BE510219A - - Google Patents

Description

       

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  ANTIBIOTIC, AND METHODS FOR ITS PREPARATION.



   The invention relates to a new chemical compound having outstanding antibiotic properties and to methods for its preparation. More particularly, the invention relates to a new antibiotic to which the name of sistomycosin has been given, and to its preparation by
 EMI1.1
 mierobiological processes.



   Sistomycosin is a neutral organic compound containing only the elements carbon, hydrogen, nitrogen and oxygen. It is a solid buff or light yellow body which appears, under a polarizing microscope, to be microcrystalline. On heating the compound begins to turn brown at about 130 C but does not melt below about 230 C. Sistomycosin is very soluble in water and methanol, somewhat less soluble in acetone. moist and aqueous mixtures of the higher aliphatic alcohols, and practically insoluble in more non-polar solvents such as chloroform, diethyl ether, ethyl acetate, benzene, petroleum ether, ,

     etc. It is extracted from aqueous solutions having a pH range of 2 to 9 with n-butanol and higher analogous aliphatic alcohols.



   Sistomycosin rapidly reduces a cold aqueous solution of potassium permanganate and a boiling Benedict's solution, it gives a positive Molisch test on carbohydrates, a negative Beilstein test on halogens and a negative test on ferric chloride. The addition of a very small amount, 1-2 mgr. antibiotic, hot concentrated sulfuric acid, gives the solution a dark cherry red to chocolate color. Addition of a small amount of the antibiotic to hot concentrated nitric acid or hydrochloric acid saturated with thymol does not produce any color change.

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   The antibiotic of the present invention is further characterized by its remarkable ultraviolet and infrared spectra. The ultraviolet absorption spectrum (Figure I) shows a maximum in the vicinity of 300 millimicrons and another at 218 millimicrons. The maximum in the vicinity of 300 millimicrons is composed of three distinct absorption peaks at 292.5, 306 and 320.5 millimicrons, and two minima between these peaks at 298 and 314 millimicrons. The main minimum in the ultraviolet absorption spectrum is located at 253 millimicrons.

   The absorption curve shown in Figure I is that obtained in water, but sistomycosin is remarkable in that this spectrum is not appreciably modified by moderate variations in acidity or acidity. the basicity of the solution; that is, from neutrality to a normality of about 0.05 N acid or base.



   Figure 2 shows the so-called "fingerprint" region of the infrared absorption spectrum of sistomycosin in a neutral mineral oil vehicle. The peaks at or near 6.88 and 7930 microns are attributable to the mineral oil vehicle. The most intense absorption bands characteristic of sistomycosin are those located at or around 6.34 and 9.44 microns. Bands of weak and moderate intensity are located at or near 7.70; 7.86; 8.55; 11916; 11, 85 and 12.4 microns with others appearing as shoulders of previous bands at or near 7.08; 7.16; 8.95; 9.89; 10.25 and 10.6 microns.

   Another characteristic band of sistomycosin which is found outside the fingerprint region is located at or near 2.96 microns.



   Sistomycosin, particularly in solutions, undergoes photochemical decomposition on exposure to light. It is decomposed by acids and alkalis, but is more stable in neutral or slightly basic solutions, ie at pH 7-9 than in solutions with an acid reaction. It is completely stable in water below 25 C and its aqueous solutions can even be heated to 50 C for an hour without loss of their antibiotic activity. In an aqueous solution at 100 ° C., it retains more than 50% of the antibiotic activity after one hour.



   Unlike known antibiotics, sistomycosin has a high level of activity against various yeasts and molds, but is practically speaking devoid of activity against gram-negative and gram-positive bacteria. For example, the product of the invention shows no activity against bacteria such as Escherichia coli, Salmonella schottnuelleri, Shigella sonnei, Klebsiella pneumoniae, Micrococcus pvogenes var. aureus, and Streptoeoceus haemolyticus, but it is extremely active and of great value in the treatment of fungal infections caused by Blastomyces dermatitidis Candida albicans, Histoplasma capsulatum, Microsporum canis Sporotrichum.

   cschenckli, Trichophyton interdigitale and Trichophyton rubrum, The table below shows more strikingly the remarkable antibiotic spectrum of sistomycosin compared to the spectra of known antibiotics produced by actinomycetes and including those characterized by antifungal activity. the.

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  - <SEP> Antibiotic <SEP> activity
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   <SEP> - <SEP>
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 + indicates that growth is arrested by less than 25 micrograms of antibiotic per ml. broth or agar.



   - indicates that growth is not stopped by 100 micrograms of antibiotic per ml. broth or agar.



   From the above table it can be seen that only the antibiotic actidione resembles sistomycosin in its inactivity against bacteria and its activity against eumycetes. However, actidione is active against Cryptococcus neoformans while sistomycosin is not, and sistomycosin is active against the remaining eumycetes mentioned above while actidione is not. It thus appears that the two are markedly different substances.



   The toxicity of sistomycosin is approximately negligible.



  In mice, the intravenous LD50 is 90 mg / Kg and the M.T.D. of 80 mg / Kg.



   In accordance with the present invention, sistomycosin is produced by the aerobic cultivation of cultures of a novel actinomycete of the genus Streptomyces which has been given the name Streptomyces viridosporus.



  This microorganism exists among others in the earth. Cultures thereof can be obtained by mixing cultures of specific eumycetes inhibited by sistomycosin with aqueous agar and adding soil containing the desired Streptomyces viridosporus. After incubating the mixture for

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 one to ten days, colonies of the desired actinomycete and other antagonists appear. The cultures of Streptomyces viridosporus are selected, transferred to a fresh nutrition medium, and are subsequently isolated in the form of pure cultures according to the usual methods. A live culture of this microorganism has been deposited in the culture office of Parke, Davis & Company in Detroit, Michigan under number 04889.



     Microscopically, the wet primary mycelium of Stre tom - ces viridosporus is glassy and profusely branched, the outer parts being curved or slightly wavy. The secondary aerial mycelium is also profusely branched. There are also primary, secondary and tertiary branches, which can be alternate, opposite and occasionally whorled. The extreme parts of the aerial mycelium subdivide into chains of conidia. The conidial chains are in the form of short open spirals having an axial length of about 10 to 20 microns. Conidia are glassy, spheroid to ovoid, and 1.3 to 1.9 microns in diameter, but average approximately 1.5 to 1.6 microns.

   When grown on glucose-tryptone-agar medium, the young, moist primary mycelium of Streptomyces viridosporus appears colorless, and subsequently turns dull tan yellow. The secondary aerial mycelium is white and bears spores which, in bulk, are dark olive green. Little or no pigment appears in the agar. Surface colonies are circular, erect, folded or radially wrinkled, with a solid to wavy margin. The table below illustrates the characteristic differences between this new actinomycete and other antibiotic-producing acinomycetes when grown on glucose-tryptone-agar medium.

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  Mycelium <SEP> Substrate
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 Microorganism Antibiotic Form of Color Color Color-for-
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 clear.

   
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The body liquefies gelatin without forming pigments and peptonizes sunflower milk with little change in pH.

   In a synthetic medium (from Gottlieb), the organism easily uses numerous carbon sources including arabinose, fructose, galactose, inositol, lactose, maltose, mannitol, rhamnose, xylose; it less readily uses sodium acetate, sodium citrate, sodium succinate and sucrose; it does not use dulcitol, inulin, raffinose and sorbitol.

   In a synthetic environment, the organism uses nitrogen sources comprising ammonium salts and nitrates; organic nitrogen such as urea, glycine, alanine, beta-alanine, valine, leucine, serine, threonine, phenylalanine, tyrosine, tryptophan, cystine, glutamic acid, aspartic acid, asparagine, lysine, arginine, histidine and adenine sulfate.



   The following table gives a comparison of the abilities of Streptohyces viridosporus and other antibiotic-producing actinomyces to use different carbon sources when growing in a medium comprising agar, inorganic salts and the tested carbon source. .

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  SOURCES <SEP> OF <SEP> CARBON
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0 = No growth or very weak growth + = Good growth
1,2 = The difference in nutrient requirements is due to a difference between crops.



   As mentioned above, sistomycosin is produced by aerobic cultivation of Streptomyces viridosporus. This is usually done by inoculating an appropriate nutrient medium with Streptomyces viridosporus and incubating the mixture under aerobic conditions at about 20 to 35 ° C for about one to eight days. In the case of aqueous nutrient media, the solid material present in the culture medium is separated and the anti-

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 desired biotic of the culture liquid by the methods described below. When using a solid nutrient medium, the culture mixture is suitably washed with a solvent for the antibiotic such as water or methanol and the sistomycosin is separated. desired from the extract.

   During the incubation phase, the temperature is preferably maintained in the region of 25 to 28 C.



  When using aqueous nutrient media, the initial pH should be between 6 and 8.2 and preferably on the alkaline side between about 7.3 and 7.7. In order to obtain the maximum yields of sistomycin, light should be excluded as completely as possible during the incubation of the culture mixture and the separation of the antibiotic. When inoculating the nutrient medium, suspensions, subcultures or culture media of Streptomyces viridosporus can be used.



   The culture of the microorganism in the aqueous nutrient medium can be carried out in several different ways. For example, the microorganism can be grown under aerobic conditions on the surface of the medium or it can be cultivated below the surface of the medium, i.e. in the submerged state, if it is at the same time supplies him with oxygen. The preferred method for the large scale production of sistomycin involves the use of submerged or deep cultures of Streptomves viridosporus.

   According to this embodiment of the invention, Streptomyces viridosporus is inoculated into a sterile aqueous nutrient medium and the latter is subjected to incubation with agitation and aeration at a temperature of between 20 and 35 ° C., preferably in the region of 25 to 28 C for about one to four days. Under these conditions, the organism develops in the form of numerous more or less separated particles dispersed in the mass of the medium, in contrast to the more or less continuous film present on the surface of the medium in the surface culture process. Because of this distribution of the organism in the mass of the medium, large volumes of the inoculated nutrient medium can be cultivated at one time in the large vats or barrels usually used in the fermentation industry.

   It has been found that fermentation apparatus with a fixed tank fitted with suitable agitation and aeration devices as well as fermenters with a horizontal rotating drum are particularly suitable in this connection. However, for the preparation of smaller amounts of the antibiotic or cultures of the microorganism, this submerged cultivation process can be carried out in small flasks which are shaken or agitated by suitable mechanical means.



   Agitation and aeration of the culture mixture can be accomplished in a number of ways. The agitation can be produced by a propellant or similar mechanical device, by rotating or shaking the fermentation apparatus itself, by various pumping devices, or by passing air or gas through the medium. other gases containing oxygen. Aeration can be accomplished by injecting air or other oxygen-containing gases into the fermentation mixture, through open tubes, perforated tubes, porous diffusion media such as sticks. carbon, carborundum, sintered glass or the like, or it can be done by spraying, blasting or pouring the wort into or through an oxygen-containing atmosphere.

   The oxygen-containing gas must be free of spores, bacteria and other contaminants before it is introduced into the culture mixture. This can be achieved very advantageously by filtration through a bed of activated charcoal. The degree of aeration resulting in optimum production of sistomycosin obviously depends somewhat on the type of apparatus used, the speed and mode of agitation, the pH, the temperature and other environmental conditions. In any case, the oxygen-containing gas should be supplied at a rate sufficient to maintain a slight positive pressure in the fermentation apparatus and thus avoid the possibility of contamination of the culture by outside air.

   In 30 liter tank fermenters an air flow rate of 10-15 liters per minute gives good results when used with a six-blade mechanical impeller of the turbine type having ten centimeters ( four inches) in diameter and rotating at the rate of. 100 to 200 revolutions per minute against four

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 25 x 400 mm peripheral vertical baffles. (1 inch by 16 inches).



     A wide variety of culture media can be used at the growth stage of the process. These media should contain an assimilable carbon source, proteinaceous material, mineral salts and traces of various nutrients, vitamins and other growth stimulants usually present as impurities in other constituents of the medium. To obtain the optimum production of sistomycosine, the culture medium must also contain a source of fat.



  An assimilable carbon source herein includes polyhydric alcohols and mono-, di- and polysaccharides, while the term proteinaceous material includes unmodified proteins and degradation products of proteins, particularly products such as these. 'shaped' by protein hydrolysis These protein degradation products include proteases, peptones, polypeptides, peptides and amino acids.



   As sources of assimilable carbon, there can be used a-rabinose, fructose, galactose, maltose, rhamnose, xylose, glycerin, mannitol, rhamnitol, etc. It is also possible to use complex materials based on carbohydrates. carbon such as dextrin starches, fermentation and distillation waste water, grain steep solids, grain steep liquor, dried fermentation residues, etc.



   Among the protein materials that can be used in the different media are beef extract, acid-hydrolyzed casein, beef peptone, oilseed soy flour, dried fermentation residues, soybeans soluble in water, defatted and partially hydrolyzed, mixtures of amino acids of natural or synthetic sources, urea, purines, etc. The use of a derivative of oilseed soybean meal or a soy protein derivative as a component of the medium is desirable because it provides a small amount of fat for use by the microorganism.



   The main mineral requirements of the medium can be met by adding 0.1 to 0.5% of one or more inorganic salts such as sodium chloride, calcium carbonate, bicarbonate phosphate, monopotassium phosphate. , etc. The other constituents of the nutrient medium, ie vitamins, growth promoters etc., are usually present in sufficient quantities in the most assimilable carbon sources and / or protein materials. impure.



   Sistomycosin can be separated from the crude culture mixture by a number of different methods. When using a solid culture medium in the growth phase, the mycelium is extracted with a sistomycosin solvent, such as water, and the extract product is isolated as described above. -after. In the case of liquid cultures, the mycelium is separated, washed appropriately with water and the filtrate and washings are used in the subsequent separation stages.

   A process for separating sistomycosin from crude culture liquid or extracts of cultures comprises extracting the solution with a non-target alcohol in water, such as n-butanol, removing the solution. solvent by distillation, extracting the residue with a fat solvent such as chloroform, discarding the extract, extracting the solid insoluble in the. chloroform by du'chloroform- containing about 10% of a lower aliphatic alcohol such as methanol, the extract obtained being poured into an ion exchanger of the inorganic silicate type (Permutite).

   The ion exchanger is washed first with chloroform containing gradually increasing concentrations of a lower aliphatic alcohol such as methanol, then the antibiotic is eluted with this alcohol. The alcohol elutriates are evaporated to dryness and the residue is extracted with water. The aqueous extract is filtered, the filtrate is frozen and the ice is separated from the product by vacuum sublimation to obtain the desired antibiotic.



   The invention is illustrated by the following examples.

 <Desc / Clms Page number 10>

 



  EXAMPLE 1.



   A mixture consisting of 180 gr. of industrial glucose, 180 gr. of a partially hydrolyzed protein derived from soybeans, 90 gr. of grain steeping liquor, 90 gr. of sodium chloride, 18 gr. of calcium carbonate and a sufficient quantity of hot water to bring the volume to 18 liters, in a fermentation apparatus of the type with a 30-liter glass fixed tank, fitted with a stainless steel cap, a propeller-type agitator, four 25 by 400 mm (1 inch by 6 inch) inner baffles and a perforated circular air distribution manifold.



   The fermentation apparatus is placed in an autoclave and the contents sterilized with water vapor at 121 ° C. for one hour. The fermentation apparatus and its contents are cooled and then removed from the autoclave. To this medium is inoculated aseptically 900 cm3 of a shaken flask subculture of two hour sptanto of Streptomves viridosporus grown on the same medium as described above and seeded from a medium of Sporulated glucose-tryptone-agar culture of Streptomyces viridosporus.



   After inoculation, the culture medium is incubated at 23-27 ° C for eighty-eight hours. During this incubation, sterilized air is introduced through the diffusion manifold at a rate of 15.6 to 17.8 liters per minute, and the agitator is rotated at a speed of approximately 150 revolutions per minute.



   Antibiotic production during incubation is monitored by taking samples from time to time and testing them against Candida albicans by the so-called cylinder-plate agar-diffusion method. Antibiotic production is indicated by the diameter in millimeters of the zone of inhibition. The table below illustrates the production of antibiotic obtained by the process described above.
 EMI10.1
 
<tb>



  Incubation <SEP> periods <SEP> Inhibition <SEP> of <SEP> Candida <SEP> albicans
<tb>
<tb> in <SEP> hours <SEP> Diameter <SEP> of <SEP> the <SEP> zone <SEP> in <SEP> mm.
<tb>
<tb>
<tb>



  40 <SEP> 16.6
<tb>
<tb>
<tb> 47 <SEP> "<SEP> '19, 2 <SEP>, <SEP>
<tb>
<tb>
<tb> 64 <SEP> 22.1
<tb>
<tb> 71 <SEP> 21.9
<tb>
<tb>
<tb>
<tb> 88 <SEP> 23.4
<tb>
 
At the end of the incubation period, 25 g are added. per liter of a cilicate filter aid and the culture mixture is filtered. The cake is washed with two 25 cc portions of water per liter of culture mixture, and the washings are added to the main filtrate.



   31.64 liters of filtrate or "beer" prepared as described above and yielding 18.5 mm are extracted twice with 10 liters of n-butanol. in the test against Candida albicans at a dilution of 1 to 2. The aqueous solution is discarded and the extracts are concentrated in the dark and in vacuo below 45 ° C. to approximately dryness. bn extract the residue by means of four portions of 50 cm3 and one portion of 100 em3 of chloroform at 40 ° C. and the extracts are discarded. The chloroform insoluble residue is shaken with 300 cm3 of water and filtered to obtain 3.39 g. of solid (Solid A) and a filtrate (Filtrate A).

   Filtrate A was frozen and the ice was removed by vacuum sublimation to obtain 3.725 g. of sistomycosin. 500 gammas of this substance in 1 cm3 of water produce 16.8 and 11.5 mm zones of inhibition against Candida albicans. at respective dilutions of 1: 1 and 1: 2. ,
Solid A (3.39 gr.) Is extracted with two 100 cm3 portions and six 150 cm3 portions of water. We filter the extracts, we

 <Desc / Clms Page number 11>

 subjected to separate tests, those with similar titers are combined and frozen. The ice is separated from the frozen extracts by sublimation
 EMI11.1
 : in vacuum to obtain a total of 395 gr, of crude sistornycosin.

   The productions and the purity of each of the crude fractions are given in the following table.
 EMI11.2
 
<tb> Weight <SEP> of <SEP> Concentra- <SEP> Results <SEP> of tests <SEP> against <SEP> on
<tb> Solid <SEP> produces <SEP> tion <SEP> of <SEP> pro <SEP> Candida <SEP> albicans <SEP>
<tb>
 
 EMI11.3
 '. '. gross (mg) raw dui t in
 EMI11.4
 
<tb> 'solution <SEP> of <SEP> Dilution <SEP> 1: 1 <SEP> 1: 2 <SEP> Il: 4 <SEP> 1: 8
<tb>
 
 EMI11.5
 stock gamma / Diameter mm. inm. nm. nrni.
 EMI11.6
 
<tb> cm3 <SEP> '<SEP> from <SEP> the <SEP> zone
<tb> of inhibi-
<tb>
 
 EMI11.7
 tion in mm ..



  B. 1623 200 17.1 14.1 10.6 C 1613 '400 20el 17.5 14.4, 12., o D 114 200 7: 5.9 - 11.3 - +
 EMI11.8
 
<tb> E <SEP> 100 <SEP> 330 <SEP> 16.0 <SEP> 13.5 <SEP> 11.6 <SEP>, <SEP> ++
<tb>
<tb> F <SEP> 60 <SEP> 550 <SEP> - <SEP> 13.9 <SEP> 11.9 <SEP> ++ <SEP> +
<tb>
 + Inhibition inside the cylinder.



   ++ Slight inhibition immediately around the cylinder.



   995 mg are extracted. of solid B for 1/2 hour using
199 cm3 of chloroform containing 10% methanol by volume 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 an exchange material based on silicate called "Florisil", previously washed thoroughly with chloroform containing 10% methanol by volume. Mixed solvents which consist of are allowed to percolate through the column in the order given. in chloroform containing 10, 20, 30 and 50% methanol by volume. The elutriates are discarded and 50 cm3 of methanol are poured onto the column. The first methanol elutriate is discarded and 2050 cm3 of methanol are poured in small portions onto the column.

   A total of eight fractions are collected, the first five of which are evaporated to dryness in vacuum. The yellow residue is extracted with a total of 35 cm 3 of water, the extract is frozen and taken up. sublimates the ice to obtain 85.4 mgr. of sistomycosin as a pale yellow solid. 400 gammas of this solid, dissolved in one cm3 of water, gives an inhibition zone of 22 mm. in diameter at a 1: 1 dilution in the Candida albicanso test. The other three fractions were combined and evaporated to dryness to give 131.1 mg. sistomycosin as a light yellow solid. 400 gammas of this substance in 1 cm3 of water produce an inhibition zone of 16 mm. at a dilution of 1:

  1 in the test against
Candida albicans. By the turbidimetric test method, '7.14 gammas / cm3
 EMI11.9
 produce 50% inhibition of the growth of Garidida'albicans.



   1.435 gr are extracted. of solid material C with 207 cm3 of chloroform containing 10% methanol by volume, for half an hour. The insoluble residue (Solid G) weighs 1.084 gr. The extract is poured into an absorption column containing 32 g. of a silicate-based ion exchange material called "Decalso" previously washed thoroughly with chloroform containing 10% methanol by volume. The following mixed solvents are percolated through the column, in portions of 700 cm 3, in the order given, chloroform containing 10, 20, 30, 40 and 50% methanol by volume.



  We reject the percolates. The column is eluted with 840 cc of methanol divided into six portions and the elutriates are combined. We evaporate the

 <Desc / Clms Page number 12>

 solution to dryness in vacuo and the residue is extracted with a total amount of 30 cm3 of water. The aqueous extract was frozen and the ice was sublimed under reduced pressure to obtain 107 mg. of sistomycosin as a buff colored solid. This substance gives an inhibition zone of 23.6 mm. at a concentration of 400 gammas / cm3 in the test against Candida albicans.



   The column is eluted with five additional 700 cc 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 to obtain 85 mgr. sistomycosin as a light yellow solid giving the Candida albicans test an inhibition zone of 20.3 mm. at a concentration of 400 gammas / cm3. In the turbidimeter test, 3.47 gammas / cm3 of the product give a 50% inhibition of the growth of Candida albicans.



     The solid G is extracted with chloroform containing 10% methanol, by volume, and the extract is poured onto a chromatographic column containing the ion exchange substance called "Decalso". The column is washed with portions of chloroform containing 10, 20, 30, 40 'and 50% methanol by volume and the washings are discarded. The column is eluted with 600 cc of methanol divided into six equal portions.



   Discard the first 50 em3, combine the remaining fractions and evaporate to dryness in vacuum. The residue was extracted with water, frozen, and the ice was sublimed under reduced pressure to obtain 92 mg. pure sistomycosin. When tested against Candida albicans, 400 gammas / cm3 of this light yellow solid produced a zone of inhibition of
23.8 mm. at a dilution of 1: 1. We can produce 60 mgr. additional. sistomycosin by washing the absorption column with more 'methanol' and treating the eluate as described above.



   Pure sistomycosin is a neutral organic compound that contains only the elements carbon, hydrogen, nitrogen and oxygen. It does not melt below about 230 C but turns brown at about 130 C.



  It rapidly reduced a cold aqueous solution of potassium permanganate and the boiling Benedict's solution. It gives a positive Molisch test for carbohydrates, a negative Beilstein test for halogens and a negative test for ferric chloride. Addition of a very small amount of the antibiotic (1 to 2 mg) to hot concentrated sulfuric acid gives the solution a dark cherry red to chocolate color. No color change occurs when a small amount of the antibiotic is added to hot nitric or hydrochloric acid saturated with thymol.



   Sistomycosin, particularly in solutions, undergoes photochemical decomposition on exposure to light. It is decomposed by acids or alkalis but is more stable in neutral or slightly basic solutions, ie having a pH of 7-9, than in solutions with an acid reaction. It is completely stable in water below 25 C and its aqueous solutions can even be heated at 50 C for one hour without losing its antibiotic activity. In aqueous solution at 100 ° C., it retains more than 50% of its antibiotic activity after one hour.



     . The ultraviolet absorption spectrum (Figure I) has a maximum in the vicinity of 300 millimicrons and another at 218 millimicrons with a
E1% of 215. The maximum in the neighborhood of 300 millimicrons consists of
1 cm three distinct absorption peaks at 292.5, 306 and 320.5 millimicrons with rpt values of 257, 340 and 295, respectively, and two microns 1% nima located between these peaks at 298 and 314 millimicrons with E1% of 230 and 217 respectively. The main minimum in the UV absorption spectra is 253 millimicrons with an E 11% of 102.

   The

 <Desc / Clms Page number 13>

 The absorption curve shown in Figure 1 is that obtained in water, but sistomycosin is remarkable in that this spectrum is not appreciably modified by moderate variations in the acidity or base of the substance. solution, i.e. from neutrality to an acidity or basicity of about 0.05 N.



   Figure 2 shows the so-called "fingerprint" region of the infrared absorption spectrum of sistomycosin in a neutral mineral oil vehicle. Spikes at or near 6.88 and 7.30 microns are attributable to the mineral oil vehicle. The most intense absorption bands characteristic of sistomycosin are those located at or near 6.34 and 9.44 microns. Bands of weak and moderate intensity are located at or near 7.70; 7.86;
8.55; 11.16; 11, 85 and 12.4 microns with others appearing as shoulders to the previous bands at or near -7.08, 7.16; 8.95;
9.89; 10.25 and -10.6 microns.

   Another characteristic band of sistomycosin outside of the "fingerprint" region is found at or near 2.96 microns.



   EXAMPLE 2.



   A nutrient medium which consists of 5 gr. of industrial glucose, 2.5 gr. of beef extract, 2.5 gr. of peptone, 2.5 gr. of sodium chloride and a quantity of water sufficient to bring the volume to 500 cm 3 is made alkaline to pH 7.7 with a 10 N solution of sodium hydroxide. The medium is placed in a Fernbach flask stoppered with a cotton ball and sterilized under a water vapor pressure of 1.26 Kgr / cm2 (18 lbs) for twenty minutes. The medium is inoculated with 1 cm 3 of a suspension of Streptomyces viridosporus spores originating from a ten-day old glucose-tryp-tone-agar culture medium. The resulting mixture is incubated at 24 ° C. for eight days.

   At the end of this time, the culture fluid below the surface mycelium has a pH of 6.8 and produces a zone of inhibition of 22 mm. in diameter when subjected to a sistomycosin test against Candida albicans by the so-called "disc-plate" method. The sistomycosin present in the culture liquid can be separated by the method described in Example 1.



   EXAMPLE 3.



   A mixture consisting of 180 gr. of industrial grade maltose, 180 gr. of grain steeping liquor, 90 gr. sodium chloride,
36 gr. of bipotassium phosphate and a sufficient quantity of water to form a volume of 18 liters, is placed in a fermentation apparatus of
30 gallons of the type described in Example 1. The pH of the solution is adjusted to 7.5 with a 10 N solution of sodium hydroxide, and 18 gr. of calcium carbonate and sterilize the fermentation apparatus and medium at 250 ° C. for two hours.

   The nutrient medium is cooled and aseptically inoculated with 10 cm3 of a Streptomyces viridosporus spore suspension prepared by stirring a seven day old Streptomyces viridosporus glucose-tryptone-agar culture medium with 10 cm3. 0.01% fine soap aqueous solution.



   The culture mixture is incubated at 28-29 ° C for 136 hours and during this time the stirrer is rotated at a speed of 150 rpm and air is passed through. , sterile in the. mixing at the speed of 7.2 to 18 liters per minute. At the end of the incubation period, the mycelium is separated from the mixture and the clear culture "beer" is subjected to an assay for its sistomycosin content by the turbidimetric method against Candida albicans. In the experiment described, cultured beer produced 50% inhibition of the growth of Candida albicans at a dilution of 1 to 135.

   Sistomycosin present in cultured beer in pure form can be separated using the separation process described in Example 1.

 <Desc / Clms Page number 14>

 



  EXAMPLE 4.



   Is introduced into each flask of a series of Erlenmeyer flasks 125 cm3 of a culture medium composed of 0.5% industrial glucose, 0.5% glycerin, 0.3% hydrolyzed casein, 0.25 % peptone, 0.1% brewer's yeast, 0.25% steeping water solids. grain, 0.25% soy flour, 0.25% fermentation residue of butanol-acetone, 0.25% sodium chloride, 0.1% calcium carbonate and water to supplement 100 %.

   The vials are covered with cotton wool covered with gauze and sterilized under a pressure of water vapor of 1.26 Kgr / cm2 (18 lbs) for twenty-five minutes, then each vial is inoculated; aseptically, 1 cm3 of a spore suspension of Streptomyces viridosporus prepared from a glucose-tryptone-agar culture medium, seven days old. The vials are shaken on a rotary type shaking machine rotating at 150 revolutions per minute for four days. The temperature is maintained at 22-4 C during the incubation. At the end of the incubation period, the cultured beer has a pH of about 6.4.

   In the turbidimetric test between Candida albicans, the cultured beer contains a sufficient amount of sistomycosin to produce a 50% inhibition of the growth of the test organism at a dilution of 1 to 1350
If desired, the culture mixture can be combined and filtered into the vials and the sistomycosin separated by the method described in Example 1.



  EXAMPLE 5.



   Is introduced into each flask of a series of Erlenmeyer flasks 125 cm3 of a culture medium composed of 1% maltose, 1% grain soaking liquor, 0.2% bipotassium phosphate, 0.5% sodium chloride and water to make up to 100% (adjusted to pH 7.5 with 10N sodium hydroxide solution) and the flasks are sealed with covered cotton wool. gauze. The flasks containing the culture medium are sterilized, inoculated and incubated as described in Example 4. At the end of the incubation period, the mycelium is filtered off and the fluids are combined. or cultured beers.

   Cultured beer has a pH of about 6.35 and by applying the turbidimetric method it has been found to contain a sufficient amount of sistomycosin to produce a 50% inhibition of the growth of Candida albicans at a dilution of. 1 to 320. Pure sistomycosin can be separated from cultured beer by the method described in Example 1.
 EMI14.1
 



  RE iT ENDIC¯A TI 0 N S .. l.- Process for the production of the antibiotic sistomycosin characterized in that one inoculates a culture medium of Streptomyces viridosporus, one subjects the inoculated medium to the. incubation under aerobic conditions at a temperature of 20 to 35 C for about one to eight days, and the sistomycosin thus produced is separated from the culture mixture.


    

Claims (1)

2. A method according to claim 1, characterized in that the culture is carried out in the submerged state and an aqueous culture medium is used having an initial pH of between 6 and 8.2 and containing an assimilable carbon source, a proteinaceous material and mineral salts. 3. A method according to claim 2, characterized in that the nutrient medium has an initial pH of 7.3 to 7.7 and the temperature is maintained at 25 to 28 C during the incubation. 4: - Sistomycosin, neutral substance, suitable for inhibiting the growth of eumycetes and having no action on the growth of most bacteria with negative gram and gram positive, which contains only the elements carbon, hydrogen, oxygen and nitrogen, is very soluble in water and methanol, less soluble in wet acetone and aqueous mixtures of higher aliphatic alcohols, and insoluble in chloroform, diethyl ether, ethyl acetate , benzene and ether <Desc / Clms Page number 15> petroleum, undergoes photochemical decomposition on exposure to light, rapidly reduces a cold aqueous solution of potassium permanganate, rapidly reduces boiling Benedict's solution, gives a positive Molisch test, gives a negative ferric chloride test, imparts a dark cherry red color to chocolate in concentrated sulfuric acid, does not color a solution of thymol in hot concentrated nitric acid, does not color a hot solution of thymol in hot concentrated hydrochloric acid, present in water characteristic maximum absorption bands in the ultraviolet region of the spectrum at 218, 292.5, 306 and 320.5 millimicrons, as well as minimum absorption bands at 253, 298 and 314 millimicrons and present characteristic absorption bands in the infrared region of the spectrum when suspended in solid form in hydrocarbon oil, at frequencies of 2.96; 6.34; 7.08; 7.16; 7.70; 7.86; 8.55; 8.95; 9.44; 9.89; 1a, 25; 10.6; 11.16; He, 85 and 12.4 millimicrons.