DE851398C - Process for the production of antibiotic substances - Google Patents

Description

Process for the production of antibiotic substances It is known to isolate substances from the metabolic products of bacteria and fungi which have valuable therapeutic properties. A great count such antibiotics have been discovered in recent years.

It has now been found that one new. antibiotic highly effective substances can be produced by Streptomycetes, in particular Streptomyces purpurascens nov. spec. grown under aerobic conditions on or in nutrient media known per se for the cultivation of streptomycetes, with antibiotic substances (rhodomycins) being formed which are orange-red in acidic reactions, purple-red in the case of neutral reactions, contain nitrogen and when heated with acids pass over into nitrogen-free compounds with elimination of nitrogenous residues. The rhodomycins are closely related and generally occurring as mixtures of nitrogenous bases which, with acids, give partially insoluble salts. They are red dyes, which are obtained as an amorphous red powder. The isoelectric point lies at a pii of 8.4 to 8.6. At the isoelectric point, the rhodomycins are soluble in many organic solvents with the exception of paraffinic hydrocarbons such as petroleum ether. Outside the isoelectric point, the rhodomycins dissolve in water, lower alcohols and glacial acetic acid acidic PH are they ora red, purple-red at neutral pH, violet-blue at alkaline pH., They form salts with z. B. picric acid, picrolonic acid, perchloric acid, stannous acid, mercury chloride, potassium chromate, platinum hydrochloric acid, -helianthin, alizarine sulfonic acid, anthraquinone sulfonic acid, iodine-iodine-iodine, phosphotungstic acid, phosphomolybdic acid, and ß-naphthalenic acid with the strong inorganic sulfuric acid, ß-naphthalenesulfonic acid. The rhodomycins are best precipitated from their aqueous solutions with picric acid. The picrates represent an amorphous yellow-red powder, which is soluble in acetone and hot alcohol, but insoluble in water, ether and benzene. By dissolving the picrates in acetone and precipitating them with the calculated amount of phosphoric acid, the phosphates are obtained as a precipitate, which can be sucked off and, in a pure state, reaches inhibition values against Staphylococcus aureus of i : 30 million.

When heated with acids, the rhodomycins also turn red on which do not contain nitrogen, are insoluble in acids and by chromatography can be separated into two main zones. The product in one zone is crystalline, has a melting point of 224 °, is nitrogen-free and contains 62.3% carbon and 5.0% hydrogen. By heating this substance in alcoholic hydrochloric acid the spectrum shifts in a characteristic way towards the long-wave area. The same shift can be achieved by adding lead acetate and zinc chloride .. The second zone is amorphous and also gives a characteristic in hydrochloric acid Base shift.

From the by heating the rhodomycins with acids after separating them the solution obtained from the insoluble red cleavage product can after neutralization and cases with styphnic acid, an amorphous styphnate can be isolated.

The. Rhodomycins as such or in the form of their salts, in particular with phosphoric acid, against gram-positive pathogens, especially Staphylococcus aureus and Bacillus subtilis effective in vitro in very large NT dilutions. Depending on the In the composition of the nutrient solution, the inhibition values of the culture filtrates fluctuate. at . the nutrient solutions described in more detail below are the inhibition values of the Culture filtrates for Staphylococcus aureus between 1: 1000 and 1: 20000.

The organism that forms the new antibiotic substance, was isolated from the Göttingen forest floor. According to its structure and mode of operation the isolated strain probably belongs to Actinomyces reticulus ruber from W a k s m a n most similar, but not identical to this tribe. The tribe received the scientific name Actinomyces purpurascens nov. spec.

The trunk shows thin, branched mycelial hyphae. The spore formation takes place en masse. The spores themselves are weakly elliptical. Often one finds Formation of white aerial mycelium takes place. When used in aerobic conditions occurs on all The crimson color appears in the nutrient media, which in the case of acidic PH is orange-red alkaline PH changes to red-violet. Submerged, unventilated mycelium is colorless. `` 'If the Streptom5-ces purpurescens is grown on agar in an inclined tube, so the agar turns a dirty brown-violet. If you use liquid culture media, so the color passes over into the culture solution and is the same as the color of the mycelium.

As with most antibiotics, this is the formation of the new dye not limited to any particular tribe. There were many others too Actinomycete strains found which are able to produce rhodomycins as a metabolic product submit. However, these strains have not yet been clearly identified and should therefore not described here «- earth.

The isolation of the trunk from the ground is carried out in the usual way Way. The soil sample from a dry, unused, grassy shell limestone slope, about 1 g, was shaken with water for ½ to ½ hour. After a short period of weaning five platinum loops were placed in a cooling, not yet solidified sterile altar tube and from this five loops inoculated into a second. Then both tubes became each poured into a sterile Petri dish and added after the altar has solidified 27v incubated. With a suitable culture medium, glycocollagar, the fungi will grow and bacteria retained so far that the slowly growing actinomycete colonies can develop. After about four days, the actinomycete colonies were able to access slant tubes to be isolated. By repeating the procedure, the strain was obtained in absolute purity achieved. The new strain was named Streptomyces purpurascens nov. spec.

In the preculture and the main culture are expedient as carbon sources Glycerin, glucose or dextrin are used. Glycocolla are suitable as nitrogen sources, the alkali nitrates, glycollester chlorohydrate or tyrosine. In the nutrient solution you want the following minerals are present as salt additives and growth substances: sodium chloride, secondary potassium phosphate, ammonium sulfate, ferrous sulfate, calcium carbonate and Hoagland's trace solution, as a substitute for the latter, yeast water or maize cluell water can also be used to serve.

A suitable nutrient solution has the following composition, for example: 20, o kg of glycerine, 1, o kg of glycollester chlorohydrate, 2, o kg of corn steep liquor, 1.5 kg secondary potassium phosphate, 1.0 kg sodium chloride, 50 g magnesium sulfate, to g Ferrous sulfate, precipitated to g, calcium carbonate.

Topped up with tap water to tooo 1.

The pH of the nutrient solution before sterilization is 6.6 to 6.8; after sterilization, the pH is 5.8 and increases during the duration of the experiment back to 6, o to 6.2. The sterilization itself is done by heating for one hour made with direct or indirect steam at too up to 12o °.

The above-described nutrient solution (toool), which is in a fermentation vat suitable for submerged cultivation of antibiotic microorganisms, which is equipped with a stirrer and a supply for sterile 1_uft is provided now provided with i to 21 vaccine from the equal nutrient liquid by inoculating Streptoniyces purpurescens was obtained. Of the Vaccine is deep red in color, it contains that 1lycel and en masse spores of the inoculated Tribal. After the sterile inoculation, the Fermentation kettle kept at 30 ° and sterile ventilated. Because of the foam formation that occurs, the The fermentation kettle shouldn't be too small, you can Also hold back foam with antifoam agents. After about 12o hours the formation of the active stoffes peaked, and the attempt is cancelled. For this purpose culture liquid and mycelium separated by a filter. The damp Mycelium weighs 18 to 20 kg and is air-dry the weight is about 3 kg The culture filtrate is 95o to 98o 1, it will under reduced pressure at 25 to 280 internal temperature reduced to about 10 to 12 1 and then further processed as described below. The rhodomycins are after the waxing tum both in the culture solution and in the mycelium found, they can come from the narrow cultural solution by clarifying with acetone and basic Lead acetate and shaking out the dye hot pH 8.6 finite butanol can be obtained. The solution of the dye in butanol is mixed with 100% vinegar acid shaken. The dye goes into the aqueous acetic acid solution Tiber. In this solution can use the rhodomycins as picrates with the help of saturated picric acid solution. the end tooo 1 culture solution is obtained with 5 g of picrate an effectiveness of about 1: 30 million to i: 100 million against Staphylococcus aureus. The rhodomycins such as can be obtained as follows: The powdered mycelium is with 8oo / oigein acetone or methanol, which o, .I n is mineral acid, worked out cold. After this The acetone is neutralized either in the Va- kuum all distilled or the solution is etherified. Impurities are made with basic lead acetate precipitated and excess lead with sulfur acid removed. The solution is provided by the isoelek- tric point (pfi 8.6) extracted with butanol and the butanol solution again with ioo / oiger Acetic acid extracted. From the aqueous vinegar acidic solution can use the picrate as above be precipitated by saturated l'ikrin acid solution. Llan receives 20 g of the rhodomycin mixture as picrate from 5 kg of powdered mycelium.

Instead of the above rule, you can also use the powder-coated Extract mycelium with acetone. The acetone extract is discarded, the mycelium suspended in alcohol and, with excess alcoholic picric acid solution added, after which the entire mass is dried at 100 °. From this material the picrates of the rhodomycins can be extracted with acetone.

The conversion of the picrate mixture into the phosphate takes place through Dissolve in acetone and add the calculated amount of phosphoric acid. The precipitation is sucked off. The inhibition values of the phosphate are about i: ioo million.

As has been found, the product consists of two components, which are determined by countercurrent distribution can be separated in the following ways.

The phosphate was made between distilled water and a mixture of butanol benzyl alcohol in the ratio i: io in that of N. G r u 1ih o v e r (Chem Ing. Techn. 195o, p. 209) described countercurrent distribution apparatus of a fractionated Subject to distribution. One fraction was in the first ten tubes of the apparatus retained predominantly in the aqueous phase, while the second fraction, the in, the mixture of organic solvents is more soluble, in the tubes 18 up to 24 found * The first fraction can be after adding the same volume of ether to the mixture of butanol-benzyl alcohol when shaking with distilled water completely transfer into this. The second fraction remains under the same conditions almost completely in a mixture of organic solvents.

Crystallized sodium perchlorate is obtained from the rock phosphate with sodium perchlorate Rhodomycin perchlorate.

Claims (1)

PATENT CLAIM: Process for the production of antibiotically active substances, characterized in that Streptomycetes, in particular Streptomyces purpurascens nov. spec. grows under aerobic conditions on or in nutrient media known per se for the cultivation of streptomycetes and extracts the rhodomycins from the cultures obtained in this way according to conventional methods.