SU352469A1 - METHOD OF OBTAINING ANTIBIOTIC COMPLEX - Google Patents

Description

This invention relates to a biological method for the preparation of antibiotics.

The known method involves the cultivation of a producer from the class of Streptomycetes under aerobic conditions on a liquid nutrient medium containing sources of carbon, nitrogen and mineral salts at pH 5-8.8.

In order to obtain a complex of libomycin, consisting of libromycin A, libomycin B and libomycin C, Streptomyces libani p. Sp. Is taken as a producer. the cultivation is carried out under aerobic conditions on a liquid nutrient medium containing sources of carbon, nitrogen and mineral salts at 22-37 ° C for 72-160 hours and the antibiotic complex is isolated by known methods.

S. libani F.I are used for cultivation. 2343, F.I. 2399, F.I. 2501 and F.I. 2521, registered with the Institute of Microbiology of the Rutger University (USA) under the numbers LM.R.U.3915, I.M.R.U. 3916, I.M.R.U. 3917 and I.M.R.U. 3918 and at the Commonwealth Musical Institute, Ferry Lane, Kew, Serrey (UK) under the numbers I.M.I. 130777, I.M.I. 130778, I.M.I. 130779 and I.M.I. 131233.,

It has been found that in shaken liquid culture, they produce the antibiotic complex of libanomycin.

Strain F.I. 2343 was isolated from the sample; soil collected in Byblos (Lebanon), 5 F.I. 2399 - from a soil sample taken from the rice floor in Piedmont, strain F.I. 2501 - from a sample of soil collected in the Nieder-Beerbach-Zexheim (Germany).

Morphological features - common for

of all strains, while culture

The V individual strata are somewhat different.

Morphological features

In ordinary culture media, vegetative mycelium consists of more or less thin, long, abundantly branched hyphae 0.4-0.8 microns thick, forming thicker hyphae 1-1.5 microns thick. On these hyphae there are short lateral and thick spiral branches alternating or located opposite each other with monopolial branching. These small branches are further transformed into chains of spores, which are connected at the beginning and then free. When observed under an electron microscope, the spores have a smooth surface, which is oval or typical reniform, and often spherical.

Table 1

Continuation

Cultural and biochemical properties

In tab. 1 and the cultural properties of each examined strain grown in test tubes and Petri dishes at 28 ° C. Observations were made at 6.15 and 25 days after inoculation. Cultural characteristics differ little from individual strains. Aerial mycelium is quite smooth in strain F.I. 2343, and sometimes the strain F.I. 2501, and vice versa, cotton-like as a result of the formation of very long sporophore hyphae in F.I. 2399 and F.L 2521.

The color of the aerial mycelium is white in all strains on some organic media and preimush, naturally light brown on other organic and synthetic media, but on the latter strains of F.I. 2399 and F.I. 2521 may also have shades of gray. In addition, the strain F.I. 2501 differs from the other three strains in that it produces a red-pink color pigment, which to a certain extent also causes the color of the vegetative mycelium and, therefore, the color of the aerial mycelium.

In addition, the strain F.I. 2501 differs from the other three strains in that it assimilates 1-arabinose and does not thin the gelatin.

These strains do not reduce nitrates to nitrites; neither melanin nor hydrogen sulfide is produced. They hydrolyze starch, liquefy gelatin and use tyrosine. Strains F.I. 2343 and F.I. 2521 peptonized and coagulated milk. Strains F.I. 2399 and F.I. 2501 coagulate milk but do not peptonize it.

Glucose, sucrose, d-xylose, meso-inositol, d-mannose, d-fructose, maltose, and raffinose are used to grow the strains.

All four strains did not grow at 50 ° C and did not produce sclerotia; in deep and shaken media they produce an antibiotic complex of libonomy.

Identification of strains

According to the given properties, the microorganisms considered can be attributed to the genus Streptomyces, Waksman et Henrici (Bargeys Mapza of Determinative Bacteriologi, 1957, 744-745; to the section “Spira no Pridham ef al.

(Appl. Microbiol., 1958, p. 5). However, these strains cannot be attributed to any of the series and, therefore, to any of the species listed in this section. In fact, in this taxon, the seri is inclusive, and the brown aerial mycelium is characteristic of strains F.I. 2343, F.I. 2399, F.I. 2501 and F.I. 2521, not considered. These microorganisms can not be attributed to any of the systematic groups proposed by Waksman

(The Actinomycetes, 1961, 2, 111, and 117), as well as to any of the groups proposed by Baldacci (Giorn. Microbiol, 1958, 6, 10), because in these taxa the series or groups include the yellow vegetative mycelium and aerial mycelium brown, absent. For the same reason, they cannot be attributed either to the systematic groups proposed by Flaig und und Kutzner (Arch. Mikrobiol, 35, 1960, 105-138), or to the species described by Krasssilnikov (Guide for the identification of bacteria and actinomycetes , 1949, Moksva).

The microorganisms in question should be attributed to a new species, for which the name Streptomyces libani n. Sp.

For successive planting, the microorganisms are stored in a suitable solid medium for or by lyophilization of their spores per mole.

The carbon source can be glucose, dextrin, starch, flour (soybean, corn, wheat, etc.), molasses, corn extract, various vegetable oils, and other substances commonly used in fermentation processes. In addition to the above-mentioned complex nitrogen-containing substances, casein, casein hydrolyzate, bard and ammonium salts such as sulfate, phosphate, ammonium chloride, and other substances commonly used for this purpose can serve as a source of nitrogen. The choice of mineral salts depends on the medium used. Calcium carbonate is almost always used. Chloride, sulphate or sodium phosphate, salts of potassium, magnesium, iron, zinc and copper can also be added.

Fermentation can be carried out in Erlenmeyer flasks, in laboratory, factory fermenters of various capacities.

Kompleks lybanomycin consists of three substances with very similar physico-chemical and biological properties. These substances are named libanomycin A, libanomycin B and libanomycin C.

The antibiotic activity of broths and raw products is determined on a sensitive microorganism and compared with the activity of samples containing significant amounts of antibiotic.

The content of the antibiotic in the feedstock is also determined by spectrophotometry of methanol solutions at 284 mmk.

At the end of fermentation, the antibiotic complex of libanomycin, which is predominantly in the mycelium, is extracted with a suitable solvent, for example, alcohol or aqueous acetone, and precipitated. The resulting precipitate of the antibiotic complex is purified and separated into libapomitiums A, B and C by chromatography.

For example, the mycelium is extracted with alcohol or aqueous acetone, the extracts are concentrated to a small volume, the crude product is precipitated, which is washed with water, dried with calcium chloride and washed with anhydrous acetone. After washing the raw product, the floating layer and the washings are poured into the filtered broths, to isolate the active product from which butyl alcohol is used at pH 7.5-8.5 or cation-exchange carboxyl resin in the H-form. The obtained butanol extracts, after washing with water and evaporation to a small volume, are treated with acetone or diethyl ether to obtain the active crude product. When using resins after adsorption and washing of the resin with water and 0.5N. ammonia solution, the active product is eluted with a water-alcohol solution of the salt, for example with a 3% solution of sodium chloride in methanol (1: 3).

butyl alcohol at pH 7.5-8.5 and precipitating the extract with acetone.

The resulting crude product (concentration) is dissolved in a low molecular weight alcohol and chromatographed on a column filled with neutral activated alumina or silica gel. Alcohol is used to elute. Libiomycin B is first obtained, and then libanomycins A and C. Antibiotics are isolated from the eluates in the form of a white amorphous powder containing 90-95% of the active ingredient.

For analysis, antibiotic samples are passed through a column filled with cellulose filling, eluting with a mixture of butanol-pyridine-water (6: 4: 3).

Libomination A is an amorphous powder, m.p. 152-154 ° C (decomposition); a 4-62.5 ° (, methanol). The absorption bands listed in Table 2 were found in the UV spectrum of the antibiotic. 2

table 2

ji%

Xjiax, MMK

Wednesday 1 cm

100, 107

244 and 284,100, 102

245 and 283

at me101

me284

70

kink at 255

Found,%: C 63.12; H 8.05; N 6.49.

Reactions with ResC1, KMp04, Br2 are positive. In the presence of conc. sulfuric acid antibiotic has a blue color that turns into purple.

The reactions of Fehling, Ningridrin and Sakaguchi are negative. In paper chromatography, antibiotic is detected by Burton's reagents for phenols and enols, and Pan-Dutcher's reagents for amides.

With Ehrlich reagents, he gives. Yellow staining.

Libanomycin A is highly soluble in aqueous debris, aqueous acetone, pyridine, DMF and DMSO; dissolves in methanol (20 mg / ml) and ethanol (4.5 mg / ml) and poorly soluble in water (1 mg / ml at 20 ° C and 5 mg / ml 0 at 50 ° C). It does not dissolve in acetone, chloroform, benzene, ethyl ether, and petroleum ether.

This product is rapidly activated in acids, stable at neutral or weakly pH, in the light, at room temperature.

Libanomycin B is a white amorphous powder, m.p. 155-160 ° C (decomposition); a -f72 ° (, methanol). 0 Found,%: C 64.34; H 8.42; N 6.77.

The UV spectrum and Jipylnie physicochemical properties of libomycin B are the same as those of libomycin A, with the exception of solubility in ethyl alcohol, which is higher for Li5 baiomycin B and is 10 mg / ml. The UV spectrum of libanomycin C does not differ from the corresponding spectra of libomycin A and B. When chromatography is performed on paper at room temperature, antibiotics reveal bioautography on you. subtilis. The reagents used and Rt for the corresponding libanomycins are listed in Table 3. Table 3 The antibiotic, either as a complex of libanomycin, or in the form of one of its three components - libanomycin A, libanomycin B and libanomycin C, is very active in animals and it is advisable to add it to feed. Pharmacology. Table 4 shows the values of the microbiological activity of LIBANOMYCIN complex (in a solution of 1000 µg, 1 ml) in solid medium. Table 4 The diameter of the stain is inhibitor Microorganism vania, mm S. aureus B. subtilis P. vulgaris Mycobacterium sp., Strain 607 Mycobacterium phlei Candida albicans Toxicity of libanomycin in mice 1 g / kg for oral administration and 25 lgg / kg for intravenous administration. Lybanomycin activity was determined empirically on 180 chickens when kept in cages. Chicks are divided into three groups of 30 chickens and petunches in each. The first receives the main feed, the second - the main feed and 1 g, 1 c of libanomycin, a third - the main feed and 2 zju, libanomycin. The composition of the main feed (in%): Corn62,118 Dehydration of an enepa alfalfa2,000 Dicalcium phosphate0,800 Calcium carbonate0,950 Sodium chloride0,400 Gabbomix P without antibiotics0,500 s / -Metionin0,032 the composition of the feed (in%): Water11,78 Crude Protein3.75 Fiber2.72 Ash5.07 Non-nitrogen Extractives55.93 Calcium0.96 Phosphorus 0.72 Duration of experience 30 days. The results are given in table. 5 .. Table 5 Example I. Two 300 l flasks filled in 60 ml of medium containing (in%): Dextrin 3.00 Casein 0.50 Corn extract 0.30 Calcium carbonate 0.40 Ammonium sulphate 0.10 Potassium phosphate 0.01 Water tap Up to 100 Flasks sterplizuyut 20 .shish at 120 ° C. RP environment after sterilization of 6.8-7. To each flask, 2 ml of spore suspension is added, which is formed by washing with 5 ml of distilled water from the culture of the plant obtained from a 20-day F.I. 2343 grown on a mixture of potato-glucose-agar in a test tube with an oblique cut. Flasks spaced on a rotary agitator (eccentricity 3 cm, speed 225 rpm) are incubated for 48 hours and 2 ml of culture are inoculated into other flasks with a capacity of goiter ml containing 60 ml of the production medium of the following composition (in%): Starch Sulfuric acid and calcium carbonate Calcium carbonate Fat-free soy Myokolyphosphate Sodium chloride Magnesium sulphate Zinc sulphate Ferrous sulphate Ferrous sulphate copper Water plumbing

eleven

The solution is sterilized for 20 minutes at 120 ° C. After sterilization, pH 5.8. The culture was incubated at 28 ° C with shaking as indicated in Example 1, after 120 hours, 150 µg / ml of product was obtained. 10 L of broth broth is filtered through a 2% Supersel, washed with water and the washing liquid is poured into the filtered broth.

The wet mycelium is extracted first with 3 liters of methanol, and then 2 liters of 80% aqueous methanol.

The mixed extracts are concentrated in vacuo to 1/10 (500 L1l) of the original volume. The precipitate is separated, washed with water, dried overnight in vacuum over calcium chloride at 20 ° C and for 4 hours at 56 ° C over phosphoric anhydride. A slurry of yellow-brown and powder in anhydrous acetone is filtered and the precipitate is dried. Obtain 1.2 g of a 70% complex of libanomycin.

The filtered broth was alkalized to pH 8.5 with 10% sodium carbonate, shaken for 1 hour, filtered and the filtrate (11 L) was extracted twice with 1 vol. butyl alcohol. The extract is washed with water, concentrated in vacuo to 50 ml with the addition of 10 vol. aceto.na. The precipitate obtained is washed with acetone and dried. - 0.500 g of a 70% active crude product is obtained from the filtered broth.

Example 2. The same operations are repeated as in Example 1, but the productive phase is carried out on a medium containing (in%):

Dextrin4.00

Casein1,00

Corn extract1,00

Ammonium sulphate0.20

Calcium carbonate 0.50

Dikaliyfosfat0,01 Water plumbing Up to 100

The solution is sterilized for 20 minutes at 120 ° C. After sterilization, pH 6.7-7. The solution was incubated at 28 ° C with shaking as in Example 1. After 120 hours, 120 µg / ml of product was obtained.

Example 3. The order of operations is the same as in example 1, but the productive phase is carried out on a medium containing (in%):

Starch2.00

Barda 1.75

Sodium Chloride0.25

Fat-free soy flour 3,00 Soybean oil0,8 ml

Piped water Up to 100.

The pH is adjusted to 7.2 with caustic soda; after sterilization, the pH is about 6.7.

The solution was sterilized for 20 minutes at 120 ° C, thermostatic at 28 ° C with shaking, as in Example 1. After 120 hours of incubation, 100 µg / ml product was obtained.

Example 4. In a 2000 ml flask with three grooves outside, forming three ribs inside the flask, and a side neck for the floor.

Cultivation is poured over 500 ml of medium specified in Example 1 for the vegetative stage and sterilized for 20 minutes. After cooling, the flask is inoculated with a spore suspension, obtained by washing with sterile distilled water with a coating of three cultures in a test tube.

The flask was thermostatted at 28 ° C and agitated (speed 120 rpm, eccentricity 45 mm) for 38 hours, 50 ml and a growing vegetative culture was inoculated with 3 liters of medium, filled into 5-liter glass fermenters and having the following composition (% ):

Dextrin4.0

Casein1,0 Corn extract

 (500 / oy) 1.0

Calcium carbonate0,5

Ammonium sulphate 0.1

Dikaliyfosfat0,1 Water plumbing Up to 100.

The broth is shaken at a speed of 400 rev / lsh and rinsed with 1 rev. air on 1 about. medium in 1 min at 27 ° C for 24 hours. At the end of this period, 3 liters of mycelial suspension are collected and inoculated in 50. / G of fermentation medium, which contains (in%):

Starch4.0

Casein1,0

Soyeva flour3.0

Beet molasses 0,2

Calcium carbonate0,4

Magnesium sulphate0.1 Water piped Up to 100.

The medium is sterilized for 30 min. With a 12GS iri and inoculated with shaking at a speed of 200 rpm and blown through 0.7 l of air per 1 vol. medium in 1 min at 28 ° C for 110 hours. 110 μg / ml of antibiotic are obtained. 50 l of the resulting fermentable broth is filtered through a 2% Supersel. The wet mycelium is extracted with 15 liters of methanol and 10 liters of 80% aqueous methanol. The extracts are evaporated to 4 liters.

5 The precipitate is separated, washed with water, dried, suspended in anhydrous acetone, filtered and dried. 11.30 g of a 60% crude product are obtained. Uterine solutions and immature waters are poured to the filtered broth. Calcium is removed, bringing the pH of the solution (55 l) to 6 with caustic li li added carbonate sodium to rp 9.2. The mixture is shaken for 1 hour, 100 g of infusorial earth is added and filtered.

5 to remove calcium carbonate. After this, the RP of the filtrate is adjusted to 6 with hydrogen chloride, passed through a column containing 2 liters of Amberlite IRC in hydrochloric form. The resin is washed with 5 liters of water.

5 liters 0.5 II. ammonia solution and 5 ug of water.

Elution is carried out with a mixture of 3 vol. methanol and 1 vol. 3% aqueous solution of chloride iatri. 6 l of active eluate is obtained, which is heated to 1.5 l in vacuum,

are double 1 vol. butyl alcohol. The extract is washed with water and concentrated to 100 lgl. After adding 10 vol. acetone is separated, the precipitate formed is washed, dried, and 3.25 g of a 60% crude product are obtained.

14.40 g of crude product are suspended in 200 g of methanol. The precipitate of the inactive fraction (1.40 g) is separated, the filtrate is passed through a column containing 300 g of neutral alumina, stirred in methanol, washed with 3 liters of methanol and eluted with 80% aqueous methanol. Collect fractions of 100 ml, followed by elution by a spectrophotometer (reading readings at 244 and-284 mmk), and then analyzed by paper chromatography, using butanol-pyridine-water as a solvent. For the manifestation of active components using the method of bioautography on you. subtilis.

The first 20 fractions contain libomycin

B (Rf 0.78), and fractions from 21 to 30 contain

a mixture of libanomycin A n B (Rt 0.65 and Rf 0.78).

The fractions from 31 to 50 contain libiomycin A and C (Rt 0.65 and Rr 0.40).

Each group of fractions is evaporated separately in vacuo to dryness. The residue is dissolved in ethyl alcohol, filtered, concentrated to a small volume and precipitated 10 vol. acetone. The precipitates are dried in vacuo over phosphoric anhydride. 1.80 g of libimycin B (90%), 1.70 g of libiomycin B and A (90%) and 3.80 g of libanomycin A n C (90%) are obtained.

A solution of 1.40 g of libanomycin A, also containing libanomycin B and C, in ethyl alcohol (96 °) is passed through 25 g of cellulose powder, dried overnight in vacuum over calcium chloride and placed in a chromatographic glass column containing 225 g of powder pulp. The mixture was eluted with a mixture of butanol-pyridine-water (6: 4: 3), collecting 10 ml fractions, which were analyzed by paper chromatography, revealing the active components by bio-chromatography of chromatograms on plates of agar inoculated by you. subtilis.

Fractions 5–7 contain libanomycin B, fractions 8–16 libanomycin A and B, fractions 17–24 libanomycin A, fractions 25–40 libonomy A and traces of lybanomycin C and fractions 41–45 libanomycin C. Distilled water is added to each group of fractions and petroleum ether. The aqueous phase is extracted with 1 vol. petroleum ether, repeat one ration three times, add ethyl alcohol, evaporate to dryness, dissolve the residue in ethyl alcohol, filter the resulting solution and concentrate to half the original volume. Added 10 about. anhydrous acetone, precipitated antibiotics in amorphous form. The precipitate is filtered off, washed with anhydrous acetone and dried over phosphoric anhydride in vacuum at 56 ° C. Get 30 mg of libanomycin B; 120 mg of libanomycin

A, containing traces of lybanomycin B, 640 mg of libanomycin A, 150 mg of libanomycin A, containing traces of libanomycin C, and 5 mg of libanomycin C.

Example 5. In a 80-liter fermenter nz stainless steel pour in 50 l of the medium described in example 4, and sterilized for 30 min at 121 ° C. At 27 ° C, the fermenter is inoculated with 500 ml of a vegetative culture grown on the same medium in the flask described in Example 4.

The fermenter is blown 0.7 vol. air on 1 about. medium and shake 25 hours. During this time, the RP was adjusted to 6.7 and a broth prepared for seeding was obtained.

The composition of the fermented medium (%):

Starch Glucose

Calcium carbonate Soya flour Magnesium sulphate Water water supply

500 liters of medium were sterilized for 20 minutes at 12 ° C, after cooling to 27 ° C, 25 liters of the above-described vegetative culture were seeded, the mixture was shaken at a speed of 210 and rinsed with 0.65 vol. air on 1 about. Wednesday in 1 min for 87 hours. Get 120 μg / ml of antibiotic.

450 liters of broth is filtered on a filter press using a 3% SuperCell. Washed with water, but still moist mycelium (86 kg) is extracted twice with 360 l of 90% aqueous metaiol. The extracts are concentrated to 100 liters in vacuo at 40-45 ° C. The RP of the concentrate obtained is adjusted to 8.5. The concentrate is extracted first with 70 liters and then with 20 liters of butyl alcohol. The combined extracts are washed with water and concentrated to 8 liters in vacuo. The precipitate is filtered, washed with acetone and dried. Obtain 180 g of a 20% crude product containing libanomycin A, B and C.

Further concentration of the resulting butaiol extract to 1 L and subsequent precipitation with 10 L of acetone makes it possible to additionally obtain 208 g of a 20% crude product consisting of a mixture of three antibiotics.

The filtered broth and wash water (500 L) are shifted, the pH is adjusted to 8.5, extracted twice with 280 L of butyl alcohol, the resulting extract is washed with water and concentrated to 25 L in vacuo. The inactive precipitate is separated, the filtrate is concentrated to 2 liters, and the volume is 10 vol. acetone, the precipitate obtained is washed with acetone, dried and obtain 75 g of a 20% crude product containing libanomycin A, B, and C.

Subject invention

1. A method for producing an angiotic complex, which includes cultivating a producer of the Streptomycetes class under aerobic conditions on a liquid nutrient medium containing sources of carbon, nitrogen and mineral salts, at a pH of 5-8.8, characterized in that, in order to obtain a complex of lybanomycin consisting of libanomycin A, libapomycin B and libanomycin C, as a producer, take Streptomyces libani p. sp., a5 cultivation is carried out at 22-37 ° C for 72-160 hours and isolate the antibiotic complex by known methods, 2. The method according to claim 1, wherein using Streptomyces libani F. strains. I. 2343, F.I. 2399, F.I. 2501 or F.I. 2521 (Societa Farmaceutici Italia).