifying avermectin B1a, further comprising contacting the insoluble matter with a solvent that is mixed with water, and then concentrating the solution from which the remaining insoluble matter has been removed to obtain a precipitate. 3. The method for purifying avermectin B1a according to claim 1, wherein the avermectin-containing substance is an insoluble substance in a fermentation broth of avermectin. 4. The method for purifying avermectin B1a according to claim 1, wherein the salt concentration after adding a solvent and a salt which are mixed with water is adjusted to 10% or more. 5. The method for purifying avermectin B1a according to claim 1, wherein, when the insoluble matter in the fermentation broth of avermectin is brought into contact with a solvent miscible with water, the concentration of the solvent miscible with water is 75% or more. . 6. The method for purifying avermectin B1a according to claim 1, wherein the concentration of the solvent mixed with water is reduced to 30% or less when the extract is concentrated. 7. The method for purifying avermectin B1a according to claim 1, wherein when the solvent miscible with water is added again, the concentration of the solvent miscible with water is increased to 50 to 70%. 8. The method for purifying avermectin B1a according to claim 1, wherein the solvent miscible with water is methanol, acetone or ethanol.