DE888918C - Process for obtaining an antibiotic active ingredient - Google Patents

Description

Method of Obtaining an Antibiotic Active Invention deals with the production of a new antibiotic, resistomycin. This new antibiotic. is valuable because of its bacterlostatic effectiveness and According to the results of the analysis, it probably has the composition C2, H "06. Because of its extraordinary stability, the product is named Resistomycin. Resistomycin crystallizes in saffron-yellow bar-shaped needles that are located at about 315 'decompose without melting, and is sparingly soluble in water at pa7, but in ether, dioxane, benzene, ethanol, methanol, acetone with yellow color, in Sodium hydroxide solution soluble in orange-red color. The alcoholic solution fluoresces Intense yellow in ultraviolet light, the acetone and benzene solutions light red. If ResistomycÜi is dissolved in concentrated sulfuric acid, an intense one is obtained yellow-green fluorescent solution. Resistomycin sublimes in a high vacuum (io-'Torr) undecomposed at 2oo to 2o5 '.

Resistomycin is exceptionally stable to acids and alkalis. Unchanged resistomycin can be recovered from the solution in concentrated sulfuric acid, which has been heated to 10 ° for 2 hours, as well as from a solution in 2N cold liquor that has been heated to go 'for 7 hours under nitrogen. Resistomycin can be heated to 25 ° for 1 hour without changing its chemical and biological properties.

The constitution of resistomycin is not yet known. Resistomycin is produced by Streptomyces resistomycificus and can be obtained from cultures, in particular this microorganism can be obtained from the mycelium.

Streptomyces resistomycificus was isolated from a soil sample. It does not grow on gelatin and Czapek agar. A dense, but very fine, smooth growth with unspoiled, colorless ivycel can be observed on meat water and peptone. If the strain is cultivated on glycerol peptone, it shows good growth with dense, granular, light olive-brown mycelium that is hardly ever transported. . Good, fine-grain growth can be seen on the potato slice with a medium to dark brown, slightly brownish-gray transported mycelium and a dark burned zone at the edge of the colonies, litmus milk agar is reddish in color, but not cleared; the colonies on this nutrient medium are large, well-grooved and well-distributed. On litmus milk itself you get a good, coarse, strongly curled, black-brown, very well transported mycelium, but like on litmus milk agar, the milk does not clarify. If starch and glycocolla are used as a nutrient medium, medium-sized, flat colonies and gray-white mycelia are produced. With Lugol's solution, a large zone with a reddish-purple, narrow edge is obtained on these plates. The starch is hydrolyzed, as was also shown in a parallel experiment with starch and potassium nitrate as a nutrient mixture. On cellulose agar, poor growth is observed, nitrate is not reduced, and hydrogen sulfide is not formed. On whole milk as a nutrient medium, good, coarse growth with unsported, light brown mycelium can be seen on the fat layer; the milk is not clarified. When cultivated on skimmed milk, the strain also shows good, light brown growth without any clarification of the milk. The trunk grows unmoved on meat water glucose in the form of a colorless, shiny mycelium.

Because of its pigment formation, Streptomyces resistomycifieus can only be placed in the Albus group of Bergey's "Manual of Determinative Baceteriology" 6th Edition or in group E i (Chromogenic Types). Of the species described in these groups, only Streptomyces albus and Streptomyces antibioticus come as relatives in question. However, the differences to both are too strong to classify the Streptomyces resistomycifieus in one of the two species. Streptomyces resistomycifie-Lis is therefore a new species.

Comparison of Streptomyces resistomycificus with Streptomyces albus and Streptomyces antibioticus. Streptomyces Streptomyces Streptomyces resistomycificus albus antibiotieus Soluble pigment brown - deep brown pigment initially brown, later pigment deep brown disappearing Gelatine: no growth Gelatine: growth and liquefaction Gelatine: dark brown growth, on the surface with brown Aerial mycelium, strong liquefaction Starch is hydrolyzed. Starch is not hydrolyzed Nitrate is not reduced. Nitrate is reduced Czapek: no growth Czapek: thin growth, thin gray aerial mycelium Potato slice: dark brown potato slice: colony and air potato slice: growth fallen Growth, pale brownish- mycelium white, brown with thin black gray aerial mycelium, narrow dark ring merging into bluish, none brown zone aerial mycelium Milk: coagulated, not clarified Milk: coagulated and clarified Milk: not coagulated, not clarified clarifies Spore formation in long chains, not in spores in rolled up chains, on spore chains in clusters, spores Bundles of standing chains, spores side branches, ellipsoidally spherical to slightly ellipsoidal ellipsoidal To obtain resistomycin, a suitable nutrient medium is inoculated with Streptomyces resistomycificus and incubated for a few days under air above conditions at slightly elevated temperatures, preferably at about 30 '. After incubation, resistomycin is found in the mycelium that has formed, and the culture filtrate generally shows only weak antibiotic activity. A nutrient medium with the following composition, for example, is suitable for the production of resistomycin: 2 0/0 glycerin, 0.25 0 /, glycocoll, o, i % sodium chloride, o, i 0 /, dipotassium hydrogen phosphate, o, oi 0, /, Ferrous sulphate, o, oi 0! '. Magnesium sulfate, 0.50 /, yeast cooking juice, traces of calcium carbonate.

Other culture media can also be used with success assimilable carbon source and a nitrogenous material alongside the rest Contain nutrients, especially salts, As an assimilable source of carbon come, among other things, carbohydrates, such as starch, glycogen, glucose, or multi-valued Alcohols, such as glycerine and sorbitol, are possible. Suitable nitrogenous materials are: peptone, glycocolla, molasses, pregelatinized corn, vasser, milk proteins, hydrolyzed Casein, soybean meal, potassium nitrate. The cultivation of the microorganism can be done both in the surface process as well as in the submerged process with the addition of oxygen or air. In both cases a brownish-gray to black-brown color is obtained colored mycelium obtained from the culture fluid by filtration or centrifugation can be separated.

Resistomycin can be extracted from the mycelium by extraction with organic solvents be won. In particular, ethers and lower ketones are suitable for this, but extraction is also possible with alcohols and other organic solvents. It is advisable to dry the mycelium before extraction and a pre-extraction with hydrocarbons, such as petroleum ether and the like Accompanying substances are eliminated.

The solubility in organic solvents is pli-dependent. Below pa 8,?, Resistomycin cannot be extracted from ether by aqueous solutions. However, if the ether solution is shaken with buffer solutions whose p.1 is between 8.2 and 12, the resistomycin is distributed over both phases. The partition coefficient depends on the pH of the aqueous phase. Above'pil 12, resistomycin passes completely from its ethereal solution into aqueous buffer solutions. This pE-dependence of the solubility in ether can be used to advantage in the pure representation.

From the extracts that contain the resistomycin, this can be win by adsorption methods in a suitable form. It is convenient to adsorption to be carried out in the form of a chromatography. As an adsorbent comes in particular Plaster of paris, aluminum oxide or silica can be considered. The eluates can be evaporated Resistomycin can be brought to crystallization.

Crystallized resistomycin inhibits the growth of Staphylococcus aureus in a dilution of: [ : io ooo ooo to i : 2o ooo ooo, of Bacillus subtilis in a dilution of i: 2ooooooo to 1: 40000000 and of Mycobacterium auberculosis in a dilution of i: 5ooooo . Example i A nutrient medium of the following composition is prepared in tap water: 2 l) /, glycerine, o,?, 5 0 /, glycocolla, o, i 0 /, sodium chloride, o, i 0 /, dikahum hydrogen phosphate, o, oi 0 /, Ferrous sulfate, o, oi l) /, magnesium sulfate, 0.5 01, yeast cooking juice, trace of calcium carbonate.

To produce the yeast cooking juice, i part by weight of yeast, in io Parts by weight of water suspended, left to stand at 3o 'for 1 hour; then the Mixture heated to 15o 'under pressure. You then decant from the sediment, the is discarded.

Suitable glass vessels, e.g. B. Erlenmeyer, are then filled with this nutrient medium about 2 cm high. After sterilization, these vessels are inoculated with Streptomyces resistomycificus. For this purpose, one assumes well-displaced oblique tube cultures, which are characterized by the browning of the agar. The vessels are then left to stand at 27 ' for about 3 weeks. The culture liquid already turns brown after about 5 days and becomes towards the end. deep dark brown. It only contains traces of the antibiotic.

The mycelium is dirty yellow-brown in color and is covered with a Kolier cloth filtered off and washed with distilled water. The washed mycelium is then dried and ground.

The grist is then extracted with petroleum ether in a Soxhlet for 12 hours. Lipoids and similar cell substances go into solution, while resistomycin remains almost completely in the mycelium. This is now extracted exhaustively with ether. The ether extract is evaporated and the residue is taken up in dry acetone. The acetone solution, which is colored orange and fluoresces weakly, is replaced by a Calcium sulfate column filtered. Thereby, smaller yellow and red ones form underneath Zones a uniform large yellow zone. This zone contains the resistomycin, which is eluted with acetone.

The eluate is filtered again and concentrated. This crystallizes Resistomycin in saffron-yellow bar-shaped needles, which are recrystallized twice can be obtained in pure form from dioxane.

The analysis found the following composition: 70.76 ° / 0 C, 4.610 /, H, free of nitrogen, sulfur and halogen. For the formula C2,111.0. calculate 7o, 760 / OC and 4,650 /, H. EXAMPLE 2 500 liters of a nutrient medium having the composition of p # I 6.8 given in Example i were inoculated under sterile conditions with approximately 1: 1 of a preculture of Streptomyces resistomycificus which had been produced in Schüttelkölbehen. Thereafter, the nutrient solution was stirred for 140 hours at 30 'with aeration. Then the solution turned dark brown, while a brown-black fungal cinycel was formed. After an incubation time of 1:40 hours, the experiment was terminated and the fungal mycelium was filtered off through a bag filter. So 20 kg of moist mycelium were obtained, which yielded 2.6 kg of air-dry mycelium. The further work-up takes place according to the procedure given in Example i and yields 10 g of crystallized resistomycin.

Claims (1)

PATENT APPEAL- i. Process for obtaining an antibiotic active ingredient, characterized in that Streptomyces resistomycificus is grown in a nutrient medium and the resistomycin is obtained from the resulting culture, preferably from the separated mycelium. 2. The method according to claim i, characterized in that the cultivation takes place with the admission of air. 3. The method according to claims i and 2, characterized in that Streptomyces resistomycificus is grown submerged. 4. The method according to claims i to 3, characterized in that the nutrient contains glycerol. 5. The method according spoke i, characterized in that resistomycin is obtained from the mycelium of Streptomyces resistomycificus by extraction. 6. The method according to claim i, characterized in that the extracts from the mycelium of Streptomyces resistomycificus are purified by adsorption methods, in particular chromatographic.