DE1058215B - Manufacture of the antibiotic C-159 - Google Patents

Description

Production of the antibiotic C-159 The invention relates to the production, Extraction and purification of the antibiotic or its salts.

The new antibiotic is used in the following description and claim consistently referred to as "C-159", as no general name has yet been proposed however, the identity of C-159 will be clear from the following description emerged. The antibiotic C-159 and its salts are characterized in that they prevent the growth of gram-positive microorganisms that they contain glycine, alanine, Contain threonine and aspartic acid and no other amino acids in alkaline made water extremely soluble and in aqueous solutions whose PH is below 4, are extremely sparingly soluble, but on the other hand in methanol soluble and insoluble in acetone, diethyl ether and butyl acetate and in purified Form practically 58.7% carbon, 7.4% hydrogen, 9.9% nitrogen and 24.0% Oxygen (as a difference) contain that, when they are dissolved in water, they are maximal Absorption in ultraviolet light at 345 m [, (E '% "= 71) and a shoulder at 260 to 280 mu and in the infrared part of the spectrum when in potassium bromide to be processed into cookies, characteristic at the following wavelengths in u Absorbance bands show: 2.90, 3.38, 5.85 (shoulder). 6.05 (strong shoulder), 6.10, 6.55, 7.23, 7.50, 8.00 (wide) and 9.40 (wide).

The preparation of the antibiotic C-159 consists in that a C-159 producing species of Streptomyces canus within an aqueous carbohydrate solution, containing a nitrogenous nutrient, incubated under aerobic conditions until the solution has significant antibacterial activity, and that then optionally the antibiotic thus formed from the fermentation liquid is won.

The fermentation medium contains a carbohydrate solution as a carbon source, z. B. a commercial sugar, a similar carbohydrate or a glyceride, and also a source of nitrogen, e.g. B. an inorganic salt such as ammonium sulfate or sodium nitrate, or an organic substance often applied in raw form like corn steep liquor, soluble still bottoms, yeast, or soy flour. The fermentation medium can also contain mineral salts and buffer substances such as calcium carbonate, contain; such media are from previous processes for the production of Antibioticis known.

The organism that produces antibiotic C-159 is in the "American Type Culture Collection «. In the present description with C-159 and C-509 designated strains of Streptomyces canus are there as ATCC 12 646 and 12 647 respectively.

Transferred specimens of Streptomyces canus (culture C-159) grow on 28 to 30 ° C on asparagine dextrose, Bennet's agar and tomato oatmeal agar. Vegetative Mycelium that grows in agar soils is colorless to flax-colored. On the second or third Day, a gray mycelium develops in the air and leads to numerous, loosely bound Sporophores (on some media). Soluble pigments are found on certain media formed from yellow to yellowish-green tint.

The following carbohydrates and organic salts can be used smoothly in synthetic Agar according to Pridham and Gottlieb can be used, which is no other carbon source contains: dextrin, starch, glycerine, arabinose, rhamnose, xylose, glucose, levulose, Maltose, lactose, cellubiose, galactose, mannitol, inositol, sorbitol, sodium acetate, Sodium citrate, sodium succinate and calcium maleate. Cane sugar, raffinose, inulin, Dulcit, 1 atrium oxalate, sodium salicylate and sodium tartrate promote growth not.

A more detailed description of Streptomyces canus (culture C-159) on a number of media commonly used for examining members of the Streptomyces species is given in Table I. The type of growth, pigment formation and other characteristics are listed in this. The media was inoculated by streaking and all cultures were incubated at 28-30 ° C for 21 days. The color names with capital initial letters used in the description correspond to those used in the book by M aerz and Paul, "A Dictonary of Color", 2nd edition, New York, MeGraw-Hill (1950). Streptomyces canus (culture C-159) produces an alkaline reaction in liquorice milk without flocculation occurring. Peptonization is slow. Gelatin was slowly liquefied, but no soluble pigment was formed. Blood agar was not hemolyzed. Peptone Iron Agar did not darken. Table I. Breeding characteristics of Streptomyces canus (culture C-159) Strength vegetative color Medium of growth growth of air mycelium soluble pigment remarks and the spurs Asparagine dextrose moderately colorless white to gray no reverse Agar flax colors Bennett's Agar good flax color gray no reverse side pale Nutrient agar moderately colorless off-white, no reverse side colorless Czapek's agar moderately thin flax-colored moderately gray no reverse gray Calcium maleate agar moderately amber white to gray, slightly yellowish, moderate clarification knows of the agar Tomato-oatmeal good flax-colored gray yellowish-green reverse side Agar bronze shimmering Potato schnitzel good cream color, wood ash color, mauve to gray The in vitro antibiotic spectrum of activity of antibiotic C-159 was examined using the test tube dilution method to determine the minimum concentrations of antibiotic at which bacterial growth was completely prevented for 24 hours. Unless otherwise stated, heart infusion broth was used as the medium for all test bacteria. The following results were obtained: Least, growth- Anti-bacterium concentration mcg / cms Micrococcus pyogenes v ar. aureus No. 209 ........................ 125 Micrococcus pyogenes v ar. aureus No. 52-79 (penicillin resistant) ... 125 Gaff kya t traga ......... ....... 125 Streptococcus pyogenes C 203 M * .. 500 Streptococcus agalactiae ........... 8 Diplococcus pneumoniae * ..... ..... 12.5 Lactobacillus acidophilus **. . . . . . . . 250 Lactobacillus Gasei **. . . . . . . . . . . . . . 500 Bacillus anthracis ................. 31 Bacillus cereus var. Mycoides ...... 250 Bacillus subtilis. .... .. .. ..... ..... 3.1 Corynebacterium xerosis .......... 4 Escherichia coli ................... 100 Shigella Bonnei ... .. .. .. ........... 500 Klebsiena pneumoniae ............ 100 Proteus vulgaris .................. 500 Pseudomonas aeruginosa .......... 500 Neisseria sp. . ... . . . . . . . ... . . . .. ... 500 Candida albicans. ... . . . . . . . . . . . . . . 500 Heart Infusion Broth - 10% serum ** Tomato juice broth C-159 was not lethal to mice when injected intravenously at 750 mg / kg in 2% aqueous solution; a dose of 1000 urg / kg was lethal.

C-159, which was dissolved in an 80% ethanol solution, protected successfully Mice immunized by intraperitoneal vaccination with a lethal dose of Diplococcus pneumoniae had become infected when administered intraperitoneally was, in an amount of 1.75 urg / kg, and if it was intramuscular at the time of infection was administered in an amount of 13.5 mg / kg. Oral administration of a At an amount of 500 mg / kg, the experimental infection was not effectively suppressed.

Of course, the manufacture of the antibiotic is not up The use of this particular microorganism is limited, but you can too those microorganisms are applied that are natural varieties, or variations Are mutants that are derived from the described organism by mutation-inducing agents, such as X-ray irradiation, j.TV irradiation and nitrogen mustard can be obtained.

The following examples illustrate the production of fermentation media that contain the antibiotic. Example 1 Streptomyces canus (culture C-509) became aerobic Incubated for 72 hours at 27 ° C in a rotating shaker. The medium (100 cc), which was in a 500 cc Erlenmeyer flask, was 30 minutes at 1 atm. sterilized and then with a 42 hour old vegetative Culture sample inoculated in an amount of 2%. The aqueous medium contained 3% polysaccharide (resynthesized dextrin), 1% corn steeping water, 0.50 / 0 K2 H P 04, 0.5 0/0 Na Cl, 0.5% Na N 03 and 1% soybean flour. The final strength of the antibiotic containing fermentation liquid obtained with the aid of the foregoing biological Examination method determined was 20.2 mm (undiluted) and 18.5 mm (3-fold diluted).

Example 2 Culture of Streptomyces canus C-509 was fermented in a 4000-1 fermenter using the growth medium described in Example 1 with a 72 hour old vegetative culture sample was inoculated. The strenght the liquid after 60 hours was 25 mm (undiluted) and 22 mm (3-fold diluted). Example 3 Streptomyces canus (Culture C-159) was made with no agitation and feed of air at a rate of 210 l / min. 120 hours at 29.5 ° C in 2250 liters of a medium incubated, the 1% dextrose, 1.0% soybean flour, 0.5% sodium chloride, 0.1% calcium carbonate and contained 0.05% soluble still bottoms.

The pg at the start of fermentation was 7.4, dropped to 6.0 and rose then down to 8.4, where it was 8.0 in the end. The final strength of the liquid was 14.7 mm undiluted and 12.5 mm when diluted 3 times. Example 4 Streptomyces canus (Culture C-159) was grown without agitation and with the supply of air in an amount of 210 rpm Incubated 100 hours at 29.5 ° C in 2300 l of a growth medium that 1.0% dextrose, 1.0% soy flour, 0.5% sodium chloride, 0.1% calcium carbonate and 0.051 / o contained soluble distillation residues. The p $ at the beginning of fermentation was 7.1 and gradually rose to 7.9. The strength of the liquid after 70 hours was undiluted 14.9 mm and thinned 3 times 12 mm. Example 5 Streptomyces canus (culture C-159) was aerobic fermented in a rotating shaker at 27 ° C for 72 hours. The growth medium (100ccm) was placed in a 500cc Erlenmeyer flask for 30 minutes at 1 atm. sterilized and then with a 32 hour old vegetative Culture sample inoculated in an amount of 1%. The aqueous medium contained 1.0% dextrin, 0.05% soluble distillation residues, 0.50 / a NaCl, 0.1% CaCO3 and 1.0% soy flour. The final thickness of the liquid was 17.4 mm undiluted and diluted 3 times 14.0 mm. The p $ was 8.0 towards the end.

Antibiotic C-159 is isolated from the fermentation fluids by this to separate the mycelium, filtered, the pH of the filtrate adjusted to about 8.5 and the antibiotic on magnesium silicate, e.g. B. 6 kg per 100 liters of filtrate, adsorbed. After the solids have been collected by filtration, the antibiotic becomes from the cake z. B. with a tenth of the volume of the original fermentation liquid a mixture of 1 part water and 1 part methanol dissolved out. The solid antibiotic is then recovered from the solution by e.g. B. by distillation in Concentrated vacuum and the aqueous concentrate by freezing or by evaporation is dried. During this narrowing, the p $ will range from 6.5 to 7.5 held.

Another method is to use the antibiotic with a mixture of 3 parts of n-butanol and 1 part of water, which is in an amount of one tenth of the Volume of the filtrate is applied, dissolved out. After repeating the combined solutions by concentration in vacuo to one tenth or one twentieth their original volume concentrated and completely dried, whereby the p $ is kept in the range 7 to 8. After filtering, the antibiotic is taken out this clear, anhydrous, concentrated solution in butanol, e.g. B. by addition mixed lower alkanes, such as. B. 4 to 5 times the volume of Skellysolve B failed.

Solvent extraction can also be used. Included the antibiotic is made from filtered fermentation liquid at pH 2.5, 6.0 or 9.0 with Extracted with the help of n-butanol, but not with chloroform or methyl isobutyl ketone. The preferred pg range for this extraction is between 8.2 and 8.5. The antibiotic is obtained from the butanol extract by concentrating it until it crystallizes out or by azeotropic distillation and drying and then by freezing out dries or precipitates due to the addition of Skellysolve B.

Antibiotic C-159 can also be used on activated charcoal from filtered Adsorb fermentation liquid and then with a mixture of equal parts Dissolve out n-butanol and water which has been adjusted to pg 3.0 with hydrochloric acid. The solids are then recovered from the solution as described above. The following examples are intended to demonstrate the extraction of the antibiotic from a fermentation liquid explain both in raw and in purified form, without thereby affecting the scope of the invention should be limited. Example 6 21401 fermentation liquid, obtained according to Example 3, were filtered with the addition of 2.4% of a filtration promoting agent. The p $ of the filtrate was adjusted to 8.5 by adding 50% sodium hydroxide and then stirred for 30 minutes with 6 kg of magnesium silicate for every 100 liters of filtrate. The cake was collected and washed with a mixture of equal parts of methanol and Water, the volume of which is one-tenth the volume of the original liquid corresponds to eluted. The resulting solution was then distilled concentrated in vacuo, 321 of an aqueous solution of the antibiotic being obtained were., the thickness of which was 19.7 mm undiluted and 17.2 mm when thinned 3 times.

A portion (1300 cc) of this aqueous concentrate was freeze-out dried to obtain 26 g of antibiotic C-159, its biological effectiveness (at 1 mg / ccm) was 12.9 undiluted and 10.0 when diluted 3 times.

Another portion (200 cc) of this aqueous concentrate was found at p $ 8.0 extracted 4 times with 50 cc n-butanol. The butanol extracts were combined and concentrated to 40 ccm by distillation in vacuo. 20 cc of this dry concentrate in butanol were added to 80 cc of “SkellysolveB”, with 170 mg of solid antibiotic C-159 failed, its biological effectiveness at 1 mg / ccm undiluted 18.2 mm and diluted 3 times was 17.9 mm. The remaining 20 cc of the butanol concentrate were distilled in vacuo with the addition of water, with 15 ccm aqueous concentrate which was then dried by freezing to give 329 mg of solid Antibiotic to get its biological Effectiveness at 1 mg / cc undiluted was 17.0 mm and diluted 3 times was 15.0 mm.

Example 7 17301 fermentation liquid, obtained according to example 4, were obtained at the pH of about 7.1 present towards the end with the addition of 2.4% of a die Filtration promoting agent filtered. The pH of the filtrate was adjusted using 500 / egg gem sodium hydroxide adjusted to 8.5 and then about 30 minutes stirred with magnesium silicate (6 kg to 100 l of filtrate). The cake was collected and by stirring twice with about 150 liters of a mixture of 3 parts of n-butanol and 1 part of water eluted. The combined solutions were distilled in Vacuum reduced to about 16 l; the solution obtained had biological activity of 17.0 mm (with 10-fold dilution). In this procedure, 185 g wet, solid antibiotic precipitated, which at 1 mg / kg had an effectiveness of 12.9 mm (undiluted) and 10.0 mm (diluted 3 times).

111 of the butanol concentrate were obtained by distillation in vacuo concentrated a volume of 1350 ccm and this with 4 times the volume of Skellysolve B added to precipitate solid antibiotic C-159 which, after drying, contained 26 g weighed and its biological effectiveness at 1 mg / ccm undiluted 19.9 mm and 3-fold when thinned was 17.2 mm.

The effectiveness of these solids was increased when they were slurried in methanol (2 cc / g), undissolved solids were removed by filtration, and 4 times the volume of diethyl ether was added to precipitate the antibiotic. The methanol-ether mother liquor contained only ineffective impurities. Example 8 A neat sample of Antibiotic C-159 was prepared in six funnels using a countercurrent flow distribution method, with one as the top phase. A mixture of 1 part of n-butanol and 1.5 parts of methyl isobutyl ketone saturated with water and water buffered with 0.02 molar phosphate at pH 6.0 and saturated with n-butanol and methyl isobutyl ketone were used as the lower phase. It was previously determined that this solvent system gave the distribution ratio of 1. 1 g of solid antibiotic C-159 was dissolved in 100 cc of the aqueous phase and placed in a separating funnel together with 100 cc of the upper solvent phase. After mixing and separating, the lower phase was transferred to a second funnel and mixed with fresh upper phase. Fresh aqueous phase was added to the upper solvent phase remaining in the funnel used at the beginning. This countercurrent process was repeated five more times in the usual manner.

The aqueous phases from tubes 2, 3 and 4 were combined and extracted with butanol. This butanol became the combined solvent phases given from the same tubes, the mixture azeotropically distilled and freeze-out dried to obtain 140 mg of purified solid antibiotic C-159, its biological effectiveness at 1 mg / ccm undiluted 23.0 mm, diluted 3 times 20.0 mm and diluted 9 times was 17.5 mm.

Antibiotic C-159 shows a UV absorption maximum at 345 in water m [. (El t = 71) and a shoulder at 260 to 280 mg. Antibiotic C-159 shows when it is processed into cookies with potassium bromide, characteristic absorption bands in the infrared part of the spectrum, namely at the following wavelengths in #t: 2.90, 3.38, 5.85 (shoulder), 6.05 (strong shoulder), 6.10, 6.55, 7.23, 7.50, 8.00 (wide) and 9.40 (wide).

Antibiotic C-159 by analysis contains 58.7% carbon, 7.4% Hydrogen, 9.9% nitrogen and 24.0% oxygen (as the difference). Like through two-dimensional Paper chromatography after hydrolysis by heating to 150 ° C in a closed Tube in the presence of 0.38 n-barium hydroxide for 1.5 hours or 20% hydrochloric acid was found for 5 hours, the antibiotic contains four amino acids, namely Glycine, a-alanine, threonine and aspartic acid and no other amino acids. Amphomycin contains histidine, aspartic acid, glycine, glutamic acid, proline, valine and a other amino acid, but no α-alanine or threonine.

Antibiotic C-159 is insoluble in acetone, ether and butyl acetate, but soluble in methanol, and becomes from methanolic solution with the addition of acetone or ether precipitated.

Antibiotic C-159 is light in weakly alkaline aqueous solutions soluble, but considerably less soluble in acidic aqueous solutions. For example 1 g was dissolved in 20 cc of water at pH 8.8 and then gradually acidified. When pH 4.4 was reached, a precipitate was formed and the biological Effectiveness of the solution reduced to about half of its original value. When the pH reached 3.7, the solution was only a tenth effective the original.

Antibiotic C-159 is an acid and can be added by adding the appropriate Bases, e.g. B. Calciumhydroxvd, sodium hydroxide, etc., to an aqueous solution of the Antibiotic converted into ammonium, substituted ammonium and metal salts become, such as B. sodium, potassium, calcium, magnesium, aluminum salts and the like. The solid metal salts are optionally, e.g. B. by freezing isolated.

Claims (1)

PATENT CLAIM; The use of strain C-159 or C-509 of Streptomyces canus nov. spec, for the production of the antibiotic C-159 in the usual biological way, obtaining the antibiotic by extraction and / or adsorption and elution and, if necessary, conversion into its metal salts.