DE1204362B - Production and extraction of the antibiotic tylosin - Google Patents

Description

Manufacture and Obtainment of the Antibiotic Tylosin The Invention relates to the production of tylosin by growing a tylosin producing strain of Streptomyces fradiae in a nutrient medium with assimilable sources of carbon, Nitrogen and inorganic salts under submerged aerobic conditions until a larger amount of tylosin has been produced, recovery of the tylosin from the nutrient medium and optionally conversion to its acid addition salts.

This invention provides a new organic base that is arbitrary here Was called tylosin. The acid hydrolysis of tylosin results in removal of one Mycarose sugar part to the formation of desmycosin. This proceeding is not an issue of this invention. Chemical, physical and other properties of the new organic Base, which will be discussed later, are used to identify the Compounds and distinguish them from compounds already known or described.

The free base form of tylosin has the following characteristic properties: It is a white solid and can be easily prepared from a number of crystallization solutions, z. B. aqueous acetone, aqueous lower alcohols such as aqueous ethanol and water, crystallize out as small platelets. The free base, the inverse solubility shows can be crystallized from water by placing them in water at about 2 ° C dissolves and the solution slowly warms to room temperature or a little above, thereby the tylosin is deposited in crystal form. The dried crystals of tylosin melt at about 127 to 132 ° C.

The free base of tylosin is in slightly acidic aqueous solutions, z. B. 5% aqueous acetic acid, soluble. It is also polar in most soluble in organic solvents; good examples of this are lower ketones such as Acetone, methyl ethyl ketone and methyl isobutyl ketone, lower alcohols such as methanol and ethanol, lower esters such as ethyl acetate and ethyl formate, halogenated hydrocarbons such as methylene chloride and chloroform, ethers such as diethyl ether, nitrogen-containing organic Solvents such as dimethylformamide, pyridine and triethylamine and heterocyclic organic solvents such as tetrahydrofuran and thiophene. It is soluble in benzene, but relatively insoluble in non-polar solvents from the group of aliphatic Hydrocarbons such as hexane and heptane. It is slightly soluble in water and in slightly alkaline aqueous solutions.

The free base of tylosin is in solution in a pH range between pH 4 and 9 relatively stable, but is unstable in strongly acidic or basic Solutions. If tylosin is treated with an acidic solution with a pH value approximately below 4, so soon the free base decomposes, with desmycosin and a carbohydrate fragment Mycarosis develops, which was mentioned earlier.

An electrometric titration of tylosin in a solution of dimethylformamide in water (2: 1 parts by volume) indicates the presence of a titratable group pK'a = 7.0 out.

The molecular weight determined with the help of the titration results is around 931 ± 30.

A solubility analysis of the crystalline base in water showed that Tylosin has a solubility level of 6.0 mg / cma at 26 ° C.

The mean of several elemental analyzes of the crystalline base, which was dried in vacuo at about 80 ° C. over phosphorus pentoxide, gave the following values: carbon. . . . . . . . . . . . . . . . . . . . 59.680 / 0 hydrogen. . . . . . . . . . . . . . . . . . . . 8.530 /, nitrogen ...................... 1.55% oxygen (from difference). . . . . . . . 30,240 / 0 The above results suggest an empirical formula C48H "N01 $, but, as one can imagine, this formula is necessarily only an approximation, and it may need some correction if and when the molecular structure of the compound is completely determined .

The IR absorption curve of the crystalline free base in chloroform is shown in the figure of the drawings. The recognizable lines in the infrared spectrum in the range from 2.0 to 15.0 #t are the following: 2.85, 3.36, 3.41 (paragraphs at 3.47, 3.52 and 3.58), 3.68, 5.80, 5.94, 6.27, 6.88, 7.09, 7.27, 7.36, 7.61 7, 90 8.45, 8.62, 8.75, 8.95, 9.28, 9.53, 9.85, 10.04, 10.13, 10.40, 10.80, 11.10, 11, 53 11.91.

The ultraviolet absorption spectrum of tylosin in water shows a strong absorption maximum at about 288 m # t with a molar absorbance of --241.

The specific optical rotation of a sample of the crystalline free base of tylosin, dried at room temperature in vacuo over anhydrous calcium chloride for about 15 hours, was found to be: c = 2% in methanol (weight / volume).

A powder X-ray diagram using unfiltered chromium radiation and an oc wavelength of 2.2896 A to calculate the interplanar spaces gives the following values: a I r / rl 7.72 0.40 6.50 1.00 6.12 0.40 5.74 0.40 5.18 0.40 4.71 0.08 4.54 0.16 4.37 0.60 4.23 0.02 4.12 0.08 3.84 0.12 3.68 0.12 3.63 0.12 3.47 0.12 3.31 0.08 3.19 0.12 3.05 0.02 2.97 0.20 2.84 0.02 2.74 0.04 2.68 0.04 2.47 0.02 2.39 0.04 2.33 0.04 2.25 0.04 2.14 0.02 2.032 0.02 1.908 0.02 Chemical studies of the crystalline free base showed that methoxy, oxy, N-methyl and C-methyl groups are present. Molisch and anthrone samples with the free base to examine the presence of hydrocarbons gave positive results. It decolorizes permanganate solution. Ninhydrin, biuret and Sakaguchi protein samples give negative results; The ferric chloride test for phenol groups and the maltol test are also negative.

Chromatography of the crystalline tylosin base on Whatman paper No. 1 gives the following values for Rf: Rf = 0.36 in a solvent of water with 7 / () magnesium sulfate (volume / weight) and 2.5% methyl ethyl ketone (volume / volume); Rf = 0.86 in a solvent of n-butanol saturated with water; Rf = 0.09 in a solvent of methyl isobutyl ketone saturated with water at 2% p-toluenesulfonic acid hydrate (volume / weight); Rf = 0.83 in a solvent Methyl ethyl ketone which is saturated with water. To determine the above values the tylosin base was applied to the paper in acetone solution.

The free base of tylosin has a growth-inhibiting effect on microorganisms, to both gram-positive and gram-negative bacteria, some of which are phytopathogenic works. But the vast majority of the organisms on which it has an inhibitory effect are gram positive. The degree to which tylosin is exemplary on the growth of organisms has a hindering effect is given numerically for the individual organisms in Table 1. The degree of inhibitor was determined by the agar dilution test or by the broth dilution test (both types are identified in the table with the letters »ad« and »bd« designated).

In the agar dilution test, the organism to be examined is exposed to different agar plates streaked or transplanted, the tylosin in different Concentration included to the minimum concentration of tylosin base in p.g / cm3 (micrograms per cubic centimeter) in the agar substrate to determine the growth of the organism for a period of 48 hours (or 72 hours in the case of phytopathogenic organisms) prevented.

The broth dilution test uses a number of tubes, the nutrient broth with tylosin contained in different concentrations, with the to be examined Inoculated to the minimum concentration of tylosin base in # tg / cm3 in the organism Brewing substrate to determine the growth of the organism for a period of about Prevented for 20 hours.

The acid addition salts of tylosin show about the same potency as those given in Table I. Table I. concentration Test organism with contraceptive effect (fL / ern 3) bacteria Staphylococcus aureus. . ....... 1.56 ad Staphylococcus albus. . . . . . . . . . 3.13 ad Bacillus subtilis. . . . . . . . . . . . . . . 1.56 ad Mycobacterium phlei. . . . . . . . . . 0.78 ad Mycobacterium tuberculosis (607) 0.78 ad Mycobacterium avium. . . . . . . . . 0.78 ad Escherichia coli. . . . . . . . . . . . . . . > 100 ad Proteus vulgaris ............... 50 ad Pseudomonas aeroginosa ....... > 100 ad Aerobacter aerogenes. . . . . . . . . . > l00 ad Klebsiella pheumoniae. . . . . . . . . 12.5 ad Salmonella enteritides. . . . . . . . . > 100 ad Shigella paradysenteriae. . . . . . . . 100 ad Brucella bronchiseptica. . . . . . . . 25 ad Vibro metchnikovii. . . . . . . . . . . 50 ad Streptococcus pyogenes. . . . . . . . 0.195 bd concentration Test organism with contraceptive effect qt / Crn 3) Corynebacterium diphtheriae. . . 0.0975 bd Diplococcus pneumoniae ....... 0.195 bd Plant pathogenic bacteria Erwinia amylovora. . . . . . . . . . . . 100 ad Agrobacterium tumefaciens ..... > 100 ad Xanthomonas campestris ....... 25 ad Xanthomonas malvacearum .... 12.5 ad Xanthomonas phaseoli. . . . . . . . . 6.25 ad Pseudomonas syringae .... . . . . . 25 ad Corynebacterium insidiosum .... 100 ad Corynebacterium sepodonicum .. - The table above shows that tylosin has a relatively broad antibacterial activity. This effectiveness is demonstrated not only in vitro but also in vivo. Sk, whether the agent is administered subcutaneously, intraperitoneally, or orally, the treatment of mice infected by various pathogenic organisms leads to immediate elimination of the infection. The EDSO value of tylosin (dose sufficient in its effect to protect 5001 of the mice used as test animals) for some exemplary infections can be seen from Table II. Table II Appointment ED6o Organism that provides mg of tylosin Infection caused by the kg of body Tylosin weight Staphylococcus aureus .... subcutaneous 3.8 (x2) Staphylococcus aureus .... peroral 42 (X2) Diplococcus pneumoniae subcutaneous 4.8 (x2) Meningopneumonitis virus intraperi- 41.5 (x 5) toneal Streptococcus Pyogenes C 203. . . . . . . . . . . . . . . . subcutaneous 2.6 (x 2) Streptococcus haemolyticus peroral 65 (x2) In mice, the administration of tylosin causes infections by Spirochaeta vovyi to be overcome, the effective dose for oral administration being 31 mg / kg body weight; if the drug is administered intraperitoneally, a dosage of about 2 mg / kg body weight is effective.

Tylosin can be made by growing new and previously found ones organism strains not described, which were obtained from soil samples from Nongkhai, Thailand, were isolated. The organisms were submerged cultures taking by fermentation aerobic conditions grown in a nutrient medium, the assimilable carbon and nitrogen sources and such inorganic salts. The organisms were isolated from the above-mentioned soil samples by removing parts of these soil samples suspended in sterile distilled water and these suspensions on nutrient agar have been deleted. The inoculated nutrient agar plates were for several days at 25 to 30 ° C incubated. After incubation, these tylosin-producing organisms became colonies Using a sterile platinum loop, transfer them to agar slanted culture media for inoculation. the inoculated slant culture media were used to produce larger quantities of inoculum Tylosin production incubated.

The new organisms with the property of producing tylosin are for permanent at »The Culture Collection of the Northern Utilization Research and Development branch of the U. S. Department of Agriculture "in Peoria, Illinois, with the numbers NRRL 2702 and NRRL 2703 have been deposited.

In some ways, the newly discovered organisms are very similar to that Species Streptomyces fradiae from the group of the Actinomycetales, determined in »Bergey's Manual of Determinative Bacteriology, 7th Edition, p.799. The new organisms NRRL 2702 and NRRL 2703 have been classified as new strains of the species S. fradiae, an organism first determined by W a k s m a n, which is now known by the »American Type Culture Collection as ATCC 10745 (Waksman 3535). A clear difference between the new strains and the Waksman strain of S. fradiae is that the Waksman strain is incapable of producing tylosin or desmycosin. The Waksman strain was grown in the preferred media described herein, but no detectable amount of tylosin or desmycosin was ever observed.

At this point, the tabular comparison of the properties of fradicin and tylosin is given: Comparison of the properties of fradicin and tylosin Property 1 Fradicin Tylosin Color ................... greenish yellow white Molecular weight. . . . . . . . . . 514 904 Formula .................. C3oH34N404 C4sH77NCi7 Melting point. . . . . . . . . . . . 180 to 300 ° C (no defined melting point 128 to 130 ° C Zen) Optical activity. . . . . . . . (a) DS = +65 [a] D5 = -45 UV absorption maxima .... 290 to 295 m # t, hexane 340, 356, 378m. 283 m # t, no absorption in the area from 300 to 400 m @ c Solubility. . ...... . . . .. ... 0.05 mg / ml in water at 25 ° C, very 5 mg / ml in water at 25 ° C, easy slightly soluble in alcohols soluble in alcohols Toxicity. . . . . . . . . . . . . . . . LDbo = 4 mg / kg intraperitoneally and LDso = 3000 mg / kg subcutaneously orally Biological effectiveness .... only antifungal LDSO => 5000 mg / kg orally Literature. . . . . . . . . . . . . . . . Proc. Soc. Exptl. Biol. Gram-positive, gram-negative, TB, Med., 73, p. 376 (1950), 113, 361 Spirochetes, PPLO (1951) The invention will be discussed in detail with particular reference to the newly found organism, NRRL 2702. It should be noted, however, that the manufacture of the antibiotic can be obtained by cultivating other tylosin organisms, e.g. B. other tylosin producing strains including NRRL 2703 or variants of NRRL 2702 and NRRL 2703 also fall under this invention. Such other organisms, strains or mutants can be obtained by known methods, e.g. B. by subjecting a tylosin-producing organism to X-rays or ultraviolet radiation or treating it with chemical agents such as mustard oils.

The following sections summarize the results of more thorough classification studies with the tylosin producing strain S. fradiae NRRL 2702 described above. The colors used in the representation agree with those in R i d g w a y: »Culture Standards and Nomenclature ”(1912), used definitions.

Microscopic morphology of tomato paste oatmeal agar (14 days at 30 ° C). The spore chains form hooks, loops and irregular tangles. Typical Spirals are rare. The spores have an almost spherical shape and are roughly 0.8 to 1.5 #t in size.

Agar with inorganic salts and starch (14 days at 30 ° C). Same microscopic morphology as observed in the tomato paste oatmeal agar became.

Agar with inorganic salts and ribose (14 days at 30 ° C). Same microscopic morphology as observed with the tomato paste oatmeal agar became.

Characteristic culture features of Czapek's agar (14 days at 30 ° C). Medium growth. No aerial mycelium. Underside almost cream-colored. Calcium aleate agar (14 days at 30 ° C). Moderate growth. Shiny creamy in the air. Underside Naples yellow. Glucose asparagine agar (14 days at 30 ° C). Very little growth. Agar with inorganic Salts and starches (14 days at 30 ° C). Strong growth. Light aerial mycelium, white to pale ocher salmon. Underside almost shiny cream. Tomato and oatmeal agar (14 days at 30 ° C). Strong growth. Light aerial mycelium, white to glossy wine-colored. Emersons' agar (14 days at 30 ° C). Moderate growth. Faint aerial mycelium, white. Underside almost shiny ocher.

Yeast extract agar (14 days at 30 ° C). Moderate growth. Faint aerial mycelium, white. Underside antimony yellow. Potato peg agar (14 days at 30 ° C). No noticeable growth. Physiology gelatin liquefaction. . . . . . . . . . . . slow nitrate reduction ....... . . . . . . . . . reduced nitrates nitrate reduction. . . ... . .. ... . . . . no HZS formation ..... ............. no starch hydrolysis. . . ... . . . . . . . . . . plenty of temperature 26 to 30 ° C .... .......... .. optimal growth 32 to 37 ° C. . . . . . . . . . . . . . . . optimal spore formation No formation of aerial mycelium at 26 ° C.

Table III lists the results of carbon utility tests performed on NRRL 2702 organism. The following symbols are used: + = growth and usefulness, - = no growth, no usefulness, = limited growth, probably poor usefulness. Table III Carbon exploitation theme for NRRL 2702 Compound I growth reaction L (+) arabinose. . . . . . . . . . . . . + L (- @ -) rhamnose. . . . . . . . . . . . . + D (+) ribose ................. - D (+) xylose. . . . . . . . . . . . . . . . + D (-) fructose. . . . . . . . . . . . . . + D (+) glucose ................ + D (- @) melibiosis. . . . . . . . . . . . . . - Sucrose ...................... + D (+) trehalose. . . . . . . . . . . . . . + D (+) MelizitWe. . . . . . . . . . . . . - D (+) raffinose. .. ... ... . . . .. - Cellulose ..................... - Inulin ............... «....... - Adonit ....... ............... - i-inositol. . . . . . . . . . . . . . . . . . . . .. - Mannitol ..................... Sodium acetate ............... + Salicin ...................... - As mentioned, tylosin can be obtained by cultivating NRRL 2702. A number of media can be used as a culture medium, since the organism can make use of many energy sources, as can be seen from the usability tests described above. For the purpose of efficient production, maximum yield of antibiotic and for reasons of isolation of the antibiotic, however, certain culture media are to be preferred. Media suitable for the production of tylosin are assimilable carbon sources, e.g. The preferred carbon sources are glucose and starch. In addition, suitable media contain an assimilable source of nitrogen, e.g. B. flaxseed meal, sewage sludge, fish meal, cottonseed meal, oatmeal, ground wheat, soybean meal, beef extract, peptones (meat or soybean), casein, mixtures of amino acids. Preferred sources of nitrogen: soybean meal, casein, and solid corn steepmeat.

Mineral salts, e.g. B. sodium, potassium, ammonium, calcium, magnesium, Supply cobalt, sulfate, chlorine, phosphate, carbonate, acetate and nitrate ions, and a source of growth factors, e.g. B. Distillery residues and yeast extract, can be incorporated into the media with good results.

As it is for the growth and development of other microorganisms is necessary, the culture medium for culturing the actinomycetes according to the invention should also be used contain effective trace elements. These trace elements are usually called Contamination added simultaneously with the other components of the medium.

The initial pH of the culture medium can vary widely. It was however, found that the desired initial pH of the medium is between 5.5 and 8.0, preferably between about 6.5 and 7.0. As observed with other actinomyces the pH of the medium increases during the entire growth period of the Organism, while tylosin is being produced, slowly and can reach a value up to to reach 7.2 to 8.0 or more; the final pH depends at least in part on the initial pH, the buffers present in the medium and the length of time during which the organism is growing.

Submerged aerobic cultivation conditions are essential to achieve good yields cheapest on tylosin. Sufficient for the production of relatively small quantities Swivel flasks and surface cultures in bottles, but for the production of large quantities submerged aerobic cultures in sterile containers are preferred. The medium in that Sterile containers can be inoculated with a suspension containing spores, but experience has shown that this is the case growth retardation occurs when using suspensions containing spores, cultures in vegetative form are preferred. This avoids this growth retardation and makes better use of the material used for fermentation. It is therefore desirable first prepare a vegetative inoculum of the organism by making a inoculates a relatively small amount of culture medium with the spore shape of the organism and the vegetative inoculum onto the large container under aseptic conditions transmits as soon as it has developed vigorously enough but is still young. That Medium in which the vegetative inoculum is grown can be the same or different than that used for tylosin production.

The organisms grow best at temperatures of around 25 to about 32 ° C. Optimal tylosin production appears at a temperature of around 26 to 30 ° C.

As is common with submerged culture procedures, sterile air is used blown through the culture medium. To promote favorable growth of the organisms and high production of tylosin tylosin is preferably used in container production an amount of air from one tenth of the volume of the culture medium per minute upwards. Good growth and an optimal yield of tylosin can be achieved if at least one 1 volume of air per minute per volume of culture medium used.

During the fermentation period you can measure the tylosin effectiveness in Easily track the culture medium by placing samples of the culture medium on it investigate how they hinder the growth of an organism from which one knows that it is inhibited in the presence of tylosin. As very satisfactory It turned out to be for this purpose if one used the organism Staphylococcus aureus. The investigations can be carried out according to the known flocculation or plate method carry out.

Generally, maximum production occurs 2 to 5 days after vaccination of the culture medium when using submerse aerobic cultures or oscillating flask cultures and 5 to 10 days after inoculation if using surface cultures.

Mycelium and undissolved solids are removed in a known manner, e.g. B. Filtration or centrifugation, removed from the fermentation broth. The antibiotic removed from the filtered or centrifuged broth by adsorption or Extraction methods. Extraction processes are used in the extraction of the antibiotic from the filtered broth is preferred as this extraction process is normally enable faster, more thorough and more efficient extraction of the antibiotic. Water is not used to extract the antibiotic from the filtered broth miscible polar organic solvents preferred, e.g. B. fatty acid esters such as ethyl acetate and amyl acetate, chlorinated hydrocarbons, e.g. B. chloroform, ethylene dichloride and trichlorethylene, water-immiscible alcohols, e.g. B. butyl and amyl alcohols, water-immiscible ketones, e.g. B. methyl isobutyl ketone and methyl amyl ketone and ether, e.g. B. diethyl ether and methyl propyl ether. Other solvents more similar Art can also be used. Chloroform and amyl acetate are currently the most common preferred solvents.

A variety of adsorbents and ion exchange resins can be used in adsorption processes be used, e.g. B. carbon, silica gel, aluminum oxide and ion exchange resins with acidic character, e.g. B. "XE" 64 and "IRC" 50 (weakly acidic cationic exchange resins), Carboxymethyl cellulose resin and "Dowex" 50 (a strongly acidic cationic exchange resin). The compound tylosin can be obtained from one of the above-mentioned adsorbents from chloroform, Acetone or benzene solution are adsorbed. The adsorbed tylosin can then be taken out eluted from the adsorbent e.g. B. by the adsorbent, the which has absorbed tylosin with a lower alcohol such as methanol or ethanol, or with a lower alcohol; that of up to about 50 per cent. of a lower one Contains ketones, for example acetone, washes.

The organic solvent extracts preferred by the Extraction process, you can easily evaporate until dry, raw tylosin receives. However, from the extracts the organic solvent wins one usually purified tylosin supplements. One wins z. B. Purified Tylosin, by taking the extract of the organic solvent, which contains tylosin, in a vacuum concentrated to a small volume, the tylosin concentrate decolorized with charcoal and the Tylosin by adding a non-polar solvent, for example a 2- to 10-fold excess of petroleum ether precipitates. The so educated Precipitate consists of solid, purified tylosin and is usually amorphous. The amorphous precipitate can be obtained with the aid of a crystallizing agent mentioned above crystallize. On the other hand, you can get tylosin from an organic one containing tylosin Obtain extract by applying adsorption chromatography and the adsorbed Tylosin eluted from the adsorbent.

The acid addition salts of tylosin can with the help of a mineral acid, for example sulfuric, hydrochloric or nitric acid, or an organic acid, for example tartaric acid, gluconic acid, oxalic acid or I`.ssigsäureged are formed- The salts can be prepared by converting the free base of tylosin in an appropriate Solvent such as acetone or ether dissolves and the solution with about an equimolar Acidify amount of desired acid; the salt usually falls out of the Solution. Assuming the salt does not separate out easily, you can you can precipitate it by evaporating the solution to a smaller volume or a miscible solvent in which the salt does not dissolve is added.

Because of their broad antibacterial spectrum and extremely effective The compounds according to the invention are suitable in manifold activity against microbes Way as a means for the sterilization of both laboratory and household appliances as well as food.

The compounds are also useful in the veterinary field for treatment from infections like turkey sinusitis. Sinusitis can be effectively combated by direct injection of a solution of 5 to 20 mg of tylosin or one of its cationic ones Put salts in both sinuses below the eyes of the diseased animal.

The invention is further illustrated by the following specific ones Examples: Example 1 Preparation of tylosin A spore culture of NRRL 2702 is made generated by placing the organism on a nutrient agar slant with the following composition breeds: yeast extract. . . . . . . . . . . . . . . . . . . . . . 1.0 g beef extract . . . . . . . . . . . . . . . . 1.0 g hydrolyzed casein (»N-Z-AMINE-Type Aa) . . . . . . . . . . . . . . . . . . . . . . 2.0 g dextrin .......................... 10.0 g cobalt chloride heptahydrate. . . . . . . . 20 mg agar ...........: ................ 20 g water .......................... 11 The pH value of the medium is adjusted by adding brought to 7.3 by sodium hydroxide.

The inclined culture medium is inoculated with spores from NRRL 2702 and 5 days incubated at about 30 ° C for a long time. The spore growth on the inclined nutrient medium is with Covered with water and carefully scraped off the spores from the nutrient slant so to produce a spore suspension.

1 cm3 of spore suspension is used to inoculate 100 cm3 of sterile vegetative culture medium with the following composition under aseptic conditions glucose ........................... 15 g soybean meal .................... 15 g solid cornstarch. . . . . . . . . . . . . . . 15 g sodium chloride. . . . . . . . . . . . . . . . . . . . . 5 g calcium carbonate. . . . . . . . . . . . . . . . . . . 2 g tap water to make up to a total volume of. . . . . . . . . . . . . . 11 The inoculated vegetative medium is incubated for 48 hours at about 30 ° C. and during this time it is shaken in a reciprocating shaker with a piston stroke of 50.8 mm at 114 periods per minute.

5 cms of vegetative inoculation material is used for the aseptic inoculation of portions of 100 cm3 of a sterilized production material, which is contained in 500 cm3 Erlenmeyer flasks and has the following composition: Soybean meal ................. 15 g casein ......................... 1 g raw glucose syrup. . . . . . . . . . . . 20 cm3 calcium carbonate. . . . . . . . . . . . . . . . 2.59 sodium nitrate. . . . . . . . . . . . . . . . . . . 3 g water to make up to a total volume of. . . . . . . . . . . 11 The inoculated culture is incubated for 100 hours at approximately 26 to 28 ° C. During this time it is shaken in a reciprocating shaker with a piston stroke of 50.8 mm at 114 cycles per minute. The initial pH of the medium is about 6.5. At the end of the incubation period, the pH of the medium increases to around 7.5.

The resulting nutrient broth is filtered to remove the mycelium and other undissolved solids. The filtered broth contains tylosin at a concentration of about 250Ag per cubic centimeter of broth. Example 2 Preparation of tylosin A spore culture of NRRL 2702 is prepared by growing the organism on an agar slant having the following composition: tomato paste-oatmeal agar tomato paste. . . . . . . . . . . . . . . . . . . . . . 20 g previously cooked oatmeal. . . . . . . . . 20 g agar .............................. 15 g water to make up to a total volume of ........ ............. 11 The inclined culture medium is inoculated with spores from NRRL 2702 and incubated for 9 days at a temperature of about 30 ° C. Thereafter, the spore culture on the nutrient medium is covered with water, and the top layer is gently scraped off to remove the spores to produce an aqueous spore suspension.

Half of the inoculum obtained from an inclined agar medium is used for the aseptic inoculation of 500 cm3 of sterilized vegetative culture medium with the following composition in a 2-1 Erlenmeyer flask: Pregelatinized corn yeast I glucose .............. ............. 15 g solid cornstarch ..... :. . . . . . . . . 5 g yeast .............................. 5 g calcium carbonate. . . . . . . . . . . . . . . . . . . 3 g water to make up to a total volume of. . . . . . . . . . . . . . . . . . . . . 11 Incubation is carried out for 48 hours at 28 ° C. while shaking with a reciprocating shaker with a piston stroke of 50.8 mm at 110 periods per minute.

0.951 vegetative inoculum from the flask is used as a vaccine aseptic to 9501 sterile medium of steeped corn yeast 1, as described above, in one Fermentation vessel made of iron with a capacity of 13251 added. 0.11 Antifoam A (a product against foaming) is added to the culture medium for prevention excessive foaming is added, and as the fermentation progresses, comes as needed even more. The inoculated medium is fermented for 24 hours at 28 ° C left. During this time it is treated with sterile air, 0.765 m3 / min and stirred with two 406 mm impellers at 160 rpm.

In an iron fermentation vessel with a capacity 45401 medium of the following composition were brought out of 64351: corn soya XII Glucose ........................ 30 kg soybean oil flour ..................... 15 kg Solid cornstarch. . . . . . . . . . . . 5 kg raw soybean oil ..................... 10 kg of calcium carbonate. . . . . . . . . . . . . . . . 2 kg of sodium chloride. . . . . . . . . . . . . . . . . . 5 kg of water to make up to a total volume of . . . . . . . . . . . 10001 The medium becomes 3631 des in the fermentation vessel cultured inoculum. The fabric is left to ferment at 28 ° C. for 4 days. The foam is made by adding z. B. »Larex« No. 1 (anti-foam agent) reduced as required. The fermentation medium is made by adding sterile air, 3.625 m3 / min, ventilated and agitated by two 610 mm impellers at 130 rpm.

272 kg of »Silflo«, a diatomaceous earth filter aid, becomes 37851 to this broth is added and the mixture is filtered. The filtrate is on a Brought a pH of 8.5 by adding 20 ° / @ total sodium hydroxide, there are still to come 19001 chloroform is added, the mixture is stirred for 30 minutes and in the form an emulsion existing chloroform layer poured off. The chloroform extraction is repeated twice using 1900 l of chloroform per extraction. the Chloroform emulsions containing the tylosin are combined and broken up the emulsion passed through a De Laval separator. The separated chloroform solution, which is obtained is concentrated in vacuo to a volume of 251, and impurities are mainly made by passing the concentrate through a column with 152 mm in diameter, which contains 10 kg of activated carbon. The carbon column is washed with 161 chloroform. The combined effluent chloroform extracts with the tylosin are concentrated in vacuo to 21, and the chloroform concentrate is slowly added to petroleum ether with stirring, whereby the tylosin precipitates will. After stirring the mixture for 15 minutes, this is to remove the white, amorphous tylosine precipitate filtered; this is dried in vacuo and weighed then about 1070 g.

The amorphous tylosin is crystallized by placing it in 355 cm3 of acetone dissolves, the acetone mixture is filtered to remove a slight haze and that filtered acetone mixture with careful stirring to 201 water with one temperature from 5'C pours. The resulting aqueous acetone solution of tylosin is left at room temperature stand and stir gently so that the acetone can slowly evaporate, whereby tylosin crystallizes out. The tylosin crystals are separated off by filtration and dried in vacuo at room temperature. The melting point is around 127 up to 132 ° C.

Example 3 Preparation of tylosin tartrate 5 g of crystalline tylosin are dissolved in 100 cm3 of acetone, and 1.5 g of d-tartaric acid, dissolved in 20 cm3 of acetone, are poured in while stirring. The solution is left to stand at room temperature, whereby the tartrate of tylosin crystallizes out of the solution. The tartrate crystals the tylosin are separated by filtration, washed with acetone and on the Air dried. A yield of 5.5 g is obtained. The crystalline salt melts at about 140 to 146 ° C. Example 4 Preparation of tylosin gluconate 1.03 g of glucon-B-lactone are dissolved in 10 cm3 of water, and the aqueous solution is heated to 85 ° C. for 2 hours heated; this causes hydrolysis of the lactone to form gluconic acid. To the aqueous solution 15 cm3 of warm methanol are added. 5 g of tylosine crystals are dissolved in 10 cm3 of methanol dissolved and added to the methanol-water mixture with stirring. The tylosin-methanol mixture left to stand overnight at room temperature. By evaporation in a vacuum becomes Removed methanol from the mixture. Then 40 cm3 of water are added to the aqueous tylosin mixture poured in. The diluted mixture is filtered and the filtrate with the active Tylosin freeze-dried; 5.3 g of white solid are obtained, consisting of the gluconate of tylosin. The tylosin gluconate melts at about 114 to 117 ° C.

Example s Preparation of tylosin hydrochloride 890 mg of tylosin will be dissolved in 200 cm3 of ether. The ether mixture is made by adding 0.082 cm3 of 12N hydrochloric acid acidified. The precipitate of the resulting tylosin hydrochloride is filtered off, washed with ether and dried in vacuo. The tylosin hydrochloride is made from a Ethanol-ether mixture recrystallized. Tylosin hydrochloride melts at about 141 up to 145'C.

Claims (1)

Claims: The use of a strain of Streptomyces fradiae, especially of the strain NRRL 2702 or NRRL 2703, for the manufacture of the antibiotic Tylosin in the usual biological way under submerged aerobic conditions, production of the antibiotic from the nutrient medium and, if necessary, conversion of the tylosin into its acidic salts. Publications considered: Chemical Centralblatt, 1952, p. 4315, and 1953, p.5198.