FR2519023A1 - MICROBIOLOGICAL PROCESS FOR PREPARING NEBRAMYCIN-2, NEBRAMYCIN-5 'AND NEBRAMYCIN-4 - Google Patents

Abstract

THE INVENTION RELATES TO A PROCESS FOR THE PRODUCTION OF FERMENTATION BROTHS CONTAINING EITHER PRACTICALLY ONLY NEBRAMYCIN-5 (6-O-CARBAMOYL-TOBRAMYCIN, OR A MIXTURE OF NEBRAMYCIN-2 (APRAMYCIN) AND NEBRAMYCIN-5-QUELONQUE REPORT DESIRE PRE-ESTABLISHED AND NEBRAMYCIN-4 (6-O-CARBAMOYL-KANAMYCIN B), THESE FERMENTATION BROTHS ARE PRODUCED FROM STREPTOMYCE TENEBRARIUS STRAIN MNG 204, POSSIBLY IN ASSOCIATION WITH TENEBRARIUS SUBSTITUTION 169 MNGUS 169 APPLICATION TO THE PREPARATION OF CULTURE BROTHS CONTAINING EITHER SUBSTANTIALLY ONLY NEBRAMYCIN-5 OR A MIXTURE OF NEBRAMYCIN-2 AND NEBRAMYCIN-5.

Description

The present invention relates to a microbiological process for

  prepare fermentation broths

  which contain almost exclusively neb-

  mycine-5 ', a mixture of nebramycin-2 and nebramycin-1

  5 'according to any predetermined desired ratio and nebramycin-4.

  It is known that nebramycin, a complex mixture

  of several antibiotics, is a biosynthetic metabolite

  Streptomyces tenebrarius tick (Stark, Hoehn and Knox, Antimicrobial, Agents and Chemotherapy, 1967, p 314;

  British Patent No. 1,178,489 and U.S. Patent 3,691,279).

  The antibiotic mixture is composed, along with several minor factors, of the following main components:

  nebramycin-2 (apramycin); nebramycin-4 (6 "-O-carbamoyl)

  kanamycin B), and nebramycin-5 '(6 "-O-carbamoyl-tobramyl)

  (Koch, Davis and Rhoades, J Antibiotics 26, 745,

  1973) U.S. Patent No. 3,853,709 discloses a

  organism producing nebramycin-7 in addition to the

  Nebramycin-2 Japanese authors in US Patent.

  No. 4,032,404, co-produce nebramycin-2 and

  nebramycin-5 'by cultivating Streptoalloteichus hindus-

  According to the Hungarian patent N 176 103, the Streptomyces tenebrarius MNG 169 and 170, grown under conditions

  submerged and aerated, produce fermentation broths

  containing either nebramycin-2; nebramycin-5 'and nebramycin-4, respectively nebramycin-2 and nebramycin-5', respectively; These antibiotics are isolated according to the technique described in the Hungarian patent

N 174 315.

  Nebramycin-2 inhibits the growth of microorganisms

  phytopatogens and zoopathogens (U.S. Patents 3,691,279, 3,853,709 and 3,876,763, while kanamycin B, obtained by hydrolysis of nebramycin-4 and tobramycin (Brulamycin), obtained by the hydrolysis of nebramycin-5 ', are both antibiotics

  broad spectrum, playing a major role in

  for the treatment of infection caused by polyresistant bacteria (eg Prestonand Wick,

  Artimtimobial Agents and Chemotherapy 1970, p 322).

  By applying the process described in the Hungarian patent N 176 106, an antibiotic mixture is obtained having the following composition Table I):

Table 1

  Nebramycin-2 strain Nebramycin-4 Nebramycin-5 '

MNG 169 77 2 21

  MNG 170 91 traces 9 # Deposited on December 28, 1977 at the National Collection of Microorganisms, National Institute for Public Health,

  Budapest and accessible to the public since 28 February 1980.

  * Deposited on April 5, 1978 in the same collection; accessible

  to the public since 28 February 1980.

  It is apparent that nebramycin-2 is the main product of biosynthesis in both strains, whereas nebramycin-5 'must be separated from the other predominant components at the cost of a highly procedure.

sophisticated and expensive.

  The composition of an antibiotic complex, a biosynthetic

  thetized by a microorganism is determined primarily by the set of cellular genetic information and

  only in a much smaller measure, by the conditions

fermentations.

  In the processes known until now, the ratio of the components in the fermentation broth

  may not be modified in accordance with the industrial requirements

  In addition, if the production of nebramycin-5 'is required, it must be separated from the predominant nebramycin-2 by extremely expensive procedures.

  such systems to multicom Dosers, the evaluation of experiments

  None of these strains, which tend to increase the productivity of the strain, require a lot of work and time. After chromatographic separation on a thin layer, each component is determined in quantity, separately by a biological assay. It would be advantageous to make these selections. of strains with a system with a comnosant allowing simple titration procedures

  Moreover, the increase in productivity in a biological strain

  If a single antibiotic is present, it is more promising, in which case the cells can use their

  entire biosynthetic capacity to form a single compound.

  It is an object of the present invention to develop a novel and simple fermentation process for preparing fermentation broths which contain either only nebramycin-5 'or a mixture of nebramycin-2 and

  5 'nebramvine according to any predetermined desired ratio.

  The Applicant noted with surprise that the

  Streptomyces tenebrarius MNG 169, treated with bromide

  dium (2,7-diamino-10-ethyl-phenyl-phenanthridinium bromide)

  provides a mutant strain that does not produce nebramycin

  2, but gives nebramycin-5 'as the main component beside traces of nebramycin-4 This phenomenon is rather unexpected and surprising to the extent that, until now, ethidium bromide had no all summer

  applied as a mutagenic agent.

  To achieve the isolation of a mutant strain

  synthesizing an antibiotic complex having a modified component ratio, spores of Streptomyces tenebrarius MNG 169 are cultured in a liquid nutrient medium. The culture grown is divided into five parts and amounts of O; 0.05; 0.5; 5.0 and 50.0 μg / l of ethedium bromide are added. The culture is continued for 24 hours, then a portion of the culture is spread on agar in a bunch after dilution, while the other portion serves to inoculate liquid media containing equal amounts of ethidium bromide and their culture is continued for an additional 24 hours. Next, the above procedure for deposition on canned agar and inoculation is repeated. The new liquid cultures obtained are returned to agar again after 24 hours, the agar bunches are incubated, some separate colonies are isolated and their productivity is evaluated according to a procedure.

described in the examples.

  From several hundred isolates from

  Streptomyces tenebrarius MNG 169, two mutant strains fail to produce nebramycin-2, but

  produce relatively small amounts of nebramy-

  In order to improve the productivity of these two mutant strains producing nebramycin-5, strain selection and medium optimization experiments are performed. mutant is lost, while the production capacity

  of the second increases by a factor of five and can be

  This strain was deposited on 2 September 1981 at the National Collection of Microorganisms (National Institute for Public Health, Budapest, Hungary), where it has been assigned the number MNG 204. In addition, the present invention relates to a method for preparing fermentation broths containing a mixture of nebramycin-2 and nebramycin-5 in any predetermined ratio by mixing the spores

  or vegetative mycelia of the In Streptomyces tene-

  brarius MNG 169 and those of the strain Streptomyces tenebrarium 204 not producing nebramycin-2, in a required ratio, and then culturing them in a mixed culture. It was surprisingly found that during this Fermentation carried out with a mixed culture, the two strains produce higher amounts of antibiotics than in fermentations in which each strain

is grown separately.

  From these data, the invention relates to a process for preparing fermentation broths which contain either substantially only nebramycin-5 'or a mixture of nebramycin-2 and nebramycin-5', according to one report. In accordance with the process of the present invention: (a) to prepare fermentation broths containing substantially only nebramycin-5 ', strain MNG 204 from Streptomyces tenerarius, or b) to prepare fermentation broths that contain nebramycin-2 and nebramycin-5 '

  in any predetermined desired relationship and nebramy-

  Figure 4, the vegetative spores or mycelia of any Streptomyces tenebrarius producing a nebramycin complex, preferably the MNG 169 strain, and those of any Streptomyces tenebrarius producing substantially only nebramycin-5 ', preferably strain MNG 204 and are mixed in a required ratio and then grown in a medium containing organic sources of nitrogen and carbon, mineral salts, trace elements, oils and / or fats of animal origin and / or plant in a submerged, aerated culture

  at 33 ° C, preferably at 37 ° C.

  According to a preferred process of the present invention, the production of fermentation broths

  containing nebramycin-5 'next to traces of nebulized

  mycin-4, is carried out by culturing the Streptomyces tenebrarius strain MNG 204 or a mutant, variant or recombinant strain. During fermentation, 10 to 180 μg / ml of nebramycin4 accumulate in the broth. Fermentation next to 2100 to 2600 pg /

  ml of nebramycin-5 ', depending on the culture conditions.

  Nebramycin-5 'is isolated from the fermentation broth and separated from nebramycin-4 and various impurities in a single chromatography step, then

  is hydrolyzed to tobramycin according to known methods.

  in themselves Eventually, nebramycin-5 'can be converted into tobramycin already in the culture and isolated

then of it.

  In accordance with another preferred method of

  present invention for producing fermentation broths

  containing, next to nebramycin-4, a mixture of

  nebramycin-2 and nebramycin-5 'according to some

  pre-established desire, spores and plant mycelia

  Streptomyces tenebrarius MNG 169 and those of Streptomyces tenebrarius MNG 204 are mixed in one ratio.

  required and grown in a mixed culture.

  The products produced are isolated by methods known per se, preferably according to the method described in Hungarian Pat. No. 174,315.

  the amount of nebramycin-2 and nebramycin-5 'bio-

  synthesized is approximately proportional to the cell numbers of Streptomyces tenebrarius strains MNG 169

  and MNG 204, mixed with identical phases of development.

  ment. It is obvious to the specialist that the foregoing considerations are valid only in

  fermentation systems in which the mixing cells

  These species proliferate at almost identical rates and the fermentation conditions are favorable for the biosynthesis of the two components.

  be moved in either direction For a specific

  cialist, the determination of the mixing ratio does not pose

no problem.

  In accordance with the present invention, the micro-

  organisms are grown in a nutrient medium containing an organic source of carbon, i.e., starch, glycerol, fructose or other analogous compounds, preferably glucose; an organic nitrogen source,

  that is to say, nut flour, mace liquor

  wheat ration, peptone, yeast extract or other similar preparations, preferably soy flour or casein, amino acids, preferably glutamic acid; a mineral source of nitrogen, preferably ammonium chloride, ammonium nitrate or ammonium sulfate; inorganic salts, preferably magnesium sulphate, zinc sulphate, cobalt nitrate or calcium carbonate; oils and / or fats of animal and / or vegetable origin, that is to say palm oil, linseed oil, sunflower oil,

  rapeseed oil, pork fat or others, preferably

  a 1: 1 mixture of palm oil and linseed oil and, in addition, trace elements essential for the growth of the microorganism under fermentation conditions.

  submerged and aerated, at a temperature ensuring the development

  microorganism, preferably between 330 and 40 ° C until large amounts of antibiotics accumulate. Antibiotics are isolated

methods known in themselves.

  The digoelements can either be present as impurities in other nutritive ingredients of the

  middle, ie be added separately in the middle.

  The intensity of the air flow is regulated according to the composition of the medium, the performance of the ventilation rotor and other technical characteristics of the fermenter. It is generally advantageous to control the oxygen input, which ensures that the content in C 02 of the gas mixture exiting does not exceed 1.0% and that the reducing sugar content becomes zero between the 96th

and the first hour in the middle.

  Steam sterilization of the medium is carried out at 121 to 1230 ° C for 30 to 60 minutes. The pH of the medium is preferably adjusted to between 7.2 and 7.4 before sterilization. During fermentation, no anti-foaming agent is added as oils and / or fats of animal and / or vegetable origin, added as nutrients in the medium, can simultaneously prevent

  the formation of foam in the medium.

  The culture itself can be carried out in shaking flasks, laboratory fermentation tanks,

factory-pilot or industrial -

  The antibiotic composition of the cultures is titrated by a bioautography according to silica gel thin layer chromatography. Quantities of 0.1 to 0.5 μg / ml of the antibiotic complex are deposited on the silica gel thin-layer plates (Merck , Darmstadt) and chromatographed in a system consisting of 25% ethanol-methylethylketone-25% ammonium hydroxide at a ratio of 1: 1: 1 After careful drying, medium containing subtilated Bacillus ATCC 6633 is spread

  on the surface of the plate and incubated at 370 C for 16 hours.

  The size of the inhibition zones is then compared with that of the zones observed on controls. This procedure is suitable for qualitative and quantitative titration.

the standard deviation being + 10%.

  The main advantages offered by the process according to the present invention are as follows: The strain of Streptomyces tenebrarius MNG 204, producing practically a single component, provides antibiotic levels higher than those of the previous related strain. can be achieved on this new strain, promise a quick success, since this strain could be free to use all its biosynthetic capacity for

  form a unique antibiotic component Biological titration

  of these fermentation broths containing a single antibiotic component is rapid, simple and accurate, the isolation of the antibiotic does not require storytelling purification procedures by mixing the cells of the two different strains and cultivating them in a mixed culture, Fermentation broths containing nebramycin-2 and nebramycin-5 'can be produced according to any predetermined desired ratio, depending on cost situations and actual industrial needs.

  additional advantage, in that during this

  combination, none of the strains lose any significant part of its productivity and both synthesize

  a higher overall level of antibiotics.

  In addition to the foregoing, the invention also includes other arrangements which will be apparent from

the following description.

  The invention will be better understood by means of the

  description which will follow, which refers to

  examples of implementation of the method object of the present invention.

Example 1

  a) Production of fermentation broths containing nebramycin-5 'in laboratory fermentation tanks. Slanted nutrient agar media prepared from distilled water and having the following composition: 1.0% dextrin yeast extract 0.1% hydrolyzate (10%) + 2.0% casein meat extract 0 , 1% cobalt dichloride hexahydrate 0.001% agar 2.5% X enzymatically hydrolyzed These systems are inoculated with spores or

  vegetative mycelia of Streptomyces tenebrarius MNG 204.

  The pH of the medium is adjusted with an aqueous solution of hydro-

  sodium hydroxide at 7.2 before sterilization, then sterilized at 121 ° C. for 20 minutes. The cultures are incubated at 37 ° C for 4 days, then the spores are removed from the surface of the medium by washing with sterile distilled water and the resulting spore suspension is used to inoculate two media of 100 ml of inoculum, prepared with tap water and sterilized in 500 ml Erlenmeyer flasks The inoculum has the following composition: soy flour 2.0% hydrolyzate (10%) * casein 3.0% ammonium nitrate 0.1% ammonium chloride 0.3% calcium carbonate 0.3% magnesium sulphate heptahydrate 0.5% 1: 1 mixture of palm oil and 0.5% glucose linseed oil (sterilized separately in the form of 50% solution) 2.0% e Enzymatically hydrolyzed The pH of the medium is adjusted to 7.2 with an aqueous solution of sodium hydroxide, and the medium is sterilized at 121 ° C. for 20 minutes. in a pressure vessel. Inoculated cultures are grown during 14

  at 18 hours at 37 ° C, in a rotary shaker apparatus (dia-

meter 2.4 cm, 280 rpm).

  20 ml of this inoculum is used completely

  developed to inoculate a laboratory fermentation tank

  10 liters volume of useful volume, containing 5 liters of the main fermentation medium, prepared from tap water, sterilized at 121 ° C. for 45 minutes and having the following composition: 3.0% hydrolyzate soy flour (10%) ) 4 casein 5.0% ammonium nitrate 0.1% ammonium chloride 0.5% L-glutamic acid 0.8% magnesium sulphate heptahydrate 0.5% cobalt dinitrate hexahydrate 0.001% 1: 1 mixture of palm oil and linseed oil 3.0% glucose (sterilized separately as a 50% solution) 4.2%

* hydrolyzed enzymatically.

  P H is adjusted to 7.2 using hydroxide amide

monium before sterilization.

  The culture is performed at 37 C with 360 rpm ventilation rotor speed and sterile airflow

of 3 liters / minute.

  At the 144th hour of culture, 260 mg of nebramycin-5 'and 9 mg of nebramycin-4 are titrated in 100 ml of

  broth None nebramycin-2 is detected.

  Other nutrient media with a different

  composition, can also be used for

  Thus, following method a), similar results are obtained using the following medium: Soy flour 3.3% Ammonium chloride 0.6% Magnesium sulphate heptahydrate 0.7% calcium carbonate 0, 5% cobalt dinitrate hexahydrate 0.001% palm oil 2.5 glucose (sterilized separately as a 50% solution Y * 2.1%

  * Depending on the geometric configuration and access

  fermentation tank used for cultivation and factors influencing cell metabolism,

  Glucose concentration can be increased up to 4%.

  At the 144th hour of culture, 100 ml of fermentation broth contain, apart from traces of nebramycin-4

  (3 mg for the highest level), 180 mg nebramycin-5 '.

  Nebramycin-2 can not be detected.

  (b) Production of fermentation broths

  nebramycin-5 'in a fermentation tank of

1000 liters of a pilot unit.

  To produce fermentation broths

  For large amounts of nebramycin-5 ', the procedure described in (a) is inoculated with the vegetative spores or mycelia of Streptomyces tenebrarius MNG 204 for 100 ml of the inoculum medium and cultured for 14-18 hours. 100 liters of a sterile inoculum medium are then inoculated with this culture and cultured at 37 ° C. with an air flow rate of 100 liters / minute and agitation at 360 rpm. After 16 hours, 1000 liters of the main medium. sterilized fermentation (composition indicated in a), are inoculated in a stainless steel fermentation tank, with 50 liters of this inoculum The main fermentation is carried out at 37 C with an air flow of 500 1 / minute with agitation of 200 rpm Changes in product content

  antibiotics are shown in Table 2.

Table 2

  Duration of the closure Component Nebramycin (ig / ml) hours 2 4 5 '

24 O O 50

48 O O 400

72 0 5 1200

96 O 30 1740

O 40 2320

144 O 60 2650

  c) Decarbamoylation of nebramycin-5 'and isolation

of tobramycin.

  22 g of sodium hydroxide are added to 3.0 liters of

  fermentation broth containing 2600 kg / ml of neb-

  mycine-5 'and 90 μg / ml nebramycin-4; we heat the

  broth with constant stirring at 90 100 C and

  hold this temperature for 25 minutes.

  The mixture is cooled to room temperature and its pH is adjusted to 2.0 with 50% aqueous sulfuric acid solution. This acidic fermentation broth is treated with 6 g of oxalic acid with stirring. constant and its pH is then adjusted to 7.0, then the mixture is stirred for an additional 30 minutes, filtered and the mycilium is washed twice with 600 ml of water The filtrate and the washings are combined and the resulting solution removed from calcium ion, is subjected to ion exchange chromatography on a column prepared from 200 ml

  of Amberlite CG-50 (NH 4 +) (Rohm and Hass Co, Philadelphia, USA).

  The column is washed with 400 ml of deionized water and eluted first with 1000 ml of ammonium hydroxide solution, 0.05 N and then with 2000 ml of such a 0.1 N solution and finally

  with 3000 ml of a 0.15 N solution of ammonium hydroxide.

  During the elution, fractions of 250 ml are collected

  Fractions 13 to 18, containing tobramycin, are collected

  the pH of the solution is adjusted to 7.0 with a solution

  50% aqueous sulfuric acid and subjected to a

  ion exchange graph on 120 ml of Amberlite CG-50 (NH 4).

  The column is washed with 200 ml of deionized water and the

  biotic is eluted first with 1000 ml of 0.05 N solution, then with 2000 ml of 0.1 N solution and 2000 ml of 0.15 N solution and finally 2000 ml of a 0.2 N solution of hydroxide

  Ammonium Fractions of 60 ml are collected Fractions

  The residue, dried over phosphorus pentoxide, gives a product containing 0.7 g (70%) of kanamycin B, 12% of component N-13 (6'-N-carbamoyltamramycin) and the mixture is evaporated under reduced pressure. ), 10% of an unknown component and 8% of tobramycin Fractions 57 to 58 are evaporated and a residue containing 0.2 g (60%) of tobramycin, nearly 40% of kanamycin B and 0.1 to 0 is collected. 2% of N-13 component The distillation of fractions 59 to 69 provides 5.7 g of tobramycin containing 0.1 to 0.15% of N-13 component. By recrystallizing this crude tobramycin

  in 96% aqueous ethanol, 4.2 g of tobromine are obtained.

pure mycine.

Example 2

  Production of fermentation broths containing nebramycin-2 and nebramycin-5 'according to a pre-established report. By applying the procedure of a) of Example 1, 10 100 ml portions of an inoculum medium such as

  described above, with a spore suspension of Strep-

  tomyces tenebrarius MNG 204 and 10 further 100 ml portions of the same inoculum medium with the spore suspension of Streptomyces tenebrarius MNG 169 The microorganisms are cultured according to method a) of Example 1, followed by 100 ml portions of a medium sterilized in 500 ml Erlenmeyer flasks and 5 liter portions of a sterilized fermentation main medium in a 10 liter fermentation tank and having the composition indicated in a) of Example 1 are separately inoculated with both strains or after

  their mixture in a ratio determined below.

  The culture is continued for 144 hours in

  the fermentation conditions described in Example 1.

  Then, the content of antibiotic components is titrated

  by thin layer chromatography, bioautography

  cutive and comparison with a control The results are

collected in Table 3.

  OP 22: Ol M him GLI him 9 x 1 op, 091 086 him M 1 itu 09 IIIA OCZ OO W t, 1 him 31 him ç L IIA M TZ Ot O 71 3 Ira 001 him 001 IA O 9 lo?, Z OC-1 2 ila 9 L TW 9?, 1 AO -9 T Ot 1 Oç Oç him 09 IIU Oç T AI Oç-il O 3 O 8 Tm foin o Nx Tu 'Ll 69-1 ONN ooç_z 06 O IW 00? O 9 01 M II OL 8 Oi ?, OOK no. OOZ 691 ONVI I UOT-4 -Qquaw 2 ad ap to An D 0 71 6 M i: uu iw "O il 091 OLIV -Lui O, t Eu O ' T 01 O 7, O 71 Ozl lm 91, TEU 6 0991 0101 OI -3 TW O "Do you O 7, 9 Otel OLI Ob '2 2 Tw ç' F T-LU 9 i el L 0691 O 3 Otiç Tw 9 2: lm oliç_ 9 0-19 T ORT On-V tw 9 ILLUSTRA TION YES 6 l O 1 llui 0111 him Oi 09?, T 5 LI Ov 9 lia 9 "OO ?, '91 \ zqlui 691 Dizi ç Ogi ?, 09 O IW 9 -Voz sky 71 Oli 091 Ot 1 O him 69 i oim 1 gnooas uooe-lj TW) (Ttu) aulo Atut-aqq 4 uesodwo DZ -a UISEU 7 C XOOJDTW 1,, a Wcj TU'e Ld IOOZDTW eanq-In D sú M 9 L 0 L aq

2 ZO 6 LSZ

s 1

  The data in Table 3 clearly show that

  Fermentation lions containing nebramycin-2 and nebramycin-5 'according to any desired preset ratio can be produced by application of Streptomyces tenebrarius MNG 204 and Streptomyces tenebrarius MNG 169. Antibiotics can be isolated from fermentation broths according to the technique that is described in the

Hungarian Patent No. 174,315.

  The bioautography of the broths obtained in fermentation tanks I to IX is represented in

figure 1.

  As is apparent from the foregoing, the invention is not limited to those of its modes of implementation, implementation and application that have just been described more explicitly; on the contrary, it embraces all the variants that may come to the mind of the technician in the field, without departing from the scope, the scope, the

present invention.

Claims (4)

  Process for preparing fermentation broths   containing only substantially nebramycin   or a mixture of nebramycin-2 and nebramycin-5 'according to any predetermined desired ratio and nebromycin-4, characterized in that: a) to prepare fermentation broths containing substantially only nebramycin-5; Streptomyces tenebrarius strain MNG 204 is used, or b) to prepare fermentation broths containing a mixture of nebramycin-2 and nebramycin-5 'in any predetermined desired ratio, use is made of them.   spores or mycelia of any strain of Streptococcus   myces tenebrarius, producing a nebramycin complex, preferably strain MNG 169 (deposited December 29, 1980) at the National Collection of Microorganisms, Budapest) and those of any strain of Streptomyces tenebrarius producing substantially only 5 'nebramycin , preferably strain MNG 204 (deposited on September 2, 1981 at the National Collection of Microorganisms, Budapest) which is mixed according to the required ratio and grown in a medium containing organic sources of carbon and nitrogen, mineral salts , oligoé 3 éments, oils and / or fats of animal and / or plant origin, in a submerged, aerated culture, between 33 and 40 C and at 37 C preferably.  Process according to claim 1, characterized in that the organic carbon source used comprises starch, glycerol, fructose and preferably clucose and that the organic nitrogen source comprises nut flour, maceration liquor wheat, a peptone, a yeast extract, preferably soy flour or casein, amino acids, preferably glutamic acid. 3 Fermentation broth containing   nebramycin-5 'or a mixture of nebramycin-2 and nebramycin   cine-5 'according to any pre-established desire ratio and   nebramycin-4, characterized in that it is prepared according to   the process described in any of the   1 or 2. 4 Nebramycin-5 'or mixture of nebramycin-2 and nebramycin-5' according to any predetermined desired ratio and of nebramycin-4, characterized in that they are prepared   according to the method of claims 1 or 2.