CH298331A - Process for the preparation of a new antibiotic. - Google Patents

Description

Process for the preparation of a new antibiotic. The invention relates to a process for the preparation of a new antibiotic, which will be called sistomycosin.

Sistomycosin is a neutral organic compound, containing only the elements carbon, hydrogen, nitrogen and oxygen. It is a buff or slightly yellow solid body which appears microcrystalline under a polarizing microscope. On heating, the material begins to brown at about 130 L, but does not melt below about 230 C. Sistomycosin is very soluble in water and methanol, slightly less soluble in acetic acid. - wet tone and in aqueous mixtures of higher aliphatic alcohols; it is practically insoluble in solvents having a more pronounced non-polar character such as chloroform, diethyl ether, ethyl acetate, benzene, petroleum ether and the like. It can be extracted from aqueous solutions of p$ 2 to 9 with n-butanol and other similar higher aliphatic alcohols.

Sistomycosin rapidly reduces cold solutions of potassium permanganate and boiling Benedict's solution, gives a positive Molisch reaction for carbohydrates, gives a negative Beilstein reaction for halogens, and a negative reaction with ferric chloride. Addition of a very small amount of the antibiotic, 1 to 2 mg, to hot concentrated sulfuric acid gives the solution a deep cherry red to chocolate color. Addition of a small amount of the antibiotic to hot concentrated nitric and hydrochloric acids saturated with thymol produces no color change.

The new antibiotic is further characterized by its remarkable ultraviolet and infrared spectra. The absorption spectrum in the ultraviolet (fig. 1) shows a maximum near 300 mu and another at 218 my. The maximum near 300 mu is formed by three distinct absorption peaks at 292.5, 306 and 320.5 m,u, and two minima between these peaks at 298 and 314 my. The steepest minimum in ultraviolet absorption is located at 253 my. The absorption curve represented in FIG. 1 is that obtained in water, and sistomycosin is remarkable in that the spectrum is not appreciably altered by moderate variations in the acidity or basicity of the solution, it is ie by variations ranging from neutral to 0.05 N concentrations of acid or base.

fig. 2 shows the region called a fingerprint in the infrared absorption spectrum of sistomycosin in neutral suspension in mineral oil. Highs at or near 6.6.88 and 7.30.0 are attributable to the mineral oil carrier. The strongest absorption bands characteristic of sistomycosin are those at or near 6.34 and 9.4-4 u. Bands of weak or moderate intensity are located at or near 7.70, 7.86, 8.55, 11.16, 11.85 and 12.4,u with others appearing as shoulders of the above. at or near 7.08, 7.16, 8.95, 9.89, 10.25 and 10.6 u. Another common feature of sistomycosin, which lies outside the so-called fingerprint region, is at or near 2.96u.

Sistomycosin, and particularly its solutions, undergo photochemical decomposition upon exposure to light. It is decomposed by acids and alkalis, but is more stable in neutral or slightly basic solutions, pg 7 to 9, than in acidic solutions. It is quite stable in water below 25°C and its aqueous solutions can even be heated at 50°C for 1 hour without loss of antibiotic activity. In aqueous solution at 100°C, it retains more than 50% of good antibiotic activity after 1 hour. Unlike known antibiotics, sistomycosin has a high degree of activity against various yeasts and molds, but is virtually devoid of activity against gram-negative and gram-positive bacteria. Thus, it is practically devoid of activity against bacteria such as Escherichia coli, Salmonella schottmuelleri, Shigella Bonnei, Klebsiella pneumoniae; Micrococcus pyogenes var. aureus and Streptococeus haemolyticus, but is extremely active and of great value for the treatment of fungal infections caused by Blastomyces dermatitidis, Candida albicans, Histoplasma capsillatum, Microsporum canis, Sporotrichum schenekii, Trichophyton interdigitale and Trichophyton rubrum. The following table shows more strikingly the remarkable antibiotic spectrum of sistomycosin in comparison to the spectra of known antibiotics produced by actinomycetes, including those characterized by antifungal activity.

Antibiotic activity Micro-organism Sisto- Chloro- Chlor- Strepto- Strepto- Hydroxy mycosin Actidione tetra- amphe- mycin thricin tetra cyclin mcol cyclin Gram-positive bacteria (most) - - + -f- + -f Gram-negative (the most) - - + + -f Acid-resistant (most) - - -E- -± -+ Aetinomycetes Nocardia asteroides - - .+ -i- + -+ Eumycetes (yeasts and molds) Blastomyces dermatitidis -f - - - - - -f- Candida albicans - - - - Means that growth is inhibited by less than 25 micrograms of antibiotic per ml. broth or agar. - Means that growth is not prevented by 100 micrograms of antibiotic per ml. broth or agar.

<I>(Table continues):</I> Hydrogy antibiotic activity. Micro_ -or ganism Sisto- Actidione Chloro- tetr- anphe- Chlor- Strepto- Strepto- tera mycosin cyclin nieol mycme thricme cyclin Cryptococcus neoformans - -f- - - - + Histoplasma capsulatum -E- - - - - Microsporum canis -E- - - - - - Sporotrichum schenckii - - - - Triehophyton rubrum -f- - - - - - -f- Means that growth is inhibited by less than 25 micrograms of antibiotic per ml. of broth or agar. - Means that growth is not prevented by 100 micrograms of antibiotic per ml. of broth or agar. It can be seen from the above table that Beul the antibiotic aetidione resembles sistomycosin in its inactivity against bacteria and its activity against cumycetes. Actidione is, however, active against Cryptococcus neoformans, while sistomycosin is not, and sistomycosin is active against the other eumycetes mentioned in this list, while aetidione is not. It therefore follows that these are two different substances.

Sistomycosin toxicity is almost negligible. For the mouse, the intravenous LD50 is 90 mg/kg and the MA.D. is 80 mg/kg. .

The process according to the invention, for the preparation of this new antibiotic, is characterized in that a nutrient medium with Streptomyces viridosporus is inoculated, the inoculated medium is subjected to incubation under aerobic conditions; at a temperature of 20 to 35°C, for at least one day, and isolates the antibiotic thus produced from the culture mixture.

Streptomyces viridosporus is a new actinomycete which is found in soils, among other places. Mixed cultures of the specific eumycetes inhibited by sistomycosin can be obtained from it with aqueous agar and the addition of soil containing the desired Streptomyces viridosporus. ApHs incubation of the mixture for 1 to about <B><U>10</U></B> days, colonies of the desired actinomyeete and other antagonists appear. The colonies of Streptomyces viridosporus are separated, transferred to a fresh nutrient medium, then isolated as pure cultures according to the usual methods. A living culture of this micro-organisin has been deposited with the Office of Culture Parke, Davis & Co., Detroit, Michigan, under number 04889.

examined microscopically, the moist primary mycelium of Streptomyces vzridosporus is hyaline, and excessively branched, the distal parts being curved or slightly undulating. The aerial secondary mycelium is also strongly branched. There are also primary, secondary and tertiary branches, which may be alternate, opposite and sometimes whorled. The distal parts of the Serian mycelium are subdivided into chains of conidia. The conidial chains present as curved RTI ID="0003.0262" WI="12" HE="4" LX="1473" LY="2059"> free spirals with an axial length of approximately 10 to 20 microns. The conidia are hyaline, spheroidal or ovoidal, 1.3 to 1.9 miron in diameter; on average approximately 1.5 to 1.6 microns. When <B>11</B> has grown On a glucose-tryptone-agar medium, the young humid Primary myceliim of Streptomyces viridosporas appears colourless, taut. later a häle ja-Lane Lerne; The secondary aerial mycglittm is blue and bears spores which, en masse, appear dark olive green. There are little or no pigments in the agar. Surface colonies are circular, domed, wrinkled or radially ridged, with a margin of undulation.

The organism liquefies the gelatine without the formation of Pigments and peptonizes the litmus milk with a slight change in p$. In a synthetic medium (of Gottlieb), the organism easily uses many sources of carbon such as arabinose, Fructose, galactose, inosite, lactose, maltose, mannite, rhamnose, xylose; it uses sodium acetate, sodium citrate, sodium suecinate and sucrose less easily; he cannot use dulcite, inulin, raffinose and sorbitol.

In a synthetic medium, the body uses inorganic sources of nitrogen, including ammonium salts and nitrates, as well as organic nitrogen compounds such as urea, glycine, alanine, ss-alanine, valine, leucine, serine, threonine, phenylalanine, tyrosine, tryptophan, cystine, glutamic aeid, aspartic aeid, asparagine, lysine, arginine, 1 histidine and adenine sulfate.

When the present method is carried out in aqueous nutrient media, the solid material present in the culture medium is removed and the desired antibiotic is isolated from the culture liquid by methods described below. When using a solid nutrient medium, the culture mixture is washed well with a solvent for the antibiotic such as water or methanol, and the desired sistomycosin isolated from the extract. During the incubation phase, the temperature is preferably maintained near 25 to 28°C. is alkaline between 7.3 and 7.7. To obtain maximum yield of sistomycosin, 1a should be avoided as much as possible. oil During incubation of culture mix and isolation of antibiotic. When the culture medium is inoculated with spore suspensions, subcultures or slant cultures of Streptomyces viridosporus can be used.

The culture of the microorganism in an aqueous nutrient medium can be carried out in different ways. For example, RTI ID="0004.0274" WI="3" HE="4" LX="1607" LY="665"> the microorganism can be cultured under aerobic conditions on the surface of the medium, or else it can be grown below the surface of the medium, ie in a submerged state, provided that oxygen is supplied at the same time. The preferred method for producing sistomycosin on a scale scale is through the use of submerged or deep cultures of Streptomyces viridosporus. In this way of carrying out the invention, a sterile aqueous nutrient medium is inoculated with Streptomyces viridosporus and incubated with shaking and aeration at a temperature between 20 and 35 C, preferably in the vicinity of 25 to 28 C for about 1 hour. at 4 days. Under these conditions, the organism grows in the form of more or less separate particles, dispersed in the medium, which is quite different from the more or less continuous films present on the surface of the medium in the surface culture method. Because of the distribution of the organism throughout the medium, the culture can be cultured at once in individual volumes of inoculated nutrient medium placed in the individual reservoirs or vats usually employed in the fermentation industries. Stationary fermentation vats, provided with suitable means of agitation and aeration, as well as rotating fermentation drums, have been found particularly useful for these purposes. However, for the preparation of small quantities of the antibiotic or of cultures of the microorganism, this method of sustained culture can be carried out in small bottles shaken or agitated by suitable mechanical means. Agitation and aeration of culture mixtures can be achieved in many ways. Agitation may be produced by propellers or other similar mechanical agitation devices, or by. by rotating and shaking the fermentation vessel itself, or by various pumping devices or by passing through the medium of air or gas containing oxygen. Aeration can be produced by injecting air or other oxygen-containing gases into the fermenting mixture, using open pipes, perforated pipes, porous diffusion means such as candles carbon, carborundum, agglomerated glass or the like; it can also be produced by spraying, projecting or pouring the mixture through an atmosphere containing oxygen. The gas containing oxygen should be freed of spores, bacteria or other contaminants before being introduced into the culture mix. This can be obtained most conveniently by filtering it through a bed of active animal charcoal: employed, intensity and type of agitation, pA, temperature and other such conditions. In all Gas, the gas containing the oxygen is preferably supplied in sufficient quantity to maintain xme Low Positive Pressure in the fermentation vessel and thus eliminate any possibility of contamination of the culture by entry of outside air. With reservoir type vessels of 210 liter capacity, it has been found that a quantity of 10 to 1.5 liters of air per minute gives good results, when used with a mechanical propeller of the turbine type with six Aales, of 10 cm in diameter, rotating at 100-200 revolutions per minute, facing four peripheral vertical deflectors 2.5 cm by 40.

A variety of nutrient media may be employed containing an assimilable carbon source, protein matter, mineral salts and traces of various nutrients, vitamins and other growth stimulants, which are generally found as impurities in the other constituents of the environment. For optimum production of sistomycosin, the nutrient medium also advantageously contains a source of fat. As an assimilable source of carbon, mention should be made here of polyhydroxy alcohols and mono-, di- and polysaccharides, while as protein material, one can mention unmodified proteins and protein degradation products, in particular those resulting from the hydrolysis of proteins. Such degradation products are proteases, peptones, polypeptides, peptides and amino acids.

As the assimilable carbon source, arabinose, friictose, galactose, lactose, maltose, rhamnose, xylose, glycerin, mannite, rhamnite and senners can be employed. Carbohydrates such as starches, dextrin, complex products such as fermentation and distillation liquors, water soaked cereals, cereal steeps, dry fermentation residues and other similar products can also be used. Among the proteinaceous materials which can be used in the various media, one can find beef extract, acid-hydrolyzed gasein, beef peptone, soybean meal meal, dry residues of fermentation, water soluble, defatted and partially hydrolyzed soybean flour, mixtures of amino acids - from natural or synthetic sources, urea, purines and the like. The use of soybean meal or soy protein derivatives as the medium component is advantageous and provides a low amount of fat usable by the microorganism.

The major mineral constituent requirements can be met by adding 0.1 to 0.5% of one or more inorganic salts such as sodium chloride, aluminum carbonate, dipotassium phosphate, monopotassium phosphate and the like. The other constituents of the nutrient medium, ie, vitamins, growth stimulants and the like, are generally present in sufficient quantity in impure assimilable carbon sources and/or protein materials. Sistomycosin can be isolated from the crude culture mixture by many different routes. When a solid culture medium has been used, the mycelium is treated for extraction with a solvent for sistomycosin such as water, and the product is isolated from the extract as described below. In the case of subsequent liquids, the mycelium is separated, washed with water, and the filtrate and washings are used in the subsequent isolation stages. A method for isolating sistomycosin from a brat culture fluid or herbal extracts comprises treating the solution, for extraction, with a water-immiscible alcohol, such as n-butanol, removal of the solvent by distillation, extraction of the residue with a solvent for fats, such as chloroform, setting aside of this extract, extraction of the insoluble solid residue with chloroform, with chloroform containing about 101 /o of a lower aliphatic alcohol, such as methanol, and passing the resulting extract over an inorganic silicate ion exchange agent. The ion exchange material is washed first with chloroform containing decreasing proportions of a lower aliphatic alcohol; such as methanol, puls the antibiotic is 61u6 with said alcohol. The alcoholic eluates are evaporated to dryness, and the residue is extracted with water. The aqueous extract is filtered, the filtrate is frozen and the ice is removed by vacuum sublimation to obtain the desired antibiotic.

The following examples serve to illustrate the invention.

Example <I>1:</I> A mixture consisting of 180 g of commercial ghteose, 180 g of a partially hydrolyzed protein derived from soya beans, 90 g of grain soaking liquid, 90 g of sodium carbonate, 18 g of calcium carbonate and enough hot water to bring the volume to 18 litres, is placed in a stationary 30-litre glass ctive equipped with a stainless steel cap, a stirrer of the type powered, four vertical interior deflectors measuring 2.5 cm by 40, and a perforated circular air diffusion ring.

The vessel is placed in an autoclave and its contents are steam sterilized at 121°C for one hour. Vessel and contents are cooled as they are removed from the autoclave. The milicus is inoculated aseptically with 900 cm3 of a subculture of Streptomyces viridosporus, prepared in a shake bottle, in 72 hours, by culture. On the same medium as described above, inoculated with a culture in tube layer of Streptomyces viridosportts sporulated, On glucose-tryptone-agar.

After inoculation, the culture medium is incubated at 23-27 C for 88 hours. During incubation, sterile air is introduced through the diffusion ring at the rate of 1.5.6 to 17.8 liters per minute and the stirrer is rotated at a speed of approximately <B> 150</B> rounds per minute.

The production of the antibiotic is monitored during incubation by means of samples which are removed from time to time and tested for Candida albicans by the agar diffusion method in circular plates. The antibiotic content is indicated by the diameter, in millimeters, of the tone of inhibition. The following table shows the production of the antibiotic by the method described above:

Inhibition of Candida albicans incubation period in hours es Zone diameter in millimeters 40 <B>16.6</B> 47 19.2 64 22.1 71 21.9 88 23.4 At the end of the period of ineubation, an adds 25 g per liter of a Silicate filtration adjuvant and the culture mixture is filtered. The filter cake is washed with two 25 cc portions of water per liter of the culture mixture and the washings are added to the Main Filtrate.

31.46 liters of the filtrate of the culture or beer, prepared as described above, and giving 18.5 mm in the test against Candida albicans at a dilution of 16.2, are extracted twice with 10 liters of n-butanol. The aqueous solution is set aside, and the extracts are concentrated in the dark and under vacuum, below 45°C, almost to dryness. The residue is extracted with four 50 cc portions and one 100 cc portion of chloroform at 40° C., and the extracts set aside. The residue insoluble in chloroform is shaken with 300 cm3 of water and filtered, which gives 3.39 g of a solid (solid A) and a filtrate (Filtrate A). Filtrate A is frozen and the ice removed by vacuum sublimation to yield 3.725 g of sistomycosin. 500 gamma of this material in 1 cm of water produces Candida albicans zones of inhibition of 1.6.8 and 11.5 at dilutions of 1:1 and 1:2 respectively. .

Solid A (3.39 g) is extrafitted with two 100 cm3 portions and six 150 cm3 portions of water. Excerpts are filtered, tried Aparement, and those of similar titles are combined and frozen. The ice is removed from the frozen extracts by vacuum sublimation and a total of 3.5 g of crude sistomycosin is obtained. The quantities and purities of the various crude fractions are indicated in the following table

Cone. of Results of tests with Candida albicans Weight of raw product Solid produced in Dilution 1: 1 1: 2 1: 4 1: 8 Raw solutions Diameter (in mg) stored of area MM MM mm mm 7# / oms of inhibition B 1623 200 17.1 14.1 <B>10.6</B> C 1613 400 20.1 1.7.5 14.4 12.0 D 114 200 15.9 11.3 *` E 100 330 16 .0 13.5 11.6 ** F 60 550 13.9 11.9 * Inhibition inside cylinder. * * Slight inhibition immediately around the cylinder. 995 mg of solid B are extracted for half an hour with 199 cm3 of chloroform containing 10% by volume of methanol, and the mixture is filtered. The insoluble residue weighs 770.5 -mg. The extract is poured onto a chromatographic column containing 21.1 g of a silicate-based exchange material, for example the product brand Florisil, previously washed thoroughly with chloroform containing 10 μm of volume of methanol. The solvent mixtures in the order listed, consisting of chloroform containing 10, 20, 30 and 50<B>%</B> by volume of methanol, are then passed through the column. The 6lts are saved, and 50 cc of methanol are passed through the column. The first methanol eluate is set aside, and about 2050 em3 of methanol is then passed through the column in small portions. A total of eight fractions are thus collected, the first five of which are evaporated to dryness under vacuum. The yellow residue is extracted with a total of 35 cm3 of water, the extract is frozen and the ice is sublimated, and 85.4 mg of sistomycosin is obtained in a slightly yellow solid form. 400 gamma of this solid dissolved in 1 cm3 of water produced a Zone of Inhibition 22 mm in diameter at a 1:1 dilution, for Candida albicans.

The other three fractions are mixed and evaporated to dryness to obtain 131.1 mg of sistomycosin in the form of a slightly yellow solid. 400 gamma of this material in 1 ems of water produced a 16 mm zone of inhibition at a 1:1 dilution in the test with Candida albicans. With the turbidimetric test method 7.14 µm/cm3 produced a 50% inhibition in the growth of Candida albicans: 1.435 g of solid C was extracted for half an hour with 287 ems of chloroform containing 10 % by volume of methanol. The insoluble residue (solid G) weighs 1.084 g. The extract is poured onto an absorption column containing 32 g of an ion-exchanged silicate material (decalso brand product), thoroughly washed beforehand with chloroform containing 101/9 by volume of methanol. . 700 em3 portions of the following solo chloroform mixtures containing 10, 20, 30, 40 and 50% by volume of methanol are then percolated through the column, and in the order indicated. 0n put aside the percolates. The column is then eluted with 840 em3 of methanol divided into six portions, and the eluates are combined. The solution is evaporated to dryness in vacuo and the residue is extracted with a total of 30 em3 of water. The aqueous extract is frozen and sublimated into ice under reduced pressure, forming a buff colored solid. This substance produced a zone of inhibition of 23.6 mm at a concentration of 400 y/cms with Candida albicans.

The column is eluted with five more 700 em3 portions of methanol and the combined extracts evaporated to dryness in vacuo. The residue is extracted with water, the extract is frozen and dried from the frozen state, yielding Good 85 mg of sistomycosin as a slightly yellow solid producing when tested with Can. - dida albicans a zone of inhibition of 20.3 mm .ä iune concentration of 400 y/em3. By the turbidimetric test, 3.47 y/emg of the product caused a 50% inhibition of the growth of Candida albicans.

Solid G is extracted with chloroform containing 10% by volume of methanol and the extract is poured into a chromatographic column containing Decalso®. The column is washed with portions of ehloroform RTI ID="0008.0276" WI="15" HE="4" LX="1220" LY="579"> containing 10, 20, 30, 40 and 50%: in your own right: of metlianol, and the washing liquids are set aside. The column is eluted with 600 em3 of methanol, divided into six equal portions. The first 50 ms are discarded, and the remaining fractions are combined and evaporated to dryness in vacuo. The residue is extracted with water, the extract is frozen and the ice is removed by vacuum sublimation, yielding 92 mg of pure sistomycosin. 400 µl/cm3 of this light yellow solid produced an inhibition tone of 23.8 mm, at a 1:1 dilution, with Candida albicans. A further 60 mg of sistomycosin can be obtained by washing the absorption column with more methanol and treating the eluate as already described.

Example <I>2:</I> A nutrient medium consisting of 5 g of commercial glucose, 2.5 g of beef extract, 2.5 g of peptorne, <B>2.5</B> g of Sodium chloride and enough water to bring the volume to 500 em3, is made alkaline to a pH of 7.7, with a 10N solution of caustic soda. The liquid is put. In a Fernbach flask closed with a cotton swab and sterilized under a vapor pressure of 1.2.5 kg/cm2 for twenty minutes. The medium is inoculated with 1. cin3 of a suspension of spores of Streptomyces viridosponis originating from a ten-day tube culture on glucose-tryptone-agar. The resulting mixture is incubated at 2.4 C for eight days. After this time, the culture liquid below the surface mycelium has a pH of 6.8 and produces an inhibition zone 22 min in diameter when tested. for sistomycosin with Gandida albicans by the circular plaque method. The sistomycosin contained in the culture fluid can be isolated as described in Example 1.

Example <I>3:</I> A mixture consisting of 180 g of technical maltose, 1,80 g of cereal maceration liquid in water, 90 g of sodium chlor-Lire, 36 g of potassium phosphate diba- siqLie et sau, sufficient to make a volume of 18 liters, is introduced into a 115 liter tank of the type described in Example 1. The pa of the solution is adjusted to 7.5 with a lON solution of caustic soda, and added 18 g of calcium carbonate; the tank and the medium are then sterilized for two hours at 250 C, then the nutrient medium is cooled and aseptically inoculated with 10 cm2 of a suspension of spores of Streptomyces viridosporus prepared by shaking a culture in 7-day-old layer of Streptomyces viridosporus on glucose-trypt.'one-agar, with 10 cm3 of an aqueous solution of 0.01% white soap, known as Castille soap. The culture mixture is incubated at 28-29 C for 13.6 hours during which time the stirrer must be rotated at a speed of 150 rpm and the mixture must be changed from Fair sterile to the mix at a rate of 7.2 to 18 liters per minute. At the end of the incubation period, the fungus is removed and the cultured light beer is tested for its content. sistomycosin by the turbidimetric method, compared to Candida albicans. In this experiment, cultured beer was found to produce a 50% growth inhibition of Candida albicans at a 1 to 135 dilution. the isolation method described in Example 1.

Example <I>4:</I> 125 cm3 of a culture medium consisting of 0.5% commercial glucose, 0.5% glycerine, 0.3"/@ hydrolyzed Gaseine, 0" 25% peptone, 0.1% brewer's yeast, 0.25% cereal steep solids in water, 0.9-51% soybean meal flour, 0.25% Fermentation residues but; anol- aetone, 0.25% sodium chloride, 0'.1% calcium carbonate, the 1st remainder being water, is divided into several Erlenmeyer flasks, which 1'0n Close with cotton covered with gauze and steam sterilized under a pressure of 1.25 kg/cm2 for 25 minutes. Each vial is inoculated aseptically with 1 ems of a Streptomyces spore suspension. viridosporus prepared with a tube culture layer of RTI ID="0009.0275" WI="7" HE="4" LX="1635" LY="690"> seven days on glucose-tryptone-agar. The vials are shaken on a rotary type shaker at 150 rpm for four days. During the incubation period, the temperature is maintained at 22-24°C. At the end of the incubation period, the cultured beer had a pdd of about 6.4. By irbidimetric assay with C-andida albicans, the cultured beer was found to contain sufficient sistomycosin to produce ≥50% inhibition in growth. of the test organism at a dilution of 1 to 135.

The culture mixtures in the vials can be combined, filtered and the sistomycosin can be isolated by the method described in Example 1.

Example <I>5:</I> 125 cm3 of a nnitritive medium composed of 19/o mallose, 1/o cereal maceration liquid. water, 0.2% dipotassium phosphate, 0.5% sodium chloride, and water up to 100% (brought to a PI of 7.5 with a lON solution of sodium hydroxide). caustic), is divided into several Erlenmeyer flasks, which Fon Mouth with cotton, covered with gauze. The flasks containing the nutrient medium are sterilized, innocuous and inflammable as described in Example 4. At the end of the incubation period, the mycelium is removed by filtration, and the culture liquids or beers are com - bins. The beer culture has a p$ of about 6.3.5 and when tested by niethode-turbidimetry is found to contain sufficient sistomycosin to provide a 50% inhibition in the growth of the beer. Candida albi-i eans at a dilution of 1 to 130. 3istomycosin can be isolated in the pure state from this cultured beer by the method described in Example 1.

Claims (2)

CLAIM: Process for the production of a new antibiotic, characterized in that Von, inoculates a nutrient medium with Streptomyces viridosportis, subjects the inoculated medium to an incubation. .use a temperature of 20 to 35 C., for at least one day, and isolate the antibiotic thus produced from the culture mixture. on the growth of the majority of gram-negative and gram-positive bacteria; it contains only the elements carbon, hydrogen, oxygen and nitrogen; it is very soluble in water and methanal, less soluble in moist acetone and aqueous mixtures of higher aliphatic alcohols, and insoluble in chloroform, diethyl ether, ethyl acetate, benzene and petroleum hydroxide; the cold aqueous solution of potassium permanganate, and the boiling aqueous solution of Benedict, gives a positive .Volisch reaction, and a negative reaction with ferric chloride; olle communzque ttne Dark color cherry red to chocolate with hot concentrated sulfuric acid, does not give color to. a hot solution of thymol in concentrated nitric acid, and likewise no color to a hot solution of thymol in concentrated hydrochloric acid; in water it exhibits bands of maximum absorption characteristic of the ultraviolet region of the spectrum, at 218, 29,2.5, 306 and 320.5 mu with bands of minimum absorption at 253, 298: and 314 m@,c, and, when in solid form in suspension in a hydrocarbon oil, it shows characteristic bands of absorption in the region of 1' intra-red, ä 6.34,<B>7.08,</B> 7.16, 7.70, 7.86, 8.55, 8.9'5., 9.44, 9.89 , 10.2!5, 10.6, 11.16; 11.85 and 12.4,cc; another Earateristic Band is found at or near 2,9,6,u. SUB-CLAIMS 1. Process according to claim, in which the culture is carried out in the submerged state, in an aqueous nutrient medium having an initial pH of 6 to 8.2 and containing an assimilable carbon source, protein substances and mineral salts. 2. Process according to sub-claim 1, in which the liquid nutrient medium has an initial pH of 7.3 to 7.7, and. the temperature during incubation is maintained at . 25-28 C.