SE178454C1 - - Google Patents

Description

Inventors: F Arcamone, A di Marco, M Ghione, P Pennella and A Grein Priority begtird Iron March 15, 1957 (France) - This invention relates to a new antibiotic, called Fl 1163, and a way to produce delta. More particularly, the invention relates to the preparation of this antibiotic by fertilizing a new strain of Streptomyces (Streptomyces lucensis), and its recovery from the substrate.

It is an object of this invention to provide the antibiotic or its salts either in solid state or in solution.

The antibiotic produced according to this invention exerts a specific action against those fungi and ihstarter which are pathogenic towards humans, animals and plants.

Many other substances are already known, which show fungicidal activity, and they are produced either by synthesis or by fertilization induced by microorganisms. However, it is possible that these substances generally show high toxicity to the higher organisms, as a result of which their practical use is severely limited. These include, for example, candidiasis, askosin, actinomycins, streptotrycin, geomycin, etc., which constitute the products of the metabolism of other streptomyces species.

As already mentioned, the antibiotic Fl 1163 is formed by fermentation of a new organism, belonging to the species streptomyces. Its strain has been isolated from a soil sample obtained from the Lucca zone (Tuscany), by suspending soil on plates in a glycerol-glycol coal medium and culturing in a thermostat at 37 ° C for 5 days.

Under part of the optical microscope, this strain exhibits branched hyphae with spiral-like branches, often with coiled spirits.

Agar potatoes: very good growth, light elongated yellow vegetative mycelium, velvety or powdery light brown aerial mycelium with diffuse, light colored tangles or zones. Loose pigment, ash happy after 3 days, then gray-brown.

Agar-Czapek: very strong growth, lost to honey-colored vegetative mycelium, powdery, white or opaque-white aerial mycelium.

The Midas lake has some loose pigment. Back Is frau fdrglos to very pale lemon yellow.

Agar-carrot: good growth, colorless vegetative mycelium, powdery, cohesive gray aerial mycelium, ingestion of loose pigment, backside From colorless to oekra-colored.

Agar starch: very good growth, vegetative mycelium from colored cheese to honey-colored, powdery aerial mycelium frail co-colored to light brown, sometimes with small white flocks. It dven assumes a malvanyans. No loose pigment. Back frail colorless to very pale yellow. Starch is highly hydrolysed vAL- Agar yeast: good growth, brown vegetative mycelium, sparse gray-brown aerial mycelium, waxes in small free colonies, loose brown pigment.

Agar-glycerol-glycocol: excellent lemon yellow vegetative mycelium with yellowish back, powdery gray-brown Inftmyeelium, a loose pigment Midas.

- Gelatin: abundant growth, brown vegetative mycelium, first white and then gray-brown aerial mycelium. Strong browning of the substrate already after 3 days. No fluidization even after 25 days.

Spatula: after 20 days very light applied in flocks or floating on the surface, strong browning of spadef.

Derma stain shows very low activity against the gram-positive spores, no activity against the gram-negative or mycobacteria, good activity against yeast species, very good activity against other pathogenic fungi or saprophytes of plants or animals in general.

The Streptomyces strain isolated from us with the above-mentioned characteristics, which has been called "Streptomyces lucensis", does not correspond to any other species, which we have found mentioned in the literature (1) and in particular in "Manual of General Microbiology" by Bergey.

The strain is maintained viii on a solid medium containing carbohydrates (such as starch, glucose, etc.), nitrogenous substances, such as amino acids and more complex substances, as well as salts.

The formation of spores in this way is always very abundant. The growing time is about 10 days at a temperature between 26 and 37 ° C, preferably at 28 ° C.

On a solid medium, the strain can be maintained for up to 3 months at a temperature of about 0 ° C. It can also be lined on a lyophilized sterile medium.

The antibiotic is prepared according to an Oman process, consisting in culturing the steptomyces strain with the above-mentioned properties in contact with a sterile liquid containing one or more materials, which contain assimilable carbon, nitrogen and salts. As starting material with assimilable carbon, dextrose, rudder seekers, dextrins or other assimilable polysaccharides can be used. Protein-containing substances can be used, such as casein, peptones or simple nitrogen compounds, such as amino acids, or vegetable extracts, vertebral extracts or other products, such as sad malting materials or protein hydrolysis products.

The fermentation can be carried out in a 100 ml to 500 ml Erlenmeyer flask under good aeration and at an optimal temperature of 25-35 ° C. The activity becomes noticeable as early as 30 hours and remains high and constant for up to about 70 hours.

The cultivation of the generating organism can also be carried out as sub-fermentation under favorable conditions with respect to stirring, aeration and temperature, using known working methods and apparatus.

The antibiotic activity of these cultures was determined by means of a test organism, which in this case carries a Debariomyces marylandii op.52 L.C.I. An appropriate suspension of this organism was added to a Sabouraud agar (cooled to 50 ° C), the plates were stored for 48 hours at 25-29 ° C and then the rings formed were determined.

This invention relates even to the extraction and purification of the new antibiotic from cultures of Streptomyees lucensis in which it is formed. The preparation of active and sufficiently pure preparations of antibiotic according to this invention takes place according to a general method, consisting in the extraction of culture filtrate and the myelium individually or of the whole unfiltered spade with solvents, such as chloroform or immiscible or partially miscible with water. alcohols. The normal butyl alcohol is preferred and the extraction of the culture filtrate and the mycelium has been carried out separately with this solvent.

The separate extraction of mycelium can be carried out with these as other solvents in which the antibiotic is sufficiently soluble, for example anhydrous or aqueous lower alcohols, aqueous acetone. The pH of the cultures is installed before filtration and extraction at about 7.

The resulting extracts are then concentrated in vacuo and the active product is precipitated with the aid of a solvent in which it is insoluble, such as petroleum ether.

This fanning, dried and powdered, gout powder with light yellow, degree yellow or light brown color.

The product can be purified using the solubility of the antibiotic in water (in the form of sodium salt in a phosphate buffer solution at pH 7 or in a sodium bicarbonate solution). However, the antibiotic is slightly soluble in water at a pH between 4 and 5. It is preferred to use a process based on this property and therefore the crude product is extracted with a phosphate buffer solution at a pH of 7 or with a sodium bicarbonate solution, and the resulting suspension is subjected to centrifugation to separate an inactive residue. The clear solutions are brought to a pH of 4.5 with the help of one. mineral acid. The resulting precipitate is centrifuged and dried.

These operations are optionally carried out in nitrogen gas medium in the absence of light and yid Mg temperature.

A further purification of the product can be effected by extraction and washing with solvents, in which Fl 1163 is soluble and, under all conditions, shows it. In the manner described above, the product obtained as the further purified product has the above-mentioned biological properties, whereby the practical use of the antibiotic is reduced.

The new antibiotic is a nitrogen-containing compound with an acidic character, it is slightly soluble in water, while its sodium salt is water-soluble, and it contains sulfur. Furthermore, it is very soluble in chloroform and ethylcellus solvents, soluble in methanol, ethanol, n-butanol, pyridine, dimethylformamide, slightly soluble acetone and ethyl acetate, insoluble in ether, gasoline and petroleum ether. With concentrated sulfuric acid, it gives a reddish-brown color. The solution of Fl 1163 in methyl alcohol shows ultraviolet spectra with. 3 absorption maxima at the following wavelengths: 290, 304 and 318 mp.

The ultra-rod absorption spectrum in KBr is shown in the accompanying drawing, where the ordinate indicates - —3. percent light transmission and abscissa wavelength in the micron.

In order to leave a more complete characterization of the new product, the Rf values obtained in chromatography experiments with Whatman paper no. 1 with ascending development and stamping by bioautography on plates inoculated with «Candida albicans» are given in the following.

Solvent system Rf Butanol-acetic acid-water 2: 1: 1 0.7 benzene-acetic acid-water 2: 1: 1 0.21 31) butanol-pyridine-water 2: 0.6: 1 0.60 4) butanol saturated with water 2% ammonium hydrate 0.06 Fl 1163 is very stable in the solid state, especially when stored in the absence of light and air.

Neutral aqueous solutions of Fl 1163, stored at + 5 ° C, retain their activity unchanged for several days. On the other hand, the activity of the solutions decreases rapidly if they are stored at room temperature.

As can be seen from the above information, the substance obtained according to this invention belongs to the group of polyena antibiotics with a tetraene chromophore. The first two representatives of this group have been described in 1951 and aro nistatin (2) and rimocydin (3,), which are produced by Streptomyces noursei and Streptomyces rimosus (strain that produces terramycin).

Later, antimycetin (4) has been described, which is very similar to nistatin, and which is produced by Streptomyces aureus, as well as chromium, which is obtained by culturing a strain similar to Streptomyces antibioticus (5).

A tetraene chromophore is even found in amphotericin A, which was recently obtained by straining Streptomyces M-4574 (6).

Due to its morphological and culture properties Or Streptomyces lucensis is a species which clearly differs from those which produce other fungicidal antibiotics and in particular frail S. noursei, S. rimosus, S. aureus, S. antibioticus and S.M.4575.

The substance produced by F11163 exhibits chemical and physicochemical properties which make it possible to distinguish this substance from those mentioned above.

The fungicidal activity of F11163 is confirmed by the following biological properties of the substance.

The values that lead to the antibiotic activity "in vitro" of Fl 1163 are reported in Table 1.

Experiments with blastomycetes and sehizornycetes have been carried out using a sputum supplemented with glucose, while experiments using what is actually called hyphomycetes have been carried out with a liquid-shaped Sabouraud medium.

The determinations were made after 8 days at 30 ° C.

Table 1.

Minimum inhibited dose (MID) in micrograms ml.

Actinomyces bostriimi A> 10 (P Nocardia asteroides> 100 / Candida albicans0,8 'Candida albicans val.0,7 Debaryomyces hudeloi0,7 6)' Debaryomyces neoformans0,6 Debaryomyces tyrocola8 Debaryomyces marylandii2 Debaryomyces guillermondiansis03 Debaryomyces guillermondiansisisis Eremothecium hashbyii Penicillium sp.

Aspergillus sp. (T) Aspergillus niger Glenospora graphii100 Glenosporella dermatitidis <Pseudomycoderma matalense1 Trichophyton faviforme ochraceum Maggini Trichophyton Trichophyton Trichophyton radiance rhodainii 21) Trichophyton plicatile <Trichophyton mentagrophytes and Epidermophyton floccosum <Sabouraudites gypseus <Histoplasma Capsulatum (myce- liumfas) <Grafting: about 1000 cells for blastomyceter and schizomyceter. Parts of young cultures for the actual hyphomycetes. Furthermore, it has been found that the presence of 10% serum in the culture medium only results in insignificant modifications of the MID value.

Toxicity: The acute toxic properties of Fl 1163 Concerns beneficial, after LD 50, determined on rats using different modes of administration, are very far from the therapeutic doses.

The following examples serve to better illustrate this invention, but the invention should not be limited to the substances or amounts set forth in the examples.

Example 1. 1. 300 ml of Erlenmeyer flasks are prepared, each containing 60 ml of a medium having the following composition (thousandths): sugar (dextrin) 20, sad malting material 10, casein 5, calcium carbonate 4, ammonium sulphate 1, potassium diphosphate 0.1 (pH 6 of the medium 7). These flasks are sterilized at 120 ° C for 20 minutes, each inoculated with 0.7 ml of a suspension of spores, obtained by washing with 10 ml of sterile distilled water of a 20 days 4. - 178.45 ° C culture in a flute-like tube. with a diameter of 25 mm.

The flasks are kept yid ° C and stirred with alternating rotation chopping and with. 220 vary per minute for 60 hours.

As can be seen from the value below, the activity silt reaches the maximum value after approximately 40 hours.

The diameter of the inhibition ring Debar.maryl. (agar plate method) mm 38 6.4 33 41 0.6 34, 47 7.8 31,; - 54 7.8 30, -61 7.0 28 Example 2.

3000 ml of a medium with the following composition are used: dextrin Sadmalting material CaCO3 (NH4) 2 S (technical) casein K2HP 04 After sterilization at 120 ° C for 60 minutes (pH 7.10 after sterilization) in a 5 liter laboratory jar, the inoculation is performed with a suspension of spores, obtained by sealing haly bottles according to Kolles.

The fermentation conditions Concerns as follows: Stirring 400 vary / min aeration1 1/1 / min temperature 27 ° C time24 h For the production step, a 10 liter jasper with 6000 ml of a medium of varying composition was used. cane sugar% dextrose 10% dextrin% sad malting material 10% CaCO3 4% K HP0 24 0.1% (NH4) 2SO4 (technical) 1% silicone (liquid) 1% The sterilization of the medium is carried out at 120 ° C for 90 minutes. Currant sugar and dextrose are sterilized separately at 120 ° C for 20 minutes. After sterilization, the pH is 7.04. The medium is inoculated with 2.5% of a 24 hour gammon vegetative mycelium.

The fermentation conditions Concerns as follows: Stirring 450 vary / min aeration1 1/1 / min temperature 27 ° C The course of the fermentation: AgePH content micrograms DS7,0 246,6200 487,0580 57.0 Example 3.

The fermentation is carried out in the same manner as in the previous example, but using a medium. with the same composition as the medium for the vegetative step of the medium in the production step.

Maximum content: 280 micrograms / ml after 48 hours.

Example 4.

The fermentation is repeated in the same manner as in Examples 2 and 3, but using a medium having the following composition of the production step: Cane sugar 213% Dextrose% NaCl 3% (NH) SO 4 (technical)% CaCO 3% Silicone (liquid) 1%. Maximum content: 230 micrograms / ml after 24 hours.

Example 5. The fermentation is carried out in the same manner as in Examples 2 ', 3 and 4, but using a medium with the following composition instead of the medium for the production step: Dextrose 20% NaCl 3% Sadmalting material 20% (NH 4) 2 SO 4 (technical) 3% CaCO3 10% Silicone (liquid) 1% Maximum content: 170 micrograms / ml after 24 hours.

Example 6. 18 liters of a jazz spade, obtained on the same salt as in Example 2, are filtered with supercell as an aid. The obtained mycelium is extracted by stirring with 3 successive per-Honer ay n-butyl alcohol, amounting to 2-1, 5-1 resp. 5 liters. The beet filtered spade (17,100 liters) is extracted with 3 butanol portions (total 6.0 liters). All the extracts, combined and washed with 2 liters of water, are evaporated in vacuo at a temperature below 40 ° C. Yield 16.6 g.

Example 7. 128 g of crude antibiotic obtained by a procedure similar to that used in Example 0 hours of culture using the pH of the culture pad are dissolved in 1250 ml of a matt sodium bicarbonate solution.

This operation is carried out under the guidance of a stream of nitrogen gas through the liquid.

After dilution with an equal volume, the suspension is centrifuged and the resulting brown solution is adjusted to pH 4.5 by the addition of 6-n hydrochloric acid.

The antibiotic precipitates as free acid, is separated by filtration and dried. Yield 95 g. Literature: S. A. Waksman and H. A. Lechevalier, "Actinomyces and their Antibiotics" - 1953.

E. L. Hazen and R. Brown, Proc. Soc. Exptl. Biol. With. 76, 93 (1951).

J. W. Davisson et al. Antibiotics of Chemotherapy 1, 289 (1951) 1) F. Raubitschek et al. Ibid. 2, 179 (1052).

S. Wakaki et al. J. Antibiotics (Japan) 5, 677 (1952).

J. Vandeputte et al. Antibiotics Annual 1955-56 pp. 587.

Claims (2)

Patent claim: Set to produce a new antibiotic substance, designated 1163, or a salt thereof, which substance is defined by the formation of water-soluble salts with alkali metals, exhibits ultraviolet absorption maxima at 290, 304 and 318 mu and exhibits the infrared absorption spectrum shown in the accompanying drawing. , consisting of a culture of a new species of Streptomyces, deposited under the name Streptomyces lucensis with Waksman Collection No. 3783, or a species obtained therefrom by mutation, which species or mutation is defined by the formation of the new antibiotic substance, grown under normal conditions, preferably by submerged fermentation, and that the antibiotic is recovered from the fermentation medium by known methods, such as extraction or adsorption and possible transfer of the antibiotic to a salt thereof. 2. A kit according to claim 1, characterized in that S. lucensis or a species thereof thriving is grown by submerged, aerated and stirred fermentation at a temperature between 24 and 30 ° C for 2-6 days. Salt according to claim 1, characterized in that the antibiotic is extracted with solvents, such as alcohols, chloroform, aqueous alcohols and aqueous acetone. 4. Set according to claim 3, characterized in that the antibiotic is extracted with n-butyl alcohol from the culture medium or mycelium or from bath veins at the same time at a pH value between 2 and 8. 5. Set according to claim 1, characterized in that the antibiotic is made soluble in water by formation of the sodium salt of the antibiotic. Salt according to claim 1, characterized in that the antibiotic is recovered from its neutral or weakly alkaline aqueous solutions by regulating the pH of the solutions to a value between 3 and 6 and separating the precipitate obtained by centrifugation or filtration. Cited publications: Stockholm 196 2. Ilungl. Boktr. P. A. Norstedt & Saner. 020089 58 0 * gi - -7 95 ')