BG64840B1 - Method for pleuromutiline preparation - Google Patents

Abstract

The pleuromutiline is an antibiotic of expressed antibacterial effect against microplasmas and other bacteria, its semi-synthetic derivative thiamuline being particularly effective. By the method, a strain producer Clitopilus pinsitus, registered in NBIMCC under No. 3910, cultivates in deep, in aerobic conditions on nutrients, containing assimilation sources of carbon, organic nitrogen and mineral salts in wt. %: glucose from 3 to 15, proflo 0.3-0.6, maize extract from 3 to 5, soya bean oil 0.3-1.5. magnesium sulphate 0.01-0.05 and calcium carbonate 0.1-0.5. The fermentation process runs at temperatures ranging from 25 to 30 degrees C, pressure 0.5-0.8 bar and pH 6.0-8.0. The mutant used has increased antibiotic titre in the cultural medium and accumulates relatively small side products in the process of fermentation.

Description

The invention relates to a method for the preparation of pleuromutilin, which is used in the pharmaceutical industry.

BACKGROUND OF THE INVENTION

The pleuromutilin antibiotic was isolated for the first time by Kavanagh et al. (Proc, Natl. Acad. Soc. 1951, 37, 570-574). It is produced as part of an antibiotic complex during deep fermentation of the basidiomycetes strain Clitopilus pinsitus. It has a pronounced antibacterial action against mycoplasma and other bacteria, and its semi-synthetic derivative thiamulin, which has been in use since 1978, is particularly effective.

A method is known for the preparation of pleuromutilin, wherein the pleuromutilin producer strain of Clitopilus passeckerianus (Pil.), NRRL 3100 or variants thereof, Clitopilus passeckerianus NRRL 3279, Clitopilus prunulus NRRL 3473, Clitopilus pinsitus culture media contains sources of nitrogen and carbon and mineral salts. The fermentation process was carried out at a temperature of 24-25 ° C, under aerobic conditions, with continuous stirring for about 120 hours. (GB 1 111 010; US 3716579)

The disadvantage of this method is the low yield of the target product.

A method for the preparation of pleuromutilin included in the composition of an antibiotic complex is known, wherein the strain Clitopilus pseudopinsitus NRRL 11179 is cultured on a fermentation medium containing absorbable carbon and nitrogen sources and inorganic salts at aerobic conditions at about 25 DEG C. 288 h. (US 4247542)

The disadvantage of this method is the production, as a result of the fermentation process, of an antibiotic complex, in which pleuromutilin is present in a negligible amount.

A method for the preparation of pleuromutilin is known, in which the Clitopilus pinsitus strain strain NBPMCK 3418 is cultured deeply under aerobic conditions, forming pleuromutilin and the concomitant antibiotic complex (BG 63800).

The disadvantage of this method is that the target product is not the end product of the fermentation process, which makes it ineffective for industrial production, as well as the productivity itself with respect to pleuromutilin.

SUMMARY OF THE INVENTION

According to the method for the production of pluromutilin, the Clitopilus pinsitus strain registered in NBPMCC under No. 3910 is cultured deeply under aerobic conditions, on a culture medium containing absorbed nitrogen, carbon and inorganic salts. The fermentation process is carried out at a temperature of 25 to 30 ° C, a pressure of 0.5-0.8 bar and a pH of 6.0 to 8.0 for a time of 250-300 h.

Clitopilus pinsitus strain NBPMCK No. 3910 was obtained from Clitopilus pinsitus strain NBPMCC 3418 by protoplastic initial and subsequent UV light irradiation (K. R. Satewart, 1986, A method for generating protoplasts from Clitopilus vinsibus. J., Ant. p. 1486-7). The resulting variant of the strain is stabilized by natural selection.

The following nutrient media were used to characterize the parent parent strain Clitopilus pinsitus NBPMKK 3418 and the improved strain obtained during selection:

1. Saburo-desctrous agar, with a composition by weight: glucose - 4.0; peptone 1.0; agar15.0; pH 5.6 ± 0.1.

2. Malt-corn agar, with a composition by weight: malt extract - 2.0; corn extract - 1.0; agar 1.5; pH 6.5 ± 0.2.

3. Saburo-maltose agar, composition by weight: meat peptone - 0.5; casein - 0.5; maltose - 2.0; agar - 1.5; pH 5.6 + 0.1.

4. Combined agar, composition by weight: malt extract -1.5; peptone - 0.075; maltose - 1.3; dextrin - 0.3; glycerol - 0.25; monocotassium phosphate - 0.1; ammonium chloride 0.1; agar - 1.5; pH 4.8 + 0.2.

5. Malt agar, composition by weight: Malt extract - 3.0; soy peptone - 0.3; agar 1.5; pH 5.6 ± 0.1.

The morphological and cultural characteristics of the two strains were made on day 14 of their development and are presented in Table !.

Table 1 Comparative morphological characteristics of Clitopilus pinsitus strains NBPMCC 3418 and 3910

No Nutrient medium Parent strain Cl.pinsitus NBPCC 3418 Improved strain Cl.pinsitus NPBMCC 3910 1 Saburodextrose agar Large colonies 20-25 mm radially furrowed with concave center; fluffy BM n7; CM bb-d5 Round colonies with irregular end 1520 mm, flat with a bud in the middle; fluffy VM dZ; CM kZ-o5 2 Malt corn agar There is no growth 2025 mm circular colonies with bud in the middle, hemispherical; VM dZ; SM kZ 3 Saburomaltose agar There is no growth Irregularly shaped colonies, 12-15 mm; fluffy VM n7; Cm kb 4 Combined agar Round colonies 15-20 mm, radially furrowed; weak VM m2; Cm2; the darker end of the colony vol Round colonies 7-10 mm with apex in the middle; weak VM about; Cm l7-sb 5 Malt agar Large irregular colonies 20-25 mm, radially furrowed with secondary growth; VM dZ; Cm2 Round colonies 1520 mm with a bud in the middle, less developed VM d3; SM kZ

The color of the aerial mycelium was determined on the Bondartsev scale (AS Bondartsev, The Color Scale, published in the USSR, 1954).

Data for absorption of different carbohydrate sources from the two strains are presented in Table 2. The assay was performed on a single carbohydrate source medium at a concentration of 1%, with a composition in weight%: ammonium sulfate - 0.026; monocotassium phosphate

0.024; dicalium phosphate - 0.56; magnesium sulfate - 0.1; saline solution * - 0.1; agar - 1.5; pH 6.8 ± 7.0.

Раз Saline solution: copper sulphate - 0.63 g; ferrous sulfate - 0.11 g; manganese sulfate - 0.79 g; zinc sulfate - 0.15 g; distilled water to 100 ml. The sugars are added in the form of sterile solutions at an agar temperature of about 60 ° C.

Table 2. Absorption of carbohydrate sources from the parent strain Clitopilus pinsitus

NBPMCK 3418 and Clitopilus pinsitus sham NBPMKK3910

No Substrate Clitopilus pinsitus NBPCC 3418 Clitopilus pinsitus NBPMKK3910 1. Control ++ + 2. Glucose ++ ++ 3. Fructose ++ ++ 4. Sucrose ++ ++ 5. Maltose ± ++ 6. Lactose ++ ± 7. Galactose + + 8. Raffinose ++ ± 9. Xylose + +

.... . and. and

Markings in the table: "+" - positive growth; "++" - very good growth; "±" - doubtful growth; "-" - lack of height.

Storage of the strain on bevel and ap at temperatures up to + 4 ° C while maintaining its properties is within 3 months. For a longer period, the strain was stored at a temperature of> 50 l below - 50 ° C using a glycerol lactose protection medium.

Fresh culture of Clitopilus pinsitus strain NBPMCC No. 3910 grown on a tube of beveled Saburo-dextrose agar is washed with

O sterile saline solution, homogenized. 0.5 ml of this suspension was inoculated onto 60 ml of the culture medium with a composition by weight: glucose 1.0 to 6, proflo 1-3, monocotassium phosphate 0.1-0.5, magnesium sulfate 0.05- 0.5, potassium nitrate 0.05-0.1, sodium chlorobenzyl chloride ⁴⁰ 0.1 -0,5.rN is adjusted in the range from 5.4 to 5.8 and the medium is sterilized at a temperature of 120 to 121 ° C in 30 min. The inoculation process takes place at 25 ° C, with continuous stirring for 120-168 h. $ ⁴

From 5 to 10% by volume of the seed is added to a fermentation medium containing a carbohydrate source of 2 to 10% by weight, organic nitrogen sources of 0.2 to 2% and mineral salts in an amount of 0.12%. The pH of the fermentation medium is maintained in the range of 6 to 8. The vegetative phase can be two-stage. The fermentation process is carried out at a temperature of 25 to 30 ° C for a period of 200 to 300 hours.

Carbon sources such as glucose, fructose or sucrose, and soybean oil may be used as sources of carbon in the fermentation medium. The following may be used as sources of organic nitrogen: dried yeast, soybean meal, corn extract, proflo, pharmamedia, peptone, tryptone, casein, alone or in combination, and as mineral salts - sulfates, phosphates, chlorides and nitrates of sodium, potassium , magnesium and calcium.

Specific to strain Clitopilus pinsitus NBPMCC No. 3910 is that it better absorbs glucose, soybean meal, maize extract and proflo from nutrient media.

The advantage of the method is the high antibiotic titer in the culture medium, as well as the relatively low accumulation of by-products in the fermentation of the mutant strain used compared to the parent strain.

Explanation of the annexed figures

The pleuromutilin obtained by the method of the invention was quantified by HPLC method. The results of the analysis are shown as follows:

1. Figure 1 shows the results of HPLC analysis of a standard solution of pleuromutilin.

2. Figure 2 shows the results of an HPLC analysis of a pleuromutilin culture fluid sample obtained by the method described in Example 1.

Figure 3 shows the results of bioautographically showing samples of culture fluid in a Micrococcus luteus test

ATCC 9341, which results in thin layer chromatography.

Examples of carrying out the invention

The invention is illustrated by the following examples:

Example 1.

Fresh culture of Clitopilus pinsitus strain NBPMKK 3910 grown on a test tube of slanted Saburo-dextrose agar was washed with sterile saline and ground into a Potter mill for homogenization. 0.5 ml of this suspension is inoculated with a 500 ml Erlenmeyer flask containing 60 ml of the seed medium of the following composition:

No Component Quantity, g / l 1. Glucose, crystalline 50 2. Proflo 10 3. Monokalium phosphate 1 4. Magnesium sulfate 0.5 5. Calcium nitrate 0.72 6. Sodium chloride 0.1 7. Tap water Up to 11

The pH was adjusted to 5.6 + 0.2; sterilization 30 min. At 121 ° C; pH after sterilization 5.7 + 0.2. Glucose was added separately sterilized to each flask before sowing. It thrives at 25 ° C on a circular shaker for 120 to 168 h.

5 to 10 vol. % of the seed to the fermentation medium with the following composition:

No Component Quantity, g / l 1. Glucose, crystalline 25.0 2. Proflo 4.27 3. Corn extract 38.6 4. Magnesium sulfate 0.32 5. Soybean oil 5.0 6. Calcium carbonate 0.9 7. Tap water Up to 11

The medium is poured into 500 ml Erlenmeyer flasks of 50 ml each, with glucose added separately sterilized to each flask before sowing. The pH was adjusted to 6.6 ± 0.2.

The fermentation was continued for 250 to 300 h 5 at 25 ° C in a circular shaker with 250 rpm and 5 cm deviation. After completion of the fermentation process, each flask was analyzed for quantitative content of pleuromutilin by HPLC method and qualitatively by 10 TLC method against the reference substance. Activity 3-5 g / l.

Example 2.

Fresh culture of Clitopiplus pinsitus strain NBPMCC No. 3910 grown on a 15-beaker test tube. Saburo-dextrose agar was washed with sterile saline and ground into a Potter mill for homogenization. 0.5 ml of this suspension is inoculated with a 500 ml Erlenmeyer flask containing 60 ml of the seed medium of the above composition. After developing for about 120 to 168 h at 25 ° C on a 250-rpm circular shake machine, 5 cm of deviation, 5 ml of the developed culture was transferred to 200 ml of the same seed medium in a one-liter Erlenmeyer flask. The development is continued under the same conditions for about 72 to 96 hours, after which the material thus prepared serves to sow a 50 liter working apparatus with the following fermentation medium:

No Component Quantity, d / 1 1. Glucose, 70% 36.5 2. Proflo 4.27 3. Corn extract 38.6 4. Magnesium sulfate 0.32 5. Soybean oil 3.0 6. Calcium carbonate 0.9 7. Tap water Up to 11

The pH of the native solution was adjusted with sodium hydroxide to 6.6-6.8, the medium was sterilized under standard conditions, and then the separately sterilized glucose solution was added. The cultivation was carried out at 25 ° C, aeration 0.5: 1 = volume: volume of medium, pressure 0.5 -0.8 bar for 250 - 300 h. The pH, glucose level, oil, dissolved oxygen and biomass accumulation are controlled during the fermentation process. The formation of pleuromutilin begins after 40 hours and is monitored by HPLC until the end of fermentation.

The product accumulated at the end of the process is in the amount of 10-14 g / l.

The HPLC method by which the quantitative content of pleuromutilin is analyzed is carried out as follows:

A standard solution of pleuromutilin was prepared by dissolving 0.060 g of standard substance in 100 ml of mobile phase and analyzed. The results of the analysis of this solution by the HPLC method are shown in FIG. 1.

A sample of the culture fluid obtained as a result of the fermentation process was prepared by transferring 10 g of the culture fluid into a volumetric flask, extracting with 40 ml of methanol for 30 min at 35 ° C. After the sample was cooled to room temperature, make up to 50 ml with methanol and filter. The resulting solution was chromatographed on a Lichrosorb RP-8 column (5 pm) 4 x 250 mm in mobile phase containing 0.02 M mono-potassium phosphate, acetonitrile and methanol, solution pH 7.5, flow rate 0.6 ml / min, detection 205 nm and an injection volume of 20 μΐ. The results of the analysis of this sample by the HPLC method are shown in FIG. 1.

The product is qualitatively proven by the TLC method against the reference substance using sorbent chromatographic plates

Merck 5553, in mobile phase: dichloromethane: ethyl acetate: cyclohexane = 70:20:10, saturation of the chamber about 1 h, distance of travel 15 cm.

Application solutions:

A solution of 1: 10 g of culture fluid was extracted with 20 ml of methanol for 30 min. Starting point “a” is applied from 1 to 5μ1 depending on the expected activity.

Solution 2: 0.010 g of the comparative substance pleuromutilin is dissolved in 10 ml of methanol. At start point "b", 5 μΐ of the solution is applied.

Manifestation: After complete removal of the solvents (2 min at 100 ° C), the plate is sprayed with ethanol solution of 5% sulfuric acid and 1% ferric chloride. Heat for 5 min at 150 ° C.

Assessment: The observed spot above the starting point "a" must be of Rf value, magnitude and intensity equal to that above the starting point "b". The Rf value is 0.5.

To conduct the bioautograph study (Fig. 3), fresh test culture grown on a medium slanted agar tube from medium A was washed with 10 ml of saline. A 150 ml agar plate was poured into agar medium A (Eur. Pharm. 2001, 2.7.2., Microbiological assay of antibiotics) contaminated at 50 ° C with 3 ml of the test suspension. The developed chromatogram was applied to the agar for 30 min, then removed and cultured for 16 to 18 h at 37 ° C. After the emergence of growth inhibition zones according to the activity of the collected samples, the agar plate was stained with 30 ml of a 0.3% aqueous solution of methylene blue for 3 min and then sequentially treated with 30 ml of 96% ethanol containing 1 ml of 6 N hydrochloric acid for 3 min and 30 ml of 96% ethanol containing 1 ml of 10 N sodium hydroxide again for 3 min. Copies of the agar plate were made on filter paper. The activity of the collected samples shall be calculated from the standard nearest to the sample by the formula:

, nxd. xd, (A, / Y, / ^Α · = - where As is the activity of the sample in micrograms per gram of culture fluid; dj and d ₂ are the diameters of the ellipse, Ast is the activity of the standard, Sst is the area of the corresponding standard calculated by the formula

KXd, Xd _a 4

The method is semi-quantitative and gives up to 10% error, which depends primarily on the exact comparison of the sample with the corresponding nearest standard.

Claims

Claims (1)

A method for the preparation of pleuromutilin comprising culturing a producer strain of Clitopilus pinsitus on a culture medium containing carbon, nitrogen and mineral salts, characterized in that the producer strain Clitopilus pinsitus registered in NBPMCC under No. 3910 is cultivated on the culture medium containing by weight. %: glucose from 3 to 15, profile from 0.3 to 0.6, corn extract from 3 to 5, soybean oil from 0.3 to 1.5, magnesium sulfate from 0.01 to 0.05 and calcium carbonate from 0 , 1 to 0.5, under aerobic conditions at 25 to 30 ° C, pressure 0.5-0.8 bar and pH 6.0-8.0.