DE1089513B - Process for the preparation of the antibiotic rifomycin and its components - Google Patents

Description

Process for the preparation of the antibiotic rifomycin and its components The production of rifomycin and its components A, B, C, D, E, in which a Streptomyces mediterranei rifomycin producing strain (A. T. C. C. No. 13 685) or a naturally occurring or artificially produced mutant thereof in an assimilable source of carbohydrates, nitrogen and inorganic salts containing culture medium is grown until the medium has a substantial has acquired antibiotic activity, and then from the medium rifomycin and can win its components.

The strain Streptomyces mediterranei was selected from a soil sample the area around St. Raphael (France) and has the following characteristic Properties common to various media in inclined culture media after exposure observed for up to 20 days at 28 ° C. The color determination was after Dictionary of Color made by Maerz and Paul, McGraw Hill, 1950. Oatmeal agar Fairly good growth with a smooth surface. The vegetative mycelium is glassy to yellowish with a pink underside. The whitish aerial mycelium is pink Colour. Traces of a yellowish soluble pigment.

Yeast Extract Glucose Agar Abundant yellowish to pink growth with rugged surface. Minor aerial mycelium. No pigmentation of the media.

Emerson Glucose Agar Abundant yellowish to pale orange in color Growth with a fissured surface. The aerial mycal turns pink (2 / B / 8). Light amber soluble pigment. Oatmeal Mix Agar (1) Good growth, smooth, yellowish with slightly orange color. Aerial mycelium whitish to pink. Amber colored soluble pigment.

Benett agar Good growth, yellowish to orange-yellow. Aerial mycelium turns pink. Light amber pigment.

Agar after Penassay (Difoo) Weak growth.

(1) Oatmeal Mix Agar contained oatmeal (20 g cooked oatmeal filtrate) : Enzymatic Casein Hydrolyzate 5, g; Sucrose 10 g; Agar 40 g, in 1000 cc H20 distilled, pg value adjusted to 6.9. Yeast Extract Molasses Agar (2) Abundant fissured growth, colorless to yellowish. Whitish aerial mycelium. Dark amber, soluble pigment (12 / H / 10).

Dextrose sucrose agar acc. To Czapek Weak growth, thin and colorless to slightly melon-colored (11 / A / 8). Traces of pink-white aerial mycelium. No soluble pigment.

Potato Agar Weak growth, thin and colorless. Traces of whitish Aerial mycelium. No soluble pigment. Potato pieces Poor development, glassy. Color of the bit unchanged.

Glucose Asparagine Agar Fairly good growth with a smooth surface. Thin vegetative mycelium of light orange, pink color (10 / B / 21) and yellowish underside. No aerial mycelium. Slightly yellowish pigment.

(2) yeast extract molasses agar contained: glycerin 7.5 g; Molasses 7.5 G; Yeast extract 5 g; Na Cl 4 g; Agar-agar 30 g; H20, distilled, 1000 cc; pn value set to 6.0. Glycerin asparagine agar Like glucose asparagine agar. Nutrient agar MOige growth with smooth surface; melon-colored to orange (2 / B / 10) with yellowish-orange underside. Luftmycol pink-white. Soluble pigment is absent.

Pridham Agar Moderate growth, smooth, colorless with lobster red (2 / G / 11) spots. Pink aerial mycelium. No pigmentation of the medium.

Starch agar Growth weak, colorless to slightly orange-pink. Starch hydrolysis doubtful. Hardly any white aerial mycelium. Dextrose Tryptone Agar Abundant growth, orange with golden yellow to orange-pink reverse side. Pink aerial mycelium. Slightly golden yellow soluble pigment.

Cobalt agar according to Hickey and Tresner Moderate growth, glassy to light pink orange. Some aerial mycelium. Slightly yellowish soluble pigment. Tyrosine Agar Weak Growth. Ca Malate Agar Growth fairly good, colorless. Aerial mycelium whitish with pink coloring. No soluble pigment. Partial digestion of calcium malate.

Dorset Albumin Agar Fairly good growth, pink. No aerial mycelium or pigmentation of the medium.

Gelatin No pigmentation. Liquefaction slow and incomplete.

Nitrate surface growth with pink aerial mycelium. No reduction too Nitrites. Broth turns yellowish.

Lakmus milk No peptonization or coagulation. Slightly alkaline Reaction.

Dextrose Tryptone Agar Abundant growth, orange with yellow gold to orange-pink back. Pink aerial mycelium. Slightly golden yellow (9 J 6) soluble pigment.

Cellulose No growth on filter paper discs on carbohydrate-free ones Mineral based agar.

Temperature optimum 28 ° C. Growth takes place at 37 ° C, but there are the characteristics less clear.

Colony morphology On Benett agar, this strain forms small ones (usually no more than 2 mm in diameter) colonies with well identifiable Outlined, with a firm but rugged surface; covered with whitish aerial mycelium. A large number of colonies planted on agar do not develop aerial mycelium, they do remain in their orange-red vegetative DZycel, which grows to a considerable thickness grows until they take on a rock-like appearance.

Sporulation ME / 83 does not usually develop spores on the above listed media. If spore formation takes place, the spores appear in long, slightly twisted chains. Spirals are not common. The spurs are ellipsoidal to elongated (0.8 to 1.0 - 3.0 to 5.0 #t). Tests using of carbon source cultures on mineral-based agar with various additional sources according to Pridham and Gottlieb showed no growth whatsoever (raffinose, Acetate, citrate, glycine). All other carbons tested showed moderate to good growth on (xylose, arabinose, glucose, rhamnose, fructose, mannose, galactose, Lactose, sucrose, maltose, glycerin, mannitol, inositol, succinate and salicin).

Streptomyces mediterranei can be characterized as follows 1. It does not belong to the Streptomycetes chromogenic group.

2. On most media, vegetative growth takes on a yellowish tinge to slightly pink-orange in color, but never an intense orange-red to cherry color.

3. The aerial mycelium is pinkish white, but never takes an orange or lavender color. Weak sporulation on most media.

4. Acetate, citrate, raffinose and glycine cannot be used as carbon sources be used.

For the production of rifomycin, the invention is not limited to Streptomyces mediterranei strain described above or others of the same Description of appropriate strains is limited, but includes the use of Variants of these strains, for example, by selecting the by treatment of Spore suspensions with mutagenic agents, such as. B. UV radiation, mustard gas, etc., formed variants or mutants are obtained.

For the production of rifomycin, Streptomyces mediterranei or any other rifomycirx producing microorganism at 25 to 37 ° C and preferably at 28 ° C in a submerged aerated culture in an aqueous culture medium cultured which contains an assimilable source of nitrogen. As a carbon source the carbohydrates and carbon derivatives listed below can be used, z. B. glucose, xylose, lactose, sucrose, maltose, glycerin, mannitol, inositol, succinate starch, Dextrin etc. Valuable nitrogen sources are, for example, amino acids and mixtures the same. Peptides and proteins and their hydrolysates, e.g. B. peptone, meat extracts, Yeast extract, soybean meal, corn steep liquor, soluble fish ingredients, aqueous Fractions of seed grain, in the distillation of brandy. obtained soluble Fabrics. The fermentation is carried out for 24 to 120 hours. The initial one pH, usually adjusted to about 7 to 8, decreases as the fermentation proceeds down from 5 to 7. Usually, the best results will be obtained with a fermentation period received from 48 to 72 hours. After this time there will be an excellent yield of the antibiotic received.

After the fermentation is complete, rifomycin can be prepared using the following method be isolated: The fermentation medium is at an Endp H value from 5.0 to 7.0 or after adjusting to 7.5 to 8; 5 at 26 to 28 ° C or less filtered. The filtrate is quickly adjusted to an acidic pH to the to ensure the best resistance of the antibiotic substance. The active substance is by water-insoluble solvents, e.g. B. butanol, ethyl, propyl, butyl or amyl acetate or chloroform. The ratio between the volume of the medium and that of the solvent can vary depending on the solvent used vary. Usually a ratio of 2: 1 to 10: 1 is used.

Part of the antibiotic substance is insoluble in an acidic medium and can be obtained directly from the medium, optionally by filtration. In any case, the filtrate must be extracted with the organic solvent, to gain most of the antibiotic effectiveness. The insoluble part can be dissolved in the same solvent and with the organic extract that comes with the extraction of the filtrate is obtained, are combined. The leftover aqueous medium still contains a microbiological effectiveness after extraction, which cannot be extracted by common organic solvents. she can but isolated by absorption on charcoal at a slightly acidic pH and through Wash out with water-soluble alcohols, such as. B. methanol or ethanol obtained will.

The mycelium also holds back some microbiological activity. These can be made from the mycelium by means of the above solvents or simply by multiples Wash with water, and extracted from the acidified aqueous solution the antibiotic activity is transferred to the above-mentioned solvents.

As soon as most of the antibiotic activity has been transferred to the solvent, this is distilled in vacuo to a small volume under a stream of nitrogen at below 30 ° C. By adding petroleum ether or other hydrocarbon mixtures, a brown substance is precipitated which is highly effective against gram-positive bacteria and mycobacteria. This crude product is an amorphous powder with a light brown to dark brown color, which sometimes has a greenish shade. It consists of a mixture of substances with antibiotic activity. Chromatography on Whatman No. 1 paper using a mixed solvent of 3 ° / ° ammonium chloride and 10 % ascorbic acid-containing water, developed on an agar plate inoculated with Sarcina luthea, shows at least five zones of inhibition, arranged in ascending order of R f Value, starting with the point of deposition of the drop, can be denoted by A to E. Districts C and D are usually larger than the other districts. The presence of other political groups cannot be ruled out. Fig. 1 shows a chromatogram obtained as described above.

Instead of precipitating the antibiotic mixture from the extraction solvent, this can also be done by means of buffer solutions at a pH of. 7.0 to 7.5 and then extracted at 10.0 to 10.5. The adjustment of the pH allows an approximate separation of the various fractions forming the entire raw powder. At a pH of 7.0 to 7.5, rifomycin B with a pronounced acidic nature is selectively extracted. The other fractions are extracted at higher pH values. Due to the poor resistance of antibiotics in neutral and alkaline medium, the use of buffer solutions with a content of 0.5 to 30 %, ascorbic acid or other substances with the same stabilizing effect is necessary. The volume ratio of the solvent to the alkaline buffer solution varies depending on the solvent used and the pH value used. From a solution in ethyl acetate with a 1% activity, three extractions are carried out with one tenth the volume of the buffer solution with a pH of 7.0 to 7.5 in order to pass fraction B into the aqueous phase, and three Extractions with a fifth of the volume of the buffer solution with a pH value of 10.0 to 10.5 and a content of 107 ascorbic acid are necessary in order to bring the other components into the aqueous phase.

The antibiotics are removed from the aqueous phase in a semi-purified Condition by acidification and extraction with a solvent, such as. B. ethyl acetate or chloroform, evaporation of the solvent and precipitation with petroleum ether. The antibiotics can also be precipitated from the aqueous phase by acidification and be won.

The purification of the raw mixture can be done by chromatography on different Adsorbents, such as. B. synthetic magnesium silicate, are made, which was previously treated with a methanolic solution of hydrogen chloride gas became. The crude mixture is dissolved in a solvent, e.g. B. ethyl acetate, dissolved, adsorbed on a column and with the same solvent or with solvent mixtures washed out with increasing polarity. The washing water with the highest microbiological Values are those that contain rifomycin C and D.

The following are the procedure for obtaining five main components of the raw antibiotic and some physical and chemical properties are described, which allow their identification.

Rifomycin A. Its Rf value is 0.00 to 0.10 on chromatography by means of the system described above. Rifomycin A is made from the crude mixture through Countercurrent separation obtained in a two-phase system. Methanol - 0.01 N hydrochloric acid - Benzene - petroleum ether (10: 5: 15: 5). After 160 passages, rifomycin A is in the Tubes 150 to 160 obtained directly from the light phase. After concentration and precipitation in petroleum ether gives rifomycin A in the form of a dark brown amorphous Powder won.

Rifomycin A is insoluble in water, but in alkaline agents soluble. When dissolved in ethyl acetate, it shows an absorption spectrum in the visible range at 460 m & ,. After shaking the ethyl acetate solution with an acidic aqueous solution 1% stannous chloride solution, the absorption maximum is 422 m # t. The same The effect can be observed when shaking with an aqueous 1% ascorbic acid solution will. The maximum at 460 mp. can by shaking the ethyl acetate solution with a 0.1 0% potassium ferricyanide solution can be obtained. Rifomycin. probably has the nature of a quinone with the possibility of reversible oxydo-reduction.

Rifomycin B. Its Rf value is 0.20-0.30 on chromatography by means of the above mixtures. Rifomycin B is very acidic and can go through Dissolving the crude mixture in a solvent such as. B. ethyl acetate or chloroform, and extracting from the organic solution with a phosphate buffer at neutral p $ value (6.5 to 7.5) can be obtained. Rifomycin. B is converted into the aqueous phase and can either be precipitated by adding acids or again from the acidified Water solution can be extracted with ethyl acetate. After concentration of the ethyl acetate fraction becomes rifomycin B in semi-purified State failed. Through more Crystallizations from ethyl acetate or benzene will give the pure product.

Rifomycin B is in the form of shiny yellow crystals. It will dark at 165 ° C and does not melt below 300 ° C. The pH of the saturated water solution is 3.6. It is very light in water and dilute mineral acid as well as in Bicarbonate solutions soluble with evolution of carbon dioxide. In concentrated Sulfuric acid, it dissolves with a red color. In methanol, ethanol, chloroform, It is weak in ethyl acetate and acetone, very weak in benzene, and in petroleum ether, It is not at all carbon tetrachloride, ethyl ether and hydrocarbons soluble. It decolorizes potassium permanganate solutions and reduces Fehling's solution and Tollen's reagent, adsorbs bromine and is catalytically reduced by hydrogen. When treated with nitrous acid and alkaline agents, an intensive one takes place Red coloring instead. It does not react with ninhydrin, although it is stronger Reacts acid hydrolysis, can not be diazotized and does not sit with aromatic Diazo derivatives. There is no Molish reaction and it reduces triphenyltetrazolium not. However, it reacts with ferric chloride and gives it a red color.

The elemental analysis gave the following mean values: C = 61.75 ° / 0, H = 6.72 ° / 0, N = 1.88 ° / 0; O = 29.22%. The following raw formula can be derived from this become: C3gH47`63N014. Rifomycin is optically active and has a rotation of [a] 26. -11 ° (0.1 methanol, determined in a photoelectric polarimeter).

A potentiometric test sample in a mixture of water and methanol shows two acidic functional groups: pgl 1/2 about 2.8 (equivalent molecular weight 780), p $ 21/2 about 6.0 (equivalent molecular weight 765). The molecular weight determined in cyclohexanol according to Rast is about 750. The UV and the visible spectrum in water shows a peak at 220 to 230 m # t (E; ro = 513) and two maxima at 305 m # t (EI- = 246 ) and 430 m # t (Eift = 202). The maximum in the visible spectrum in ethyl acetate is 415 m # t. The UV and visible spectrum of rifomycin B in buffer phosphate at a pH of 7.3 shows maxima at 223 m # t ( I EI-_ = 555), 304 mp. (Ei m = 275) and at 425 m # t ( 1 E , ' #. = 220). The spectrum is shown in FIG.

The infrared spectrum of the rifomycin is shown in FIG. It shows Absorption maxima at the following values in cm-1: 3465, 3330, 1742, 1730 (peak), 1705 (peak), 1673, 1602, 1575, 1550, 1445, 1342, 1300, 1267, 1240 (shoulder), 1205, 1180, 1165, 1102, 1073, 1048, 1020, 975, 948, 915, 905, 850; 795, 760, 730, 680.

Rifomycin B, which is acidic in nature, forms with both inorganic as well as salts with organic bases. The mono- and disodium salt was produced, the former being weakly and the latter freely soluble in water. The solutions have a pH value of 5.0 or 7.0. These solutions are very persistent.

Salts with the following organic bases were produced: dibenzylamine, Quinine, 1-p-chlorobenzyl-2-pyrrolidyl-methylbenzoimidazole. All of these have salts have poor solubility in water and appear useful for therapeutic purposes to be.

Rifomycin C and D. These fractions show firmly identical Rf values for the chromatographic mixture described (Rf = 0.45 to 0.55). The inhibition zones adjoin each other and are connected by a long neck. Rifomycin C and D are in the semi-pure state from the raw mixture by countercurrent division in a two-phase system obtained from methano1-0.01 n-hydrochloric acid-benzene-petroleum ether (10: 5: 15: 5) exists. At 85 passes, the maximum of rifomycin C is between the 55th and 65th tube and that of rifomycin D between the 35th and 45th tube. By Extract the tube contents with benzene or ethyl acetate or chloroform or others Similar solvents., Concentration of the extracts and precipitation with petroleum ether or other hydrocarbon mixtures, the two rifomycins are considered amorphous Obtain powder with a high degree of purity.

Rifomycin C and D appear as dark brown powders with a greenish tinge Nuance. They are soluble in common organic solvents, in water but practically insoluble. An aqueous solution in a buffer agent is obtained at a pH of 8.0 to 10.0 by adding ascorbic acid as a stabilizing agent. The UV and visible spectrum of rifomycin C and D are identical. The maxima at 385 to 390 and 460% t in methanol and ethyl acetate are very characteristic. The maximum at 460 m # t in ethyl acetate is after shaking the ethyl acetate solution of rifomycin C or D with a 0.1% aqueous solution of stannous chloride or a 1.0% aqueous solution of ascorbic acid to 422 m # t. To Further shaking with a 0.1% aqueous potassium ferricyanide solution shows the spectrum again a maximum at 460 m # t. This shows that rifomycin C and D, like quinone, are substances with reversible oxido-reducing properties.

Properties of rifomycin C. Elemental analysis: C = 61.51 () / o, H = 6.73%, N = 4.21 ') / "O = 27.55% (as the difference). Rifomycin C results with the reagents according to Tollens and Fehling, with ferric chloride, iodoform and permanganate, positive reactions. The ninhydrin reaction is negative but becomes positive after strong acid hydrolysis. The visible spectrum in ethyl acetate shows maxima at 388 m # t (F-1111 = 78) and at 460 mV. (E '% = 134). Rifomycin C is in water, mineral acids, and bicarbonate insoluble, soluble in sodium carbonate, methanol, ethyl acetate, chloroform and acetone and again insoluble in ethyl ether and petroleum ether.

Properties of rifomycin D. Elemental analysis: C = 62.17%, H = 6.58%, N = 3.53 "/ a, 0 = 27.72 0/0 (as difference). Rifomycin D gives positive reactions with the Reagents according to Tollens and Fehling, with ferric chloride, iodoform and permanganate. The ninhydrin reaction is negative before and after hydrolysis. The visible spectrum in ethyl acetate shows maxima at 388 m # L (Eid. = 97) and at 460 m # t (Egg ö , = 115) It is insoluble in water, mineral acids, bicarbonate, ethyl ether and petroleum ether, and soluble in sodium carbonate, methanol, ethyl acetate, chloroform and acetone.

Rifomycin E. This fraction shows an R f value of about 0.7 to 0.8 on the chromatography strip. It is divided from the crude mixture by countercurrent flow obtained with the following mixture: methanol-0.01 N-hydrochloric acid-benzene-petroleum ether (10: 5: 15: 5). With 160 passes, most of the rifomycin E is in the first ten tubes by extraction of the acidified aqueous phase with ethyl acetate, concentration of the extract and precipitation with petroleum ether. The Eist faction is easy brown amorphous powder The visible spectrum in ethyl acetate shows a maximum at 400 mp, that after shaking with dilute solutions or potassium ferricyanide or Stannous chloride remains unchanged.

As stated above, rifomycin and its components show a high degree of effectiveness against gram-positive bacteria and mycobacteria. Biological tests were carried out separately with rifomycin: B and with a mixture of rifomycins A, C, D and E, which one hereinafter referred to as the "rifomycin complex" is designated, carried out.

The following table illustrates the antibacterial activity of rifomycin and the rifomycin complex in vitro on a large number of gram-positive and gram-negative microorganisms. The numbers indicate the minimum inhibitory concentration in y / cm8. Strain rifomycin rifomycin- B complex Micrococcus pyogenes var. aureus ATCC 6538 .... .... 0.025 0.01 Micrococcus pyogenes var. aureus AT CC 13301 ....... 0.1 0.025 Micrococcus pyogenes var. aureus ATCC 9144. . . . . . . . 0.025 0.01 Micrococcus pyogenes var. albus ATTC 12228. . . . . . . . 0.025 0.01 S. pyogenes var. Aureus gray Weinstein ... ............ 0.1 0.05 S. faecalie ATCC 10541 ...... 0.5 0.05 S. hemolyticus C 203. . . . . . . 0.025 0.0025 S. mastitidis ATCC 7077 .... 0.5 0.05 S. bovis ATCC 9809. . . . . . . . . 0.25 0.025 Neisseria catarrhalis ATCC 8176 .................... 0.5 0.05 Neisseria gonorrhaeae ATCC 9826 ............ **** 1 * '** 2 0.1 Diplococcus pneumoniae XXXVII L. . . . . . . . . . . . . . . 0.05 0.01 Sarcina lutea ATCC 9341 ..... 0.5 0.025 Sarcina subflava ATCC 7468 .. 0.5 0.025 M. flavus ATCC 10240 ....... 0.25 0.01 B. substilis ATCC 6633 ...... 2.5 0.25 B. cereus ATCC 10876 ....... 5 0.25 B. anthracis ............... 1 0.05 Clostridium perfringens ATCC 3226. . . . . . . . . . . . . . . 0.5 0.05 Klebsiella pneumoniae ATCC 10031. . . . . . . . . . . . . . > 200 20 Klebsiella pneumoniae capsulata ...............> 200 100 Escherichia coli McLeod 10536> 200. 50 Pseudomonas aeruginosa Gottlieb. . . . . . . . . . . . . . . . . > 200 75 Pseudomonas fluorescens ATCC 11251 ....... ....... > 200 25 Proteus vulgaris X 19. H ATCC 881 ............> 200 50 Proteus morganii ATCC 9237 ..> 200 50 Proteus rettgeri ATCC 9919. > 200 50 Shigella sonnei ATCC 9290 ... > 200 75 Shigella dysenteriae ATCC 9583 ....................> 200 20 Salmonella typhi. . . . . . . . . . . . > 200 150 Salmonella paratyphi ATCC 9150 .....................> 200 150 Salmonella schottmuelleri ATCC 9149. . . . . . ... . . . . . . > 200 150 Brucella abortus. . . . . . . ... . 150 10 Brucella melitensis ATCC 75 10 4309 .................... Pasteurella pestis ATCC 87 NIH ................. 100 10 Mycobacterium ranae. . . . . . . . 50 10 Mycobacterium phlei ATCC 10142 ................... 0.05 0.5 Mycobacterium minetti ....... 100 10 Mycobacterium app. 607 ..... 100 10 Strain rifomycin rifomycin- B complex Norcardia asteroides .........> 200 50 Micobacterium tuberculosis var. heminis H 37 E. v ATCC 9360 ..................... 0.05 2 Candida albicans ATCC 10231>200> 200 Tricophyton mentagrophytes ATCC 8757. . . . . . . . . . . . . . . >200> 200 From the above figures it can be seen that the antibiotics are particularly effective against gram-positive microorganisms with a certain degree of effectiveness against bacilli.

The acute toxicity of rifomycin B has been studied and found to be extremely low, especially in view of the fact that the antibiotic has a very high level of effectiveness. The following table shows the results: LDso Animal Administration- - (Lethal Dose) mg / kg - intravenous 2040 Mouse .......... intraperitoneally> 2000 subcutaneous> 2000 intravenous 1680 Rat .......... intraperitoneal> 2000 subcutaneous> 2000 The acute toxicity of the rifomycin complex is not that favorable, but it is still very low and offers a high degree of safety. Intravenous administration to mice gave an LDSO (lethal dose) of 262 mg / kg.

The activity of the rifomycin complex and rifomycin B in vitro was examined with very satisfactory results in mice to which several pathogenic gram positive microorganisms had been injected.

All of the injections listed here were by intraperitoneal injection a lethal dose of 10 to 100 of a 15 to 20 hour old culture of the pathogenic Microorganism made. While the control animals always 2 to 3 days after the Infection died, the ultimate survival of the treated animals was each Detected 12 days after the start of the experiments.

Some of the experiments are given in the table below.

It appears that the rifomycin complex has a very strong activity the inhibition of Streptococcus and Staphylococcus infections in mice and in general shows in gram-positive bacteria. A 100% dose of 50 mg / kg appears for an antibiotic with an intravenous lethal dose of 50 of the order of magnitude from 250 to 300 mg / kg very low.

Although rifomycin B is less active, it shows nonetheless still get a better therapeutic coefficient taking into account his very low toxicity. In humans, blood levels of the antibiotics were good obtained with intramuscular injection of daily doses of 500 to 1000 mg.

The antibiotic has been shown to be extremely valuable in patients who have abscesses of various localizations, such as: Mastitis, purulent otitis media, etc. have. The prescribed daily doses are generally about 2 g administered with two intramuscular injections. There was always a rapid drop in temperature and a decrease in the inflammatory activity of the rifomycin complex and of rifomycin B in vivo when administered subcutaneously for 7 days - Live daily Infection after antibiotic dose administrations mg / kg _ 12 days per day 100 2 20/20 100 75 2 20/20 100 Streptococcus haemolyticus 6203. . . . . . . . . . . . Rifomycin complex 50 2 20/20 100 30 2 19/20 95 20 2 13J20 65 10 2 4/20 20 100 2 20/20 100 75 2 20/20 100 Staphylococcus aureus Weinstein. . . . . . . . . . . . Rifomycin Complex 30 2 19/20 95 20 2 9/20 45 10 2 2/20 10 400 4 20/20 100 300 4 20/20 100 300 4 16/20 80 Streptococcus haemolyticus E203. . . . . . . . . Rifomycin B 200 4 6/20 80 400 2 20/20 100 300 2 19/20 95 300 2 13/20 65 200 2 4/20 20 Rifomycin B 350 4 10/20 50 350 2 4/20 20 Diplococcus pneumoniae XXXVII L:. . . . . . . . Dibenzylethylene diamine salt of Rifomycins B 350 2 20/20 100 Process recorded in no more than 3 to 4 days of treatment. Topical application to infected wounds of various types with the use of aqueous solutions of the antibiotic was also successful and resulted in a rapid decrease in pus formation and secretion in general with subsequent rapid healing of the wounds.

These results have often been achieved after undergoing treatment other antibiotics had proven unsuccessful.

The following are two examples of the fermentation of Streptomyces mediterraneus indicated. Example 1 A culture of Str. Mediterraneus was grown from 6 to Maintained at 28 ° C. for 8 days on Bennet agar. The organism was from the The agar slant rinsed and inoculated aseptically into a 500 cm3 bottle 100 cm3 of a medium with the following composition contained meat extract ....................... 5 g yeast extract ......................... 5 g peptone ................... ..: ........ 5 g casein hydrolyzate. . . . . . . . . . . . . . . . . . . 3 g glucose ............................. 20 g NaCl ............................... 1.5 g H20 ........... ............... until Re 11 The pH was adjusted to 7.3 with NaOH. The bottles were vaccinated Shaken for 48 hours at 28'C on an interchangeable shaker. A bottle of that inoculated material was planted in a 10-1 pre-fermenter, the 41st of the above Medium contained. Fermentation was carried out at 28 ° C and 800 rpm. of the agitator and an aeration of 1 / vol / vol / m. After 24 hours the mycelium had a badly torn look. The volume of the packed cells was 3 to 5 ° / o.

In the next step, a 20-1 glass jar was used for fermentation, which contained 101 of the fermentation medium consisting of the following ingredients: Soybean meal .................... 5 g ( NH4) 2S04. . . ... . . . . . . . . . . . . . . . . . . 7 g MgS04-7H20 ....: ............... 1g glucose ......... »............. .... 50 g KH, P04 .... *** '' .... . . . . . . . . . . . . . 3 g CaCO3 ............................ 9g CUS04 5 H20. . . . . . . . . . . . . . ... . . . . 3.3 mg FeS04 7 H20. . . . . . . . . . . . . . . . . . . . . 10 mg ZnS04 - 7 11.0. . . . . . . . . . . . . . . . . . . . . 50 mg MnS04-4 H20. . . . . . . . . . . . . . . . .... 4 mg H20. . . . . . . . . . . . . . . . . . . . . . . . up to 1 1 The pH was adjusted to 7.0 with Na OH. The sterilization was carried out at 120 ° C for 30 minutes.

100 % of the contents of the pre-fermenter were used as inoculum.

The fermentation was carried out at 28 ° C with stirring at 800 rpm. of the agitator and an aeration of 0.8 vol / vol / m and in the presence of an antifoam agent. In the course of the fermentation the medium took on a characteristic orange-red color at. After 48 hours of growth, a volume of 20-25% became packed cells obtain. The pH was 5.6 to 5.4. The highest antibiotic effectiveness was obtained after 40 to 50 hours (300 to 400 y / cc antibiotic mixture).

Example 2 A culture of Str. Mediterraneus was produced in a shaken bottle as described in Example 1. posed. For pre-breeding, it was placed in a 10-1 glass fermentator, which contained 41 of the following medium: Lactose ............................ log Glucose ............................ log (N H4) zS04 #. . . . . . . . . . . . . . . . . . . . . . . . 7 g Soybean meal ..................... 5 g M9S04 - 7 H20. . . . . . . . . . . . . . . . . . . . . 1 g KH1P04 .... ................... .... 1 g Zn S 04 - 7 H, 0. . . . . . . . . . . . . . . . . . . . . . 20 mg CUS04 - 5 H20. . . . . . . . . . . . . . . . . . . . . . 5 mg After 24 hours of growth, the volume was packed cells 6 to 80 /,). A 10% inoculum was for one. 20-1 glass fermenter used, the 101 one consisting of the following components Fermentation medium contained: Corn steep water. . . . . . . . . . . . . . . . . . . . . 20 g Soybean meal ..................... 5 g (NH4) 2S04. . . . . . . . . . . . . ... . . ... . . . . 6 g M9S04 - 7 H20. . . ... . . . . . . . . . ... . . . 1 g Glucose ............................ 50 g KHZP04. . . . . . . . . . . . . . . . ... . . . . . . . . 3 g CaC03 ... . . . . . . . . . . . . . . . . . . . . . . . . . . 9 g. Zr-S04 7 H20. . . . . . . . . . . . . . . . . . . . . . 2 mg CUS04 - 5 H20. . . . . . . . . . . . . . . . . . . . . . 5 mg H20 ......................... up to 11 The pH value was corrected to 7.0 with Na OH. The sterilization was carried out at 120 ° C for 30 minutes. The fermentation lasted 60 hours at 28 ° C. The pH of the fermentation liquid was 5.6 to 5.8 when it was obtained. At the end the antibiotic activity was 400 y / ccm.

Claims (1)

PATENT CLAIM: The use of Streptomyces mediterraneus and its mutants and variants for the production of the antibiotic rifomycin in the usual fermentative way, extraction of the antibiotic from the fermentation medium and, if necessary, separation of the antibiotic into the individual components A, B, C, D and E.