CS218032B1 - Process for preparing monensin or its salts, in particular factor A - Google Patents

Abstract

The invention relates to a method for preparing monensin or its salts, in particular factor A. The starting strain Streptomyces cimamonensis 179 CCM 3504 is fermented at a temperature of 28 °C to 40 °C in a submerged culture in three stages. 1. the inoculation medium contains glycerol, peptone, meat extract and NaCl, 2. the inoculation medium contains glucose, dried brewer's yeast Pangamin, soy flour and calcium carbonate and the fermentation medium in the third stage contains in mass concentration vegetable oil 2 to 10% glucose 1% soy flour 0.5 to 2% calcium carbonate 0.5% potassium hydrogen phosphate 0.01% after which the resulting product is isolated in a known manner.

Description

The invention relates to a method for preparing monensin or its salts, in particular factor A. The starting strain Streptomyces cimamonensis 179 CCM 3504 is fermented at a temperature of 28 °C to 40 °C in a submerged culture in three stages. 1. the inoculation medium contains glycerol, peptone, meat extract and NaCl, 2. the inoculation medium contains glucose, dried brewer's yeast Pangamin, soy flour and calcium carbonate and the fermentation medium in the third stage contains in mass concentration vegetable oil 2 to 10% glucose 1% soy flour 0.5 to 2% calcium carbonate 0.5% potassium hydrogen phosphate 0.01% after which the resulting product is isolated in a known manner.

The invention relates to the preparation of monensin, in particular factor A, or to the preparation of an antibiotic and coccidiostatic complex of monensin-type substances.

The method of preparing monensin complex (antibiotic A 3823) is known from US patent No. 3,501,568. The preparation is based on fermentation of the Streptomyces cinnamonensis ATCC 15,413 strain under submerged aerobic conditions in a culture medium containing assimilable sources of carbon, nitrogen and inorganic salts.

The description of the cited invention does not state the yield of the claimed process, but factors A and B predominate in the obtained complex (Example 4). After lengthy chromatography on a silica gel column (450 fractions), 40% of factor A can be obtained in a pure state, while 50% of the same complex is eluted as a mixture of factors A and B.

The antibiotic activity of factor A against Bacillus subtilis is more than twice as high as that of factor B. The coccidiostatic activity of the individual factors is not known, but according to a preliminary test, the activity of factor A is higher even in this case. Therefore, it is advantageous to ferment the production strain under conditions in which factor A is predominantly formed without reducing the biosynthetic activity of the strain.

The production strain grows well on nutrient media containing assimilable carbon and nitrogen sources, such as mono- and polysaccharides including their hydrolysates, molasses, ammonium salts, nitrates, amino acids, urea, various types of protein meals of plant and animal origin including their hydrolysates. The nutrient medium also contains mineral salts of phosphates, chlorides, sulfates and carbonates, containing potassium, magnesium, sodium and calcium ions. Trace elements such as cobalt, copper, boron, etc. are usually already present in the nutrient components of the culture medium as impurities.

According to the cited US patent, the strain is cultivated either in submerged aerobic culture at 28 ^° C to 38°C for 3 to 6 days or for up to 12 days in surface fermentation.

Under these conditions, the strain Streptomyces cinnamonensis 179, deposited in the Collection of Microorganisms of the J. E. Purkyně University in Brno under no. CCM 3504, produces a monensin complex in which factors A and B predominate. Their mutual ratio ranges from 30 to 63% factor A to 43 to 70% factor B, both during fermentations in flasks and in tanks.

The subject of the invention is a method for preparing monensin or its salt, in particular factor A, the essence of which is that the Streptomyces cinnamonensis 179 CCM 3504 strain is fermented at a temperature of 28 °C to 40 °C in a submerged culture in three stages, namely in the first stage on the 1st inoculation medium, which contains in mass concentration glycerol peptone meat extract sodium chloride up to 3% 0.25%

0.2 to 0.7% 0.1% in the second stage on the 2nd inoculation medium, which contains in mass concentration glucose 2 to 6% dried brewer's yeast Pangamin 1% soy flour 0.5 to 2% calcium carbonate 0.2% and in the third fermentation stage on the medium, which contains in mass concentration vegetable oil 2 to 10% glucose 1% soy flour 0.52% calcium carbonate 0.5% potassium hydrogen phosphate 0.01% after which the resulting product is isolated in a known manner.

The advantage of this method is that by the composition of the fermentation media and the fermentation conditions, a monensin complex can be prepared in which factor A predominates and which contains only a very low proportion of factor B. During fermentation in flasks, the content of factor B was found to be less than 10% of the total content of the monensin complex. During fermentation in a pilot tank under the same conditions, the content of factor B was even lower.

Individual factors A and B were identified by nuclear magnetic resonance and mass spectroscopy. The spectra obtained agree with published spectra.

The content of the monensin complex in fermentation broth, extracts, etc. is determined by a known biological titration on agar plates, using Bacillus subtilis as the test microorganism. With a higher content of factor B in the complex, the results of the determination are lower than the actual content due to the lower sensitivity of the test microorganism to factor B.

Individual factors can be determined by thin-layer chromatography on silica gel. Ready-made silica gel plates for thin-layer chromatography (Silufol — k. p. Kavalier) are used for the determination. Chromatograms are developed in the system ethyl n-heptanoctane-methanol in the volume ratio 5:4:1. Red spots of monensin appear after spraying the chromatogram with vanillin reagent (3 wt. % vanillin in ethanol containing 1.5 wt. % sulfuric acid) and subsequent heating to 110 °C. With a suitably selected dosage of both samples and standards, the accuracy of the determination corresponding to the usual biological titration of antibiotics is achieved. In contrast to the biological determination of monensin218032

The results are not adversely affected by the content of factor B in the complex.

The components of the monensin complex are lipophilic in nature. They dissolve easily in organic solvents, but only slightly in water, both in the form of the free acid and the sodium or potassium salt. The antibiotic complex of monensin can be obtained by extraction using known methods, both from the filtrate of the fermentation liquid and from the filter cake, which is a mixture of mycelium and the rest of the solids of the fermentation broth. Before filtration, the pH of the fermentation liquid must be adjusted to a value of 8 to 9 with a solution of soda or other suitable neutralizing agent. Extraction from the filtrate can be carried out with less polar solvents immiscible with water (e.g. chloroform, ethyl acetate, butyl acetate, etc.) or their mixtures with non-polar solvents (aliphatic or aromatic hydrocarbons). The active complex from the filter cake can also be extracted with organic solvents. Suitable solvents are, for example, ethanol, methanol, acetone, ethyl or butyl acetate. Extraction can also be performed with chloroform or its mixture with some non-polar solvents.

When S. cinnamonensis is fermented on vegetable oil media, the filtrate of the fermentation liquid contains approximately half of the monensin complex formed. During extraction, the remaining oil that has not been metabolized also passes into the organic phase. This oil is removed from the extract after distilling off the solvent by shaking the residue between a non-polar solvent (n-hexane, petroleum ether, etc.) and a mixture of methanol with sodium chloride solution (10% by weight) in a volume ratio of 4:1. After shaking, the majority of the active complex is contained in the more polar weight (at least 80% of the total content). It is obtained from the solution by shaking into chloroform or its mixture with carbon tetrachloride and evaporating the residue under reduced pressure. The obtained residue contains 20 to 50% of the active complex, depending on the level of production during fermentation.

This residue can either be directly processed into a monensin premix for the preparation of medicated feed for fattening in large-scale farms, or further purified. In this case, the dissolved residue is first decolorized with activated carbon and the filtrate is then further purified by column chromatography on a suitable stationary phase. Silica gel chromatography is preferred. Benzene, chloroform, ethyl acetate and their mixtures with acetone or methanol can be used as eluents.

The progress of the column chromatography is monitored by the thin layer chromatography described above. The eluate fractions containing the same factor are combined and evaporated under reduced pressure. The residues are subjected to crystallization.

The following are examples of the method for preparing monensin according to the invention.

β

Example

Preparation of monensin by culture in flasks Spores of the production strain Streptomyces cinnamonnesis 179 are inoculated onto a nutrient agar slant with the following composition:

yeasty extract 4.0 grams malted extract 10.0 grams glucose 4.0 grams Oxide agar 30.0g distilled water to 1.0 1

The inoculated medium is incubated at 28 °C for 10 days. The culture is transferred to the 1st inoculation medium of the following composition by weight: glycerol 2.0% peptone 0.25% meat extract. 0.5% sodium chloride 0.1% and shaken for 48 hours at 37 °C on a rotary shaker at a frequency of 2.8 Hz. The inocula thus obtained are inoculated with the 2nd inoculation medium of the following composition by weight: glucose 4.0% soy flour 1.0% dried brewer's yeast Pangamine 1.0% calcium carbonate 0.2% ml of this medium in a 590 ml boiling flask is inoculated with 20 ml of the 1st inocula. The inoculated medium is shaken for 30 hours at 37 °C on a rotary shaker at a frequency of 2.8 Hz.

Fermentation medium contains by weight-

low concentration soybean oil 2.0 % glucose 1.0 % soybean flour 1.0 % calcium carbonate 0.5 % hydrogen phosphate potassium 0.01%

ml of fermentation broth is inoculated with 2 ml of the 2nd inoculum. The inoculated fermentation broth in 500 ml boiling flasks is cultured at 37 °C on a rotary shaker for 196 hours.

After the fermentation is complete, the fermentation liquid is decanted and the pH is adjusted to 8 to 9 with a 13% soda solution. This mixture is filtered on a Buchner funnel. 400 ml of the filtrate is extracted with 200 ml of chloroform and twice with 150 ml of chloroform. The combined extracts are evaporated under reduced pressure and dried. The filter cake is suspended in 100 ml of methanol and stirred for 1 hour. The mixture is then filtered on a Buchner funnel and the cake is washed with a small amount of methanol. The cake is resuspended in 80 ml of methanol and the procedure is repeated as in the first extraction. The extraction of the cake is repeated twice more. The filtrates are combined, the solvent is distilled off under reduced pressure and the residue is dried.

The residues obtained from the filtrate and cake are dissolved in 230 ml of methanol, the solution is mixed with 200 ml of 10% aqueous sodium chloride solution and the mixture is extracted with 200 ml of petroleum ether. The extraction is repeated three more times. The petroleum ether extracts are combined and shaken four times with 200 ml of a mixture of methanol and 10% sodium chloride solution in a volume ratio of 4: 1. The aqueous-methanol extracts are combined and mixed with 10% NaCl solution, so that the resulting mixture contains 40% by volume of NaCl solution. This mixture is extracted with 430 ml of chloroform and then 2 times with 300 ml of chloroform. The chloroform layer is evaporated and dried under reduced pressure. The brown residue — 1150 mg — contained 24% of monensin according to TLC; of this, less than 2.4% is factor B.

Example 2

Preparation of monensin in a fermentation tank.

1 sterile 1st inoculation medium described in Example 1 is inoculated with a spore suspension from Slant agar, which is obtained by washing the culture with 15 ml of sterile distilled water. The inoculated medium is stirred in a laboratory fermenter for 48 hours at a rotation frequency of 10 Hz, a temperature of 37 °C and an aeration rate of 1 l/min. This culture is used to inoculate 15 l of the 2nd inoculation medium, described in Example 1, in a 35 l fermentation tank. The suspension is cultivated for 30 hours at 37 °C, a stirring frequency of 4.66 Hz and an aeration rate of 8 l/min. 135 l of fermentation medium is inoculated with this culture. The soil contains 5% soybean oil, and in other components it is identical to the soil described in Example 1. The actual fermentation takes place for 190 hours at a temperature of 37 °C, a rotation frequency of 4.66 Hz and an aeration rate of 50 1/min.

After the fermentation is complete, the pH of the fermentation liquid is adjusted to 8 to 9 with a 10% soda solution and then the soil is centrifuged in a centrifuge (Sharples). The supernatant (95 l) and the wet solids (9.9 kg) are obtained. The supernatant is mixed with 31 l of chloroform and 9 l of carbon tetrachloride and the mixture is stirred at a frequency of 5 Hz at 30 °C for 1 hour. The mixture is discharged into a separator and left to stand until the phases separate. The heavier phase is discharged into a container and the aqueous phase is discarded. (Monensin was not detected in the aqueous phase sample by thin-layer chromatography (TLC). Solvents are distilled off from the heavier phase separation under reduced pressure. The distillation residue (about 2.5 l) is mixed with 11 l of petroleum ether and this mixture is extracted 5 times with 4.5 l of a mixture of methanol and 10% sodium chloride solution in a volume ratio of 4:1. The aqueous-methanolic extracts are combined and mixed with 5 l of petroleum ether. The layers are separated and the aqueous-methanolic is mixed with 11 l of 10% NaCl. This mixture is further extracted twice with 11 l of a mixture of chloroform and carbon tetrachloride in a volume ratio of 3:1 and a third time with 6 l of the same mixture. The solvents are distilled off from the heavier phase under reduced pressure and the residue is dried. 111.3 g of the brown residue contained 31% monomensine. According to TLC, at least 95% factor A is present, while factor B is less than 5%.

Another portion of monensin is obtained by extraction from the solid portion after centrifugation of the fermentation broth. This is suspended in 25 l of methanol and the suspension is stirred for 1 hour. The extract is filtered and the filter cake is suspended twice more in equal volumes of methanol. The filtrates are combined and the methanol is distilled off under reduced pressure. The distillation residue is extracted with petroleum ether (5X 1.2 l). The petroleum ether layers are combined and extracted 5X 2 l of a mixture of methanol and 10% NaCl solution in a volume ratio of 4:1. The combined aqueous-methanolic extracts are mixed with 2.5 l of petroleum ether. After phase separation, the aqueous-methanolic layer is mixed with 3.5 l of a 10% NaCl solution. This mixture is then extracted 2X 4 l and a third time with 2 l of a mixture of chloroform and carbon tetrachloride in a volume ratio of 3:1. The solvents were distilled off from the extract under reduced pressure and the residue was dried. The above procedure yielded 119.5 g of brown residue, which according to TCL contained 25% monensin, of which more than 95% was factor A.

The obtained residue can be used directly for the preparation of a mecensine premix for the production of medicated feed for fattening in large-scale farms. If necessary, pure factor A can be obtained. First, the dissolved residue is decolorized with activated carbon and the filtrate is then further purified by chromatography on a silica gel column according to a known procedure. Chloroform and its mixture with methanol are used successively for elution. Based on the results of thin-layer chromatography, the fractions containing the desired factor A are combined and the pure product is obtained from the residue by crystallization from a mixture of n-hexane and chloroform.

Claims (2)

SUBJECT OF THE INVENTION Process for the preparation of monensin or its salts, in particular factor A, characterized in that the strain Streptomyces cinnamonensis 179 CCM 3504 is fermented at 28 ° C to 43 ° C in submersible culture in three stages, in the first stage on the first inoculation medium, which contains, by weight, glycerol peptone mass extract sodium chloride 1 to 3% 0.25% 0,2 to 0,7% 0,1% in the second stage on the second inoculum medium containing 2 to 6% by weight of glucose concentration Pangamin 1% soya flour 0,5 to 2% calcium carbonate 0,2% and in a third fermentation step on a soil containing, by weight, vegetable oil glucose soybean flour calcium carbonate dibasic potassium phosphate 2 to 10% 1% 0.5 to 2% 0.5% 0.01% after which the resulting product is isolated in a known manner.