DD207222A1 - PROCESS FOR PREPARING A POLYETHERANT ANTIBIOTICS COMPLEX - Google Patents

Abstract

The invention relates to a process for the preparation of a novel antibiotic complex which is active against Gram-positive bacteria and some fungi and protozoa and poteniel l is suitable for use as a coccidiostat and / or ergotricant in livestock husbandry. In particular, the invention relates to a method for obtaining the polyether antibiotic complex A-111 using the microorganism Actinomyces hygroscopius, strain 111. The aim of the invention is the production of a potentially coccidiostatic and / or ergotropic antibiotic complex, consisting of 4 components so far not described polyether antibiotics. The object is achieved by the fact that by aerobic submerged fermentation of the strain 111 in media with suitable starting materials of the polyether-antibiotic complex A-111 formed, isolated by suitable methods from the mycelium, purified and, if necessary, subsequently in the components A-111-1 , A-111-2, A-111-3 and A-111-4.

Description

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Title of the invention Process for the preparation of a polyether antibiotic complex Field of application of the invention

The invention relates to a method for producing an antibiotic complex which belongs to the group of polyether antibiotics which are active mainly against Gram-positive bacteria and protozoa and because of their ooooidiostatisohen, ergo tropic and cardiac stimulating effect in livestock and for veterinary medicine are of use "(Westley, JL # r · Appl. Miorobiol. 22, 177-223 (1977)).

Characteristic of the known technical solutions

Polyether antibiotics belong to a class of compounds called ionophores because of their ability to react with Alkalibzw. Alkaline earth ions to form lipid-soluble complexes, which allows the penetration of ions through the lipid barrier of the cell membrane ».

There are about 40 polyether antibiotics known in the literature, including monensin (J * Amer Ghem · soc, 8, 5737, 1967), Nigeria (Biochenu Biophys, Ees, Commun, 22, 29, 1968), Dianemyol (J). Antibiot., 2χ, 814, 1974), X-537A (J, Chem. Soo. Chem. Commun. 1971, 927), A 204A (Amer. Chem. SooG £ 3399, 1973), salinomycin (Tetrahedron Lett. 4, 4955, 1973). So far, the polyether antibiotics have been isolated from various members of the genus Streptomyoes (Aotinomyoes), e.g. B. hygrosoopicus, Str. Albus, Str. Griseus, Str. Aureofaoiens, Str. Cinnamonensis u. a · ·

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Monensin, Lasslooid (X-537a) and Salmonomyoin are currently used as cocoidiostats from the range of polyether antibiotics listed above and are important agents in industrial poultry production. In addition, monensin in polygastric animals and more recently salinomycin in monogastric animals (especially pigs) are used as ergotropics · The worldwide use of these introduced into animal nutrition antibiotics carries the risk of diminishing the effect due to resistance of microorganisms of the intestine or rumen flora. Therefore, it is necessary to seek new representatives of this very effective substance class, which represent chemically new structures or derivatives of known compounds. "

Object of the invention

The aim of the invention is the production of an antibiotisoihen complex) which consists of 4 components, which belong to the group of the polyether-antibiotics. Duroh this new antibiotic-complex the range of this Wirkatoff group is extended and supplemented »

Explanation of the essence of the invention

The invention has for its object to describe a method by which a new polyether-antibiotic complex can be obtained with the aid of a previously unknown microorganism from readily available and inexpensive Sinsatzstoffenv

According to the invention, this object is achieved in that under aerobic and sterile culture conditions in a liquid nutrient medium with appropriate carbon and nitrogen sources and mineral salts, a microorganism of the species Aotiaomyoes hygrosoopious or its mutants or variants are fermented at a temperature from 25 ⁰ C to 32 ⁰ C, preferably 28 ⁰ C, carried out during a period of 2 to 8, preferably 6 legends * The new polyether antibiotic complex formed in the name

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A-111 is extracted from the wet% cel and then concentrated and precipitated by known methods. If appropriate, this complex is finally separated by fractional sedimentation with acetone and / or subsequent chromatography into the four individual components 1-111-1, A-111-2, Δ-111-3, A-111-4. The microorganism used in the method according to the invention, which is described in detail below, was isolated from soil in the north of the PR of Bulgaria and is deposited in the Bulgarian ML KrOben collection in the State Institute for Drug Control (DIKLS) Sofia under number 111 *

Morphological characteristics of the strain 111 »The stem has spiral spore carriers densely arranged in the form of grapes with three or more turns« The spores have a rough surface *

Cultural palerism of strain 111 on different media: The actinomycete strain grows well on synthetic and organic media. Depending on the carbon and nitrogen sources and the composition of the salts, the color of the aerial mycelium varies from white to light gray, dark gray and dark gray-purple and lyses to black. The substrate mycelium changes from cream-colored, pale-yellow to brown-yellow. The color of the aerial and substrate mycelium was determined according to the color scale of A. S. Bondarzev (Publishing House of the Academy of Sciences of the USSR, Moscow, 1954 ·).

Yeast Extract-Malt Extract Agar (ISP # 2): Good Growth. The aerial mycelium in young cultures is white (d3). Later it turns pale gray (a6). The substrate mycelium is pale yellow (j5) to coffee brown (d4). No soluble pigment · Oatmeal agar (ISP No. 3): Growth very good. · The aerial mycelium is pale gray Ca6) ^ to dark gray and dark gray-violet (a2-n2). The substrate mycelium yellow to pale brown (p5 - ^> · No soluble pigment.

Starch Salt Agar (ISP # 4): Growth very good. Aerial mycelium gray, from dark gray to dark gray-violet (w4-a2-n2) _e substrate my-

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yellow to pale brown (p5-w4). No soluble pigment · glycerol-asparagine agar (ISP No. 5) ι growth very good, aerial mycelium pale gray, ash gray to dark gray (a6-k2-a2) · substrate mycelia yellow-green to yellow-brown (b3~d4). No soluble pigment. Peptone Yeast Extract Sage Agar (ISP No. 6): Growth mediocre. Aerial mycelium white to pale gray (cH-a6), substratum yellow-brown (d4) · soluble pigment pale brown (b4)). Tyrosine Agar (ISP No. 7): Mediocre Growth * Sparse aerial mycelium - pale gray (a6) · Substrate mycelium Yellow-brown to dark brown (b7- & 5) · Forms soluble pigment pink on the 7th day of cultivation and later dark brown to black. ;

SRI-glucose Aggar CKrassilnikov N.A., 1950): Growth very good · aerial mycelium dependent on age gray, dark gray to black (w4-a4-al). Substrate mycelium yellow, yellow-brown to brown (p5-d4-w77 · no soluble pigment

Tmeralagar No. 1 (Ga ^ -se, G.F., and Mltarb., 1957): growth very good. · aerial mycelium pale-gray in young cultures and pale-gray in long-cultivated dark gray and black (a6-a2-a1) substrate mycelium. yellow to light brown (j6-b4) \ No soluble pigment · Afeisextrakt-Agar: Growth very good · aerial mycelium gray, ash-gray, (w4 ~ k2). Substrate mycelium yellow-brown «Μ · · ·. No soluble pigment · Capek-agar CBaccharose): Growth is not favorable · aerial mycelium white to white-gray (d1-a6) · substrate mycelium colorless to creamy colored C06) «, no soluble pigment.

Glucose-asparagine agar: growth very good. Aerial mycelium ash gray, gray, dark gray (k2-w4-a2) · Substrate mycelium yellow-brown (d4) · No soluble pigment ·

Haltose-Trypton-Agart: Growth good »aerial mycelium white to light gray (d1-a6). Substrate mycelium off-white to yellow (b6-p5) · No soluble pigment.

Casein Starch Agar: Growth very good · aerial mycelium gray, dark gray; dark gray-violet (w4-a2-n2) · substrate mycelium yellow-brown (d4). No soluble pigment.

 Benett agar: growth good. Aerial mycelium pale gray to dark gray

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(a6-a2) · Subetratmyoel cream to pale sand (b6- »k3). No soluble pigment «

SR II (Krassilnikov N "A", 1950): growth sohwaoh * aerial mycelium white (d3). Substrate mycelium off-white (b6) · No soluble pigment «

Potato agar with 1 $ glucose; Growth good «aerial mycelium gray to dark gray (w4-a2) _a substrate juniper, yellow-brown (d4)« no soluble pigment »

ileisohpepton agar: Growth good · No aerial mycelium · Suhstratmyoel yellow to pale brown (p5-d4) · No soluble pigment «Physiological-biochemical properties Aotinomyoeten strain 111 liquefies gelatin and peptonizes milk to form brown pigment · Br hydrolyzes well starch, decomposes but no cellulose «Br reduces nitrate to nitrite and forms melanin · The strain makes good use of glucose, galactose, fructose, xylose, mannose, eamnose, arabinose, mannitol, maltose, lactose, saffinose, sorbitol, glycerine · The utilization of sucrose, dulcitol, Inositol, inulin and exulin are low or completely absent

Antibiotic activity: The antimicrobial spectrum is determined against a certain number of microorganisms using the agar diffusion method after 6 days of cultivation of the image generator on three nutrient media - soybean agar, mineral agar and SRI with glucose. Actinomyoes 111 inhibits the development of the following test cultures: Staphylococcus aureus 209 Baoillus subtilis L-2, Bacillus idosus, Bacillus myoides, Bacillus pseudoanthracis, Microooccus luteus 9341 (formerly sarcina lutea 9341), Kyoobacterium B5, Candida albioans, Penisillium chrysogenum, Botrytis oinerea, Pusarium graminearum A comparison of minimum inhibitory concentrations (MIC ) of polyether antibiotic Complex A-111 with monensin and salinomycin (Table 3) shows that A-111 is characterized by significantly higher activity against Gram-positive bacteria than the control preparations. "The strain 111 is mesophilic and aerophilic. The optimal temperature for its evolution is 25 to 32 ⁰ C. The strain becomes kept on the culture medium SRI and maize agar · The Actinomyoes 111 is similar to some morphological and physiological-biochemical properties of the species Streptomyces hygroscopicus. In recent years, in the literature under the airtype

tomyoes

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hygroscopicus many strains have been described that differ in the morphological, cultural and especially physiological-biochemical properties, but especially in the exploitation of various carbon sources and their antibiotic spectrum, from the first described Actinomycetenstamm Actinomyces hygroscopicus, Jensen, 1931 very significant also applies to Actinomyces 111 * Comparing the data obtained, Actinomyces 111 is closest to Streptomyces hygroscopicus Shogi et al., 1968, Syn. Actinomyces polyetherinus (Krassilnikov N, A, 1970). However, it differs in some morphological, Cultural and physiological-biochemical properties. In contrast to Actinomyces polyetherinus, it has spores with a rough surface. It forms melanin, utilizes sorbitol and produces various antibiotic substances. The strain m is as variable as the strains of the species Streptomyces hygroscopicus described in the literature. Up to 5 are obtained on some nutrient media and more variants · In some variants, the aerial mycelium is very well developed and typical of the species hygroscopicus; it is gray, dark gray to black as a result of the lysis occurring · in the other variants it is white or light gray · asporogenous variants or those with poorly developed aerial mycelium have been found ·

Based on the obtained morphological, cultural and physiological-biochemical data, we expect the Actinomyces 111 to be the collective species Actinomyces hygroscopicus »

Actinomyces III produces the following total antibiotic substances:.

1 »polyether complex consisting of 4 individual components · 2 · azalomycin B: identical to the description in the literature (Arai, M., J. Antibiotics 13 (1960), 46-50, 51-56)) 3 · Fungistatic.es antibiotic; Hexaen antibiotic according to data from UV spectrometry ·

4. Complex with antibacterial and fungistatic action consisting of 5 components ·

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In a further expansion, the process for the preparation of the polyether Kompiexes consists of the following steps: In 300 ml Erlenmeyer flasks 50 ml liquid medium are filled and inoculated after sterilization with a spore suspension of 10 to 20 days cultures of Actinomycetes strain 111. The cultivation is carried out on a shaker at 180 to 200 rpm and a temperature of 2? to 32 ⁰ C in the course of from 36 to 40 h · performed vegetative The recovered material is transmitted in ^ l-Alder ^ flasks filled with 500 mL culture medium. The cultivation is carried out under the above conditions for 6 days. The perforation process is terminated after the maximum antibiotic activity has been reached, which is determined by the agar diffusion method with the test cultures Bacillus subtills and Staphylococcus aureus. To obtain the wet-type mycelium containing the antibiotic complex, the pH of the culture liquid is adjusted to pH 2.0 to 3% with inorganic acid, followed by filtration, centrifugation or separation. The fresh mycelium is suspended in acetone and the suspension is adjusted to pH 8.5 to 9.5 with 1 N NaOH. The antibiotic substances, which are in the form of sodium salts, are extracted from the% cel three times with acetone. The combined extract is concentrated in vacuo until complete removal of the acetone.

The resulting aqueous phase is extracted with butanol or chloroform. The butanol or chloroform extract is concentrated under vacuum to a yellow-brown syrup. From this extract concentrate εφ the polyether-antibiotic complex A-111 can be obtained by precipitation with petroleum ether and optionally by column chromatography. To separate the individual components of the complex, a certain fifenge acetone is added to the yellow-brown syrup, whereby a yellow-green precipitate is obtained, which contains two of the antibiotic substances of the complex, the components A-111-3 and A-111- 4 · Components A-111-1 and A-111-2 remain in the acetone solution · The yellow-green sediment is then washed three times

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Hydrocarbons (pent ^ i, hexane or petroleum ether) extracted, with the components--111-3 and A-111-4 go into solution. Both the acetone extract containing components A-111-1 and A-111-2 and the hydrocarbon extract containing components A-111-O and A-111.-4 will be subjected to further purification and separation with the aid of subjected to column chromatography (see Scheme 1).

The Koiaplex A-111 is a colorless substance which is used in lower alcohols and ethers, in chloroform, in lower hydrocarbons, in dimethylformamide, in acetonitrile and in cyclohexanol. is soluble. The complex is not soluble in water, neither as a free satire nor as a sodium salt. The UV spectrum shows only final absorption. The thin-layer chromatographic characterization of the components of the complex A-111 by various solvent systems is shown in Table. 1 and 2 • The summary of these data gives rise to the complex to the subgroup of monovalent polyether antibiotics which form with alkali metal ion complexes · The molecular weights and the empirical formulas of the individual components that form the polyether complex , were determined by mass spectrometry *

Characterization of the components of the complex:

Component A-111-1

External form: colorless, amorphous substance

Solubility: soluble in lower alcohols, dimethylformamide, chloroform, acetone, ether, pentane, heptane? insoluble in water, neither in the form of acid nor as sodium salt ·

UV spectrum: only final absorption

Elemental composition: carbon, hydrogen, oxygen, sodium · halogen and nitrogen are not included ·

The molecular weight is according to the data of the lassenspectrometrie 74-6 · According to the elemental composition results

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from the high-resolution mass spectrum the empirical formula ^C 40 ^H 67 ° 1if ^{ia The ΚοΠ} * Ρ ° & ^Θ & * β A-111-1 differs from the component A-111-2 only in terms of its R _f value in the various Solvent systems (see Table 1). Component A-111-1 has a higher polarity than component A-111-2. The identical molecular weights, empirical formulas and data of spectrometry provide the basis for the assumption that component A-111-1 is an isomer of component A-111-2 »

Component A-111-2

External form: colorless, amorphous substance

Solubility: soluble in lower alcohols, acetone, esters of acetic acid, chloroform, ether u · a ·; not soluble in water, neither in the form of acid nor as salt of salt «UV spectrum: only final absorption

Elemental composition: carbon, hydrogen, oxygen and sodium · Nitrogen, sulfur and halogens are not included ·

iVblekul weight: 746; empirical formula C ^ Hg ^ O ^^ a, according to the results of high-resolution mass spectrometry thin-layer chromatography of component A-111-2: s. Tab. 1 ·

These data support identity with the polyether antibiotic Nigericin. However, the comparison between component A-111-2 and standard nigericin revealed some differences, which may vary. a, state that in the mass spectrum of component A-111-2, the fragment ion m / e 728 occurs, which is due to splitting off of a water molecule · In the lass spectrum of the above standard nigericin, such a mass peak does not occur #

Component A-111-3

External form: colorless, amorphous substance

Solubility: soluble in chloroform, acetone, petroleum ether, ether, pentane, hexane, lower alcohols; not soluble

- .10 -

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in water, neither as free acid nor as sodium salt ·

OT spectrum: final absorption

Elemental composition: carbon, hydrogen, oxygen and sodium; Nitrogen, sulfur and halogens are missing »

Molecular weight: 788, Irish formula C ^ HggO ^^ a, according to the results of the high-resolution jfcissen-

spectrometry · The investigation of fragmentation in the ivfens spectrum. indicates an acetyl derivative of component A-111-2

So far no antibiotic of the polyether group with this molecular weight has become known. This shows that the component A-111-3 is a new antibiotic. Thus an acetylated polyether antibiotic synthesized by microorganisms was observed for the first time.

Component. A-111-4

External form: colorless, amorphous substance

Solubility: soluble in chloroform, acetone, petroleum ether, pentane, hexane, lower alcohols; insoluble in water, neither as a free acid nor as a sodium salt.

UV spectrum: final absorption

Elemental composition: carbon, hydrogen, oxygen and sodium ·, nitrogen, sulfur and halogens are missing • . ' .:.

ivular weight: 728; empirical formula G ^ H ^ O ^ a, according to the data of the high-resolution ifessenspectrometrie » '-f -'- . v. ; .- · · ·.

^R values in various solvent systems (see Tgto. 1) · The component AW- ^ is a derivative of the component A-111-2, which is derived from this by splitting off a water molecule. This compound can be obtained from the component JM11-2 also by heating to 200 ⁰ C under vacuum, but it can not be won from Niger in. The biological

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Activity of component A-111-4 is lower than that of the other components of the antibiotic polyether complex »

Ausführungsbeispie1

The following exemplary embodiments are intended to explain the method in more detail.

1 · The Actinomycetes strain 111 is on ifeisnährboden or SRI culture medium with glucose for 10 days at a temperature of 28 ⁰ G cultured · Mt of the spore suspension are • 1000 ml Erlenmeyer flask inoculated (500 ml culture medium of the following composition g / l) · contain: soybean meal 10.0, glucose 10.0, NaCl 5.0, CaCO-1.0. The pH after sterilization at 115 ⁰ C is 6.8 to 7.2. The cultivation is carried out on a shaker with 200 U / min at 28 ⁰ C in the course of 6 to 7 days to the maximum yield of the polyether compiexes. The antibiotic activity is determined with the test germ Bacillus subtilis L-2.

2. The strain m is cultured in two stages.

1st stage: 2 ml of Brlenmeyer flasks containing 500 ml nutrient medium of the composition mentioned in Example 1 are inoculated into a spore suspension. The cultivation with shaking lasts for 36 to 48 h at 28 ^° C.

2 stage; The recovered vegetative% cel is used to inoculate a 101-permentor containing 6 liters of nutrient medium of the same composition. The process proceeds at 28 ⁰ C, with a stirring speed of 325 U / min and a ventilation of 1 1 air / 1 culture medium / min. After 6-7 days of cultivation, maximum activity is achieved

3. The cultivation is carried out in the manner as in Example 2 in two stages. · In the 1st stage, the same nutrient medium is used to prepare the inoculum. For the

2nd stage becomes a nutrient medium of the following composition

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used (in g / l): glucose 20.0 _; Soy flour 10.0; % isextract 5.0x, NaCl 5.0; GaCO ₃ 3.0.

4. "As in the schematic diagram" already stated, is after

End of the perforation with maximum antibiotic activity the pH of the culture fluid is adjusted to 2.0 to 3.0 with 1N HCl, after which the celery is centrifuged off. The wet% cel is suspended in acetone. The pH of the suspension becomes The Mjrcel is extracted twice more with acetone in a ratio of 1: 3 (v / v). The combined acetone extracts are concentrated in vacuo to the aqueous residue, which is then combined with Butanol is reextracted. The solution extract is concentrated in vacuo to a yellow-brown syrup which is converted to a solid precipitate by addition of five times Isfenge acetone (v / v). Centrifugation gives a yellow-green sediment containing the components A -111-3 and A-111-4 contain »In the acetone solution, the components A ~ 111r-1 and A-111-2 remain» If the precipitation takes place with a larger acetone volume, however, all 4- Components in solution and the sediment is inactive «

5. The yellow-green sediment recovered according to Example 4 is treated three times with the hydrocarbon hexane. The hydrocarbon extract is filtered, concentrated in vacuo to a small volume, then column chromatography with a magnesium trisilicate (60 to 100 mesh) as the adsorbent acetone-heptane in the ratio 10: 4 is used as the mobile phase. The active fractions of the column are combined and separated by preparative thin-layer chromatography with ethyl acetate as mobile phase.

The acetone extract, which is separated off from the yellow-green sediment by centrifuging as described in Example 4, is treated with activated charcoal and concentrated in vacuo to a small volume. The concentrate is added to the column chromatography.

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tography on silica gel (particle size 0.063 to 0.2 mm) subjected. The elution is carried out stepwise with chloroform ivfe-

THANOL mixtures. The active fractions from the column are combined and chromatographed on a second column of% tritium trisilicate as the adsorbent with the mobile phase acetone-heptane (10x4) .The active fractions are combined and concentrated in vacuo to give the components A-111- 1 and A-111-2 are obtained ·

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Scheme 1

Scheme for extracision, purification and separation of an antibiotic complex from Actinomyces III

culture fluid

- Adjust with 1N HCl to pH 2.0 i - centrifuge

%; cel (active)

- suspend in acetone

Adjust to pH 8 with 1N NaOH

- triple extraction with acetone

- Centrifuge

culture

Ac e t on-Extr akb

-> concentrate in a vacuum

- Extract the water phase with n-butanol

Butanol extract

- concentrate in a vacuum yellow-brown syrup

- Digestion with acetone

Backlog (inactive)

Ac e t on-Extr akt

(contains components A-111-1 and A-111-2)

- Decolorize with activated carbon

- Column chromatography on silica gel

Active fractions

Residue

(contains components A-111-3 and A-111-4)

triple extraction with pentane

pentane extract

- column chromatography

l magnesium trisilicate

Active fractions

ι

[Component] IA-111-1

discolor with activated charcoal

Column chromatography on (!? Lorisil) magnesium-active fractions trisilicate

Component A-111-2 components / 5 A-111-3

Component A114

^ 6

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Table 1

Lösungsfflittelsysteia Sf value of A-111-2 Koi ^ component A-111-4 Mgeric in 1. benzene-ethyl acetate = 1: 1 A-111-1 0.10 A- * 111-3 0.38 0.10 2 x Chlorofora-Bthylacetat = 2: 3 0.07 0.34 0.20 0.55 0.34 3 »benzene-acetone = 9: 1 0.08 O 0.48 O 0 4 »benzene-acetone ~ 1j1 O 0.64 O 0.86 0.64 5. Ethyl acetate 0.43 0.42 0 _t 82 0.59 0.42 6 · heptane-ethyl acetate = 4:10 0.23 0.33 0.52 0.57 0.33 7, petroleum ether-chloroforaw ifethanol = 10: 10: 2 0.18 0.60 0.56 0.85 0.60 8 · eeptane-ChloroforHl · »ethanol * 2O: 2O; 11 0.43 0.26 0.72 0.52 0.26 0.17 0.38

The color reactions of the 4 Eofi ^ onenten on silica gel are given in Tab »2»

Table 2

Parbreactions on silica gel

R e a g e η ζ

a r / b e

1 · vanillin-sulfuric acid 2 * anisaldehyde

3 · p-DiaJethylaiainobenzalde-. hyd

4. phosphoric acid

5. Anti-trichloride

6 * 2,4-dinitrophenylhydrazine 7 · ninhydrin 8. Ferrichloride

10 »Dragendorff

brown spots, white background

reddish brown spots, white ground

yellow spots, white background

yellow spots, white background brown-yellow spots, light yellow

underground

negative negative negative negative negative /

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Table 3

Comparative data for the minimum inhibitory concentration (MIC) of the polyether antibiotic complement A-111 with monensin and salinomycin in the agar diffusion plate test

Test culture HHZ (/ Ug / ml) A-hi - monensin salinomycin

Staphylococcus aureus 209 P Bacillus subtillis ATCC Bacillus bubtilis L-2 Bacillus mycoides "*") Sarcina citrina ⁺ ) Bsoheriohia ooli BTCC 0111 Candida alobans 562 Sacoharomyoes oerevisiae ⁺ ) Fenloillium chrysogenum ⁺ ) Alternaria tennis *) Botrytis cinerea lusarium lini ⁺ )

1.0 15.0 12.5 0.5 7.0 10.0 0.5 2.0 7.0 1.0 17.0 10.0 1.5 17.0 15.0 100.0 100.0 100.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0 50.0

⁺ ) These strains were taken from the Bulgarian Type Culture Collection (BTCC) without distress

Claims (4)

in ι * ο invention claim 1. A process for the preparation of a PolyVner antibiotic complex by microbiological means, characterized in that the Äcroorganism Actinomyces hygroscopicus , strain 111, under aerobic conditions in liquid nutrient media, the carbon and nitrogen sources and ^ Lneralsalze contain, at a temperature cultured between 25 and 32 ⁰ C over a period of 2 to 7 days and the resulting polyether-antibiotic complex A-111 extracted from the wet cel, then concentrated by known ^ methods and precipitated and optionally further by fractional sedimentation with Acetone and / or subsequent chromatography in the Binzel components A-111-1, A-111-2, A-111-3 and A-I11-4 is separated · 2 »method according to item 1, characterized in that the wet effiycel with the complex A-111 from the culture solution after acidification with inorganic acids to pH 2.0 to 3» 0 in a conventional manner by centrifugation or separation is separated · 3. The method according to item 1 and 2, characterized in that the complex A-111 is extracted from the% cel after adjusting the pH to 8.5 to 9.5 by adding alkali metal hydroxide in the form of the alkali metal salt with organic solvents » 4 · Method according to items 1 to 3, characterized in that the fractionated sedimentation by acetone addition is carried out in such a way that in the sediment, the components A-I1ir3 and A-111-4, in contrast, the components A-111 -1 and A-111-2 are included.