FI74995B - FOERFARANDE FOER MICROBIOLOGICAL FRAMSTAELLNING AV FERMENTERINGSMEDIA, SOM INNEHAOLLER ANTINGEN NEBRAMYCIN-5 'ELLER EN BLANDNING AV NEBRAMYCIN-2, NEBRAMYCIN-4 AND NEBRAMYCIN-5'. - Google Patents

Description

74995

The present invention relates to a microbiological process according to the preamble of claim 1 for the preparation of fermentation broths containing either practically nebramycin-5 'or a mixture of nebramycin-2, nebramycin-4 and nebramycin-5. considering nebramycin-5 'alone or a mixture of antibiotics having TO in a predetermined ratio of nebramycin-2, Nebra-mycin-4 and nebramycin-5a.

It is known that Streptomyces tenebrarius produces nebramycin as a biosynthetic metabolite, a complex mixture of several antibiotics, (Stark, Hoehn 15 and Knox, Antimicrobial Agents and Chemotherapy, 1967, p.

314, GB Patent 1,178,489 and U.S. Patent: 3,696 1,279). In addition to several of its minor ingredients, the antibiotic mixture contains the following main ingredients: nebramycin-2 (apramycin), nebramycin-4 (6 '' - 0-carbamoyl-kanamycin 20 B) and nebramycin-5 '(6 "-O-carbamoyl-tobramycin) [ Koch, Davis and Rhoades, J. Antibiotics 2 & (1973), 7453. U.S. Patent No. 3,853,709 discloses a microorganism that produces nebramycin-7 in addition to nebramycin-2. 032 404 Japanese inventors simultaneously produce nebramycin-2, and nebramycin-5 'by culturing Streptoalloteichus hindustanus According to the HU patent:' · 176 103, Streptomyces tenebrarius MNG 169 and 170 pin-under: under aerated conditions cultured to produce fermentation broths containing either nebramycin-2, nebramycin-5 'and nebramycin-4 or nebramycin-2 and nebramycin-5 1, respectively, These antibiotics are isolated according to HU patent publication 177995.

Nebramycin-2 inhibits the growth of microorganisms pathogenic to plants and animals (U.S. Pat. Nos. 3,691,279, 3,853,709 and 3,876,763), while kanamycin B, obtained by hydrolysis of nebramycin-U, and tobramycin (Brulamycin ), obtained by hydrolysis of nebramycin-51, are broad-spectrum antibiotics that play an important role in the treatment of infectious diseases caused by polyresistant bacteria in humans (e.g., Preston and Wick, Anti Microbial Agents and Chemotherapy 1970, p. 322).

Following the method of HU Patent 176,103, the following antibiotic composition is obtained (Table 1):

Table 1 strain nebramycin-2 nebramycin-U nebramycin-5 '*!' per cent 20 MNG 169+ 77 2 21, · MNG 170 ++ 91 small amount 9 + Recorded 28 December 1977 in The National Collection of Microorganisms, National Institute for Public Helth, Budapest, and available since 28 February 1980.

+ + Recorded 5 April 1978 in the same collection and ’available since 28 February 19Ö0.

: It is apparent that nebramycin-2 is a nan biosynthesis, while nebramycin-5 ', 30 must be distinguished from the other predominant components by very complex and expensive procedures.

The composition of the antibiotic mixture synthesized by the microorganism is determined primarily by the genetic information contained in the cell and only to a small extent by the fermentation conditions.

In the methods known to date, it is not possible to convert the proportions of the ingredients contained in the fermentation broth into actual commercial requirements. In addition, if nebramycin-51 is to be obtained, it must be distinguished from the prevailing nebramycin-2 by very expensive measures. For such multi-component systems, it is very laborious and time consuming to try to increase productivity by finding and selecting suitable strains. In this case, the components are separated by thin-layer chromatography and quantified by biological assays. It would be advantageous to perform these strain selections on single-component systems, making it possible to use simple assay methods. In addition, increasing productivity with a strain that biosynthesizes only one antibiotic is more promising because in this case, the cells can use their entire biosynthetic capacity to produce a single compound.

It is an object of the present invention to provide a new and simple fermentation process for the preparation of fermentation broths containing either nebramycin-51 alone or a mixture of nebramycin-2 and Nebra-rayin-5 'in any desired predetermined manner. in relation to.

In our experiments, we surprisingly found that Streptomyces tenebrarius MNG 169 treated with ethidium bromide (2,7-diamino-10-ethyl-phenylphenanthridine bromide) produces a mutant strain that does not synthesize nebramycin-2 but produces nebramycin-5 '. as a major component and a very small amount of 30 nebramycin-L. This phenomenon is quite unexpected and surprising, as so far ethidium bromide has not been used as a mutagen at all.

To isolate a mutant that biosynthesizes an antibiotic mixture with an altered ratio of 35 components, Streptomyces tenebrarius MNG 169 strains were grown in liquid nutrient medium.

The culture was divided into five portions to which 0, 0.05, 0.5, 5.0 and 50.0 pg / ml ethidium bromide were added, respectively. The culture was continued for 24 hours, after which a portion of each culture was diluted and plated on agar plates, and the other portion was inoculated with a liquid medium containing the same amount of ethidium bromide and the culture was continued for another 2 U hours.

The plate application and transfer-10 sieving described above were then repeated. The resulting new cultures were plated repeatedly every 2 hours and incubated, some individual colonies were isolated and tested for productivity by the methods described in the examples.

15 Of the several hundred mutants isolated from Streptomyces tenebrarius strain MNG 1 69, ... were two that did not produce at all! nebramycin-2, but instead produced relatively-; small amounts of nebramycin-5 * and very small amounts of nebramycin-4. To improve the productivity of these two nebramycin-5α-producing mutants, strain selection and nutrient solution optimization experiments were performed. During the experiments, one of these mutants was lost, while the other increased productivity by five-fold and could be maintained at this level. 1981 The National Collection of Microorganisms (National Institute for Public Helth, Budapest, Hungary), where it was given the number MNG 20h.

’· * ·· 30 The following tables provide information on the general and biochemical characteristics, carbon utilization spectrum, nitrate utilization, sodium chloride tolerance, antibiotic susceptibility and antibiotic susceptibility of Streptomy ces tenebrarius strains MNG 169» MNG 170 and MNG 204.

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Table 2. General and biochemical characteristics of Streptomyce3 tenebrariue strains MNG 169, MNG 170 and MNG 204 MNG 169 MNG 170 MNG 204 5 ------------------------ --------------------------- SPORTS COLLECTION branching, highly branching, helical, slightly helical, 30 to 60 spores 30 to 60 spores in spores 10

Soluble red brown red, material color red brown red 15 -------------------------------------- --------------: Growth ρΗ above pH 5.0 and below pH 8.6

For optimal growth 37 - 50 ° C

temperature 20 -----------------------------------------------

Melanin formation negative

Antibiotic- nebramy- nebramy- nebramy- 25 production of busin busin busin-5 »complex complex

Sporulation in light negative 30 ---------------------------------------------- ---- 6 74995

Table 2 (Continued):

Coal utilization spectrum

Sugar MNG 169 MNG 170 and NG 204 5 Glucose +++ +++ +++

Cellulose - Starch +++ +++ +++

Glycerin +++ +++ +++

Lactose ± ± * 10 Melibiosis -

Salicin ± ± ±

Trehalose +++ +++ +++

Arabinose - - _

Dulcite - - _ 15 Fructose +++ +++ +++

Inositol +++ +++ ++ +

Mannitol -

Raffinose -

Rhamnose - 20 Sucrose t ± ±

Xylose -

Adonite +++ +++ +++

Markings: +++ = fairly good exploitation, + = poor exploitation, 25 ± = questionable exploitation, - = no exploitation

Nitrate positi- positii- positi- * '' · utilization vinen vinen vinen. NaCl tolerance 4% 4% i | (

Antibiotic- Resistant to aminoglycoside-type anti-30 susceptibility to biotics

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Table 3. Cultivation characteristics of strains MNG 169. 170, 1a 20U.

features

Agar Substrate mycelium Aerial mycelium 5 Czapek agar poor growth, yellowish- poorly-reddish felt, poor spores- Vasto, pale reddening substance dissolves

Inorganic moderate-good poorly - fate salt-agar growth, yellowishly developing brown color, flattened, good sporulation color: browning: Bennett agar well developed moderately - 15 substrate mycelium, well developed pale yellow aerial mycelium, insoluble good - moderate · sporulation of the material

Glucose - moderate growth, greyish yellow, 20 asparagine light yellow moderate iti mycelium, barely nocturnal soluble color

The present invention relates to a process for the treatment of such products as a method of fermentation of Ca-malt agar in a moderately to moderately well-developed, developed airy-reddish mycelium, light brown red yellowish-yellow mycelium, solid color. containing nebramycin-2 and nebramycin-5 'in any predetermined ratio by mixing the spores of the original Streptomyces tenebrarius MNG 169 strain and the new Streptomyces tenebrarius MNG 20 ^ strain or vegetative mixture in the desired ratio. mycelium and then grown as a mixed crop. During the fermentation carried out with the mixed culture containing both strains, surprisingly higher amounts of antibiotics are produced than in the fermentation broths obtained by culturing the strains separately.

In view of the above, the present invention relates to a process for the preparation of fermentation broths containing either nebramycin-5 'or an antibiotic mixture having a predetermined ratio of 0.5 to 77 t of nebramycin-2, 2.7 to 8.6 % nebramycin-4 and 20-95% nebramycin-5 'a. According to the method of the invention a) for the preparation of fermentation broths containing nebramycin-5a, 20 new strains of Streptomyces tenebrarius - 20 mNG 20iJ, or b ) for the preparation of fermentation broths containing 0.5 to 77% nebramycin-2, 2.7 to 8.6% nebramycin M and 20 to 95% nebramycin 5a in a predetermined ratio, Streptomyces tenebrarius MNG 169- spores or vegetative mycelium of strain 25 and the new strain Streptomyces tenebrarius MNG 204 mixed 100: 1 to 1: 100 are cultured with organic carbon and nitrogen sources, inorganic salts, trace elements and animal and / or plant oils, and / O in a fat-containing medium as an underground culture aerated at 33 to 40 ° C, preferably at 37 ° C.

According to a preferred method of the present invention, fermentation broths containing nebramycin-5 'and a very small amount of nebramycin-4 are prepared by culturing a strain of Streptomyces tenebrarius MNG 204. fermentation,

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9 During 74995, 10 to 180 ug / ml nebramycin-h and 2100 to 2600 ug / ml nebramycin-5 'accumulate in the fermentation broth, depending on the culture conditions. Nebramycin-5 'is separated from the fermentation broth and nebramycin-k and various impurities are removed by a single chromatographic separation and then nebramycin-5' is hydrolyzed to tobramycin by methods known per se. If desired, nebramycin-5 'can be converted to tobramycin already in culture and isolated therefrom.

According to another preferred embodiment of the process according to the invention, fermentation broths are prepared which contain a mixture of nebramycin-2 and nebramycin-5 'in any predetermined ratio and nebramycin-U, such that ... The spores or vegetative felts of Streptomyces tenebrarius MNG 169 and Streptomyces tenebrarius MNG 20U are mixed together in the required proportions and the mixed culture is cultured. The antibiotics formed are separated by methods known per se, preferably according to HU patent patent 17-315. the quantitative ratio of nebramycin-2 to nebramycin-5 'is almost proportional to the number of cells of Streptomyces tenebrarius strains MNG 169 ”and MNG 20U when added at identical growth stages.

For anyone with a basic knowledge of the field, ·. it is clear that the situation described above only occurs in fermentation systems in which the mixed cell species grow and divide at almost identical rates and in which the fermentation conditions of '30 are favorable for the biosynthesis of both components. Otherwise, the relationship may shift in either direction. For an expert, determining the mixture ratio cannot present any difficulty.

According to the present invention, microorganisms are cultured in a medium containing an organic carbon source such as starch, glycerol, fructose and / or other similar compounds, preferably glucose; an organic nitrogen source such as nut flour, corn leachate, peptone, yeast extract and / or other similar preparations, preferably soy flour, casein and / or amino acids, preferably glutamic acid; inorganic nitrogen source, preferably ammonium chloride, ammonium nitrate and / or ammonium sulphate: inorganic salts, preferably magnesium sulphate, zinc sulphate, cobalt nitrate and / or calcium carbonate; animal and / or vegetable fats and / or oils, such as palm oil, linseed oil, sunflower oil, rapeseed oil, lard oil and the like, preferably as a 1: 1 mixture of palm oil and linseed oil; and, in addition, the trace elements necessary for the growth of the microorganism, and the cultivation is carried out in an aerated subsurface culture at a temperature guaranteeing the growth of the microorganism, preferably at 33 DEG-U0 ° C, until a considerable amount of antibiotics has been accumulated. The antibiotics are separated by methods known per se.

Trace elements may either be present as impurities in other nutrients or may be added to the nutrient medium separately.

25 The air flow rate is controlled according to the composition of the medium, the performance of the mixer and other technical characteristics of the fermenter. It is usually advantageous to regulate the oxygen supply in such a way that it is possible to::: ensure that the CO 2 content of the exiting gas mixture remains below 1.0% and that the reducing sugar is depleted from the medium between 96 and 110 hours .

No antifoam is added during fermentation, as oils and / or fats of animal and / or vegetable origin added to the medium prevent the culture from foaming.

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The culture itself can be performed in shake flasks, laboratory, pilot plant or industrial fermentors.

The antibiotic mixtures in the cultures are separated into their components by thin layer chromatography on silica gel and determined bioautographically. Silica gel thin-layer plates (Merck, Darmstadt) are spotted with antibiotic solutions containing 0.1 to 0.5 μg / ml of antibiotic and chromatographed using ethanol-methyl ethyl ketone ($ 25) ammonium hydroxide as eluent. mixture (1: 1: 1). After thorough drying, a growth solution containing Bacillus substilis strain ATCC 6633 is applied to the surface of the plate and incubated for 6 hours at 37 ° C. The size of the 15 zones in which growth is inhibited is then compared to known sizes. In this way: a qualitative and quantitative determination can be carried out, V: —10% as standard deviation.

The main advantages of the method according to the invention are the following: Streptomyces tenebrarius MNG20, which produces only one antibiotic grade, produces higher antibiotic concentrations than its previous parent strain. In addition, selection work with this new strain promises rapid results, as this strain may be free to use its full biosynthetic capacity to form a single antibiotic component. The bioassay of these fermentation broths containing a single antibiotic component is fast, simple and accurate, and the separation of the antibiotic does not require expensive cleaning procedures. When cells of two different strains are cultured in a mixed culture, fermentation broths containing nebramycin-2 and nebramycin-51 in any predetermined mixture ratio can be produced according to actual commercial and economic requirements. An additional advantage is that during this mixed fermentation, neither of the 12 74995 strains loses significant productivity and both synthesize higher levels of antibiotics overall.

The invention is illustrated by the following -5 wool examples.

Example 1 a) Preparation of fermentation broth containing nebramycin-5 'in laboratory fermentors 10

Agar surfaces were prepared using distilled water and contained the following ingredients

dextrin 1.0 JS

yeast extract 0.1% casein hydrolyzate (10%) + 2.0% meat extract 0.1% cobalt dichloride hexahydrate 0.001% agar 2.5% + enzymatically hydrolysed • '• 20 and inoculated with spores or vegetative mycelium of Streptomyces tenebrarius strain MNG 20U. The pH of the medium is adjusted to 7.2 with aqueous sodium hydroxide solution and then sterilized for 20 minutes to 2 hours at 121 ° C.

The cultures are incubated for 1+ days at 37 ° C, then the spores are rinsed off the surface with sterile distillate-V; water and transfer the resulting spore suspension to inoculum media (2 x 100 ml) prepared in 5 ° ® 5 ml ® ml Erlenmeyer flasks using tap water. The composition of the inoculum medium is as follows: "soy flour 2,0% casein hydrolyzate (10% ) + 3.0% ammonium nitrate 0.1% 35 ammonium chloride 0.3% calcium carbonate 0.3% magnesium sulphate heptahydrate 0.5%

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13 74995 1: 1 mixture of palm oil and game- 0.5% linseed oil glucose (sterilized separately in 2.0% 50% solution) + enzymatically hydrolyzed

The pH of the medium is adjusted to 7.2 with aqueous sodium hydroxide solution and then sterilized for 20 minutes at 121 ° C in a pressure vessel.

The inoculated cultures are grown for 1U to 18 hours at 37 ° C in a rotary shaker (diameter 2, h cm, speed 280 l / min).

200 ml of this advanced inoculum are inoculated into a laboratory fermenter with a working volume of 10 liters and containing 5 l of a main fermentation medium prepared in tap water, sterilized for 1 + 5 minutes at 121 ° C and having the following composition: 'soybean meal 3,0% casein hydrolyzate (10%) * 5.0% ammonium nitrate 0.1% 7 ammonium chloride 0.5% L-glutamic acid 0.8% calcium carbonate 0.5% magnesium sulfate heptahydrate 0.5% cobalt dinitrate hexahydrate 0.001% 25 1: 1 mixture of palm oil and pellet - 3.0% glucose anilylated separately 1+, 2%: 50% in solution) + enzymatically hydrolysed

The pH of the medium is adjusted with ammonium hydroxide to 30 ...

7.2 before sterilization.

The growth is performed at 37 ° C using a stirrer speed of 360 l / min and a sterile air flow rate of 3 l / min.

After culturing for 11 + U hours, measure 260 mg of nebramycin-5 * and 9 mg of neb Ramy s i ini-1 + per 100 ml of fermentation broth. Nebramy si ini-2 is not detected.

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Other media of different composition can also be used in the main fermentation. Thus, proceeding according to a) using the following medium 5 soybean meal 3.3% ammonium chloride 0.6% magnesium sulphate heptahydrate 0.7% calcium carbonate 0.5% cobalt dinitrate hexahydrate 0.001% 10 palm oil 2.5% glucose (sterilized separately 2.1% 50 % as a solution) * ♦ Depending on the geometry and accessories of the fermentor used in the culture, 15 as they affect the metabolism of the cells,. increase glucose concentration to 4.0%.

similar results are obtained.

After culturing for 144 hours, 100 ml of inoculum contains very small amounts of nebramycin-4 (up to 3 mg) and 180 mg of nebramycin-5: Nebramycin-2: he is not detected.

b) Preparation of fermentation broths containing nebramycin-51 · a in a 1000 liter pilot plant fermentor

Fermentation broths containing high levels of nebramycin-5 'shall be prepared according to the procedure in (a). 100 ml of inoculum medium is inoculated with spores or 30 vegetative mycelium of Streptorayces tenebrarium strain MNG 204 and grown for 14 to 18 hours and then inoculated with 100 liters of sterile inoculum medium at 37 ° C using 100 l / min air flow and 360 1 / rain mixture. After 16 hours, 35 liters of sterilized fermentation broth (same composition as in (a)) are inoculated in a stainless steel fermenter with 50 liters of this inoculum. The main fermentation is carried out at 37 ° C 11 15 74995 using a flow of 500 l / min and a stirring of 200 l / min. Changes in antibiotic concentration are shown in Table 2.

Table U

5 Fermentation time (h) Nebramycin quality (yg / ml) 2 U 5 '10' 2 U 0 0 50 U8 0 0 U 00 72 05 1200 15 96 0 30 17 ^ 0 120 0 U0 2320 1UU 0 60 2650 20! c) Decarbamoylation of nebramycin-5 'and separation into tobramycin 22 g of sodium hydroxide are added to 3.0 liters of fermentation broth containing 2600 ug / ml nebramycin-5' and 90 ug / ml nebramycin-U, broth heated cont-; with stirring to 90-100 ° C and kept at this temperature for 25 minutes. After completion of the reaction, the mixture is cooled to room temperature and adjusted to pH 2.0 with 50% aqueous sulfuric acid. To this acidic fermentation broth is added 6 g of oxalic acid with constant stirring and then the pH is adjusted to 7.0, the mixture is stirred for a further 30 minutes, filtered and the mycelium is washed twice with 600 ml of water.

The filtrate and washings are combined and the resulting calcium-non-ionic solution is subjected to ion exchange chromatography on a column prepared from 200 ml of 16 74995

Amberlite CG-50 (NH 4) (Rohm and Haas Co., Philadelphia, USA). The column is washed first with 100 ml of deionized water and eluted first with 1000 ml of 0.05 N, then with 2000 ml of 0.1 N and finally with 3000 ml of 0.15 N ammonium hydroxide-5 solution. During the elution, 250 ml fractions are collected. Fractions 13 to 18 containing tobramycin are combined, the pH of the solution is adjusted to 7.0 with 50% aqueous sulfuric acid and ion exchange chromatography is performed on 120 ml of Amberlite CG-50 (NH 4). Column 10 is washed with 200 ml of deionized water and the antibiotic is eluted first with 1000 ml of 0.05 N and then with 2000 ml of 0.1 N and 2000 ml of 0.15 N and finally with 2000 ml of 0.2 N ammonium hydroxide solution. . Collect 60 ml fractions. The fractions hj> - 56 are combined and evaporated under reduced pressure. Drying of the residue with phosphorus pentoxide gives a product containing 0.7 g (70%) of kanamycin B, 12% of component N - 13 (6'-N-carbamoyl-tobramycin), 10% of unknown component and 8% of Tobra-hygromycin. Evaporation of fractions 57 to 58 gives a residue containing 0.2 g (60%) of tobramycin, almost 1 + 0% of kanamycin B and 0.1 to 0.2% of component N- 13.

When fractions 59-69 are distilled, 5S7 g of tobramycin containing 0.1-0.15% of component N-13 are obtained. Recrystallization of crude tobramycin from 96% ethanol gives 1.2 g of pure tobramycin.

Example 2

Production of fermentation broths containing nebramycin-2 and nebramycin-5 'in a predetermined ratio

Following the procedure of Example 1 (a), 10 batches of 100 ml of said inoculum are inoculated with a spore suspension of Streptomyces tenebrarium MNG 20l + and 10 batches of 100 ml of the same 35 inoculum are inoculated with Streptomyces tenebrarium 11 17 74995 MNG l69. The microorganisms are grown according to Example 1 (a), then 100 ml aliquots of the fermentation broth are sterilized in 500 ml Erlenmeyer flasks and 5 liter aliquots of the fermentation broth 5 are sterilized in a 10 liter fermentor (composition according to Example 1 (a)) and inoculated with both strains either separately or by first mixing the strains in a specified ratio.

The culture is maintained for 1 UU hours under the fermentation conditions described in Example 1 10. Concentrations of antibiotic grades are then determined by thin layer chromatography by first separation and then bioautography and comparison to standard sizes. The results are summarized in Table 3.

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Table 5 ---- 1 - Growth Microorganism 1 Microorganism 2 Nebramycin quality (ml) (ml) __ (yg / ml) _ 4 Z »

Shake flask · 1 10 1 MNG 169 5 ml 5040 180 1310 2 MNG 204 9 ml 0 SO 2190 3 WIG 169 4 »9 ml. MNG 204 0.5 ml 5640 175 1280 4 4.0 ml 1.0 ml 5140 195 1370 15 5 3.6 ml 1.5 ml 4740 130 1610 ..... 6 3.0 ml 2.5 ml 3140 240 1690 7 3.5 ml 2.5 ml 2850 170 1340 8 2.0 ml 3.0 ml 2310 190 1820 9 1.5 ml 3.5 ml 1320 240 2430 2 &: | 10 1.0 ml 4.0 ml. 470 120 2340 11 0, c ::: 1 4.5 ml 1 50 95 2340 j

. i I

Fermen- I

tori 25 I MNG 169 200 ml 3900 210 870 j II MNG 204 200 ml 0 90. 2300 j

--f I

-j III MM G 1 69 175 ml MNG 204 25 ml 4320 240 1130 | IV .. 150 ml 50 ml 30 50 340 1530!

. i I

30- V 125 ml 75 ml. 2450 220 1640: VI 100 ml 100 ml 2320 420 2150. VII 75 ml 125 ml 1420 300 2470 VIII 50 ml 150 ml 920 160 2540 j IX 25 ml 175 ml 230 140 2310 35 1 -! -: - 1

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It is clear from the results of Table 3 that culturing Streptomyces tenebrarius strain MNG 201 + and Streptomyces tenebrarius strain MNG169 can produce fermentation broths containing nebramycin-5 2 and nebramycin-5a in any predetermined ratio. Antibiotics can also be separated from the fermentation broth according to HU patent 17-315.

The bioautography of the broths obtained in fermenters I to IX is shown in Figure 1.

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Claims (2)

74995 20 A method for the microbiological preparation of fermentation broths containing either nebramycin-5 '(6 "-O-carbamoylobramycin) or an antibiotic mixture, wherein 5 is 0.5-77% of nebramycin-2 in a predetermined ratio, 2.7- 8.6 l of nebramycin-4 and 20-95% of nebramycin-5 ', characterized in that a) for the preparation of fermentation broths containing nebramycin-5a, 10 new strains of Streptomyces tenebrarius of the species Streptomyces tenebrarius, or b ) for the preparation of fermentation broths containing in a predetermined ratio 0.5 to 77 t of nebramycin-2, 2.7 to 8.6% of nebramycin-4 and 20 to 95 l of nebramycin-5a, Streptomyces tenebrarius MNG 169- spores or vegetative mycelium of strain 15 (deposited on 28 December 1977 with The National Collection of Microorganisms in Budapest) and the new strain Streptomyces tenebrarius MNG 204 (deposited on 2 September 1981 with the National Collection of Microorganisms, Budapest) 20 sec 100: 1 to 1: 100, cultured in a medium containing organic carbon and nitrogen sources, inorganic salts, trace elements and oils and / or fats of animal and / or vegetable origin as a subsurface culture aerated at 33 to 40 ° C, preferably 25 to 37 ° C :in. Process according to Claim 1, characterized in that the organic carbon source used is starch, glycerol, fructose and / or preferably glucose, and the organic nitrogen source used is nut flour, corn steep liquor, peptone, yeast extract, preferably soy flour, casein and / or amino acids, tamiinihappoa. Il