DE1129259B - Process for the production of a new antibiotic - Google Patents

Description

The present invention relates to the preparation of a new, water-soluble antibiotic which is im the following is designated with A 22765, its components, its salts and derivatives.

The antibiotic A 22 765 results from the culture of a new actinomycete strain belonging to the species Belongs to Streptomyces aureofaciens. It was isolated from a soil sample collected near Lucknow, India and is carried out in the inventor's laboratories as well as in the Federal Institute of Technology, Institute for special botany, under the designation strain A 22 765 and in the United States Department of Agriculture, Agricultural Research Service, Northern Utiligation Research and Development Division, Peoria, Illinois, stored under the designation strain NRRL 2858.

Streptomyces aureofaciens NRRL 2858 forms spores with a smooth to slightly warty surface. the Spore chains are monopodially branched, with open, irregular spirals of usually three to four turns. The mature aerial mycelium is colored ash gray. Caused when growing on peptone-containing culture media the strain NRRL 2858 no melanoid discoloration.

For further characterization, the growth of Streptomyces aureofaciens NRRL 2858 described on various nutrient media. The nutrient media 1 to 7 and 10 were according to W. Lindenbein, Arch. Mikrobiol., 17, p. 361 (1952).

Process for the manufacture of a new antibiotic

Synthetic agar:

Growth initially punctiform and white-yellow, later wrinkled and light yellow.

2. Synthetic solution:

Sediment punctiform to pustular, milky white.

3. Glucose agar:

Growth wrinkled, initially yellowish brown, later light brown; Substrate discolored light brown; Aerial mycelium velvety to sometimes not very woolly, white-gray.

4. Glucose asparagine agar:

Growth thin, veil-like, white-yellow; Aerial mycelium initially chalk white, later ash gray and forming a regular coating.

5. Calcium malate agar:

Growth thin, veil-like, initially milk-white, later white-yellow.

6. Gelatin prick (18 ⁰ C):

Very sparse growth, only slight liquefaction.

7. Starch plate: so growth pustular, milky white; Only traces of starch hydrolysis after 11 days.

Applicant: CIBA Aktiengesellschaft, Basel (Switzerland)

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

Claimed priority: Switzerland of March 17, 1960 (No. 3040)

Dr. Ernst Gäumann, Zurich,

Dr. Ernst Vischer, Basel,

and Dr. Hans Bickel, Binningen (Switzerland), have been named as the inventor

8. Potatoes:

Growth very slow, lichen-like, light brown; Aerial mycelium sparse, dusty, white-gray; Substrate partly discolored black-brown.

9. Carrots:
Growth sparse.

10. Litmus milk:

Growth slow at first, later very good; Ring growth black-brown, as does the substrate; weak peptonization, no coagulation observed; Color of litmus no more detectable because of the black discoloration of the medium.

The following Streptomyces species are known to be an ash-gray aerial mycelium and spirally twisted Form spore chains: S. noursei Brown et al., S. echinatus Corbaz et al., S. albogriseolus Benedict et al., S. macrosporeus Ettlinger et al., S. griseoflavus (Krainsky) Waksman et Henrici, S. pilosus Ettlinger et al., S. flaveolus (Waksman) Waksman et Henrici, S. hygroscopicus (Jensen) Waksman et Henrici, S. aureofaciens Duggar, S. parvulus Waksman et Gregory and S. galilaeus Ettlinger et al. (see Ettlinger et al., Arch. Mikrobiol., 31, p. 326 [1958]). Most of these species can be compared with one another electron micrographs of the spores as different from strain NRRL 2858. While the strain NRRL 2858 has spores with smooth to warty surfaces, one finds

209 5797259

Spores with appendages in the species S. noursei, S. echinatus, S. albogriseolus, S. macrosporeus, S. griseoflavus, S. pilosus and S. flaveolus. Also differs very clearly from strain NRRL 2858 S. hygroscopicus, namely through the distinctly closed, often almost knot-like spirals of the spore chains. S. galilaeus is also easy to distinguish from the strain NRRL 2858, since this species has both in the type of spirals (openly irregular in strain NRRL 2858, openly regular in S. galilaeus) as well as in the ability to form melanin on peptone-containing culture media (for strain NRRL 2858 absent, present in S. galilaeus) behaves differently. The two remaining species (S. parvulus and S. aureofaciens) are compared to strain A 22 765 in the following compilation:

S. parvulus smooth most of time S. aureofaciens Strain NRRL 2858 Surface of the spores ash gray smooth to warty smooth to warty Color of the air mycelium open, regular;
more than six
Turns ash gray ash gray Spore chain morphology
(Spiral type) is missing open, irregular;
usually less than four
Turns open, irregular;
usually three to four
Turns Melanin formation on peptone-containing
Culture media is missing is missing

It can be seen that the species S. parvulus and S. aureofaciens are essential in the morphology of the Distinguish spore chains. On the other hand, there is almost perfect agreement in all groups of characteristics of strain NRRL 2858 with S. aureofaciens. For this reason the strain NRRL 2858 provisionally included in the species S. aureofaciens Duggar.

A number of other strains isolated from various soil samples that also contain the antibiotic A 22765 produce, according to the above classification, also belong to the genus Streptomyces aureofaciens, for example the strains A 22083, A 22141, A 22652, A 22931, A 23269, A 23344, A 23978, A 24034, A 24066, A 24292, A 25536, A 25699, A 26043, A 26354, A 26392, A 26467 or A 26581, which are also listed under the mentioned Designation at the Swiss Federal Institute of Technology, Zurich, Institute for special Botany, are deposited.

It is already known that individual representatives of the genus S. aureofaciens produce antibiotics. So Tetracyclines (U.S. Patent 2,482,055), Spiramycins (Antibiotics Annual, 1954-55, p. 724), Miamycin (Antibiotics and Chemotherapy, 7, p. 37 [1957]), Heptaene (Antibiotics and Chemotherapy, 8, P. 491 [1958]) and congocidin (Riass. Della Comm. VI Congr. internat. Microbiol. [Roma], 1, p. 241 [1953]) produced by representatives of this species. As below is shown, but the new antibiotic A 22 765 differs from all these known substances in a characteristic way.

The present invention is not limited to the manufacture of antibiotic A 22 765 Use of Streptomyces aureofaciens NRRL 2858 or others as described Strains restricted, but also affects the use of variants of these organisms, such as them z. B. by selection or mutation, especially under the action of ultraviolet or X-rays or from nitrogen mustard oils.

The properties of Streptomyces aureofaciens are used to produce the antibiotic A 22 765 NRRL 2858 exhibiting Streptomycetenstamm in aqueous, containing a carbon source, nitrogenous compounds and inorganic salts Nutrient solution grown aerobically until it shows a substantial antibacterial effect and the antibiotic A 22765 insulated on it.

As an assimilable carbon source z. B. carbohydrates such as glucose, sucrose, lactose, Mannitol, starch, dextrin and glycerin are in question. As nitrogenous nutrients and possibly growth-promoting Substances are mentioned: amino acids, peptides and proteins as well as their breakdown products, such as Peptone or tryptone, also meat extracts, water-soluble parts of cereal grains such as corn and Wheat, distillation residues from alcohol production, yeast, beans, especially the soy plant, of seeds, for example the cotton plant, etc., but also ammonium salts and nitrates. The nutrient solution can contain other inorganic salts, for example chlorides, carbonates, sulfates of alkalis, alkaline earths, magnesium, iron, zinc and manganese.

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 18 and 40 ° C. An essential antibacterial The nutrient solution generally shows effect after IVa up to 5 days.

To isolate the antibiotic A 22765 you can z. B. serve the following procedures: The mycelium is separated from the culture feed, after which the main amount of the antibiotic is found in the culture feed.

Nevertheless, significant amounts of the antibiotic remain adsorbed on the mycelium. It is therefore advantageous to wash out the latter well. Water and aqueous organic solvents are suitable for this purpose, such as alcohols, e.g. B. aqueous methanol.

There are various methods of removing the antibiotic from the culture feed and purifying it suitable, which can be used individually or in combination with one another. It turned out to be Proven beneficial to the culture solution during these operations to a pH between 4.0 and 6.0 keep.

1. One can use adsorbents, e.g. B. activated carbons, such as Norit, activated earths, such as Fuller-

earth or floridine, or resin adsorbers such as asmit. The adsorbates are expediently eluted with Mixing of water-miscible organic solvents with water or aqueous acids, z. B. with water-methanol, water-pyridine, dilute acetic acid-methanol or water-methanol-glacial acetic acid-butanol mixtures. The mixture of water (2 parts by volume) and methanol has proven to be particularly advantageous for the elution of a noritol adsorbate (1 part by volume), glacial acetic acid (1 part by volume) and n-butanol (2 parts by volume).

2. A second method for separating the antibiotic from the culture nitrate is that the antibiotic is adsorbed on ion exchange resins, particularly those containing acid groups Resins such as Amberlite IRC-50 are suitable. The elution is best done with diluted acids,

z. B. with methanolic hydrochloric acid.

3. The basic antibiotic can also be precipitated directly from the culture filtrate, e.g. B. by reaction with an organic acid of the picric acid type. By treating this Precipitations with salts of organic bases, e.g. B. with triethylammonium sulfate, or with dilute acids the antibiotic is obtained in the form of the corresponding salt. You can do this both in aqueous Medium as well as in a water-miscible solvent, such as methanol or acetone, work. This conversion of the sparingly soluble salts into easily soluble salts of the antibiotic is either with mineral acids or with ion exchange resins,

z. B. on Amberlite IRA-400.

4. An enrichment of the antibiotic is also achieved in that one aqueous or alcoholic aqueous solutions of the salt with an excess of organic, water-miscible solvents such as acetone, dioxane, etc., added, the Salts are precipitated in solid form.

5. Another method of isolating and / or enriching the antibiotic consists in extracting aqueous solutions of the same with solutions of phenol in chloroform, both the pH of the aqueous solution and the phenol content of the chloroform solution being varied. So is the antibiotic z. B. with a distribution between a solution containing ^{100 g of phenol per 100 cm 3 of} chloroform, and an aqueous phase of pH 6 to 6 almost exclusively in the organic phase. If one understands by the distribution coefficient of the antibiotic the ratio of the concentration in the organic phase to the concentration in the aqueous phase, it follows that the distribution coefficient increases with increasing phenol content of the organic phase and decreases with decreasing pa of the aqueous phase. Since it is thus possible to set any arbitrary distribution coefficient of the antibiotic in this system, a large number of inactive impurities can be separated off by combining a few distribution operations.

6. Another enrichment method for the antibiotic is chromatography, such as adsorption chromatography on various materials, e.g. B. Norit, aluminum oxide, magnesium silicates, Silica gel, calcium sulfate, as well as partition chromatography with cellulose, starch, silica gel and the like as carrier substances or chromatography on ion exchange resins, e.g. B. at Dowex 50, Amberlite IRC-50, and the like.

7. Further, the antibiotic cannot, due to the countercurrent distribution according to Craig between two, not miscible solvent phases are enriched.

The antibiotic A 22765 belongs in addition to other well-known iron-containing antibiotics, such as. B. Grisein, albomycin and the ferrimycins, to the class of the sideramycins. Its antibiotic effects As with the other representatives of this class mentioned, the growth substances ferrioxamine (cf. HeIv. Chim. Act, 43, p. 2118 [1960]). As As can be seen from the table below, antibiotic A 22765 differs from the others Sideramycins clearly from paper chromatography:

Solvent system
(Volume ratio)

(in two-phase systems, the
organic phase used)

n-butanol — glacial acetic acid — water

(4: 1: 5)

n-butanol — glacial acetic acid — water

(4: 1: 2)

n-propanol — pyridine — water

(60: 4: 40)

n-propanol-acetic acid-2.5% ig ^it

NaCl in water (10: 1: 8) ... n-propanol — glacial acetic acid — water

(25: 2: 25).

n-butanol — ethanol — glacial acetic acid—

Water (25: 25: 3: 47)

Acetone — glacial acetic acid — water

(60: 3: 37)

Methanol-0, In-HCl in water

(3: 1)

n-butanol — glacial acetic acid — water ■

(1: 1: 2)

Rf values

the antibiotics

Ferric

A22765

0.25 0.35 0.50 0.65 0.69 0.70 0.72 0.75 0.85

mycm

0.48 0.56 0.52 0.76 0.73 0.75 0.75 0.62 0.87

Albomycin

0.13 0.18 0.31 0.50 0.54 0.50 0.59 0.52 0.77

The detection of the antibiotics on the paper is carried out bioautographically with Staphylococcus aurens or Bacillus subtilis.

The fortified antibiotic A 22765 is a light brown powder that can be found in water and others highly polar solvents such as methanol, dimethylformamide, dissolves well. It is also soluble in Phenol. It has basic properties and therefore forms salts with acids.

During electrophoresis on Whatman paper No. 1 in ¹ _{l 3} η-acetic acid, the antibiotic migrates 4.5 cm (ferrimycin A 8.1 cm; albomycin 4.5 cm) in 3 hours at 220 volts and 1.7 mA Direction of the cathode in relation to a neutral reference substance (fructose).

The IR spectrum in Nujol is shown in FIG.

The salts of the antibiotic A 22765 and its components are derived from the known inorganic ones and organic salts, for example from hydrochloric acid, sulfuric acids and phosphoric acids, acetic, propionic, valeric, palmitic or oleic acid, succinic acid, citric acid, mandelic acid, Glutamic acid or pantothenic acid. They represent neutral or acidic salts. Their production takes place by the action of the corresponding acids on the free base or by double conversion of Salts, for example of A 22765 sulfate with pantothenic acid calcium.

The free base of the antibiotic A 22765 is easily accessible from its salts, from the sulfate z. B.

by. Reaction in an aqueous medium with barium hydroxide, Neutralization of the excess barite with carbon dioxide and separation of the barium carbonate and sulfate precipitate and isolation of the free base by means of freeze-drying. Easier the preparation is carried out from the salts using a strongly basic anion exchanger, e.g. B. the OH form of the product sold under the name Dowex-2.

The antibiotic differs in characteristic Way of the above-mentioned known substances that are derived from strains of the genus Streptomyces aureofaciens are produced. The spiramycin and miamycin possess in contrast to

example 1

The cultivation of Streptomyces NRRL 2858 is carried out according to the submerged method. One uses a nutrient solution containing 20 g lactones, 20 g distillers solubles, 1 g Contains sodium nitrate and 5 g sodium chloride. The nutrient solution is in the inoculation flask or in the Fermenters sterilized for 20 to 30 minutes at 1 atm. The sterilized nutrient solution shows a Ph from 7.5 to 8.0. The tapping takes place with up to 10% of a partially sporulating vegetative culture of the organism. Incubate with good shaking or stirring at 27 °, with cultures in fermenters

new antibiotic very good solubility in 15 with about 2 volumes of sterile air per volume of solution

non-polar solvents, while the heptaenes are practically insoluble in water. The tetracyclines and congocidin are highly effective against gram-negative germs, which antibiotic 22765 is not the case is. Furthermore, none of these known antibiotics belong to the class of the sideramycins, d. that is, their antibiotic effect is not affected by the ferrioxamines (cf. HeIv. Chim. Act., 43, P. 2118 [1960]) antagonized.

The antibiotic A 22765 has a high antibiotic effectiveness against various Test organisms. In the so-called agar cross-line test, it is active against the following test organisms: Micrococcus pyogenes, var, aureus, Streptococcus viridans, Streptococcus faecalis, Bacillus megatherium and Bacillus subtilis.

The antibiotic A 22765 is also effective in vivo. When administered subcutaneously five times of 10 mg / kg infected with Staphylococcus pyogenes, var. aureus, or with Streptococcus haemolyticus Mice become 87 and 50% uod at one dose, respectively of five times 3.5 mg / kg, 50% survivors were observed in both cases. If you have mice that are with Pneumococcus type III are infected, applied five times 33 mg / kg subcutaneously or orally, one obtains 100 or 25% survivors.

The toxicity of the antibiotic is low. So z. B. the subcutaneous application of 1000 mg / kg of Mice endured without harm. Higher doses have not yet been tested.

The antibiotic A 22765, its components, its salts or derivatives can be used as remedies, z. B. in the form of pharmaceutical preparations, use. These contain the compounds mentioned in a mixture with a pharmaceutical suitable for enteral, parenteral or local application organic or inorganic carrier material. For the same, such substances come into question, who do not react with the new connections, such as B. gelatin, lactose, starch, magnesium stearate, Talc, vegetable oils, benzyl alcohol, gum, polyalkylene glycols, petroleum jelly, cholesterol or others known excipients. The pharmaceutical ■ preparations can z. B. as tablets, coated tablets, powder, 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. The temperatures are in degrees Celsius specified.

ventilated per minute. After 48 to 120 hours Incubation, the culture solution has the greatest inhibitory value against the test organisms (B. subtilis, B. megatherium, Micrococcus pyogenes, var. Aureus). Now interrupts the culture and separates the mycelium as well as other solid components from the solution containing the majority of the antibiotic Filtration or centrifugation, if necessary, the culture solution before the filtration about 1% a filter aid, e.g. B. Hyflo Supercel is added. The filter residues are washed with it Water and with aqueous methanol and combined the washing liquids with the culture filtrate.

If, instead of the nutrient solution given above, one uses those that per liter of tap water contain the following nutrients, culture filtrates are obtained after analogous cultivation and processing of similar high antibiotic effectiveness.

a) Rapeseed meal 20 g

Glucose 10 g

K ₂ HPO ₄ 0.2 g

Calcium carbonate Ig

b) glucose 10 g

Soy flour 10 g

Corn steep liquor ... 20 g

Sodium chloride 5 g

Sodium nitrate Ig

Calcium carbonate 10 g

c) glycerin 20 g

Soy flour 10 g

Sodium chloride 5 g

Sodium nitrate Ig

Calcium carbonate 10 g

d) glucose 10 g

Soy flour 10 g

Sodium chloride 5 g

Sodium nitrate Ig

e) lactose 20 g

Distiller solubles 20 g

Sodium chloride 5 g

Sodium nitrate Ig

If instead of Streptomyces NRRL 2858 the strain S. aureofaciens A 22 083, A 22 141, A 22 652, A 22 931, A 23 269, A 23 344, A 23 978, A24 034, A 24 066, A 24 292, A 25 536, A 25 699, A 26 043, A 26 354, A 26 392, A 26 467 or A 26 581 grown under the conditions described above, so cultures that are also antibiotically active are obtained after 48 to 120 hours.

Example 2

3 l of a culture filtrate obtained according to Example 1 are stirred with 60 g of Norit at 20 ° for 30 minutes. The mixture is then filtered and the antibiotic inactive filtrate is discarded. The norit residue is washed twice with 11% strength methanol and twice with 11% acetone with stirring. The washing solutions do not contain any activity. The antibiotically active material is then eluted from the charcoal by stirring three times with 200 cm ^{3 of} a mixture of n-butanol-glacial acetic acid-methanol-water in a volume ratio of 2: 1: 1: 2. The combined eluates (600 cm ³ ) are mixed with 600 cm ^{3 of} ethyl acetate and shaken, the entire antibiotic activity being in the excreted aqueous phase. This is concentrated in vacuo at 30 ° to a small volume and then lyophilized. 3.08 g of a brown, water-soluble powder are obtained which contain all of the antibiotic activity present in the culture filtrate. The enrichment with respect to the lyophilized culture filtrate is 40-fold.

Example 3

1 g of an antibiotic preparation obtained according to Example 2 is dissolved in 100 cm ^{3 of} water. ^{5 cm 3 of} saturated aqueous sodium chloride solution are added to this solution and the mixture is extracted three times with a mixture of phenol-chloroform (1 g: 1 cm ³ ). The entire activity goes into the phenol-chloroform phase. The phenol extract is filtered through Celite, mixed with 10 cm ^{3 of} water, 50 cm ^{3 of} ether and 100 cm ^{3 of} petroleum ether and shaken. The organic phase is washed with a few cubic centimeters of water. The combined aqueous solutions, which now contain the antibiotic activity, are washed with benzene and lyophilized. 171 mg of the enriched antibiotic A 22 765 are obtained in the form of a beige, water-soluble powder which has an antibiotic activity that is about four times greater than that of the starting material. The paper chromatographic behavior of antibiotic A 22 765 is listed in the following table. For comparison, the Rf values of the antibiotics albomycin ■ and ferrimycin A are also listed. The substances can be detected bioautographically with Staphylococcus aureus and Bacillus subtilis.

Example 4

20 g of a of Example 3 obtained antibiotic preparation (phenol-chloroform extract of culture filtrate) is dissolved in 400 ml of upper and lower phase of a system consisting of 2 kg of phenol, chloroform and 101 101 buffer p _H 5.9 (1244 g NaH ₂ PO ₄ · 2H ₂ O and 348 g of K ₂ HPO ₄ , made up to 101 with water), dissolved and distributed over 18 stages in a Craig apparatus. 400 ml of water are added to the contents of each unit, the mixture is stirred with 8 liters of petroleum ether and the aqueous phase is washed five times with 200 ml of phenol-free ether. The solutions are checked for Staphylococcus aureus in the plate test. The majority of the antibiotic activity is found in fractions 1 to 8. The solutions that belong together are mixed with 10% table salt. 200 ml of the aqueous phase are extracted six times with 10 ml of phenol-chloroform (1 g + 1 ml) each time and washed four times with 50 ml of 0.01 N hydrochloric acid 10% sodium chloride solution each time. The extract is dried over Celite, mixed with Hyflo and the active substance is precipitated with the addition of 2 volumes of ether and 5 volumes of petroleum ether. The Hyflo is washed phenol-free with ether, the antibiotic is eluted with methanol and the solvent is removed in vacuo. From elements 1 to 8, 817 mg of the enriched antibiotic A 22 765 are obtained in the form of a light brown, water-soluble powder which has an activity that is 8 times greater than that of the starting material used. In the UV ίο it shows final absorption at 220 to 230 ΐημ and a weak band at 330 πιμ (IR spectrum see Fig. 1). The substances isolated from elements 9 to 18 show lower activities than the starting material.

Example 5

According to Hirs (Hirs, Moore, Stein, J. Biol. Chem., 219, p. 623 [1956]) prepurified cation exchange resin Dowex 50 WX 2 (100/200 mesh) becomes in the ammonium form through sedimentation

ao brought a column 1.5 cm in diameter. Layer height: 45 cm. The resin is 4 days with 0.1 molar Ammonium acetate buffer pn 5.0 (37.0 ml glacial acetic acid, 37.5 ml 25% ammonia on 51 water). 350 mg of an antibiotic preparation obtained according to Example 4 are used as a 5% solution in 0, 1 molar buffer is applied to the column and the chromatogram is developed with this buffer, with this previously passed a feed vessel of 11 capacity. Throughput speed: 18 ml / hour. Fraction volume: 9 ml. By adding a buffer with a higher molarity to the above-mentioned mixing vessel a slow increase in ion concentration is achieved. For Fraction 86 and Fraction 134, the Concentration of the incoming buffer increased to 1 molar or 2 molar. The evaluation is carried out by Activity control in the plate test for Staphylococcus aureus. The extraction is on the same principle as carried out in the case of countercurrent distribution. 13 mg are isolated from fractions 83 to 117 143 to 197 20 mg of a light brown powder. An approximately three-fold increase in activity is achieved with both preparations. They show that described in Example 4 Absorption in the UV.

45 solvent system
(Volume ratio)

(in two-phase systems, the
organic phase used)

65 n-butanol — glacial acetic acid — water
(4: 1: 5)

n-butanol — glacial acetic acid — water
(4: 1: 2)

n-propanol — pyridine — water
(60: 4: 40)

n-propanol-acetic acid-2.5% ig ^it
NaCl in water (10: 1: 8).

n-propanol — glacial acetic acid — water
(25: 2: 25).

n-butanol — ethanol — glacial acetic acid — water (25: 25: 3: 47)

Acetone — glacial acetic acid — water
(60: 3: 37)

Methanol-Oin-HCl in water
(3: 1)

n-butanol — glacial acetic acid — water
(1: 1: 2)

Rf values

the antibiotics

Ferric

A 22 765

0.25
0.35
0.50
0.65
0.69
0.70
0.72
0.75
0.85

mycin

0.48
0.56
0.52
0.76
0.73
0.75
0.75
0.62

0.87

Albomycin

0.13 0.18 0.31 0.50 0.54 0.50 0.59 0.52 0.77

209 579/259

Claims (1)

Claim: The use of Streptomyces aureof aciens A 22 765 (NRRL 2858) or one of its properties exhibiting microorganism to produce production of the antibiotic A 22 765 or its components by conventional biological breeding, Obtaining the antibiotic from the culture filtrate, if necessary dividing it into components and pure display. 1 sheet of drawings © 209 57W25S 5.62