FI91084B - Process for manufacture of salinomycin antibiotic - Google Patents

Description

91084

This invention relates to an improved method for preparing a salinomycin-5 antibiotic by fermentation of a microorganism species Streptomyces albus deposited in the microorganism microorganism collection American Type Culture Collection, Rockville, USA, No. ATCC 21838 and the microorganism collection Fermentation Research Institute, Chibe, FERM-P No. 419, Japan.

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It is well known that salinomycin is produced by various species of the microorganism Streptomyces albus under aerobic growth conditions in nutrient media containing an organic carbon source such as starch, soybean oil, Glu-15, a nitrogen source such as ammonium sulfate, CaCl 2, ammonium chloride, and mineral Kl ^ PO ^ a,

NaCl and MgSO 4.

The small amounts of salinomycin antibiotic in the fermentation broth 20 and the high cost of the nutrient medium composition constantly encourage the search for better solutions that achieve higher yields and cheaper production.

The salinomycin molecule is very complex and belongs to 25 so-called cyclic polyether structures with a relatively large number of methyl and ethyl side radicals.

Chemically, polyether antibiotics are derivatives of branched polyhydroxy fatty acids. The synthesis of salinomycin bio-30 is likely to begin with the polyketide pathway from acetate, propionate, and butyrate precursors. Of course, other similar routes are not excluded. Thus, various oils are the basis of nutritional supplements in the cultivation of said microorganism. Due to the large amount of oils in the nutritional medium, strong aeration and mixing is required, which causes strong foaming, which can only be reduced with a special silicone-type defoamer.

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The salinomycin antibiotic and the microorganism ATCC 21838, which produces said antibiotic, were first obtained in 1972 by the Japanese company Raken Chemical Co. (JP Patent 21553). Variations of the first method have also been patented. However, the yields are very low, only 100-500 μg / ml.

A new, advanced multi-stage process with new variants with yields of up to 10 60,000 pig / ml was later patented. The nutritional supplements contained up to 25% oil, which is very unusual for all known fermentations (EP patent 0 000 037). A special silicone-type defoamer KM-68-2F (Shinetsu Chemical Industry Co. Ltd) was used in said process.

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In the present study, it has been found that some of the methyl side chains are derived from the methionine amino acid. Microorganisms produce said acid in common nutritional media by hydrolysis of soybean meal. These 20 studies have shown that salinomycin biosyntheses can be strongly stimulated by methionine. Stimulation is much stronger in the presence of vitamins that are essential for transmethylating and carboxylating enzyme reactions. Said vitamins are preferably Vita-25 amine D-biotin and pyridoxine (vitamin Bg).

Based on the above invention, a fermentation broth composition was prepared containing a carbon source (soybean oil, molasses, starch), a nitrogen source (soybean meal, (NH4) 2SO4), inorganic salts (KCl, NaCl, CaCO3, KH2PO4), DL-methionine amino acid and possibly either vitamin H, i.e. biotin D, or vitamin Bg. With such a nutrient medium composition, higher yields, up to 100% yields are obtained in the same time and under the same conditions, while foaming can be reduced by means of cheaper defoamers.

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Interestingly, the simultaneous presence of both vitamins in a nutritional medium has a inhibitory effect on salinomycin biosynthesis.

The method for educating the antibiotic salinomycin by fermentation of the microorganism Streptomyces albus ATCC 21838 is characterized in that said microorganism is submerged under aerobic conditions at pH 5.5-6.5 and 26-29 ° C, first in the growth phase and then at 10 ° C. fermentation stage in a nutrient medium consisting of a carbon source, a nitrogen source, mineral salts, 0.01-0.15% DL-methionine amino acid and possibly either vitamin D-biotin 10-20 μg / ml or vitamin Bg 5-10 μg / ml. Vitamin D biotin and vitamin 15 Bg must not be present in the nutrient medium at the same time.

In the growth phase it is grown for e.g. 40-60 hours and in the fermentation phase e.g. 110-210 hours.

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The following table shows the yields of salinomycin on a laboratory scale.

Nutrient medium Yield: μg / ml / 200 h 25 ---------------------------------------- --------------------

Standard medium 1 3000

Standard medium + 0.01% methionine 4000

Standard medium + 0,01% methionine + 20 μg / ml D-biotin 6000 30 Standard medium + 0,01% methionine + 10 μg / ml vitamin Bg 4500

Standard medium + 0.01% methionine + 20 μg / Inl D-biotin + 10 μg / ml Vitamin Bg 1700 35 Composition of medium as in Example 1.

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In the case of stronger foaming of the fermentation medium, various defoamers are used, such as vegetable oil, silicone-type defoamers, etc.

5 The antibiotic is isolated from the fermented broth by a standard method.

As mentioned above, the patent and other technical literature advises that foam can only be successfully destroyed with a special silicone-type defoamer (KM-68-2P, Shinetsu Chemical Industry, Japan).

In this method, the composition of the medium is such that no strong foaming occurs; however, if foaming occurs, it can be destroyed by other, less expensive antifoams.

The concentration of the salinomycin antibiotic in the culture medium is determined by a bioassay method against the microorganism S. cereus avul-20 la.

The present invention is illustrated by the following examples and is not limited thereto in any way.

25 Example 1

The microorganism Streptomyces albus 80614 (ATCC 21838) is cultured on oblique agar in a culture medium having the following composition: 30 meat extracts 10 g yeast extract 4 g glucose 4 g agar 20 g water up to 1000 ml pH = 5.8-6.0; after sterilization 6.2-6.4.

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The food medium has been pre-sterilized at 120 ° C for 20 minutes. A suspension of Streptomyces albus spores is added to the nutrient medium thus prepared, and said suspension is incubated at 28 ° C for 8-10 days. The sporulating mycelium, which grows on oblique agar, is resuspended in 0.1% Tween 80 solution (10 ml). The suspension thus prepared is added to a growth medium having the following composition: glucose 4% 10 soybean meal 1% yeast extract 0.5% ground CaCO3 3 0.2% water up to 100% 15 The pH of the nutrient medium after sterilization is 6.6-7 , 0.

The growth phase of the growth of the microorganism lasts up to 48 hours at 28 ° C at 220 rpm and is used as an inoculum; the pH of the inoculum is 6.4-6.8.

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5-6% by volume of growth culture is inoculated into the fermentation broth. The fermentation broth has the following composition: soybean oil 10% 25 soybean meal 1% corn starch 0.5% (NH4) 2SO4 0.3% KCl 0.2%

NaCl 0.2% 30 KH2PO4 0.02%

CaCO3 0.5%

MgSO 4 0.01% DL-methionine 0.01% water up to 100%

The fermentation medium is sterilized at 120 ° C for 30 minutes and the pH after sterilization is 7.5. At the end of the fermentation, i.e. after 200-210 hours at 28 ° C, the concentration of salinomycin in the culture medium is 400 pg / ml. During the fermentation, the oil content in the nutrient medium is constantly maintained between 3-4% and the pH at 5.8 with 10% ammonia solution. The foam is destroyed with a silicone type antifoam 1510 Dow Corning.

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Example 2

Proceed as in Example 1 except that the fermentation broth additionally contains vitamin D-biotin at a concentration of 20 μ9 / ιη1: η. At the end of the fermentation in 200 hours, the yield of salinomycin is 6000 .mu.g / ml.

Example 3

Proceed as in Example 1, except that the fermentation broth additionally contains vitamin Bg at a concentration of 10 μg / ml. At the end of the fermentation, the yield of salinomycin is 6000 μg / ml in 200 hours.

Example 4

Proceed as in Example 2 except that the composition of the fermentation medium is as follows: soybean oil 10% s o i j auxiliary flour 1% maize dissolution liquid 1% 25 molasses 1% DL-methionine 0.01% D-biotin 0.002% NH4Cl 0.11%

CaCO3 0.5% 30 NaCl 0.2% KCl 0.2% KH2PO4 0.02%

MgSO 4 0.01% water up to 100%

At the end of the fermentation in 190 hours, the yield of salinomycin is 8000 μg / ml.

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Claims (4)

91084 A method for producing a antibiotic of saquinomycin by fermentation of the microorganism Streptomyces albus ATCC 21838, which is submerged under aerobic conditions at pH 5.5-6.5 and 26-29 ° C, first in the growth phase and then in the fermentation step in the culture medium , consisting of a carbon source, a nitrogen source and mineral salts, characterized in that DL-methionine amino acid of 0,01 to 0,15% and possibly either vitamin D-biotin of 10 to 20 μg / ml are added to the nutrient medium , or vitamin Bg at 5-10 μg / ml. Process according to Claim 1, characterized in that starch, vegetable oils and / or molasses are used as the carbon source. Process according to Claim 1, characterized in that the nitrogen source used is soybean meal, (NH 4) 2 SO 4, maize steeping liquor and N 2 Cl 2. 20 Process according to Claim 1, characterized in that sulphates, phosphates, chlorides and / or carbonates of sodium, potassium, calcium and / or magnesium are used as mineral salts. 25