DD263082A5 - PROCESS FOR PREPARING AN ANTIBIOTIC COMPLEX - Google Patents

Abstract

The invention relates to a process for the preparation of an antibiotic complex. According to the invention, a novel antibiotic complex is prepared in a microbiological way, the grividomycin I and II of the formula I a; Grividomycin III of the formula I b; Neoviridogrisin IV, neoviridogrisin II and optionally griseoviridine, neoviridogrisin I and III. Formulas I a and I b

Description

Field of application of the invention

The invention relates to a process for the preparation of an antibiotic complex.

In particular, the present invention relates to the novel microorganism Streptomyces species Y-8240155, a process for the preparation of novel streptogramin-type antibiotics using this strain and / or its mutants and variants, and the use of the novel antibiotics as medicaments and agents for improvement the nutrient utilization and acceleration of the growth of livestock, especially pigs, cows and poultry.

Characteristic of the known state of the art

The microorganism strain Y-8240155 was obtained from a soil sample collected in Panchgani, Maharashtra, India using a nutrient medium at pH 6.5 to 8.5. Variants and mutants of the strain Y-8240155 are in known manner, for. B. by using a mutagen such as N-methyl-N-nitro-N'-nitrosoguanidine available. The microorganism Streptomyces sp. Y-8240155 belongs to the order Actinomycetales, the family Streptomycetaceae and the genus Spreptomyces. Several Streptomyces species producing streptogramin antibiotics are known and described in the literature (J. Antibiotics 32: 575-583, 1979; GB-A 1575533). Streptomyces sp. Y-8240155 is considered to be a new strain because it differs from the known strains in some of its morphological and physiological properties as well as its breeding behavior, as will be apparent from the following description. It is also considered a new strain because, as shown in the following description, it forms new antibiotics called Grividomycin I, II and III. The strain Streptomyces species Y-8240155 was deposited on January 15, 1986 with the "German Collection of Microorganisms" in Göttingen under the accession number DSM 3640.

By breeding Streptomycessp.Y-8240155 (DSM 3640), in addition to the known neoviridogrisin IV (US-A-023204), new antibiotics are obtained, which i.a. are suitable to accelerate the growth of livestock in the form of feed additives.

Object of the invention

The aim of the invention is to provide new antibiotics with good activity against a number of microorganisms.

Explanation of the essence of the invention

The invention has for its object to find suitable components for a novel antibiotic complex. The new antibiotics correspond to the general formula I.

H ₀ C CH ₀ R ₀

\ / ³ I ²

CH CH

OH CH «CHo CHo

Η I. Ί *

Nil ^ ^G0 ~ ^NH - ⁰ H- ⁰ O-NE - ° H - CO -

CH - COH ₃ C-CH "N-CH ₃

CO CO

CH - N - CO - CH - KH - CO - CH - Ή - R. Ii ι 4

CH ₃ R ₃ _ CH-R ₁

in which the radicals R 1 to R _{4 have the} following meanings:

Ri R ₂ R ₃ R ₄ Grividomycin I (II) CH ₃ OH H CH ₃ Grividomycin III CH ₃ OH CH ₃ H

Grividomycin I and II are a pair of diastereoisomers of the same chemical formula.

The novel antibiotics of the present invention are active against many microorganisms such as Gram-positive bacteria and mycoplasma. Such microorganisms include the species Staphylococcus aureus. Streptococcus pyogenes, Diplococcus pneumoniae, Mycoplasma gallinarum and Mycoplasma pulmonis. The novel antibiotics of the invention are also effective in strains that have become resistant to other antibiotics.

The novel compounds of the invention may be used as remedies or feed additives in any bioavailable form, alone or in combination with one another or with the known neoviridogrins I, II, III and / or neoviridogris in group IV IV and / or streptogramin-like antibiotics such as griseoviridine. The nutrient medium used to recover the microorganism consists of carbon and nitrogen sources, inorganic nutrient salts and solidifying agents. As carbon sources are glucose, starch, dextrin, glycerol, sucrose or molasses into consideration. Suitable nitrogen sources are peptone, yeast extract, broth, malt extract or casein. A suitable solidifying agent is, for example, agar. Suitable inorganic nutrient salts are sodium, potassium, magnesium, calcium salts or phosphates or sulfates.

The new Grividomycins I, il and IM are Streptograminantibiotika. The latter belong to the group of depsipeptide compounds and are divided into two main groups-A and B- (D. Vazquez, Antibiotic III, "Mechanism of action of antimicrobial and antitumor agents" Springer-Verlag Berlin-Heidelberg-New York, 1975, page 521- 534) and are formed by microorganisms as a complex of these two groups, group A and group B antibiotics act at closely related sites on ribosome 50 S subunits.

Since the binding of the group Α antibiotics is stronger in the presence of group B antibiotics, group A antibiotics show a marked synergism with group B antibiotics in their activity against Gram positive bacteria. Group A includes the following antibiotics griseoviridin, mikamycin A, osteogrycin A, virginiamycin M ₁ , pristinamycin II _A , synergistin A ₁ ; Osteogrycin G, virginiamycin M ₂ , pristinamycin II B, dihydroosteogrycin A, madumycin I and II. Group B is subdivided into the subgroups viridogrisein (neoviridogrisein) and vemamycin B (mikamycin B). The representatives of the subgroup viridogrisein (neoviridogrisein) correspond to the formula I with the following meanings for R ₁ , R ₂ and R ₃ :

R ₁ R ₂ R ₃ CH ₃ OH CH ₃ H OH CH ₃ CH ₃ H C ₂ H ₅ CH ₃ H CH ₃ CH ₃ OH C ₂ H ₅

(R ₄ = CH ₃ ) designation

Viridogrisin I etamycin, F1370A neoviridogrisin IV viridogrisin II neoviridogrisin I neoviridogrisin II neoviridogrisin III

The neoviridogriseins (NVG) I, II and III a process for their preparation using the strain Streptomyces species P 8648 (FERM-P 3562) and their use as medicaments or feed additive are already known from DE-A 2725163. Neoviridogrisein IV is known from US-A 3,023,204 as an antibiotic with activity against actinomycetes, bacteria, rickettsia and viruses, in particular for the treatment of mastitis

 The subgroup vernamycin B (micamycin B) is in formula II

CH l

0 R ₁ 0 CH ₀

HI ¹ J ²

CH, CH - CH - C - NH - CH - C - N

O = C-CH <4

NH-

-σ

CH ι

c = o

NO

in which R ₁ , R ₂ , R ₃ , R ₄ and X have the following meanings:

R ₁ R ₂ R ₃ XR ₄

C ₂ H ₅ CH ₃ N (CH ₃ ) ₂

CH ₃ N (CH ₂ I ₂

0 -C-CH- N-

I i

CHp CHp O = C - CH "

(4-Oxopipecolinsäure)

(4-Oxopipecolinsäure)

C ₆ H ₅

C ₆ H ₅

designation

Ostreogrycin B Mikamycin B

Streptogramin B Synergistin B-1 Vemamycin B ₂ ¹ Pristinamycin I _A fOstreogrycin B ₁ J Pristinamycin 1 _c L, Vernamycin Bi

CH ₃ NHCH ₃ (4-Oxopipecolinsäure) C ₆ H ₅ C2H5 CH ₃ N (CH ₃ I ₂ 3-hydroxy-4-oxopipe- C ₆ H ₆ C ₂ H ₆ colin acid CH ₃ H proline C ₆ H ₅ C ₂ H ₅ CH ₃ H pipecolic C ₆ H ₅ C ₂ H ₅ CH ₃ NHCH ₃ 4-Oxopipecolinsäure C ₆ H ₆ CH ₃ CH ₃ N (CHa) ₂ aspartic acid C ₆ H ₅ C ₂ H ₅ CH ₃ H 4-Oxopipecolinsäure C ₆ H ₅ C ₂ H ₆ H H 4-Hydroxypipecolin- C ₆ H ₅ C ₂ H ₅ acid CH ₃ H 3-hydroxy-4-oxopiper C ₆ H ₅ C ₂ H ₅ colin acid CH ₃ H 4-Oxopipecolinsäure C ₆ H ₅ CH ₃ CH ₃ H 4-Hydroxypipecolin- CH ₃ C ₂ H ₅

designation

Ostreogrycin B ₂ Pristinamycinie Vernamycin B Ostreogrycin B ₃

Patricin A Patricin B Vernamycin B Vernamycin C Virginiamycin S Virginiamycin S ₂

Virginiamycin S ₃

Virginiamycin S ₄ Virginiamycin S ₅

acid

According to their physico-chemical and spectroscopic data, the above-mentioned new antibiotics formed by Streptomyces species Y-8240155 belong to the viridogrisine subgroup of strepto-gamma group B, but differ from the known neoviridogris I, II, III and IV in their thin-layer chromatogram (see FIG accompanying drawings), high performance liquid chromatogram, their physicochemical and spectroscopic properties. The solvent system used for thin layer chromatography (TLC) was 3% methanol in chloroform and detection was at 366nm / 254nm.

The microorganism according to the invention forms elongated colorless aerial mycelia of branched substrate mycelia. Spiral spore chains are formed on the aerial mycelia. Neither swirling nor ascospore formation is observed. The characteristics of the microorganisms in culture on various agar media are described as follows.

1. Yeast extract-malt extract agar

Growth: Good

Aerial mycelium: Abundant, whitish, velvety cotton-like, occasionally water droplets on the surface.

Back: light brown

Soluble pigment:. None

2. Oatmeal agar Growth: Airy mycelium: Back: Soluble pigment:

Well

Good, whitish, velvety Weak yellow to light brown None ·

3. Inorganic Salts Starch Agar Growth: Good

Luftmyzel: Good, white

Back: light brown

Soluble pigment: None

Starch hydrolysis: positive

4. Glycerol asparaginagar growth:

Air mycelium: Back: Soluble pigment:

5. Tyrosine agar Growth: aerial mycelium: Reverse side: soluble pigment:

6. sucrose-nitrate agar growth: aerial mycelium:

Back: Soluble pigment:

7. Glucose-Asparaginagar Growth: Airy Mycelium:

Back: Soluble pigment:

Moderate

Weak or none. If present then knows.

Light yellow

None

Well

Moderate, white

brown

Brown, dark pigment formed

Moderately none

Slender yellow None

Moderate

Weak, white Weak yellow None

8. Nutrient agar Moderate Growth: None aerial mycelium: tan Back: Weak brown Soluble pigment:

9. Peptone Yeast Extract Iron Agar

Growth: Moderate

Luftmyzel: None,.

Back: brown

Soluble pigment: brown

The optimum growth temperature range for the microorganism of the present invention is 25 to 35 ^° C. The strain liquefies gelatin to a very small extent into glucose-peptone gelatin medium, hydrolyzes starch in inorganic salt-starch agar, and peptonizes skimmed milk.

The formation of dark pigment is observed in tyrosine agar, peptone / yeast extract iron agar and tryptone yeast extract broth.

The carbon source assimilation scheme of this microorganism is as follows (in Pridham-Gottlieb medium):

Positive D-xylose, D-glucose, L-arabinose, l-inositol, rhamnose, raffinose, D-mannitol

Slightly positive: sucrose, D-fructose

With regard to the formation of known streptogramin antibiotics such as Mikamycin A and B, virginiamycins, eastern reticulocytes, etamycin, vernamycin, viridogrisein, griseoviridin and pristinamycins, the following known microorganisms should be compared to Streptomyces species Y-8240155:

Streptomyces sp. P8648 Streptomyces griseus NRL 2426 Streptomyces griseoviridus NRL 2427 Streptomyces sp., Etamycin former Streptomyces conganensis Streptomyces ostreogrisius Streptomyces mitakensis Streptomyces loidensis Streptomyces griseoroseus Streptomyces lavendulae

The published data on the breeding and physiological properties of said microorganism show clear differences between the microorganism of the invention and the above-mentioned known microorganisms. For example, Streptomyces griseus NRRL 2426 differs in that it belongs to the yellow row, Section Rectiflexibiles, and does not form dark pigments, whereas the microorganism according to the invention belongs to the white row, Section Spirales, and forms a dark pigment. Streptomyces griseovirides NRRI 2427 can be differentiated from the microorganism according to the invention on the basis of the breeding properties on yeast extract malt extract agar, oatmeal agar, glycerol, asparagin agar and the formation of dark pigment. Streptomyces sp. P8648 shows clear differences from the microorganism of the present invention in its carbon source assimilation scheme and the growth characteristics on czapek agar, inorganic salts, starch agar and oatmeal agar. Moreover, strain P8648 does not form a dark pigment whereas strain Y-8240155 forms it. The results of a taxonomic comparison between the two strains are summarized in Table I below:

Table I

Streptomyces sp. Streptomyces sp. P8648 (published Y-8240155 clear data) Color of the air White to gray White aerial mycelium mycelium white aerial mycelium will be on the mei will be on the mei most ISP media most ISP media moderate to rich only bad ge formed. forms. white Air mycelium will on yeast extract malt extract agar amply formed. Color of the substrate Slight yellow or Slight yellow to mycelium hellgelbbisgrau- light brown until brown on the mei brown on the mei most ISP media. most ISP media.

Streptomyces sp. Streptomyces sp.

P8648 (published Y-8240155.

clear data)

Soluble pigment No dark pigment Dark pigment formed. Usually formed. Island no further pigment cases observed, but weakly formed in brown pigment rare cases.

badly educated.

yellow pigment.

Utilization of L-arabinose-L-arabinose +

Carbon-1-inositol-inositol +

swell raffinose raffinose +

As can be seen from the above data, between Str. Sp. P8648 and the streptomycetes according to the invention confirmed clear differences in the physiological and breeding properties and the carbon source utilization scheme. In addition, the fermentation of the microorganism of the present invention mainly forms neoviridogrisin IV (viridogrisin I), besides grividomyin I ₁ II and III and griseoviridine, while Streptomyces sp. P8648 only neoviridogriseins 1,11, III, viridogrisein (neoviridogrisin IV) and griseoviridine, but not grividomycin I, II and III forms. In addition, the proportion of griseoviridine in the fermentation product of the new microorganism is low (S2%), while the known Streptomyces sp. P8646, Streptomyces griseus NRRL 2426 and S. griseoviridis NRRL 2427 consistently produce significant amounts (> 20%) of griseoviridine. From the above results, the microorganism of the present invention is considered to be a new Streptomyces species.

The present invention also provides a process for the preparation of compounds belonging to the class of streptogramin antibiotics which is characterized in that Streptomyces sp. Y-8240155 (DSM 3640) or its natural and artificial variants and mutants by fermentation at a pH between 6.5 and 8.5 and a temperature between 20 and 40 ⁰ C under aerobic conditions in a carbon and nitrogen sources, inorganic 'nutrient salts and nutrient medium containing trace elements and the compounds are isolated in a conventional manner from the culture broths.

The carbon sources used in the nutrient medium to produce the new antibiotics can be glucose, starch, dextrin, glycerin, sucrose, molasses, and oil. Suitable nitrogen sources used in the nutrient medium for the preparation of the new antibiotics are soybean meal, yeast extract, broth, malt extract, corn steep liquor, peptones or casein. Suitable inorganic nutrient salts are sodium chloride, magnesium sulfate, ammonium sulfate and potassium carbonate. Trace elements are iron, manganese, copper, zinc and cobalt.

Streptomyces sp. Y-8240155 (DSM 3640) is preferably cultured at 25-3O ⁰ C and pH 6.5-7.0. The fermentation is advantageously stopped after 40 to 50 hours, after which maximum yields of the compounds are obtained. The fermentation is preferably a submerged fermentation.

The course of fermentation and the formation of the Streptograminverbindungen can be controlled by the antibacterial activity of the culture fluid and the mycelium against Staphylococcus aureus 209P.

In the fermentative breeding of Streptomyces sp. Y-8240155 DSM 3640 is mainly formed by neoviridogrisin IV, besides grividomycin I, II and III and neoviridogrisin II, as well as optionally griseoviridine and neoviridogrisin I and III. The proportions of the individual components can vary within wide limits, depending on the composition of the culture medium. Typically, the isolated fermentation product contains about 90% or more NVG IV, while the remaining ingredients together do not make up more than 10%. The fermentation products may be added at pH 4.5 to 7.5 - preferably at pH 6.5 - from the culture filtrate as a mixture using conventional methods such as extraction with water immiscible solvents such as chloroform, methylene chloride, n-butanol, ethyl acetate or butyl acetate (preferably ethyl acetate) are isolated. Grividomycins I, II and III can be separated from neoviridogrins I, II, III, IV and griseoviridine using routine chromatographic methods. Such methods may be gravity, medium pressure silica gel column chromatography, Sephadex ^IF "-LH-20 chromatography, droplet countercurrent chromatography with a suitable solvent system, preparative high performance liquid chromatography on LiChrosorb ^(RI- RP-18 or thin layer chromatography Mycelium, the antibiotic-effective complex can be obtained by extraction of the mycelium cake with an organic solvent, preferably acetone.The acetone extract is concentrated, the aqueous layer adjusted to pH 4.5 to 7.5 - preferably pH 6.5 - and mixed with water immiscible organic solvents such as chloroform, methylene chloride, n-butanol, ethyl acetate or butyl acetate, with ethyl acetate as the solvent being preferred, extracted The ethyl acetate extracts of the mycelium and the culture filtrate are combined or worked up separately.

The Grividomycine I, II and III are colorless amorphous powders and in methanol, ethanol, n-propanol, isopropanol, n-butanol, ethyl acetate, n-butyl acetate, acetone, ethyl methyl ketone, methyl isobutyl ketone, benzene, toluene, methylene chloride, chloroform, dimethylformamide and dimethylsulfoxide soluble, hexane, petroleum ether (40-60), diethyl ether and water, difficultly or insoluble. They have the following melting points:.

Grividomycin I: 141-145 ⁰ C

Grividomycin II: 153-157 ° C

Grividomycin III: 197-198 ° C

The molecular weight of the three Grividomycine is 864.

The spectroscopic data are shown in the accompanying drawings as follows: FIG. 2: IR spectrum of Grividomycin III (in KBr) FIG. 3: Mass spectrum of Grividomycin III FIG. 4: IR spectrum of Grividomycin II (in KBr)

FIG. 5: Mass spectrum of Grividomycin II FIG. 6: IR spectrum of Grividomycin I (in KBr) FIG. 7: Mass spectrum of Grividomycin I

In the thin layer chromatogram f silica gel precast plate), the Grividomycins, I, II and III have the following R _r values:

Rf 7,5Vc Methanol in 0.41 chloroform 0.48 3% 0.59 0.18 0.23. 0.29

Rf 10 ° / c Methanol in 0.25 Essigester 0.34 5% 0.40 0.15. 0.21 0.28

Grividomycin III Grividomycin II Grividomycin I

In analytical high performance liquid chromatography (HPLC), the Grividomycins I, II and III show the following

Retention times:

Column filling: LiChrosorb ^ml -RP-18/7 μ particle size [0.4 χ (25 + 3) cm]

Flow: detection: solvent:

Grividomycin III Grividomycin II 7 Grividomycin I J

1 ml / min

at 305 η m

Acetonitrile / 25 mM ammonium acetate /

Acetic acid (150: 100: 3)

retention time

7.0 min

6,8 min

(The mixture could in the above

Solvent system can not be separated.)

Antibacterial activity

Grividomycins I, II and III are antimicrobially active against bacteria, mycoplasmas and actinomycetes in laboratory tests. They show an in vitro activity against the susceptible and resistant strains of Staphylococcus aureus and against strains of Streptococcus pyogenes, Diplococcus pneumoniae, Sarcina lutea, Bacillus subtilis, Mycoplasma gallinarum and Mycqplasma pulmonis. The minimum inhibitory concentration of the new antibiotics was determined on various microorganisms. The results are shown in the table below in comparison to NVG II and NVG IV.

tables NVG Il NVGIV MIC values ^ g / ml] Grividoll Grivido III test organism 0.4 1.6 Grividol 0.4 6.4 0.4 1.6 6.4 3.2 3.2 S. aureus 209 P 0.4 1.6 25 6.4 6.4 S. aureus 3066 Meth. ^R 1.6 6.4 25 50.0 50.0 S. aureus R85Tet ^R 0.2 1.6 > 50 12.5 25.0 S. aureus R 85 / M Em ^R 0.8 3.2 25 12.5 50, O ^- S.aureus712Meth ^R 12.5 12.5 25 50.0 > 50.0 S. aureus 789 Meth ^R 6.4. 25 > 50 •> 50.0 50.0 Str.faecalisO2DODU1 Mac ^R 25 25 > 50 50.0 > 50.0 Str.faecalisUD8b 0.4 1.6 > 50 0.4 6.2 Str.faecalis Eder Mac ^R 0.4 1.6 12.5 3.2 12.5 Micrococcus luteus 0.2 0.4 12.5 1.6 12.5 Sarcina lutea > 50 > 50 3.2 > 50.0 > 50.0 Bacillus subtilis > 50 > 50 > 50 > 50.0 > 50.0 E. coli 9632 > 50 > 50 > 50 > 50.0 > 50.0 E. coli Bss 2231 > 50 > 50 > 50 > 50.0 > 50.0 Alkaline faecalis > 50 Ps. Aeruginosa M35

Meth ^R = methicillin resistant; Tet ^R = tetracycline resistant;

Em ^R = erythromycin resistant; Mac ^R = macrolide antibiotic resistant.

Growth-promoting effect

The present invention substantially obtained antibiotic complex consisting of Neoviridogrisein IV as the main constituent together with the new Grividomycinen I, II and III show a marked growth-promoting effect in feeding experiments in animals, eg. As in poultry, pigs, cattle, sheep and rabbits. This also applies to the individual components, in particular for neoviridogrisin IV, for which a growth-promoting effect has hitherto not been described. Also, the laying performance of poultry, in particular the egg weight and milk yield of dairy cows, sheep and goats is positively influenced by the antibiotic complex or its individual components.

To achieve the effect used concentrations of 2 to 100 ppm, preferably 2-50 ppm, in particular 5-20 ppm, based on complete feed. Accordingly, the concentrations are higher when the antibiotics are administered as an adjunct to feed.

Exemplary embodiment

The invention is illustrated by the following examples:

Example I Recovery of Streptomyces Y-8240155 from a soil sample

(a) Preparation of a nutrient medium

Medium 1: Glucose · 1.0g glycerin 1.0g L-arginine 0.3 g K ₂ HPO ₄ 0.3 g MgSO ₄ -7H ₂ O 0.2 g NaCl 0.3 g yeast extract 2.0 g Fe ₂ (SO ₄ I ₃ 10mg CuSO ₄ -5H ₂ O 1 mg ZnSO ₄ -7H ₂ O 1 mg MnSO ₄ -7H ₂ O 1 mg agar 15.0 g distilled water 1 liter pH 6.5 Medium 2: glycose 2.0 g L-asparagine 1.0g K ₂ HPO ₄ 0.5 g MgSO ₄ -7H ₂ O 0.5 g soil extract 200 ml agar 15.0 g distilled water 800 ml pH 8.0 Medium 3: Strength 10.0 g casein 0.3 g KNO ₃ 2.0 g NaCl 2.0 g K ₂ HPO ₄ 2.0 g MgSO ₄ -7H ₂ O 0.05 g CaCO ₃ 0.02 g FeSO ₄ 0.01 g agar 15.0 g distilled water 1 liter , pH 7.2-7.5

The media were sterilized at 121 ^° C. for half an hour

(b) Preparation of the soil suspension

1 gram of soil was suspended in distilled water and shaken well. The soil was allowed to settle and the supernatant was used to inoculate each of the above isolation media.

(c) inoculation of the isolation medium

1 ml of the soil suspension was inoculated onto 50 ml of Petri dishes containing any of the above-mentioned isolation nutrient media.

(d) Isolation of Streptomyces sp. Y-8240155

The inoculated peti-shell was incubated for two weeks at 30 ⁰ C and Streptomyces sp. Y-8240155 was obtained from the growing microorganisms.

Example Il Breeding of Streptomyces sp. Y-8240155 (DSM 3640) for the fermentative production of the antibiotic complex

Streptomyces sp. Y-8240155 was grown on yeast malt agar of the following composition:

malt extract 10.0 g yeast extract 4.0 g glucose 4.0 g agar 15.0 g Distilled water 1 liter pH 7.0

The medium was dispensed into test tubes and sterilized at 121 ^° C. for 30 minutes. The glasses were cooled in an inclined position for the production of slanting agars. The slants were inoculated with the culture and incubated for 10 to 15 days at 25 ⁰ C,

after which good growth and sporulation were observed. A suspension of the spores of a slant agar in distilled water was used to inoculate five 500 ml Erlenmeyer flasks each containing 100 ml of the seed culture medium or a 5 liter aspirated flask containing 11 of the same seed culture medium.

Composition of the seed culture medium

Glucose 15.0g

Soybean meal 15.0 g

Corn steep liquor 5.0 g

CaCO ₃ 2.0 g

NaCl 5.0 g Distilled water 1 liter

pH 7.0

Each 100 ml portions of the above medium were dispensed into 500 ml Erlenmeyer flasks or 1 liter portions in 5 l aspirated bottles and sterilized at 121 ^° C. for 30 minutes. The flasks / flasks were cooled, inoculated with spore suspension and incubated at 240 rpm. M. shaken for 72 hours at 30 ⁰ C on a rotary shaker with a rash of 3.8cm (1 ^1/2 inches). The resulting growth was used to inoculate two 10 L glass fermenters, each containing 101 of a 8-10% by volume seed culture medium, to make the second stage seed culture. The fermentation was carried out with stirring at 160-180 rpm and an aeration amount of 6bis7lpm 24 hours at 27 ⁰ C (± 1 ° C). The resulting well grown second stage seed culture was used to inoculate the preparation medium.

Composition of the production medium

glucose 15.0 g Soluble starch 20.0 g soy flour 3.0 g peptone 3.0 g CaCO ₃ 2.0 g NaCl 2.0 g Corn steep liquor 2.0 g (NH ₄ I ₂ SO ₄ 0.5 g Distilled water 1 liter pH 7.0

The fermenter content was treated with 0.025% Desmophen® as a defoamer.

280I of the above medium was placed in a 390 L fermenter. The medium was sterilized for 28 minutes at 121 ° C by indirect and direct steam. The fermenter was cooled and inoculated with the second stage of seed culture (7% v / v). The fermentation was carried out at 27 ° C (± 1 ° C) with stirring at 100-200 r.p.m. Performed for 40-44 hours. The aeration was carried out at a rate of 170 liters per minute. At the end of the fermentation after 40 to 44 hours, the antibiotic concentration was 40-60 mg per liter, and the pH of the culture liquid was in the range 6.5-7.0. The mycelium weight was 5-6 (% by volume).

The harvested culture broth containing the antibiotic complex was centrifuged to separate the mycelium from the culture broth. These were then processed according to the flowchart in the following diagram:

Claims (1)

claims: A process for the preparation of an antibiotic complex comprising Grividomycin I and II of the formula Ia GH, GH, OH V ³ CH, OH ₂ CO-NH-CH-CO-NH-CH-CO - N CH-CH- So CH-NH-CO-CH ₂ -NH-Co-CH CH ₃ CH-CH ₃ CH-CH ₃
CH ₃ f ₂ -CH-CO N-CH. CH ₂
CO
N-CH. Grividomycin III of the formula I b
-OH CH. CH. OH OC CH
I CH, CH, CH ₂ CH-CO CH. in, CO NH CO-NH-CH-CO-NH-CH-CO - N CH-CH ₃ CO CH-NH-CO-CH-NH-CO-CH ι i Io CH-j CH-CH ₀ 3 3 ι 3 CH-CH ₃ Neoviridogrisin IV, neoviridogrisin II and optionally griseoviridine, neoviridogrisin I and IM, characterized in that the microorganism Streptomyces sp. Y-8240155 (DSM 3640) or one of its mutants or variants at a pH of 6.5 to 8.5 and a temperature between 20 and 40 ⁰ C under aerobic conditions in a carbon and nitrogen sources, inorganic nutrient salts and nutrient medium containing trace elements cultured to achieve a substantial antibiotic activity and the fermentation product isolated from the culture medium in a conventional manner. For this 7 pages drawings