DE1070781B - - Google Patents

Description

The invention relates to the production of the new antibiotic danubomycin, its components B 1, B 2, B 3 and B 4 and salts and mixtures thereof.

The antibiotic danubomycin arises from the culture of a strain of the species Streptomyces griseus, which was isolated using known methods from a soil sample collected near Donaueschingen (Germany). It is stored in the inventor's laboratories and at the Swiss Federal Institute of Technology, Institute for Special Botany, Zurich, under the name Danubomycin.

The Streptomycete strain A 9990 forms a yellowish greenish gray aerial mycelium. He has chains of conidia which are a typical feature of the genus Streptomyces and which do not form spirals in this strain show, but are branched irregularly. The individual spores are smooth. Next are peptone-containing Culture media from strain A 9990 not discolored black-brown melanoid. The growth is relative little dependent on temperature; Both at 18 ° C and at 36 ° C the fungus will develop well, but the optimum is 25 to 32 ° C

For further characterization, the growth of strain A 9990 on various Culture media described. The nutrient media 1 to 7 and 10 were according to W. Lindenbein, Arch. Mikrobiol., 17, p. 361 (1952).

1. Synthetic agar: growth thin, haze-like. Aerial mycelium velvety, initially chalk white, later yellowish greenish gray. Substrate not discolored.

2. Synthetic solution: sediment, flakes milk white to greenish yellow. Pellicle initially punctiform, later wrinkled, light yellow or partly chestnut brown. Aerial mycelium sparse, velvety, light yellow. Substrate initially not discolored, after 2 weeks reddish brown.

3. Glucose agar: growth thin, veil-like, yolk yellow. Aerial mycelium sparse, velvety, light yellow to yellowish greenish gray. Substrate discolored light yellow.

4. Glucose asparagine agar: growth thin, veil-like, yolk yellow to deep yellow. Velvet mycelium, yellowish greenish gray.

5. Calcium malate agar: growth thin, veil-like, light brown to brownish yellow, later deep yellow, after 14 days brownish, copper-red. Air mycelium velvety, yellowish greenish gray.

6. Gelatin prick (18 ° C): growth good. Pellicle light yellow to reddish brown. Velvet mycelium, λvite yellow to greenish gray. Substrate golden yellow to reddish brown. Liquefaction after 27 days 10 to 12 mm.

7. Starch plate: growth thin, veil-like, white-yellow. Aerial mycelium velvety, light yellow, when ripe. Production of the antibiotic
Danubomycin

Applicant:

CIBA Aktiengesellschaft, Basel (Switzerland)

Representative: Dipl.-Ing. E. Splanemann ₁ patent attorney, Hamburg 36, Neuer Wall 10

Claimed priority: Switzerland from October 29, 1957

Dr. Ernst Gäumann, Zurich, and Dr. Walter Voser, Binningen (Switzerland), have been named as inventors

would be greenish-gray. Hydrolysis after 10 days 1.4 cm.

S. Potatoes: Growth good, brownish to rust-brown. Aerial mycelium velvety, yellowish greenish gray, partly shimmering light yellow-red through the color of the substrate. Substrate not discolored, partly but carmine.

9. Carrots: Very sparse growth, with white-yellow aerial mycelium.
10. Litmus milk: growth in the form of a pellicle with velvety, light yellow aerial mycelium. Substrate red. Peptonization and slow coagulation.
The strain A 9990 grows, examined according to the methodology of TG Pridham and D. Gottlieb, J. Bacteriology, 56, p. 107 (1948), using different carbon sources as follows:

Glucose + Raffinose + L -Xylose Inulin + L -arabinosis + D -Mannit + L -rhamnosis + D sorbitol D -fructose + Meso-inositol (+) Sucrose + Salicin +

* 5 where:

+ good growth, safe use of the re.

Carbon source.
(+) weak growth, use of the relevant carbon source questionable.
- no growth, no use of the relevant carbon source.

The most important characteristics of the strain A 9990, namely spore contour, aerial mycelium color, morphology

909 688/383

the spore chains and chromogenicity agree with those of Streptomyces griseus (Krainsky) Waksman match. In contrast to Streptomyces griseus, strain A 9990 forms a bad diffusing, red-brown pigment. Despite this difference, strain A 9990 is provisionally the Species Streptomyces griseus included.

It is known that some representatives of the species Streptcmyces griseus produce antibiotics, such as streptomycin (S.A. Waksman, Streptomycin, Verlag Williams and Wilkins, 1949), rhodomycetin (G. Shockman and S.A. Waksman, Antibiotics and Chemotherapy, 1, pp. 68 to 75 [1951J), Actidion (B.B. Leach, Η. J.Ford and A. J. Whiffer, J.A.C.S., 69, p.474 [1947]), Candicidin (H.Lechevalier, R.F. Acker, C. T. Corke, C. M. Haenseler and S. A. Waksman, Mycologia, 45, pp. 155 to 171 [1953]), Grisein (D. M. Reynolds and S. A. Waksman, J. Bacterid., 55, pp. 739-752 [1948]) and streptocin (S.A. Waksman, D.A. Harris, A. B. Kupferberg, H. O. Singher, and H. Stvles, Proc. Soc. Exp. Biol. Med., 70, pp. 308 to 312 '[1949]). It will be shown below that the new antibiotic Danubomycin is characteristically different from these already known antibiotics.

The present invention does not address the manufacture of the antibiotic danubomycin restricts the use of strain A 9990 or other strains corresponding to the description, but also relates to the use of variants of these organisms, as z. B. by selection or mutation, especially under the action of ultraviolet or X-rays, or obtained from nitrogen mustard oils.

The properties of Streptomyces griseus are used to produce the antibiotic danubomycin A 9990 containing strain in aqueous, inorganic salts containing a carbon and nitrogen source Nutrient solution grown aerobically until it has a significant antibacterial and / or fungicidal effect shows, and then the antibiotic danubomycin is isolated from the culture filtrate and / or from the mycelium.

The nutrient solution contains as inorganic salts, for example, chlorides, nitrates, carbonates, sulfates of Alkalis, alkaline earths, magnesium, iron, zinc, manganese. The main sources of carbon are carbohydrates, such as glucose, sucrose, lactose, starch, and alcohols such as glycerin and mannitol. As nitrogenous Examples of compounds and growth-promoting substances are: amino acids and their mixtures, peptides and proteins and their hydrolysates, such as peptone or tryptone, meat extracts, water-soluble components of cereal grains such as maize and wheat, of distillation residues from alcohol production, of yeast, beans, in particular the soybean plant, of seeds, for example the cotton plant.

The cultivation takes place aerobically, for example in dormant surface culture or preferably submerged while shaking or stirring with air or oxygen in shake flasks or the known ones Fermenters. A suitable temperature is between 20 and 36 ° C. An essential antibiotic The nutrient solution generally shows an effect after IVs of up to 5 days.

To isolate the antibiotic Danubomycin example, the following methods can be used: the mycelium with an unchanged p _H is separated from the culture filtrate, after which the principal amount of the antibiotic is found in the culture. Nevertheless, significant amounts of the antibiotic remain on the mycelium absor-

beer. It is therefore advantageous to wash out the latter well. Organic, at least partially water-soluble solvents, such as alcohols, e.g. B. methanol, ethanol and butanols, or ketones, e.g. B. acetone and methyl ethyl ketone, or organic or dilute inorganic acids such as acetic acid, hydrochloric acid or sulfuric acid. These mycelium extracts are added to the culture filtrate either directly or after prior concentration in vacuo. It then the mixture was extracted advantageous in a p _H greater than 7, preferably between 8 and 10 with a water-immiscible organic solvent such as lower fatty acid esters, such as ethyl acetate or amyl acetate. Hydrocarbons, e.g. B. benzene, chlorinated hydrocarbons, e.g. B. ethylene chloride, methylene chloride or chloroform, ketones, e.g. B. methyl propyl ketone, methyl amyl ketone or diisobutyl ketone, alcohols such as butyl alcohols or amyl alcohols, ethers, e.g. B. ethyl ether, diisopropyl ether, dibutyl ethers or glycol ethers and the like. If one, the resultant culture broth to p _H 4 and then separates from the mycelium, the entire antibiotically active substance passes into the culture filtrate, which is then extracted into the just mentioned manner at a p _H 8 to 10 The mycelium extract is practically inactive. Instead of solvent extraction of the cultures or in combination with such as a further cleaning operation, the antibiotic can also be obtained by adsorption, for example on activated charcoal, on activated earths such as fuller's earth or floridine, or on suitable ion exchangers such as IRC-50, and subsequent extraction of the adsorbate z. B. with an organic solvent which is at least partially soluble in water, such as acetone, butanol or methyl ethyl ketone, optionally with the addition of low molecular weight organic or inorganic acids.

The cultures can also be cultivated directly, without prior separation of the mycelium, in the manner indicated and extract way.

Further enrichment can be achieved in that one repeats attenuates the antibiotic-containing organic extracts washed with an acidic aqueous solution with a p _H less than 5, wherein the majority of the antibiotic activity passes over into the aqueous phase. A smaller amount of activity remains in the organic phase. The combined acidic aqueous solutions are then extracted again in the manner described with a water-immiscible organic solvent at a pn above 7. This procedure can be repeated several times. Dilute acids, such as acetic acid, hydrochloric acid or sulfuric acid, or buffer solutions, such as citrate or phosphate buffers, are suitable as acidic aqueous solutions.

The raw bases thus obtained are expediently again in an organic solvent, z. B. in methanol, ethanol, acetone or chloroform, added and insoluble, antibiotic ineffective Separated substances. After removing the solvents, the crude antibiotic is obtained Danubomycin in the form of a reddish-brown amorphous powder. This is in most of the organic solvents such as alcohols, ketones, esters, chlorinated hydrocarbons, aromatic Hydrocarbons, readily soluble, on the other hand almost insoluble in petroleum ether and water. In the treatment yellow salts which are readily soluble in water are formed with acids. In alkaline media there will be a color change

Orange-red to Vioktt observed. The antibiotic shows an absorption band in the ultraviolet spectrum at 243 ιημ with a shoulder of 260 πιμ.

The paper chromatographic behavior with various solvent systems is from the following Table can be seen, the Rf values by means of the antibacterial effect (Bacterium megatherium and Micrococcus pyogenes, var.aureus):

Solvent Systems Rf

n-butanol, saturated with water .... 0.65
n-butanol, saturated with water + 2%

p-toluenesulfonic acid + 2% piperidine 0.90
Methyl isobutyl ketone, saturated with

Water ..'. 0.00

80% ethanol with 1.5% sodium chloride, paper impregnated with

0.95 molar sodium sulfate solution

and 0.05 molar sodium bisulfate

solution 0.5

Butanol - methanol - water 4: 1: 2 0.67
Water saturated with methvlisobutvl-

ketone 0.05 and 0.70

Water, saturated with methyl isobutyl

ketone + 1% p-toluenesulfonic acid .. 0.80 75% water + 25% of a mixture

of 3 parts of methanol and 1 part

Acetone, with ammonia to p _H 10.5

brought up with phosphoric acid

p _H 7.5 truncated 0.05 and 0.70

The raw antibiotic danubomycin can be administered using various methods, e.g. B. Chromatography Cellulose, aluminum oxide and the like, or distribution between water and one with water not or only partially miscible organic solvents, optionally with the addition of water-soluble ones Acids, alcohols, ketones and the like, can be separated into several components. As particularly suitable The following solvent systems, individually or in combination, have proven to be suitable for countercurrent distribution can be used:

a) 5 parts by volume of petroleum ether (boiling range 40 to 70 ° C), 5 parts by volume of benzene, 8 parts by volume of methanol, 2 parts by volume of water;

b) 7.5 parts by volume of petroleum ether, 2.5 parts by volume of benzene, 7.5 parts by volume of abs. Ethanol and 2.5 parts by volume Water.

The distribution is expediently carried out by the countercurrent process in appropriate apparatus. The individual components differ in their physico-chemical and biological properties. They can be obtained in pure form by crystallization or reprecipitation from organic solvents, such as from acetone, methanol, ethyl acetate, acetone-methanol mixtures, acetone-ether mixtures, acetone - Petroleum ether - mixtures, ethyl acetate - ether - mixtures, ethyl acetate-petroleum ether mixtures or from Aqueous-organic solutions such as dilute alcohols, dilute acetone, etc. can be obtained.

The raw antibiotic danubomycin can be broken down into an active and a practically inactive component in system a) over ten stages. The active component is in levels 4 to 10 and can be separated in system b) over 375 levels into at least four components, which are referred to as danubomycin B1, B2, B3 and B4. The maxima of the four components are at level 40, 64, 124 and 164. Component B 1 crystallizes from ethyl acetate, components B 2 and B 3 from acetone.

The antibiotic danulxjmycin bears no resemblance to the others from Streptomyces griseus produced antibiotics. In contrast to the antibiotic danubomycin are streptomycin and grisein readily soluble in water, while rhodomycetin is insoluble in dilute acids. Candicidin is in the insoluble in most organic solvents and shows a different absorption in the ultraviolet spectrum. Streptomycin and Actidion also differ from the antibiotic danulximycin and streptocin for its own color, for the neutral actidion and for the acidic rhodomycetin and Streptocin in terms of chemical character. Grisein also contains iron and sulfur, which are found in antibiotics Danubomycin cannot be detected.

The salts of the antibiotic danubomycin and its components B1, B2, B3 and B4 are derived from the known inorganic and organic acids, for example from hydrochloric acid, the Sulfuric acids, acetic, propionic, valeric, palmitic or oleic acid, succinic acid, citric acid, Mandelic acid, pantothenic acid, ascorbic acid or amino acids such as glutamic acid, cysteine They represent neutral or acidic salts. They are produced by the action of the appropriate Acids to the free base or by double conversion of salts, for example of Danubomycin sulfate with pantothenic acid calcium.

The antibiotic danubomycin has an antibiotic activity against various test organisms. If one uses as a test method in vitro dilution series (powers of ten) in glucose broth, which are incubated for 24 hours at 37 ° C, the following still inhibitory results result Concentrations:

Inhibitory Test organisms Conc
tration
/ ig / cm ³ Micrococcus pyogenes, var. Aureus 10 Micrococcus pyogenes, var. Aureus Penicillin Resistant 10 Streptococcus pyogenes 1 0.1 Streptococcus faecalis 10 0.1 100 Salmonella typhosa 100 100 100 Klebsiella type A 100 100 Vibrio cholerae el Tor 100 10 10 10 100 <0.1

* Cultivated in Kirchner's synthetic medium with 5% bovine albumin; Read the growth after 2 weeks. ** In bouillon with 10% horse serum.

The development of influenza virus on isolated membranes of the chickens. R-chorioallantois of 14-day hatching eggs is still inhibited ^{by the antibiotic danubomycin in a concentration of less than 10 μg / cm 3.}

1

The antibiotic danubomycin is also effective in vivo. Become sexually mature female hamsters Infected intravaginally with Trichomonas fetus and becomes the resulting chronic trichomonads infection locally with a 0.3% aqueous suspension of the antibiotic danubomycin for 3 weeks treated, the trichomonads are made to disappear in four out of four animals.

The present invention also relates to the production process for the antibiotic Danubomycin, its components and, for its neutral and acidic salts, also the compounds mentioned themselves, in particular the sulfates, the hydrochlorides, acetates and pantothenates, furthermore the fission products, how they z. B. obtained in the hydrolysis.

The antibiotic danubomycin, its components B1, B2, B3 and B4, their salts and derivatives, the above-mentioned cleavage products or mixtures thereof can be used as remedies, e.g. B. in the form of pharmaceuticals Preparations, A ^ application. These contain the compounds mentioned in a mixture with a pharmaceutical organic suitable for enteral, parenteral or local application or inorganic carrier material. For the same purpose, those substances come into question that are compatible with the new ones Connections not responding, such as B. gelatin, lactose, starch, magnesium stearate, talc, vegetable oils, benzyl alcohol, gum, polyalkylene glycols, petrolatum, cholesterol or other known ones Drug carrier. The pharmaceutical preparations can e.g. B. as tablets, coated tablets, powders, ointments, Creams, suppositories or in liquid form as solutions, suspensions or emulsions. If necessary, they are sterilized and / or contain auxiliary substances such as preservatives, stabilizers, Wetting or emulsifying agents. They can also contain other therapeutically valuable substances.

The invention is described in the following examples without implying any restriction of the subject matter of the invention is intended. The temperatures are given in degrees Celsius.

example 1

A nutrient solution of the composition 20 g glycerine, 10 g soy flour, 5 g sodium chloride, 1 g sodium nitrate, 10 g calcium carbonate and 1 liter of tap water is prepared and it is adjusted to pH 7.3. This or a multiple thereof is ^{filled into 500 cm 3} Erlenmeyers (with 100 cm ³ nutrient solution each) or in 500 l fermenters (with 300 1 nutrient solution each) and sterilized at 1 atm for 20 to 30 minutes. Then up to 10% of a partially sporulating, vegetative culture of the Streptomycete strain A 9990 is inoculated and incubated with good shaking or stirring and in the fermenters with aeration (with about 1 volume of sterile air per volume of nutrient solution per minute) at 27 °. After 70 to 120 hours of growth, the cultures are filtered with the addition of a filter aid, depending on the volume, through a suction filter or through a filter press or a rotating filter, thus removing the mycelium and other solid components from the antibiotic aqueous solution.

Example 2

If the nutrient solutions a), b), c), d), e) or f) described below are used instead of the medium given in Example 1, 781 is obtained

after similar sterilization, inoculation with the Streptomycete strain A 9990, incubation at 27 ° and filtration of aqueous antibiotic solutions.

a) 10 g raw glucose, 5 g peptone, 3 g meat extract (Oxo Lab Lemco), 5 g sodium chloride, 10 g calcium carbonate and 1 l tap water; p _H before sterilization 7.5.

b) 20 g of lactose, 20 g of distillers solubles, 1 g of sodium nitrate, 5 g of sodium chloride and 1 liter of tap water; Ph before sterilization 7.5.

c) 20 g of mannitol, 20 g of soy flour and 1 liter of tap water; p _H before sterilization 7.5.

d) 10 g lactose, 10 g soy flour, 5 g sodium chloride, 10 g calcium carbonate, 1 g sodium nitrate and 1 l tap water; p _H before sterilization 7.5.

e) 20 g mannitol, 20 g "Distillers solubles", 3 g sodium chloride, 1 g sodium nitrate and 1 liter of tap water; p _H before sterilization 7.5.

f) 10 g glucose, 10 g soy flour, 5 g sodium chloride, 1 g sodium nitrate, 10 g calcium carbonate and 1 l tap water; p _H before sterilization 7.5.

Example 3

The filter residue of a 150-1 batch obtained according to Example 1 or 2 is stirred with 25 l of acetone and filtered again. This is repeated twice, whereupon the antibiotic-containing acetone solutions are combined, concentrated in vacuo to 5 liters and combined with the culture filtrate. This solution is then extracted at _pH 8.5 with 70 1 ethyl acetate in Westfalia extractor, wherein the total antibiotic activity passes over into the organic phase. The orange-red extract is washed with water, evaporated to 5 l in vacuo and then extracted several times with 0.5 η-acetic acid. The ethyl acetate solution shows little antibiotic activity, while most of the activity is found in the acetic acid solutions. These solutions are combined, brought with 2N-sodium carbonate solution to p _H 8.5, with the color from yellow to orange-red turns, and extracted with ethylene chloride. The ethylene chloride extract is extracted again with 0.5 η-acetic acid, whereupon this, as described, is made alkaline and extracted. Finally, the ethylene chloride solution is dried over sodium sulfate and evaporated in vacuo. The brown-red colored, antibiotic raw bases are thus obtained in the form of an amorphous powder. This is readily soluble in methanol, ethanol, acetone, ethyl acetate, chloroform, ethylene chloride, benzene and dilute aqueous acids. It is hardly soluble in water and petroleum ether. Its ultraviolet spectrum shows a maximum at 243 ιημ with a shoulder at 260 ιημ.

Its behavior in the paper chromatogram in various solvent systems is as follows:

Solvent Systems Rf

n-butanol, saturated with water .... 0.65

n-butanol, saturated with water + 2%
p-toluenesulfonic acid + 2% piperidine 0.90

Methyl isobutyl ketone saturated with

Water 0.00

"80% ethanol with 1.5% sodium chloride, paper impregnated with
0.95 molar sodium sulfate solution
and 0.05 molar sodium bisulfate solution 0.5

Butanol - methanol - water 4: 1: 2 0.67

Claims (1)

Water, saturated with methyl isobutyl ketone 0.05 and 0.70 Water, saturated with methyl isobutyl ketone + 1% p-toluenesulfonic acid .. 0.80
75% water + 25% of a mixture of 3 parts of methanol and 1 part Acetone, with ammonia to p _H 10.5 brought up with phosphoric acid p _H 7.5 truncated 0.05 and 0.70 Example 4 32 g of the crude bases obtained according to Examples 1 and 3 are subjected to a ten-stage countercurrent distribution using the following solvent mixture: 5 parts by volume of petroleum ether (b.p. 40 to 70 °), 5 parts by volume of benzene, 8 parts by volume of methanol and 2 parts by volume of water. After evaporation of the contents of the individual distribution vessels in vacuo at 30 °, an activity and substance maximum is found in levels 4 to 10 , while the main amount of inactive accompanying substances is in levels 0 to 3 . After combining steps 4 to 10 , 8.5 g of the enriched antibiotic danubomycin are obtained in the form of an orange-red powder. Ultraviolet absorption: maximum at 243 ιημ with shoulder at 260 ιημ. Example 5 8.5 g of the enriched product obtained according to Example 4 are subjected to 375-stage countercurrent distribution, the following solvent mixture being used: 7.5 parts by volume of petroleum ether, 2.5 parts by volume of benzene, 7.5 parts by volume of abs. Ethanol and 2.5 parts by volume of water. The contents of the individual distribution vessels are evaporated in vacuo at 30 °. Activity and substance maxima are found at levels 35 to 45 (component B1), 60 to 70 (B 2), 115 to 135 (B 3) and 155 to 175 (B 4). The solutions from these vessels are combined and evaporated to dryness in vacuo at 30 °. This gives components B 1, B 2, B 3 and B 4 of the antibiotic danubomycin as red to orange-red Powders that crystallize when treated with ethyl acetate or acetone. Example 6 The culture solution of a 950-I batch obtained according to Example 1 or 2 is, with the addition of Hyflo is freed from mycelium as a filter aid and the filtrate and filter residue are further processed separately. a) The culture is cooled with sodium hydroxide solution to p _H 8.5 to 9.0 and in the ratio 3: 1 with Essigester extracted in the extractor, wherein the total antibiotic activity in the organic phase transforms. The extract is directly adsorbed on a 1 Amberlite IRC-50 (acid form, dehydrated with methanol and conditioned on ethyl acetate). The flow is antibiotic and effective. Other inactive accompanying substances can be washed out η methanol. The antibiotics danubomycin is then eluted with 0.4 η methanolic hydrochloric acid. Approximately half of the active eluates has a p _H between 4 and 6, whereas <ίο other half a p _H of about 1.5 has Last <-45 are IR entiäre on a column of Amberlite The combined methanolic eluates are there with 1 η methanolic hydrochloric acid to a p _H 3.5 d "and in a rotary evaporator at max. 30 ° on small <volume concentrated (200 to 300 cm ³⁾ and stirred in a volume IOfaches Essigester. I precipitate is suction filtered, washed with ethyl acetate and ether and in vacuo at 20 to 25 °; dries. Yield: 5 g of yellow-brown antibiotic ao danubomycin in the form of the hydrochloride. b) The moist filter residue, consisting of; Mycelium and Hyflo (about 170 kg) is twice 1 170 1 of acetone stirred aktivi The antibiotic is (form säu »5, air conditioned to 70% acetone) on a column of Amberlite IRC-50 adsorbed. I as under a) obtained hydrochloride of the Ai biotic Danubomycin is contaminated with quite a lot of inorganic salts. Yield: 29.4 g gel powder. Example 7 6700 1 culture solution of a batch obtained according to Example 1 or the mixture is adjusted to Ph 4 <with hydrochloric acid. If the p _H Vt hour no longer changes, it is filtered after adding 2% Hyflo. I tor culture filtrate is at _pH 8.5 to 9.0 in a Extr with Essigester in the ratio 3: extracted. 1 'Antibiotic activity is, as described in Example 6, a), adsorbed on 4 1 Amberlite IRC-50 1 eluted. The yield is 85 g of yellow-brown Hyc chloride of the antibiotic danubomycin. More active material can be obtained from c filter cake by extraction with acetone. The spical rotation is [a]? = + 223 ° ± 8 (c = 0 ,: in methanol). The infrared spectrum recorded in Nujol. is shown in FIG. Claim: The use of Streptomyces gris A 9990 or one of its properties the microorganism of the genus Streptom) for the production of the antibiotic Danubomj by conventional biological cultivation, obtaining the antibiotic from the culture filtrate, pure < position and manufacture of its salts. 1 sheet of drawings