HU192388B - Process for preparing nurzeotrycine further obtaining thereof in salt form or in adsorbed state - Google Patents

Abstract

Prodn. of nourseothricin (I) or its salts or adsorbates is effected by culturing a (I)-producing Streptomyces strain under submerged aerobic conditions in a medium contg. C and N sources and mineral salts, with the following improvements. - (a) respiratory inhibitors of the alkali metal azide, pyrocatechol and/or ethyl isoamyl barbiturate type and/or amino acid transport inhibitors of the water-soluble Zn salt and/or beta-alanine type are added to the main culture; (b) the prodn. medium is heat-sterilised in the presence of phosphate-precipitating substances (esp. Zn, Fe, Al, Ca, Mn or Mg salts), sulphate ions and CaCO3, opt. with addn. of alkali, to give a pH of 7.2-8.2 after sterilisation. - (c) fermentation is effected with a phosphate supply of 0.1-10 mg/l.hr, a pH of 5-6.5, an O2 partial pressure of 30-80 (esp. 40-60)%, and with further substrate limitations, esp. with a wt. ratio of glucose units (as glucose or oligo- or polysaccharides) to NH4 N of 5-15; 0.015-0.2; (d) (I) is isolated as an adsorbate by adding an adsorbent, esp. bentonite, or is isolated by adsorption on a weakly acidic cation exchanger, elution with a dil. polybasic acid, concn., neutralisation, purificn. and pptn. in MeOH or drying.

Description

The present invention relates to the production of nurzeotricin and its recovery in the form of a salt or an adsorbed state.

Neurzeotricin is an antibiotic belonging to the streptomycin family. When used in livestock feeds, it can accelerate liveweight gain in ergotropic doses, while also improving feed utilization. The antibiotic is used in animal husbandry, and the pharmaceutical industry formulates it.

Characterization of familiar technical solutions

Nurzeotricin isolated from a culture of a variant of Streptomyces noursei ATCC 11455 shows in vitro activity against Gram-positive and Gram-negative bacteria and mycobacteria (Brandler, G. and Thrum, H .: Nourseothrocin A and B, Zwei neue antibacterial anti-Streptomyces Streptomyces n. -Variante. Zschr. Ailgem. Microbiol. 3, 105-112 (1963). Nurzoethricyl is a water-soluble and lower-alcohol-soluble base, and its salts are preferred for handling. The molecule of nurzeotricin (see Figure 8) is made up of the amino sugar of gulosamine, as well as of streptoline and γ-lysine amino acids. The individual components of nurzeotricin differ in the number of peptide-linked / 3-lysine groups present in the molecule and in the meaning of R, R and R. Almost 90% of the nurzeotricin complex is represented by the major components F and D, which are present in about the same proportion, while 10% are the proportion of minor components C and E (Graph, U., Becker, H. Reinhard, G., and Thrum, H: Regulatory Beeinflussung Nursothircinbiosynthesis Durch o-Aminobenzoesaure in Cultures Des Streptomyces noursei JA 3890b Zschr. Al. Microbial 14, 659-673 (1974).

The strain of Streptomyces noursei, which produces the antibiotic nurzeotricin, is deposited with the ZIMET JA 3890b at the Central Institute of Microbiology and Experimental Therapy, Central Academy of Sciences, currently designated ZIMET 43716.

In the prior art procedures (Bocker, H. and H. Thrum, Stimulation of Nitric acid production by aminobenzoic acids, Herold, M.M. and Gabriel Z., Antibiotics - Advances in Research, Production and Clinical Use. London 1966, p. 584,587). the culture is performed under aerobic conditions. Said Lyophilized spores of Streptomyces strain applied to a suitable agar medium and the culture was incubated for 6-10 days at 28-30 ° C ^{to this,} will be used for increased sporulated mycelium to inoculate sterile liquid. For inoculation, the nurseotricin-producing strain can be grown in immersion culture and then canned in gelatin-dried mycelium. The liquid medium required for culturing the vaccine in a submerged culture contains carbon and nitrogen sources and inorganic salts as substrates. The carbon source is glucose and / or glycerol, the nitrogen source is mainly soybean, various amino acids and / or ammonium salts. The most preferred starting pH for the vaccine is between 6.0 and 6.7. The culture is fermented at 28-30 ° C for 24-48 hours.

The resulting vaccine is used to inoculate sterile liquid medium and will be the main culture. The medium contains carbon starch and nitrogen sources of corn starch and / or glucose, while the nitrogen source is mainly soybean meal, various amino acids and / or ammonium salts. The most preferred starting pH for antibiotic production is from about 0 to about 7.5. The fermentation takes place at 26-32 ^o C, preferably at 28-30 o C and lasts for 150 hours,

Streptomyces noursei strain ZIMET JA 3890b is cultured in a well-known manner, in the form of a shake culture, or in a fermentor equipped with mixer and aeration, without addition of substrate or pH control.

It is known (Btaderer, G. and H. Thrum: Noursethricin A and B, zwei neue antibacterielle Antibiotics Euer Streptomyces noursei-Variante, Czech. Ailgem. Microbiol. 3, 105-112 (1963)) that nurzeotricin has a relatively low yield of aminoaryl , especially

It can be increased by addition of 5-10 mmol of o-aminobenzoic acid. The compound added at the start of the main culture, under known conditions, provides a 5-10 fold yield (see Bocker and Thrum, supra).

It is known that o-aminobenzoic acid and / or other amino 'arylcarboxylic acids selectively suppress the formation of α-type cytochromes (cytochrome oxidases), thereby indirectly inhibiting the transport of amino acids into cells and the oxidative deamination of amino acids in cells. This partly removes the repression of secondary metabolism caused by the overgrowth of nitrogen catabolites in the cell and, on the other hand, prevents the amino acids required as a precursor of nurzeotricin from being used upstream of the producing microorganism. During the formation of nurzeotricin, these amino acids are available for secondary metabolism because, as biomass grows, the microorganism prefers an inorganic nitrogen source (ammonium nitrogen).

Although the addition of aminoarylcarboxylic acids results in a substantial increase in yield, the production of nurzeotricin by fermentation is not suitable for large-scale production because of its cost and wastewater treatment problems.

It is also known that the biosynthesis of most secondary metabolites is greatly inhibited by excess phosphate (for review see Martin, J.F. and Demain, A.L., Controlling for antibiotic biosynthesis, Microbiol. Hev. 44, 230-251 (1980)). This is why, technically, fermentation processes for the production of secondary metabolites, such as antibiotics, are carried out at a concentration of phosphate that is not optimal for the production strain. The production of secondary metabolites by fermentation involves the use of complex media consisting of animal and vegetable components (starch, soybean meal, corn jam, molasses, meat peptone, etc.). The components contain different amounts of phosphate, depending on their origin and pretreatment, and the availability (absorption) of the phosphase varies. This condition makes it difficult to typify the medium.

The initial concentration of soluble and uptake phosphate, however, is extremely important for the yield of the desired secondary metabolite, whereas on the one hand some amount of phosphate is required for the growth of the producing microorganism and on the other hand too high a starting phosphate concentration inhibits the formation of the secondary metabolite.

No. 155,239. According to the GDR patent, the concentration of phosphate ions present in culture broth is used as a regulator in fermentation! program to stabilize. However, this process only applies to biomass production, with a nominal concentration of 20-60 mg / l phosphorus. To date, the method has not been used to control the biosynthesis of secondary metabolites in batch microbiological cultures.

In improving the efficiency of microbiological processes for the production of secondary metabolites, the microorganism and the medium are mutually coordinated so that a compromise is reached between the two opposing regulatory influences of phosphate. However, this step of stepwise increase in performance is lengthy and costly.

It is also known that besides altering the composition of the medium, there is another way to increase the fermentation yield: measuring all the parameters of the fermentation and thereby regulating the process (Sukatsch, DA and G. Nasemann, Automation Parameters for Industrial Industries, Chemie-Technik 6,261 / 1977 /).

However, in the case of the above mentioned control method, the primary control parameter is not the concentration of the substrate (usually only after a longer analysis), but the continuously measurable global parameters, such as pH, PM values, dosing volumes, composition of the exhaust gas. , heat generation).

Finally, in the isolation of nurzeotricin it is known to adsorb the active ingredient from the mycelial filtrate to suitable cation exchangers, elute with dilute acid, and after neutralization and concentration of the eluates, the crude product is repeatedly recrystallized from methanol / acetone (see above) and dried. .

Object of the invention

SUMMARY OF THE INVENTION The object of the present invention is to provide a process for the production of nurzeotricin active ingredient in high yield over time and volume.

Summary of the Invention

It has been found that the biosynthesis of nurzeotricin can be stimulated by the introduction of respiratory inhibitors, such as sodium azod or other alkali metal azide, pyritechnin, and amate (ethyl isoamyl-barbituric acid) and / or trans amino acids in living cells at start-up into the main culture. a substance such as beta-alanine or water-soluble zinc salts.

When a high starch disintegrating strain and polymer source of carbon, especially corn starch, is used, an additive that inhibits respiratory processes and / or intracellular amino acid transport is not required.

The metabolic activity (absorption, acid formation, glucose and ammonium uptake, antibiotic production) of nurzeotricin-producing cultures depends primarily on the availability of phosphate rather than on the supply of carbon and nitrogens. It is important to reduce the initial concentration of soluble and bioavailable phosphate during sterilization of the medium, based on the concentration of inorganic salts or metal ions which make the precipitate poorly soluble with phosphate and sulfate ions. It has been found that when sufficient sulfate ions are present, the reason for the decrease in initial concentrations is the reversal of the pH, the calcium sulfate precipitates and removes the acidic sulfate ions, shifting the pH towards the basic range. Thus, the concentration of soluble or absorbable phosphate can be reduced by adjusting the alchemical ρΗ at the beginning of sterilization. Raising the pH provides the conditions under which the metal ions in the medium form an insoluble precipitate with the phosphate. Precipitation of the phosphate-containing precipitate is enhanced by metal ions such as ictnk (II) and / or iron (III), aluminum (III), magnesium), calcium (II), manganese (H). Particularly advantageous in terms of precipitation of phosphates is the use of superheated water vapor sterilization in most industrial fermentations in the presence of calcium carbonate. Calcium carbonate is added for pH buffering.

In principle, the phosphorus content of the medium can also be reduced by reducing the amount of phosphate-containing nutrients. This also reduces the cost of the procedure.

Under these conditions, the initial concentration of dissolved or bioavailable phosphate is reduced, that is to say, lower than the upper phosphate concentration typical of the producing strain. (Due to the phosphate sensitivity of the secondary metabolite synthesis, nurzeotricin is no longer produced above a certain concentration of phosphate.) These low phosphate concentrations allow for sterile phosphate-typed medium to be defined after sterilization with a defined dosage of phosphate. In addition, the rate of phosphate addition can control the phosphate metabolism of microorganisms in the direction of the desired high yield. The phosphate may be added in the form of a solution, suspension or solid state in the form of free phosphate ion or chemically or physically bound phosphate. Depending on the fermentation technology, the phosphate-containing substances or the phosphate are added to the medium after sterilization or during fermentation in single or multiple or continuous addition. The amount per unit time may vary during continuous dosing.

Said phosphate is added to the phosphate released reversibly from complex phosphate sources such as soybean meal, peanut meal, potato starch or by enzymatic decomposition, or from inorganic sediment. The hydrolysis of said organic materials can be enhanced by basic sterilization. Raising the concentration of hydrogen ions (within physiological limits) results in a better release of phosphate from inorganic substances and consequently an increase in metabolic activity.

As a precondition, it must be ensured that no substrate limitation (essential nutrient depletion) that is detrimental to the formation of nurzeotricin occurs.

Sterilization is thus in the basic range of 7.4 and

PH 7.8, final pH range 7.2-8.8. It has also been found that the formation of nurzeotricin can be favorably controlled over a longer period of time if the ratio of glucose to inorganic nitrogen during the fermentation process at pH 5.0 to 6.5 is 5-15.0.015-0.2 (g / l glucose). g / l inorganic nitrogen) and the man-31 is performed at 30-80%, preferably 40-60% partial oxygen pressure (pO-). (The partial oxygen pressure is a relauv value, the saturation value is measured with an oxygen sensor before inoculation in aeration mixed with aeration medium [- 100% /. After inoculation, the growing microorganism consumes some dissolved oxygen, so the sensor measures a smaller value for saturation). The method is described in Moser, Bioprozesstechnik, Springer-Verlag Wien New York 1981).

Surprisingly, it has been found that, with the addition of an appropriate concentration of ammonium hydroxide solution (in the range in which it is effective as a physiological regulator), the time course of ammonium hydroxide administration with the physiologically effective phosphate is . The scale of the representation depends on the lower limit of the 'pH' nominal value. The fading of pH during the first hours of fermentation and hence the start of the addition of ammonium hydroxide depends on the initial nutrient buffering.

Said relationship can be used as a sufficiently sensitive continuous input signal to control direct and / or indirect phosphate addition and thus to regulate the metabolic activity of the fermentation culture.

The direct addition of phosphates takes the form of external storage of phosphate-containing materials, whereas the indirect addition of phosphates results from the enzymatic or hydrolytic degradation of the phosphate complex substrates. It has been found that the transitions between direct and indirect phosphate addition are marked by a significant increase or decrease in the ammonium hydroxide addition curve. The extent of the change is determined by the phosphate supply.

It has now been found that the amount of ammonium hydroxide that is actually added can be varied in the feed of carbon sources, additional nitrogen sources, and other effector substances that can be used as regulatory signals, and the direct addition of phosphate can be varied toward a favorable amount of product. . Thus, under apparatus-dependent oxygen delivery conditions, with a certain amount of aeration, a high final product level, efficient utilization of the substrate within the desired fermentation time, and limitation of biomass production to a workable amount can be achieved.

Further, it has been found that the Cb / Cl2 gold of the reaction heat and / or the exhaust gas mixture follows the course of metabolic activity over time. Therefore, the respective reaction heat as well as said composition can also be used as a control signal for the addition of phosphates and other substances.

Among the nurzeotride-producing Streptomyces varieties of the present invention, preferably those of the species Streptotnyces noursei, which meet the following requirements:

- the formation of nurzeotridn should be sensitive to the high concentration of soluble phosphate in the nutrient of fermentation,

- the micro-organism must be capable of utilizing complex phosphate-containing carbon and nitrogen sources, - have amylolytic activity for the digestion of polymeric carbon sources.

The specific implementation source for the process described depends on the degree to which the strain used meets the above three requirements.

In the case of high sensitivity of the producing strain to phosphates, sufficient amtibiotic production can be achieved only if the initial concentration of soluble phosphate bound phosphorus in the medium is achieved by using the above-described measures (heat sterilization under basic chemistry and / or dosing of phosphate precipitants). we.

The amount of complex phosphate-containing substrates present in sufficient amounts in the medium can be reduced to zero with the addition of phosphate during fermentation of well-digested whey.

If the nurzeotricin-producing Streptomyces strain is resistant to higher concentrations of phosphate, the addition of phosphate precipitating agents to the producing culture can be reduced to zero. According to our experiments, such Streptomyces strains simultaneously have strong amylolytic activity.

Surprisingly, during the processing of the culture broth to obtain the active ingredient, it has been found that nurzeotridn can be isolated in the form of mycelium-containing adsorbate. After the fermentation is stopped, the pH of the culture medium is adjusted to a slightly acidic pH by addition of dilute sulfuric acid, preferably between 6.0 and 6.2. A slightly acidic aqueous suspension of a physiologically acceptable adsorbent, such as natrified bentonite, is then admixed to the culture broth, making sure that the pH is between 5.5 and 6.5. Underflow - nurzeötrlcintartalmú solid is separated by filtration or centrifugation and then dried in flowing hot air in a maximum of 70 ⁶ C product temperature. The resulting mycelium-containing adsorbate is 1-10% based on the nurzeotridine base,

Contains 4-7% active ingredient.

Surprisingly, it has also been found that certain salts of nurzeotridine are poorly soluble in 90-95% aqueous methanol and can therefore be isolated and purified as detailed below. The adsorbate of nurzeotridine with a weak acid ion exchanger is eluted with a dilute polyhydric acid which forms the insoluble salts of nurzeotridine in methanol. Polyvalent inorganic cations present in the eluate as a co-agent are precipitated in the form of their water-insoluble salts. To remove monovalent inorganic cations, the eluate is treated with a sulfonic acid-type, H-form, highly cross-linked cation exchanger, and the liberated acids are neutralized with an OH-form anion exchanger. The eluate was concentrated in vacuo, the impurities adsorbed on charcoal, and the pure salts of nurzeotridine were recovered by precipitation with methanol or gentle drying.

The sulfate of nurzeotridn forms an amorphous, white, water-soluble, poorly soluble powder in methanol and most organic solvents. The elemental composition of the product (C = 32.46, 32.31, H = 6.65, 6.22, N = 15.73, 16.00, S = 7.74, 7.60) is substantially consistent with that of streptyltridine. A 1: 1 mixture of D-sulfate (C CΗΗΗΝΝΝ, ΟΟΛ δ, δ K ₂ H0 ₄ .H-10) and streptothricin F-sulfate (Cj9H34NNONO,, 5 H2SO4 .H2O),

Oxalate of nurzeotridine, amorphous, white, freely soluble in water, in alcohol and other organic solvents

192 388

- a poorly soluble powder, having a composition (C 40.31 40.44, H 6.42, 6.21, N 16.21, 16.49) and an oxalic acid content (20.12, 20.28) of streptotricin-D- oxalate (C} ihr ₈ N ₂ O _in 2.5 C ₂ H ₂ O _{4 .L-3H?} O) and D-streptotrian oxalate (CjaH ^ Ngog T 5 C ₂ H ₂ O _4, l-3 _H2 O) 1: 1 mixture of approach the composition mix.

Nurzeotricin phosphate forms an amorphous, white, water-soluble powder, poorly soluble in alcohols and other organic solvents. Elemental composition (C = O 31.59, 31, 85, H = 6.25, 6.63, N = 14.95, 15.18, P = 10.08, 10.36) a, Streptotricin-D- phosphate _(s8 CaiH N _{2 O! 0th} HaPCb 4 0-1 H> O) and D-phospha-streoptotridn ^t (CMH ₃₄ N ₈ Og. 2 Η ₂ ΡΟ _fourth 0-1 H ₂ O) 1 : 1 in the composition of the mixture! same.

The relative antimicrobial activity of the obtained salts was determined microbially by a diffusion assay on a well plate, using Bacillus subtilis ATCC 6633 as an experimental microorganism. Reference is made to purified nurzeotricin sulphate having the active ingredient content of a mixture of streptotricin-D-sulphate and streptotricin-D-sulphate and streptotricin-F-sulphate; 702 pg base / mg. The microbiological assay conditions are chosen such that 0.5 ml of the 10 pg / ml solution produces a 19 ± 1 mm inhibition zone with a 9 mm hole diameter.

Execution examples

Example 1

Spray lyophilized spore preparation of ZIMET 43716 from Strcptomyces noursei was sprayed onto Emerson agar. The plates are incubated for about 7 days at 30 ° C. After that, pieces of about 2 cm ¹ surface area were cut from the nucleus and inoculated 1 to 1 culture of the first preculture.

The composition of the medium of the first preculture is as follows; 40.0 g of glucose per liter, 15.0 g of extracted soybean meal, 0.3 g of KH ₂ PO ₄ , 5.0 g of NaQ, 3.0 g of CaCO ₃ , added with 1000 ml of tap water, pre-sterilization pH; 6.5-6.9.

50-50 ml of the medium is filled into 500 ml glass-stoppered glass jars and sterilized by autoclaving at 121 ° C for 35 minutes. The inoculated cultures were incubated on a shaking table at 29 ° C for 48 hours. 3 to 3 ml of the thus obtained pre-culture are inoculated into each vessel of the main culture (productive culture). The production culture medium contains the following two basic nutrients per liter:

Medium BO-30: 29.0 g glucose, 13.0 g extracted soybean meal, 5.0 g NaCl, 3.0 g CaCO ₃ , tap water 1000 ml, pre-sterilization pH: 6.5

Medium BO-31: 30.0 g corn starch, 3.0 g glucose, 7.0 g extracted soybean meal, 5.0 g NH ₄ NO ₃ , 2.0 g MgSO _4, 5.0 g NaCl, 3.0 g g CaC0 _3, tap water to 1000 ml pH before sterilization: 6.0.

80-80 ml of the medium is filled into 500 ml glass jars. The dishes are sealed with cotton swabs and sterilized in an autoclave at 121 ° C for 30 minutes. At the time of inoculation, a sterile filtered, aqueous, neutral solution of the additives listed in Tables 1 and 2 is added (up to 3 ml / 80 ml medium). The cultures were incubated at 26 V for 72 to 120 hours at 180 min ^-1 frequency vibrating table. During incubation, the maximum concentration of nurzeotricin was determined using the Bacillus subtilis ATCC 66330 as an experimental microorganism with said well plate assay. Tables 1 and 2 show the relationship between the additive concentration and the maximum nuzeotrldn concentration. The thyme juice is processed according to Examples 8-13.

Example 2

Lyophilized clays substrate spórakészítményét variant of Streptomyces noursei 13-14 marked strain ZIMET 43716 labeled NG sprayed Emerson's medium and incubated for about 7 napog 30 ^& C.

• 0 Then cut out about 2 cm ^{2 of the} neccelium and inoculate each preculture vessel with each piece. The pre-culture medium contains 40 g of glucose, 15 g of extracted soybean meal, 0.3 g of KH ₂ PO ₄ , 5 g of NaCl and 3 g of CaCO _{3 in} 1 liter of buckwheat. Prior to sterilization, the pH was adjusted to 6 5. 50-50 ml of the medium is filled into 500 ml bottles, the bottle is a cotton ball. v; d was sealed and autoclaved at 121 ° C for 35 minutes (s).

Inoculated pre-cultures were shaken at 29 ° C

Incubate for 48 hours. 150 ml of the resulting pre-culture is inoculated into a 2500 ml main culture in a lab fermenter.

For the production culture, Bo-34 medium is used, which contains the following l · omponents per liter of tap water: 32 g potato starch,

1.9 g glucose, 11 g extracted soybean meal, 11 g (NH ₄ ) ₂ SO ₄ , 2 g MgSO ₄ , 7H ₂ Ο, 1 g NaCl, 6 g CaCO ₃ and 0.5 g ZnSO ₄ .7H ₂ O 2500 ml To the medium was added 1 ml of anthaphron as a silicone-based antifoam. Prior to sterilization, the pH was 6.0. The fermentor culture vessel was sterilized in an autoclave at 121 ° C, then cooled to 30 ° C and the pH of the medium was adjusted to 6.8a with 10% sterile sulfuric acid solution under germ-free conditions. The inoculated cultures' (rotation speed 800 min C with stirring ^-1) was fermented for 30 hours at 168, 2500 mL of air per minute aeration. One-and-a-half hours after inoculation, potassium hydrogen phosphate is added, 1.8 ml of an hourly solution of 4 g of KH ₂ PO _{4 in} 1 liter of distilled water are added. This is continued for 5 hours and 3.5 ml of solution is added every hour for the next 21 hours. During the fermentation, samples are taken under sterile conditions and the samples contain Nurzeotricin, glucose, ammoniacal nitrogen and solids. The partial oxygen pressure, pH and carbon dioxide content of the exhaust air are recorded throughout the process. The pH of the culture was maintained above 5.8 by addition of 5% 45% sterile sodium hydroxide solution.

If the glucose concentration drops to 5 g / l or the ammoniacal nitrogen concentration drops to 0.2 g / l (this is noticed by an increase in pH and cq of partial oxygen pressure and a decrease in the CO ₂ content of the exhaust air) 25 g of glucose and 10 g of (NH ₄ ) ₂ SO ₄ were added in a single, sterile solution.

Control fermentation under the same conditions

It was also made with the exception that potassium dihydrogen phosphate, glucose and ammonium sulfate were omitted. When the fermentation was interrupted, the medium still contained a clearly detectable amount of free glucose and free ammonia.

The nurzeotricin content (calculated as base) of the additive fermented and control cultures is given in Table 3.

-5.1

Example 3

The skeletal forming agent of the nurzeotricin-producing strain used in Example 2 was suspended in glucose and gelatin (about 300 mg) in 3 ml of 0.9% sodium chloride solution. 0.5 ml of the suspension was inoculated with 400 ml preculture

The composition and pH of the pre-culture medium were the same as in Example 2. 400-400 ml of the medium is filled into 2500 ml bottles, sealed with cotton swabs and sterilized in an autoclave at 121 ° C for 35 minutes. The room temperature and inoculated with frozen cultures fermented at 29 DEG C. for 48 hours at 180 min ^-1 frequency vibrating table. 1200 ml of the first preculture thus obtained was used to inoculate the second preculture, 150 liters of medium of the same composition (sterilized for one hour at 121 ° C). The second preculture is fermented in a stainless steel fermenter of 250 liters equipped with sterile aeration equipment at 27-29 ° C for 240 minutes at ¹ rpm, aeration of 1 liter of air per minute.

The productive cultures are finally developed in stainless steel fermenters equipped with a sterile aeration unit of 710 liters. 500 L of culture medium BO-34 (recipe: see Example 2) was used per fermenter and 0.5 g / L antaphron was added as an antifoam. Prior to sterilization, the pH was adjusted to 6.7 to 7.

For sterilization, the glucose-free medium is heated to 70 ° C with the fermentor heating jacket and then directly to 121 ° C by direct injection of water vapor and sterilized at this temperature for 15 minutes. Glucose was added to the medium cooled to 30 ° C under sterile conditions, which was separately sterilized by pre-sterilization in a 50% aqueous solution for 30 minutes at 121 ° C. Prior to inoculation, the pH is adjusted to 6.9 to 7.1 by addition of sterile aqueous 10% sulfuric acid.

The production cultures are inoculated with 5% lnokulummal in the second preculture and aeration at 28 to 30 ° C per minute to 500 liters of 0.14 to 0.16 MPa pressurized air mixed at ¹ 320 rpm stirrer was fermented.

To one of the two cultures, from the second hour after the inoculation, an aqueous solution of 87 g of KH ₂ PO _{4 was} added at a constant rate using a metering pump. When the pH drops below 6.3, it is restored with 25% aqueous ammonium hydroxide solution.

Samples from both cultures were collected during fermentation under germ-free conditions. The samples measure the concentration of the nurzeotricin base, as well as the amounts of glucose, ammoniacal nitrogen and solids. The pH and CO ₂ content of the exhaust air are recorded over the entire process.

If the glucose concentration is 5 g / l or the ammonium nitrogen concentration is lower than 0.2 g / l (this is noticed by the increase of the pH value or the decrease of the CO 2 content in the exhaust air) 5000 g glucose or 1 000 g ammonium sulfate in the form of concentrated sterile aqueous solutions.

No phosphate, glucose or ammonium sulfate was added to the other culture, and the pH was not adjusted. Continuous pH measurement is between 6.4 and 7.2. At the end of the fermentation, the control fermentation medium still contains more than 5 g / l glucose and 0.5 g / l ammonium nitrogen.

The nurzeotridn content of the filtrate of the two culture broths is shown in Table 4 as a function of time of concentration.

example

Dried mycelium from a high-performance strain of Sptreptomyces noursei was suspended in physiological saline and inoculated with the medium of the first preculture. The medium contains the following components in 1 liter of tap water:

glucose, 15 g extracted soybean meal, 0.3 g KH ₂ PO ₄ , 5.0 g NaCl, 1.0 g CaCO ₃ , pre-sterilization pH:

6.5 6.9 The medium is filled into 50 ml vials per 50 ml and sterilized at 120 ° C for 35 minutes. The first preculture is fermented on a rotary shaker at 28 ° C for 48 hours.

For the second pre-culture, use a 2000 ml shake flask and 250 ml medium per flask. The first pre-culture is used as an inoculum, namely 1 volume of inoculum to 25 volumes of medium. The second preculture is fermented on a rotary shaker for 24 hours.

800 liters of inoculum was inoculated with 600 ml of the second preculture. The culture medium of the inoculum culture is as follows: 15 g glucose, 15 g extracted soybean meal, 0.3 g KH ₂ PO ₄ , 5.0 g NaCl, 1.0 g CaCO 3, 5 g anthaphone, 37.5 g sunflower oil, ad 1000 ml. Prior to sterilization, the pH is adjusted to 7.2 and 7.4, after sterilization it is between 6.8 and 7.0. The medium was sterilized at 120 ° C for 60 minutes. Fermentation was carried out at 28 [deg.] C. After about 30 hours, 60 mg / L soluble phosphate was consumed from the culture, while 12-15 g / L biomass was formed. 800 liters of inoculum is sufficient for approx. For the inoculation of a volume of 18 m ³ .

Composition of the production culture medium of the following: 15.0 g / 1 soybean meal, 1.0 g NaCl, 32.0 g butyl gonyakeményítő, CaCOj 6.0 g, 0.5 g ZnS0 _4th .7H ₂ O, 2.0 g MgSO ₄ .7H ₂ O, 3.0 g sunflower oil, 0.3 anaphron, 29.0 glucose, 6.0 g (NH ₄ ) ₂ SO ₄ , tap water 1000 ml. Glucose and ammonium sulfate were sterilized separately at 120 ° C for 30 minutes and then added to the medium.

The medium is sterilized at 115 ° C for 60 minutes before the pH is adjusted to 7.5 to 7.8, after sterilization to pH 7.2 to 8.2, after addition of the separately sterilized components. , To a pH of 0 to 7.4. The culture was fermented at 28 ° C with 0.3 volumes of air per minute at a volume of 0.02 MPa and a volume of 60% at a peripheral Οι pressure.

When the glucose concentrate drops below 15 g / l and the concentration of ammoniacal immonium nitrogen is below 0.2 g / l, these components are added in a 50% sterile solution at pH 5.2 and 200 mg / l ammonium nitrogen. until you reach concentrate.

The additional ammonium nitrogen is introduced into the culture by addition of ammonia water at pH < 5.5. After 130 hours of fermentation, the culture broth had a concentration of 8,000-11,000 ng / ml.

Example 5 Sterilized medium containing 4 g of glucose per liter, 15 g of extracted soybean meal, 0.3 g of KH ₂ PO ₄ , 5 g of NaCl, 3 g of CaCO 3 and ad 1000 ml of tap water

400 ml to 400 ml were placed in 2.5 liter vials and then inoculated with 0.5 ml of an aqueous suspension of a mycelium derivative of a variant of Streptomyces noursel Z1MET 43716. The inoculum was fermented on a swinging shaker at 29 ° C for 48 hours.

800 ml of the first preculture thus obtained is inoculated with 100 ml of the same composition of the second precursor, sterilized at 12 for 45 minutes. The second preculture in 240 liter stainless steel fermentor was fermented (speed: 240 ^min-1, aeration: 1, v-1, v-air iész portion of fermentation broth per minute) for 40 hours at 28-29 ° C.

For the production culture (main culture), 4200 liters of stainless steel fermenters are filled with 2500-2500 liters of medium containing 62.5 g of corn starch, 2.5 g of glucose, 16 g of extracted soybean meal, 11 g (NH ₄ ). ₂ SO ₄ , glycol, tap water give 1000 ml. In addition, 0.5 g of ZnSO _{4 was} added to one fermenter. Zinc salt, 7H ₂ O, and 0.1 g of Fe ₂ (SO ₄ ) ₃ iron sulfate are added to the second. Prior to sterilization, the pH was adjusted to 6.2. The glucose-free medium is sterilized by direct injection of steam at 120 ° C for 15 minutes. Separately sterilized glucose in 50% aqueous solution is added to the medium cooled to 29 ° C under sterile conditions. Prior to inoculation, the pH of the medium is adjusted to 6.8 ± 0.1 with sulfuric acid solution.

The productive cultures were inoculated with 4% v / v inoculum (second pre-culture) and fermented at 29 ° C for 140 hours. During fermentation the agitator speed is 180 min ' ¹ . With an overpressure of 0.03 MPa, 0-20. The culture is aerated with 0.5 volumes of air per hour when 1 volume of fact is observed, and from 1 hour with 20 volumes of air per hour. When the pH reached 6.0 ± 0.1, 25% ammonium hydroxide solution was kept constant at this value.

The following nurzeotridn concentrations were measured in the culture solutions by biological assay. time of sampling Nurzeotricin concentration mg'ml

(..hours) ZnSO ₄ .7H ₂ O Fe ₂ (SO ₄ ) ₃ 92nd 4.9 5.1 116th 7.1 6.8 140th 8.3 8.0

Example 6

For the preparation of the vaccine, into Streptomyces bottles containing 400-400 ml of sterilized medium (15 x extracted peanut crumbs, 10 corn jam, 40 g maltose, 5 g ammonium sulfate, 6 k calcium carbonate, tap water per 1000 ml). A 0.5 ml aqueous suspension of a mycelial suspension of a variant of noursel Z1MET 43716 strain was added and the cultures were fermented on a shaking table at 29 ° C for 48 hours.

800 ml of the first preculture obtained was inoculated with 150 liters of the same precursor culture medium, steam-sterilized at 120 ° C for 30 minutes, fermented in 240 liters of steel fermentation at 28-29 ° C for 40 hours, 240 minutes. ^{At 1} rpm, 0.01 MPa of air is cultivated at a volume of 1 volume per minute, aerated at 1 volume per minute.

For the production culture, a stainless steel fermentor (720 L) was added, containing 1000 ml of corn starch, 25 g of extracted peanut crumb, 10 g of corn jam, 10 g of ammonium sulfate, 5 g of chalk powder, 3 g of sunflower oil and tap water. Prior to sterilization, the pH was adjusted to 6.8-7.0. The medium was sterilized at 120 ° C for 30 minutes.

A 5% v / v second pre-culture is added to each fermenter as an inoculum. During fermentation, 30-30 g of corn starch is added twice in the form of a 30% suspension (prepared by aqueous suspension of corn starch and amylolytic degradation of the suspension, for example by brewing enzyme). The pH of the cultures was maintained at 6.2 ± 0.1 with 25% aqueous ammonium hydroxide solution. The tényezeteket 1440 29 hours at ⁴ ° C fermentation (foidulatszám: 320 ^min-1, aeration rate: 1 volume of culture medium per minute, 0.75 part by volume of sub-air pressure: 0.03 MPa). 580 liters of broth are obtained containing 6.85 mg of nurzeotricin per ml.

Example 7

In a stainless steel fermentor with 4200 liters of central stirrer, add 2500 liters of the following medium: 32 g potato starch, 60 g glucose, 11 g extracted soybean meal, 11 g ammonium sulfate, 2 g magnesium sulfate heptahydrate, 1 g sodium chloride, 6 g of calcium carbonate and 0.5 g of zinc sulfate, and tap water gives 1000 ml. As an antifoam, 0.5 g antaphron NM40 per liter of broth is added. Prior to sterilization, the pH was adjusted to 6.2. The glucose-free medium is sterilized by direct injection of water vapor and the sterilization is controlled using the actual (instantaneous) value of the sterilization function as an input control signal. The function riévértéke St = 35 (the impurities brought probability I0 ^'3, before the sterilization cell density of 5.16 ⁶ cells / ml media). This nominal value is controlled by the amount of steam introduced at a ceiling temperature of 120 ° C, the actual value of the sterilization function

St = _o ? Based on [A, E, T (r)] dr, the relationship between the sterilization temperature over time (f / - / = Arrhenius function), the nominal value of the sterilization function and the concentration of acid soluble phosphates in the cooled sterilized medium is determined. Figure 2 shows the relationship between said nominal value and the pH of the medium. The nominal value of St = 35 is 5 µl / l starting phosphate concentrate and pH 8.0. The required pH (7.4) is adjusted by the addition of sulfuric acid solution after the addition of sterilized sterile 50% solution (120 minutes at 120 ° C) under germ-free conditions. The culture medium of the production culture was inoculated with 4% by volume of the second culture according to Example 5 and then fermented at 29 ° C for 120 hours at a pressure of 0.03 Pa. 1250 liters of air per minute; 2500 liters of air per minute hour is introduced into the fermentor, the agitator speed to 180 minutes' ^first *

The fermentation processes are described in Figures 3-7. 3 is a graph showing the time evolution of nurzeotricin and biomass formation (expressed as bionnase dry matter), Figure 4 showing the time course of pH and the glucose and ammonium nitrogen cono-concentrate, respectively. Figure 5 shows the rate of addition of ammonium hydroxide and the time course of phosphate and dissolved oxygen concentration.

Figure 6 shows the evolution of the oxygen and carbon dioxide content of the exhaust air as well as the specific amount of heat generated, while Figure 7 shows the rate of specific product formation and specific growth.

During the fermentation, the regulatory intervention in the form of direct phosphate or glucose addition is carried out batchwise by hand, according to the rate of ammonium hydroxide addition. The allowable pH range is 6.2 ± 0.05, so the lower limit is 6.15 and this value is reached at 38 hours of fermentation.

Sterile aqueous KH ₂ PO ₄ solution is used for direct addition of phosphate. During the first 32 hours, enough of the solution is added so that the culture contains a total of 42 mg of phosphate phosphorus per liter to stimulate metabolic activity.

At the time determined by the chosen pH value and the medium buffering state, in this case, the addition of 25% ammonium hydroxide begins at 38 hours. The decrease in the rate of dosing (recognizing the slope of the curve in Figure 5) causes a direct dose of phosphate at 47 to 48 hours, ranging from 53 to 54 hours, in an amount of 8.5 mg of phosphate phosphorus / Uter culture solution.

The delivery rate of ammonium hydroxide reaches its peak at 62 hours and then begins to decrease, as the direct addition of phosphates is replaced by release of the phosphates from the substrate. For the next 20 hours, the fermentation is complete at the optimum feed rate of about 230 [ml / m ³ / h] 25% ammonium hydroxide. The addition of ammonium hydroxide is sufficient to keep the nitrogen concentrate within the physiologically favorable range and therefore no additional nitrogen source is added to the culture.

The carbon source is also regulated by the rate of addition of ammonium hydroxide. After the second dose of phosphate, the rate of anionic hydroxide dosing is greatly reduced, which is considered a signal for the administration of 48 g / L glucose (56 hours). After 95 hours, 18 g / L glucose was added.

The direct addition of phosphate has no effect on the reconstituted oxygen.

The composition of heat generation and exhaust air indicates the stages of fermentation. Continuous monitoring of the two parameters avoids substrate deficiencies. The final product level of the culture solution (10.5 ml / ml) is achieved by maintaining the specific rate of product formation at 8.10 ³ (hours ^-1 ) for 45 hours. The added glucose is fully utilized and the amount of biomass does not exceed the level of processability.

Example 8

For the isolation of the antibiotic, a saturated aqueous solution of 7.6 kg of oxalic acid is added to 750 liters of culture broth (2900 pg / ml of nurzeotride base). The pH of the solution is adjusted to 4.2 with ammonium hydroxide, the solution is heated briefly to 70 ° C, the solid phase is separated in a separator, the filtrate is neutralized with ammonium hydroxide, re-separated to remove suspended solids and then 25 l / h. from bottom to top through two columns filled with 5 liters of Na-form Wofatit CP ion exchanger. Each column is then washed first with 30 L of water, then with 20 L of 0.1 N acetic acid, then with 15 L of water, and then the antibiotic (per column) is eluted with 20 L of 0.5 N sulfuric acid followed by 10 L of water. Eluates with a pH below 3.0 were neutralized with the OH-form Wofatit AD 41 ion exchanger.

Diammonium hydrogen phosphate solution and ammonium hydroxide solution were added to the eluate of column I (32.8 L) with stirring until no further precipitate formed at pH 7.5-7.8. The precipitated inorganic phosphates were removed by filtration and the clear filtrate was desalted at 20 L / h through 10 L of Hof form Wofatit KP 16 ion exchange resin. The ion-exchange resins are washed with 20 L of water, the water is added to the filtrate, and about 55 L of liquid is concentrated under vacuum at 30-40 ° C to 6.5 L and then clarified with 100 g of activated carbon. The filtrate was acidified to pH 4.0 to 4.5 with sulfuric acid and added dropwise to 75 L of methanol with stirring. The nurzeotridn precipitates in the form of an amorphous precipitate. The precipitate was filtered off, washed with methanol and dried in vacuo at 40 ° C. The eluate of column II is similarly processed. Yield:

Column I: 745 g of nurzeotricin sulfate [.alpha.] D @ 25: 5 DEG (c = 1, water), sulfate ash; ash remaining after digestion with concentrated sulfuric acid

Bioavailability 2.2%: 687 μ & ίπΆ

II. column: 579 g of nurzeotridium sulfate sulfate ash: 2.0% bioavailability: 470 mg / mg

Example 9

9.6 L of the purified and then desalted eluate according to Example 8 are concentrated under vacuum at 30-40 ° C to a volume of 440 ml, the concentrate is clarified with 12 g of activated carbon, filtered and acidified to pH 6.0 with sulfuric acid. There are two ways to process the concentrate.

spray drying

The 220 ml concentrate (dry matter content 33%) was spray-dried in a Mini Spray Dryer Büchi 190 Lab Sprayer with an inlet air temperature of 165-175 ° C and an outlet air temperature of 85-90 ° C.

Yield 58.4 g nurzeotridn sulfate · [α] ^{ζ θ} = -32.8 ° (c = 1, water), sulphate ash: 0.4% Water content: 6.0% determined microbiologically efficiency: 980 Pg / mg

ANALYSIS:

Found:% C, 37.87; H, 6.48; N, 16.99; S, 5.64; C, 37.01; H, 6.66; N, 16.99; S, 5.71.

freeze-drying

The 220 ml concentrate is dried in a freeze dryer TG-5 (manufactured by VEB Hochvakuum Dresden). The product had an initial temperature of -20 ° C and a pressure of 3 at the end of the process _; 99966 Pa Yield: 71.5 g of nurzeotricin sulphate microbiologically determined efficacy: 994 [α] D ^: -34.2 (c 1, water), water content: 3.0%

192 388

Elemental Analysis calculated: C 36.85 H 6.32 N 16.89 S 5.87 Found,% C 36.98 H 6.32 N 17.16 S 5.85 Elemental analysis estimates approximately astreptotransdn-D-. sulfate and streptotricin F-sulfate in a 2: 1 ratio.

Example 10

75 liters of the filtered culture broth obtained in Example 8 (48.46 mg / ml nurzeotrlcin) was passed through a column containing 2 liters of Na-form Wofatlt CP ion exchange resin at 4 l / h. The column was then washed with 15 L of distilled water, 9 L of 0.1 N acetic acid, then again with 10 L of distilled water, followed by elution of the antibiotic with 14 L of 0.5 N phosphoric acid. The eluate at pH 3-4 is neutralized by mixing with 2 L of Wofatlt OH 41 ion exchange resin. The precipitated calcium phosphate is filtered off with the ion exchange resin, and the filtrate is basified to pH 7.7 with ammonium hydroxide solution to give an ammonium magnesium phosphate which is also filtered off. Then, to remove the monovalent cations, the nurzeotricin phosphate solution is mixed with 1 liter of Hoforms Wofatit KP 16 ion exchange resin. The solution (17 liters) was then concentrated in vacuo at 1.5 ° C to 1.5 liters, acidified to pH 4.5 with phosphoric acid and clarified with activated carbon. The clear filtrate was added dropwise to 20 liters of methanol. Colloidally the product remains in solution in 25% dimethyl.

the precipitate was filtered off, washed with methanol and dried in vacuo over calcium chloride. Yield: 169.5 g of nureotricin phosphate [α] ²⁵ D): -29.9 ° (c = 1, water), sulfate ash: 1.06% microbiological efficiency: 818 pg / mg

Example 11

75 liters of purified culture broth (3000 pg / ml nurzeotricin) according to Example 8 was passed at a flow rate of 4 L / hr from the bottom to a 2 L column filled with Wofatit CP ion exchange resin (Na). After adsorption, the column was washed with 15 L of distilled water, 10 L of 0.1 N acetic acid, then again with 10 L of distilled water, and the antibiotic was eluted with 10 L of 0.5 N oxalic acid. All liters of eluate were neutralized by mixing with 2 liters of Wofatit AD 41 ion exchange resin OH. The precipitated salts are filtered off with the ion exchange resin and the clear filtrate is mixed with 2 liters of Hof form Wofatlt KP 16 ion exchange resin to remove monovalent cations. The solution was neutralized with 0.5 L of Wofatit AD 41 ion exchange resin OH and concentrated in vacuo at 800C to 800 mL. The concentrate was clarified with 7 g of activated carbon, filtered and added dropwise to 12 liters of methanol. Colloidalize the remaining product in solution with 25% dimethylamine solution. The filtered precipitate was washed with methanol and dried in vacuo over calcium chloride.

Yield: 162 g of nurzeotricin oxalate [α] 125 I 41.8 ° (c 1, water), sulfate ash: 0.9% microbiological efficiency: 809 mg / mg

Examples of effects

In a pig fattening experiment, feeding a premix mix of 40 mg / kg dry matter supplemented with nurzeotricin, resulted in a 6% improvement in mean liveweight gain during the 70-day trial period. In another experiment, 21 mg / kg of nur3Q zeorridine, in the form of mycelium adsorbate, was added to the feed together with the feed. 70 days of fattening! during the experiment, the average daily weight gain was 23% higher than that of the control, while the treated animals consumed about 7% less feed.

Nurzeotricin was used in the form of a myelium adsorbate at various doses (maximum dose 52.5 mg / animal / day) for rearing calves. The antibiotic had a positive effect on the health of the animals, as seen in the reduction in the number of animals killed and the drastic reduction in diarrhea days. The beneficial effect of live weight gain on calves was mainly observed up to 4 weeks of age. Nurzeotricin added at 35 mg / animal to the feed resulted in a 25% improvement in live weight development over 4 weeks of fattening, with 10 weeks of fattening! at the end of the experiment, this parameter was 13%.

\ nurzeotridn mycelium in animal studies is apparently non-toxic. A single dose of 5000 mg / kg Nurzeotricin Micebum administered by gavage to Wistar rats of both sexes, did not affect body weight gain or cause death.

Example 12

As a premixture containing Nurzeotricin, we make a mellelium bentonite adsorbate. Immediately after the fermentation was terminated, 0.1 liter of formalin per liter of fermentation broth was added and homogenized by stirring for 15-30 minutes. Subsequently, the pH was adjusted to 0.0 by addition of 25% sulfuric acid.

Prior to preservation of the culture broth, a bentonite suspension is prepared in a suitable time by mixing 2 kg of naphthrified (i.e., activated by fine crystalline sodium carbonate) bentonite, 15 liters of water and 0.2 liters of 25% sulfuric acid. The suspension is allowed to swell for at least 12 hours.

The prepared bentonite suspension is mixed with 100 mL of slurry (5000 mg nurzeotricin / mL), usually at a pH of 6.0-6.5. If necessary, adjust the pH to the adjusted range with 25% sulfuric acid and 30% sodium hydroxide solution. During the adsorption, the suspension is constantly stirred. After curing for at least 60 minutes, the solid is separated by filtration or separation. Particularly preferably, the separation is carried out with a vacuum rotary filter. Prior to filtration, a pre-treated calcium sulphate is formed on the filter surface as an auxiliary layer.

7.10 At a pressure reduced to ⁴ Pa, the filter power can reach 250 l / m ² . From 100 liters of culture broth (about 115 liters of liquid together with the bentonite suspension), 93-98 liters of filtrate are separated, the filtrate containing 5-10% of the initial activity.

The filtered solid weighed 12-14 kg with a residual water content of 58-65%. For filtration of a suspension of 110-115 liters, 2.0-2.5 kg of filter aid is used. The separated wet solid was immediately dried.

The drying is carried out in a stream of hot air so that the temperature of the product does not exceed 70 ° C. From 12-14 kg of wet product, 4.5-5.0 kg of dried product was obtained having an active ingredient content of 75-85 g / kg (based on the microbiological assay described at the beginning of the specification, using Baeillus subtilis ATCC 6633 as the experimental microorganism). determined.

In the case of a ring-flow dryer, a fine-grained product is obtained which can be added directly to the compound feed. The product dried in a tray dryer, strip dryer or fluid bed dryer is ground before use.

The nurozeotricl is added at a rate of 5-100 mg / kg of feed mix to the Deficiency for the dry matter of the feed.

. example

A mycelial mouthwash is prepared as a Nurzeotridn-containing premix. After completion of the fermentation, formalin was added to the culture broth as described in Example 12 and the pH was adjusted to 6.0 with sulfuric acid. The pre-treated culture broth is evaporated in a suitable evaporator (vacuum rotary drier, 4.10 ⁴ -6.10 ⁴ Pa, 70 ° C) and the concentrate is dried on a vacuum drying roller to a fine fluffy final product. During the drying at the specified pressure, the product temperature should not exceed 70 ^° C. The final product should have a residual moisture content of less than 3%. From 100 liters of cultured juice originally containing 5000 mg / l of nurzeotridn, a dry matter containing 3750 g of mycelium solids, calculated on a dry matter basis, is obtained, which must be ground before use.

Feeding of the product mixture feed is as described in Example 12,

The advantages of the present invention will be demonstrated by the following animal experiments.

400 Tetra-B broiler chickens, unclassified, were divided into a subgroup of 8-8 animals. Each treatment was performed with 10 repetitions. At the start of the experiment, the average weight of a chicken was 38 g. The composition of the diet was as follows:

64.5% corn meal 2.5% minerals 21.0% extracted soybean meal mixture (54 g P / kg)

6.0% fish meal 1.0% active ingredient mixture

2.0% Feed Yeast (Antibiotic Free)

3.0% rape

The active ingredient is pure nurzeotrine hydrochloride.

The effects of different doses of nurzeotricin on body weight gain, dietary intake and nutritional utilization are shown in Table 5,

The experiment was repeated with broiler chickens kept in bunk cages. 3000 ungraded chickens (tetra B) were divided into subgroups of 100-100 animals. Each experiment was performed with 6 replicates. The mean live weight of the animals at the start of the experiment was 37.5 g. The composition of the diet was the same as described, using pure nurzeotridium hydrochloride as the active ingredient. The results are shown in Table 6.

In another experiment, 55 pigs (crossbreeding animals currently used in pig production) were divided into five groups. At the start of the experiment, the live weight was 10.5 kg / animal.

The feed composition of the mixture used was as follows:

to kg live weight:

45.0% wheat 33.0% barley 5.0% fishmeal 5.0% skimmed milk powder 10.0% extracted soybean meal 1.0% mineral mixture (50 g P / kg)

1.0% active ingredient mixture (antibiotic-free) over kg live weight.

40.0% wheat 48.0% barley

7.0% extracted soybean meal 3.0% fish meal

1.0% Mineral Mixture (35 g P / kg)

1.0% blend of active ingredients (antibiotic free)

The diet was supplemented with different doses of different ergotropics. The nurzeotride was added in the form of pure hydrochloride. Table 7 shows the effect of ergotropics on live weight gain, feed intake and feed utilization.

In a further feeding experiment, 48 piglets (crossbreeding pigs currently used in pig production) were divided into subgroups of 12 to 12 animals.

At the start of the experiment, the mean live weight of the animals in one group was 10.7 kg (group A) and in the other group.

12.6 kg (Group B). The experimental animals were supplemented with mycelium containing bentonite adsorbate. The effect observed in the 70-day experiment is shown in Table 8.

weekly feeding experiment with 36 calves (black-and-white cattle) in 4 groups. At the start of the experiment, the animals had an average live weight of 54 kg. The mycelium-containing bentonite adsorbate was added to the drinker along with the 100 g / l milk replacer. In addition, the animals received ad libitum feed concentrates of the following composition:

30.0% wheat groats

38.0% for April

20.5% extracted soybean meal 4.0% dried beet pulp 3.0% dried fodder yeast 1.5% vitamin concentrate (Mileipan).

Age-appropriate, but maximum 0.5 kg hay was also given.

The effects of different doses of nurzeotricln on animal health and weight gain are shown in Table 9.

-101

192 388

1; Table

Streptomyces noursei ZIMET 43716; content of nurzeotrldn in shake culture of strain strain under different stimulating additives (/ rg / ml culture broth) in BO-3 medium

The additive concentration mmol Sodium azide catechol what you dnkszulfát 0 HEREBY RECOMMEND 0,025 183 _ ._ - 0,050 230 - - - - 0,075 291 - - - - 0,100 330 - - - - 0,125 458 - - - 0,150 544 - - 388 - 0,175 372 - - 399 - 0,200 230 - 440 0,250 224 263 185 469 210 0,500 277 221 560 329 0.750; -r 294 228 702 311 1,000 _ 396 291 552 258 1,250 _ 412 301 439 255 1,500 , _ 382 206 437 212 1/750 - 250 174 124 157 control 95

2, Church

Streptomyces noursei ZIMET 43716; strain nurzeotrldn content of shake culture under different stimulant (jjg / πύ) in Bo-31 medium

The additive concentration mmol Sodium azide catechol zinc sulphate 0,050 961 0,100 1135 _ 0,125 1426 0,150 1891 0,175 1085 _ 0,200 657 941 0,300 - 1401 887 0,400 - 1984 1711 0,500 - 1166 1934 0,750 - 986 2158 1,000 - 868 2585 1,250 - 713 2604 1,500 - 2108 2,000 - 781 control 620

-111

192 388

Table 3, 4, _TábJázat

Streptomyces noursei ZIMET 43716 / NG13-14. Cultures of 5 strains fermented in a laboratory fermentor with or without potassium hydrogen phosphate, glucose and ammonium sulphate, containing nurzeotricin over time (as in Example 2)

The nurzeotridal beverage of the exemplification in Example 3 versus time with and without dosing

Fermentation! Time ..dk hours administration without dosing pg / ml %. pg / mL % 24 1035 249 415 100 48 2980 165 1805 100 72 5412 294 1840 100 96 8311 213 3905 100 120 8800 195 4520 100 144 9230 213 4005 100

116

144

225

1600

4858

4903

6862

7781

216

228

299

208

191

200

104

702

1627

2362

3585

3885

100

100

100

100

100

100

Table 5

Effect of administration of different doses of nurzeotridn on livestock weight gain, feed intake and feed utilization in flat-reared broiler chickens

Nruzeotridn Day 14 Day 28 52.nap · HCl. mg / kg g% g% % LW / animals ί. ϊ sr- sr ar a 0.0 200 100 525 100 163 100 5.0 190 95 532 101 1469 100 10.0 208 104 548 104 1478 101 30.0 210 105 593 113 1548 106 50.0 214 107 588 112 1543rd 196 ^T, 0.05 14 51 feed consumption by animal / animal 77 0.0 ' 1135,100 360 100 5.0 1087 98 3430 96 10.0 1183 107 3505 99 30.0 1146 104 3522 99 50.0 1100 100 3476 98 feed utilization (kg feed / kg live weight) o, o 2.0 100 5.0 2.4 96 10.0 . 2.4 96 30.0 2.3 * 92 50.0 ^T 0.05 2.3 0 (2 - 92

-121

192 385

Table 6

Effect of administration of different doses of nurzeotridn on liveweight gain, feed intake and feed utilization in broiler chickens

Nurzeotridn Day 14 Day 28 Day 52 · HCl mk / kg 8 % g% g % -::; : ς r z - x LW / animals 0 203 100,575,100 1477 100 20 196 97 59 <- 103 1489 101 30 215 106 60? ' 105 1567 106 50 208 103,547.95 1544 105 ^T, 0.05 feed consumption / animal 25 114 0 113,100 3562 100 20 1186 105 3605 102 30 1157 102 3641 102 40 1045 93 3530 99 50 1061 94 3400 96 utilization of feed (kg feed / kg live weight) 0 2.5 100 20 2.5 100 30 2.4 96 40 2.2 88 50 92

Table 7

Effect of various ergotropics on daily weight gain, daily feed intake and feed utilization in piglets

Ergo tropikum '8-45 kg 45-120 kg 8,120 kg 8 % g% 8 % dnkbadtradn 120 mg / kg dnkbadtradn 40 mk / kg 510 Daily weight gain 105 (82.8) 62) 101 (86.0). 581 101 (70.7) kormogrizln 40 mg / kg 559 115 578 94 (61.3) (74.4) 574 ~ Ϊ0Ϊ (49.8) nurzeotridn xHCl 10 mg / kg nurzeotridn xHCl 40 mg / kg 503 587 101 602 98 567 (57.8) (55.0) 121 617 101 602 (36.6) (63.7) 100 (40.2) 106 (47.7) control 484 100,613,100 568 100

-131

Table 8

Effect of different doses of nurzeotricin on livestock weight gain, daily feed intake and feed utilization in piglets

Treatment A mission units Group B g % g % Live weight gain in g / animal.day nurzeotricin 7 mg / kg (496) 104 564 97 (140) (92). 14 mg / kg 548 114 596 102 (41) (118) 21 mg / kg 580 123 616 106 (118) (50) control 479 100 584 100 (104) (85) daily feed consumption in kg / animal nurzeotricin 7 mg / kg 1.38 104 1.66 97 14 mg / kg 1.56 117 1.62 94 21 mg / kg 1.55 117 1.65 98 control 1.33 100 1.72 100 utilization of feed kg / kg nurzeotricin 7 mg / kg 2.78 100 2.94 100 14 mg / kg 2.85 102 2.73 93 21 mg / kg 2.63 95 2.73 93 control 2.78 100 2.94 100

standard deviation of measured values

-14192,388

Table 9

Effects of different doses of nurzeotridn on animal health and livestock weight gain in calves

nurzeotridn mg / animal .day parameter o, o 17.5 35.0 52.5 sr ski s «. »W« a S S SB B 75 51 r β SS S - • S S S S S SS S SS s initial weight in kg 55.0 53.0 53.0 55.0 diarrhea days number. 58.0 17.0 11.0 9.0 dead animals number 1.0 0.0 0.0 0.0 live weight gain g / animal .day 1-4 ..weeks 488 (100%) 552 (113%) 611 (125%) 587 (120%) 1-10. seven 663 (100%) 708 (107%) 742 (113%) 729 (110%)

Claims

Claims (4)

Claims A process for the preparation of nurzeotridine of formula (I) wherein R and R 'are hydrogen and R' is carbamoyl or R and R 'are hydrogen or carbamoyl and R is hydrogen and n is an integer from 1 to 7 - for the preparation of a mixture thereof, salts of the mixture and adsorbates thereof, by aerobic culturing and isolation of the streptomyces strain producing nurzeotricin in a culture medium containing carbon source, nitrogen source and mineral salts, as well as cellular respiratory inhibitors, Streptomyces noursei Z1MET 43716; strains, mutants or variants of the strain, the cellular respiratory inhibitors, namely alkali azide, pyrocatechin and / or ethylisoylamylbarbuturic acid and / or water-soluble dns and / or O-alanine, which inhibit amino acid transport, are added, - phosphate-depleting substances, preferably dnk (II), iron (III), aluminum (III), calcium (II) or magnesium (II) salts, and sulfate ions and kaldum carbonate in the production culture medium. in the presence of the former, if necessary after prior addition of the alkali, is subjected to controlled heat sterilization so as to adjust the pH to between 73 and 8.8 at the end of _{3 £} - 0.1-10 mg / L phosphate-bound phosphorus per hour at pH 5 and Is controlled by addition of alkali in the range of 6.5 to 30-80%, preferably 40-60% relative atmospheric oxygen pressure, - the carbon-to-nitrogen ratio is kept within the limits of 5-15 g / l glucose, 0, 015O, 2 g / l recovering the antibiotic as an adsorbate by adding an adsorbent, such as bentonite, or adsorbing it on a weakly acidic cation exchange resin, eluting with a polyhydric acid such as sulfuric acid, oxalic acid or phosphoric acid, concentrating, neutralizing, , (18.1.1984). 2. The method of claim 1, wherein the rate of addition of the alkali is used to control the pH, control the addition of carbon sources or sources of nitrogen, and / or control the delivery of effector agents and phosphate. (X 25, 1983) Method according to claim 1 or 2, characterized in that the heat generated is used as a control signal. (X 25, 1983) 4. The method of claim 1, wherein the thermal stabilization is controlled by controlling the amount of heat introduced based on a sterilization function. (X. 25, 1983).