DE1039198B - Process for the production and extraction of antibiotics - Google Patents

Description

Method of making and obtaining antibiotics The invention refers to the antibiotics distamycin and its components as well as distacin and its object is their production by breeding the new Streptomyces species "Streptomyces distallicus".

The antibiotics mentioned have not yet been described. Preparations, which contain these new antibiotics are highly effective against several pathogens Fungi and schizomycetes.

These new antibiotics are produced by a new strain of Streptomyces, whose strain could be isolated from a soil sample from Campania (Province of Naples). The generation takes place in different cultivation media, which for the development of the producing microorganisms and for the production of antibiotics by them. are cheap.

From the following description it appears that the one isolated here Strain does not correspond to any of the species which are specified in Bergey's manual or by W a s k s m a n -Lechev elier classified in "Actinomycetes and their Antihiotics" (1953) have been. This new species is called "Streptomyces distallicus". One Culture was held at the Commonwealth Mycological Institute, Ferry Lane, Kew, Surrey, and at the National Mycological Institute. Teddington, Mi.ddlesex. deposited.

The producing microorganism shows the following properties: Under the optical microscope shows fairly straight, barely branched, long hyphae. On yeast agar, long and elongated hyphae appear with eddies from short elongated ones sympodic hy- phen. On potato agar. Beet agar and asparagine agar forms see a vegetative llycel whose color, on the one hand, turns honey yellow as it ages to brown, on the opposite side from pale brown to maroon brown. The aerial mycelium is only weakly developed in the mentioned breeding media and has a dusty, satin-like appearance and one in the first few days of growth gray-white and later a pale cream-gray color; it shows one on asparagine agar pale purple-brown tint.

On starch agar and czapekagar, the color of the vegetative changes Mvcels from colorless to honey yellow while the reverse is yellow. On Czapek is the aerial mycelium greyish-white and, on starch agar, slightly wine-red to pink-red, always dusty and satin-like.

In meat broth and peptone-glucose broth becomes an abundant vegetative Mycelium is produced, which is mostly completely submerged in the form of flakes. at Meat broth also grows on the surface in a ring shape, one side being honey yellow, the opposite side is yellow. One aerial mycelium is missing. Also done on gelatin an abundant growth; the vegetative mycelium is brown to honey yellow with yellowish brown Back: the aerial mycelium changes from greyish white to gray; it shows up diffuse brownish black pigment, while no pigmentation on other substrates occurs. It liquefies the gelatin quite strongly and hydrolyses after 25 days Strength considerable. The most suitable growing medium to achieve good growth of mycelium and a correspondingly higher antibiotic effect consists of carbohydrates, nitrogenous substances and salts.

As a source of carbohydrates, glucose, sucrose, maltose, starch can be used and also the carbohydrates contained in the grain softener are used. as Nitrogen sources can be used: cereal softeners, soybean meal and ammonium salts. In addition, N a C1, Ca C 03, K2 H P 04, Mg S 04, Fe S O4, etc. are added. Of the The pH of the nutrient solution is around 6.6. The fermentation can take place in a 300 cem Erlenmayer flask with good ventilation and at 28 ° C. The highest activity arises after 100 to 120 hours at a pH of about 7.6.

The cultivation of the microorganism can also be done by submerged fermentation take place with stirring and aeration, as is familiar to the person skilled in the art. Around to achieve a good growth of the cultures, one can use a nutrient medium, which is one or more sources of assimilable carbon and nitrogen as well as salts of the type described.

The invention also relates to the isolation of the antibiotics, which by the microorganism grown under the aforementioned conditions be generated.

Some experimental details of this can be found in the examples Part of the invention. The active products are generally isolated from the cultures by the fact that the mycelium separated from the culture fluid with different liquids, especially methyl, ethyl and n-butyl alcohol, extracted. The use of butyl alcohol has proven to be best. The extraction is generally carried out so that the mycelium is sequentially in small amounts of the solvent shakes. The extracts are made by centrifugation or filtration separated, washed with water and evaporated in vacuo. From your so received Oily concentrate turns into a yellow or yellow-brown precipitate on treatment with ether obtained, which is dried. This product (distainycin) shows an ultraviolet absorptioil spectrum, as is characteristic of a conjugated polyene. The maxima in the ultraviolet spectrum found at 235, 290, 305, 318, 333, 350, 380 and 405 mg. The maxima at 318, 333 and 350 inlt are typical of a penta-type chromophore. With more concentrated Sulfuric acid gives the product a deep purple color. It contains nitrogen.

The product is in lower alcohols, Äthvlcellosolve, and pyridine other aliphatic or aromatic bases as well as soluble in glacial acetic acid. Partially it dissolves in chloroform, dilute acids and bases, while it is insoluble in ether is. It is thermolal> il, especially with acidic and basic PH, in methanolic Solution, however, stable.

Chromatographic Rf values: Aqueous butanol 0.15; finite butanol saturated water 0.3; Butanol-pyridine-water (1: 0.6: 1) 0.6; Butanol-acetic acid-water (2: 1: 1) 0.73; 30% ammonium chloride solution 0.04; 60% acetone 0.8; Benzene-acetic acid-water (2: 1: 1) 0.00 (bioautographically developed on Candida albicans).

The infrared absorption spectrum shows a maximum at 2.9411 with inflection points at 3.00 and 3.05 g, two sharp maxima at 6.04 and 6.33 h with inflection points at 6.10, 6.24, 6.52, 6.58 g, a less pronounced band at 5.82 L and a high one Absorption band between 6.8 and 7.3 [x.

Those from the cultures of Streptomyces distallicus on the basis of the described Process or another equivalent process, optionally also under Use of a solvent other than butanol, e.g. B. another alcohol, of chloroform, acetone, solutions of aliphatic, aromatic or heterocyclic Products obtained by bases, etc., all show remarkable inhibitory effects against various pathogenic fungi and bacteria. In particular, this is through extraction product obtained with butanol (distamycin) with the properties mentioned above is active against S. lutea, B. subtilis, Mycobacterium tuberculosis strain 607, actin. boströmi, O. albieans, Tric. mentagrophites, Sab. gypseus, Epid. floccosum and Deb. Hudeloi (see below). Your distamycin can be converted into different types in a number of ways Obtain products with remarkable antibiotic effects. These new connections can already be present in the crude product or formed by conversion of the same will. A very active compound can be obtained by using a methanolic Distamycin solution filtered through a clay column. This new product was called "Distamycin A" is called. Its ultraviolet spectrum shows maxima at 304 and 240 mg. The product gives a when treated with concentrated sulfuric acid wine-red color. It is insoluble in chloroform, ethyl acetate, acetone, and ether dilute-n bases; It is hardly soluble in a 1: 3 methanol-ether mixture.

Distainvcin A is more effective than the 1 =) istamvcin from which it is obtained will. It shows a special inhibitory effect against certain schizomycetes as well versus actin. boströmi, O. all) icans, Trie. mentagrophites, Sab. gypseus, G1. graphii, Epid. Floccosum and Deb. Hudeloi (see below).

By fractionation with suitable solvents (chloroform, alcohols. Alkoliol-ether mixtures) one can obtain further active products. For example a methanolic solution of a distamycin product with an appropriate amount of ether failed, you get two new products. namely di.staniycin B, which is found in Methanol-ether is insoluble, its 1ltraviolet spectrum maxima at 230, 318, 333, 350, 380 and 405 mg shows, and the finite concentrated sulfuric acid one violet color results, as well as distamycin C, which is soluble in the methanol-ether mixture and its ultraviolet absorption spectrum through a significant 'maximum at 236 mg and a lower absorption compared to the other products is marked at 300 to 350 nig. This product results in more concentrated Sulfuric acid a wine-red color. It is in chloroform and using dilute bases yellow color almost completely soluble.

All compounds produced by this or any other fractionation due to extraction with solvents (alcohols, chloroform, acetone, etc.), optionally obtained with subsequent precipitation show an antibacterial and fungicidal activity equal to or superior to that of distamycin.

Distamycin and the distamycins A, B and C turn out to be due their properties as different from all others in the metabolism of Acti.nomyeeten resulting fungicidal product which has been isolated or sufficiently characterized to date have been. In particular, they are different from fungichromine and philipine; H. Substances that contain a pentaenic chromophore.

Another product with a fungicidal effect can be obtained from the ethereal mother solution of the distamycin precipitation, which is called "distacin". This product can be purified by crystallization from aqueous acetone. It is a yellow crystalline substance that is insoluble in water, but soluble in many organic solvents, melting point 154 to 156 ° C, [al D = -17 (in methanol). The ultraviolet spectrum of distacin shows maxima at 255 mg (Eia,. = 640) and 340 mg (Eta. @ = 470); his anal values correspond to a connection with the 'gross formula C-22 H25 (), N.

The TV Al) sorptioiissltelctra of the here described and claimed Antibiotics are in the diagram of the Drawing indicated; in it means A is the curve for distamycin and B that for distacin, while A ', .4 "and A' denote the curves for fractions A. B and C of distamycin.

To further characterize the method according to the invention, let mentions that the filtrate of the cultures of Streptomyces distallicus also has an inhibitory effect against schizomycetes and fungi. The antibiotic - effectiveness of the Filtrate is proportional to the production of distamycin by the microorganism.

The antibiotic spectrum of distamycin and its derivatives is shown below to illustrate their activity against various bacteria. Antibiotic spectrum of distamycin and the fractions A, B and C obtained from it Minimum inhibitory dose in @ µg% ccm; Dilution in yeast suspension Strains I Distamycin Fraction AI Fraction B Fraction C Staph. 114 ................> 50 50> 50 50 S.lutea .................. 50 <10 50 ± 10 B. subtilis. . . . . . . .. ... . . . . 50 <10 50 50 Mvcob. 607 . . . . . . . . . . . ... > 50 ± 50> 50 ± 50 E. coli. . . . . . . . . . . . . . . . . . . . >50>50>50> 50 Ps.aeruginosa ............ <50>50>50> 50 Actin. boströmi A. . . . . . . , ± 10 <10 ± 10 ± 10 O. albicans. ... . . . . ... . . . . <10 ± 10 <10 ± 10 Tr.mentagrophytes ....... ± 10 ± 10 ± 10 ± 10 Sab.gypseus .............. <10 <10 <10 <10 G1. graphii. . . . . . . . . . . . . . . . 50 -! - 10 50 50 Epid. Floccosum. ... . . . . ... <10 <10 <10 <10 Deb. hudeloi. . . . . . . . . . . . . . ± 10 -! - 10 ± 10 ± 10 Distacin in a dose of 100 1 tg / ccm in agar Sabouraud culture has an inhibitory effect on Epidermophyton floccosum, Tricophyton mentagrophytes, Glenospora graphii and Sabouraudites gypseus as well as a subinhibitory effect on Pseudomonas aeruginosa and Sarcina lutea. Example 1 To 3000 cc of a sterile medium containing 2% dextrose, 2% corn steep liquor, 1% CaCO 3. 0.3% (N H4) 2 S 04 and 0.3% Na C1, gives inan a spore suspension, corresponding to the washing of a culture on solid nutrient medium, as it is contained in a bottle of the Roux type ("Kolle") .

The fermentation is continued for 40 hours, with 150 up to 250 rev / min. stirred and at a flow rate of 1 to 2 l / minute and aerated per liter of culture medium.

300 cc of vegetative mycelium are used to inoculate 6000 cc of a similar sterile culture medium for the production phase. This production phase lasts 85 to 100 hours, with a speed of 350 to 400 rev / min. stirred and aerated at a flow rate of 1 liter / minute and per liter of culture medium. Example 2 To a 171 as in Example 1 by submerged fermentation culture obtained are admixed with an auxiliary substance, and the whole was filtered. The mixture of mycelium and auxiliary substance is shaken with 2500 cc of butanol for 1 hour. This treatment is repeated twice.

The butanolic extracts are combined, washed with water, concentrated to an oily consistency and treated with ether. 8.9 g of one are obtained yellow-brown product (Distainyci.n). The essential mother solution is 5 to 6 days left standing for a long time. The crystalline precipitate is collected and made from 75% Acetone crystallizes, melting point 154 to 156 ° C (distacin). Example 3 Solve 1.0 g of distainycin in 70 cc of methanol and the turbidity is removed by filtration. the yellow-orange solution is filtered on a column containing 100 g of acidified clay contains (according to Wieland), and washed with methanol. Elution takes place with the same solvent. The first 100 cc of the after the appearance of the yellow color Eluates collected in the outflow yield 0.2 g of distamycin after drying by evaporation A.

Example 4 1.0 g of distamycin is added for 30 minutes with two servings stirred by 50 ccm of methanol each time. Practically complete dissolution occurs (Balance 50 mg). The extracts are concentrated to 25 cc, from a minor one Separated precipitate (20 mg) and treated with three times the volume of ether. The precipitate (distamycin B) is dried. The yield is 0.4 g. the ethereal mother solution is completely evaporated and again absorbed with ether. The remaining product is collected and dried; Yield 0.33 g (distamycin C).

Claims (4)

PATENT CLAIM: 1. Process for the manufacture and extraction of the Antibiotics distamycin and its components and distacin, characterized in that that one Streptomyces distallicus or a Streptomyces strain similar to it or cultivates their mutants in conventional nutrient media, expediently submerged in a known manner, the distamycin from the mycelium and components A, B and C of the distamycin as well the distacin is isolated from the mother liquors. 2. The method according to claim 1, characterized characterized in that the mycelium with the help of an organic solvent, in particular an aliphatic Alcohol with less than 5 carbon atoms, extracted, the extract concentrated in vacuo and the distamycin by precipitation with ether is isolated. 3. The method according to claim 2, characterized in that from the after the precipitation of distamycin, the ethereal mother solution obtained is distacin, Melting point 154 to 156 ° C [a] ö = -1? in methanol. 4. Procedure according to claim 2, characterized in that for extracting distamycin from Cultures or mycelium or halogenated for fractionation as a solvent Derivatives of lower aliphatic hydrocarbons, acetone, heterocyclic Bases or solutions of aliphatic, aromatic or heterocyclic bases can be used in a suitable solvent.