DE1026481B - Process for the production of tetracycline by fermentation technology - Google Patents

Description

Process for the production of tetracycline by fermentation technology The invention relates to a method for the preparation of tetracycline, a new one therapeutically useful antibiotic substance with a broad spectrum of activity fermentation technology and its extraction and purification.

It has been found that in the cultivation of organisms of the Streptomyces species or their mutants in a fermentation medium under suitable conditions antibiotic substance is formed in high concentration, which is different from already known antibiotic, in particular from the one already mentioned by means of organisms Species produced antibiotic chlortetracycline, differs.

When performing the method according to the invention, an organism of the genus Streptomyces grown under aerobic conditions in an aqueous nutrient medium, which contains sources of carbon, nitrogen and mineral salts and their chlorine ion content has a value at which with the organism used in each case and the respective constituents of the medium the formation of chlortetracycline is limited and at the end of the fermentation a higher proportion of tetracycline than of chlortetracycline is obtained will.

Using a strain of Streptomyces found in the ground aureofaciens as an organism and a practically chlorine ion-free nutrient medium already obtained good results, however, mutants allow this organism to die Production of the antibiotic substance in even higher concentrations in one Medium that has a substantial content of chlorine ions.

The new antibiotic substance was called tetracycline because its structure has been shown to be identical to that of the common molecular part of aureomycin and terramycin (Brunings et. Al., Journ. Amer. Chem. Soc., 74, 1952, pp. 4976, 4977) . It differs from chlortetracycline in its absence of chlorine and in other respects from oxytetracycline in its behavior.

Tetracycline has the gross formula C "H2, N201 and the following structural formula It is amphoteric and forms salts with organic and inorganic acids, e.g. B. the hydrochloride, hydrobromide, sulfate, borate, nitrate, phosphate, ascorbate, citrate, succinate, acetate, sulfamate, and mono- and dibasic salts with bases, eg. B. the bases of the alkali or alkaline earth metals or ammonium and amine bases. In addition, tetracycline forms complex salts with basic salts of boric acid, for example sodium borate.

Such tetracycline salts are suitable for isolation and purification, as they are converted into the amphoteric substance by neutralization or by double conversion can be converted from one form to the other. Performed in vitro Tests have shown that both the salts of tetracycline with acids and bases as well as the free base against a large number of bacteria, including gram-positive and gram-negative bacteria, are effective. For differentiation of tetracycline of previously known antibiotics are many options Disposal. Based on the chemical analysis, the free base contains carbon, Hydrogen, nitrogen and oxygen and is free of sulfur and chlorine.

The properties of crystallized tetracycline depend as at many organic compounds to a certain extent on the degree of purity and the crystal formation process away. When precipitating from aqueous solutions, the free base precipitates as a tribydrate, which can be converted into the anhydrous form by drying, but normally is used directly as the anhydrous form is quite hygroscopic.

The trihydrate obtained by recrystallization from aqueous methanol at room temperature was colored yellow and its crystal habit was isodiametric to tabular; Refractive indices a = 1.530 IL 0.003, β = 1.646 ± 0.003, y = 1.798 IL 0.003; optical angle 2 V = 80 to 85 ', optical sign positive.

The optical rotation of very pure crystallized tetracycline base in methanol is [a] - '= -2340 D (c = 0.82 0 /, in methanol).

Tetracycline is soluble in methanol and ß-methoxyethanol (about 200 mg / ml), somewhat soluble in ethanol and n-butanol, sparingly soluble in water and relatively insoluble in solvents such as ether, benzene and petroleum ether. Tetracycline hydrochloride is soluble in water to 100 to 200 mg / ml, fairly soluble in methanol and ethanol, sparingly soluble in 2-methoxyethanol and n-butanol and relatively insoluble in other common organic solvents. The calcium salt of tetracycline is soluble in water to about 890 y / cc (pH 8.55) and the magnesium salt is about 1000 y / cc (pEE 7.0). The following Table I shows the approximate solubilities of very high purity tetracycline trihydrate in various solvents. Table I. Solvent solubility in 100 ml Methanol ............... at least 3 g Ethyl acetate ............ 25 mg Chloroform ..... ........ 20 mg Water ................. less than 10 mg Benzene ................. less than # l * 0 mg Diethyl ether ............ less than 10 mg As with most organic substances, the melting point of anhydrous crystallized tetracycline depends, among other things, on the degree of purity and the way in which the crystals are heated. Crystals of very pure anhydrous base on a slide were brought to a hot stage of 100 ° C. The temperature was increased at a rate of 20 ° C./minute to 145 ° C. and then at 1 ° C./minute until the crystals melted. When heated under these conditions, the melting point is in the range from 160 to 168 ° C (decomposition).

A sample of the trihydrate in a melting point capillary was placed with a thermometer in a test tube containing a liquid at 150 to 155 ° C. This test tube was immersed in a bath of 160 to 165 ° C. made of the same liquid, which was then heated with stirring in such a way that the temperature rose 1 to 2 ° C./Iffinute. The sample slowly expanded and turned yellow as the temperature of the inner test tube rose to 160 to 165 ° C. At 170 to 175 ° C , darkness began, which became more pronounced as the temperature increased further. A sample of the anhydrous base behaved accordingly.

The infrared spectrum of the anhydrous tetracycline base, suspended in hydrocarbon oil, shows a strong OH or NH band at 3300 to 3400 cm-1, a possible amide carbonyl band at around 1655 cm- ', a possible C = C stretching vibration at around 1613 cm-', a possible NH buckling oscillation at about 1580 cm- 'and other characteristic maxima at about 703, 722, 785, 862, 950, 1178 and 1228 cm- ' and reveals the close relationship between tetracycline and chlortetracycline. Both compounds differ, however, in their absorption spectra: chlorotetracycline, for example, shows a strong doublet at around 950 cm -'- and a strong triplet at around 800 cm- ', where tetracycline has only weak points of inflection.

The ultraviolet absorption of tetracycline base and hydrochloride was determined in the Beckmann quartz spectrophotometer (model DU) . A solution of 1.0 mg of base in 50 ml of methanol gave the following maxima and minima: Ell at 235 mp. = 188, at 269 m # t - 271, at 298.5 m # t = 128 and at 365 m # L # 272.

The ratio of the E ' "% .- values at 269 and 365 m [t is 1.00 and is characteristic of the purified antibiotic.

A solution of 1.10 mg tetracycline hydrochloride in 50.0 ml methanol gave the following maxima and T minima: E 1.2. at 234 mp. = 179, at 270 m # t = 301, at 298.5 m # L = 131 and at 363 m # t = 274.

The ratio of the values Eilj # mp at 270 and 363 is 1, 10, and is characteristic of the antibiotic. Tetracycline can be distinguished from other antibiotics, including those produced by microorganisms of the Streptomyces species, by means of countercurrent distribution according to Craig (journ. Biol. Chem., Vol. 150, 1943, p. 33). When distributing in a 49-plate apparatus according to Cr ai g using the system n-butanol / 2.5% aqueous acetic acid at room temperature, the highest tetracycline concentration is found in tube 18. Under the same conditions, the highest concentration is found of chlortetracycline in tube 28, of chloramphenicol in tube 47, and of oxytetracycline in tube 16. The relationship to oxytetracycline is very close, but the constancy of the results shows that the difference of two panels is significant.

Ehrlich's reagent can also be used to differentiate between tetracycline and oxytetracycline or chlortetracycline: a solution of 5 mg of substance in 5 ml of 1.2N hydrochloric acid was mixed with 1 ml of a 2% (weight / volume) solution of p-dimethylaminobenzaldehyde added in 1.2 N hydrochloric acid and left to stand overnight. In the process, chlortetracycline turned canary yellow, oxytetracycline deposited a bluish-green precipitate with a similarly colored supernatant solution, and tetracycline turned deep yellow with a tinge of orange. If 5 mg of chlorotetracycline were added to the oxytetracycline solution in addition to the reagent, then, as with the addition of reagent alone, a bluish-green precipitate and a supernatant liquid formed, whereas when the reagent and 5 mg of tetracycline were added, neither precipitate nor color change occurred. Color differences are also obtained with sulfuric acid. Chlortetracycline in concentrated sulfuric acid (about 25 mg / ml) forms a purple-colored solution that turns greenish-black after a few seconds; oxytetracycline a stable characteristic cherry red solution (F. Monastero et. al., journ. Amer. Pharm. Assoc. Scientific Ed., 40, 1951, pp. 241 to 245) and tetracycline a stable purple solution. Furthermore, the spectrum of antibacterial activity can be used to differentiate the antibiotic. The following Table II shows the respective amounts of tetracycline trihydrate, chlortetracycline hydrochloride, oxytetracycline hydrochloride and chloramphenicol required for the inhibition of a number of representative organisms in pg / ml according to the "agar strip method" by Waksman and Reilly (Ind. Eng. Chem Anal. Ed., 17, 1945, pp. 556 to 558) These data show that tetracvcline is a defined antibiotic substance. Table II Test organism A 13 CD Staphylococcus aureus PCI 209-P .................. 0.292 6.25 0.39 0.58 Staphylococcus aureus PCI 1248-A .................. 0.39 6.25 0.78 0.78 Stfeptococcus pyogenes ATCC 8668 .................. 0.29 1.17 0.24 0.24 Streptococcus mitis ATCC 6249 ..................... 0.147 2.34 0.147 0.292 Streptococcus faecalis ATCC 7080 ................... 0.39 6.25 0.78 1.95 Mierococcus flavus PCI 16 .......................... 0.292 1.56 0.39 0.39 Diplococcus pneumoniae ATCC 6301 ................. 0.098 2.34 0.147 0.292 Sarcina lutea ATCC 9341 ........................... 0.147 1.56 0.39 0.39 Bacillus subtilis ATCC 6633 ......................... 0.195 6.25 0.39 0.39 Escherichia coli ATCC 9637 ......................... 1.45 12.5 1.17 1.17 Klebsiella pneumoniae PCI 602 ...................... 0.29 1.17 0.58 0.58 Aerobacter aerogenes ATCC 8508 .................... 1.17 3.12 2.34 1.56 Proteus vulgaris ATCC 6380 ........................ 4.6 3.12 3.9 3.12 Pseudomonas aeruginosa ATCC 9027 ................. 25.0> 100 25.0 14.0 Salmonella schottmulleri ........................... 3.12 3.12 2.34 3.12 Salmonella typhosa ................................ 1.17 4.6 2.34 1.56 Brucella bronchiseptica ATCC 4617 .................. 0.292 6.25 0.78 1.17 Mycobacterium tuberculosis ATCC 607 ............... 0.147 6.25 0.195 0.29 Mycobacterium friedmanu ATCC 14 ................. 0.122 6.25 0.24 0.292 Mycobacterium. smegmatis ATCC 361 ................ 0.073 12.5 0.147 0.292 Mycobacterium smegmatis ATCC 10143 .............. 0.073 6.25 0.147 0.122 Mycobacterium sp. ATCC 9820 ...................... 0.58 6.25 0.58 2.34 Candida albicans ATCC 10231 .......................>100>100>100> 100 A = chlortetracycline hydrochloride D = oxytetracycline hydrochloride B = chloramphenicol PCI = penicillin control immunology C = tetracycline trihydrate ATCC = American type culture controi. Table III shows the breeding characteristics of two isolates derived from the breeding of the aforementioned Streptomyces aureofaciens and used in the production of tetracycline. These characteristics are identical to those which occur in a known breed of Streptomyces aureofaciens. Table III Glucose broth ................... Yellowish growth settled to the bottom, acidic reaction; Lactose broth ........... ....... yellowish growth settled to the bottom, no acidic reaction; Glycerine broth .................. yellowish growth settled to the bottom, no acidic reaction; Sucrose broth ................ yellowish growth settled to the bottom, acidic reaction; Litmus milk .................. surface growth, slight lightening, no change in pB value; Gelatin stick .................... good growth on the surface, no liquefaction, yellow pigment; Asparagine meat extract agar .... weak yellow pigment, good growth, aerial hyphae white, turning gray; Czapek-Dox-Agar ............... weak growth, flat, colorless mycelium, no aerial hyphae, no soluble Pigment; Emerson agar .................. strong growth, brownish mycelium, no aerial hyphae, brownish reverse side, slightly soluble brown pigment; Nutrient glucose agar .............. as under Emerson agai; Glycerine asparagine agar ......... strong growth, white aerial hyphae, turning gray, reverse greenish yellow, slightly soluble yellow pigment; Starch agar .................... flat, colorless mycelium, no aerial hyphae, no soluble pigment, hydrolysis; Calcium malate agar .............. strong growth, aerial hyphae, white, turning gray, brownish pigment. The strains, variations or mutants of Streptomyces aureofaciens preferred in the practice of the present invention are those which give relatively high yields of tetracychn in the fermentation medium, in particular those which produce more than 500 µg / ml. An example is the strain UV-8, which when cultivated on Waksman agar (Journ. Bacteriol, 1952, 7, pp. 339 to 341) shows a strong mycelial growth that is initially whitish, turns yellow and gradually turns into a powdery Coats aerial mycelium, which later forms spores. Old slant cultures show a jet black color with small spots of white mycelium. This black growth, which has not yet been reported in cultures of Streptomyces aureofaciens and is not a characteristic of the species, consists of a mass of bodies in short chains that easily break apart. These bodies are variable in size and shape in the range from about 0.5 to 4.5 μm and vary in shape from oval to slightly angled spherical. The average size is slightly larger than that of Streptomyces aureofaciens (NRRL-2209) and there is more variation in size and shape.

Both natural and induced mutants of Streptomyces aureolaciens can produce tetracycline in varying amounts in addition to other antibiotics. By choosing a natural or induced mutant that is a comparatively high ratio of tetracycline to other antibiotics available, and by cultivating the organism under controlled conditions a comparative large yield of tetracycline can be obtained.

The classification of milao organisms is often one in mycology difficult problem causing different mycologists to different classifications for the same organism. The appearance of Streptomyces aureofaciens can vary widely under different conditions. Morphologically indistinguishable strains of Streptomyces aureofaciens vary widely with regard to their tetracycline and chlortetracycline formation and with regard to the Relationship between these two substances.

Tetracycline was created by growing many natural isolates of Streptomyces aureofaciens generated. The general appearance and the detailed examination these isolates vary considerably.

Natural and induced mutants or variants were obtained with growth colors in various shades of red, orange, yellow, green, brown and gray; they varied in their growth appearance, especially in liquid media in shake flasks, from threadlike to short hyphae elements and showed a pasty, yeast-like appearance. The mutants can differ in terms of vitality and growth rate, so that maximum growth can occur in submerged cultures over a period of about 1 day to over 1 week. The rate of growth does not necessarily increase with antibiotic production.

It has proven to be useful to carry out the aerobic cultivation of the above-mentioned organisms in the submerged process with suitable ventilation and agitation, for example in a flask on a shaking machine or in a fermentation vessel equipped with a stirrer that is equipped with a device for the continuous introduction of a finely divided air stream . Apparently, the temperature is not critical within the range of 25 to 35 ° C, but the range of 30 to 33 ° C is preferred. The medium should be nearly neutral at the beginning, but the antibiotic will also be produced in media with original pn7 values down to 5.0 or up to 8.5. Usually the medium is used. a buffer added.

As the carbon source, a soluble carbohydrate such as sucrose or an insoluble carbohydrate such as starch, e.g. B. corn starch or dextrin can be used. In contrast to sucrose, which is quite satisfactory, lactose and glucose, two other soluble sugars, are relatively unsuitable. The amount of carbon source can vary within wide limits from about 0.5 to 10.00 per cent of the total weight of the fermentation medium.

Inorganic ones are suitable as a source of assimilable nitrogen Ammonium salts, e.g. B. ammonium sulfate, ammonium phosphate, etc. It is also possible to use organic nitrogen sources such as amino acids and various protein-like natural substances.

Mineral salts found to be beneficial include potassium salts, Phosphates, magnesium salts and trace elements. Phosphate can be called ammonium phosphate or as metal phosphate, for example as potassium phosphate, and magnesium as sulfate are present. A source for kahum, if it is not already z. B. as potassium phosphate is present, also added.

The addition of small amounts of heavy metals is when they are not are already present in other ingredients, expedient. To these as impurities existing or added trace elements include copper, zinc, Manganese, iron and chromium.

To buffer to the pH value in the permissible range, you can use -below other caleium carbonate and salts of organic acids, for example citrates, Acetates and lactates, can be used. The organic acids can also be used as Serve carbon source for the microorganism. The use of a defoamer is useful for fermentation on an industrial scale; the foaming is here no particular problem and can be achieved by using conventional defoaming agents, such as octadecanol in lard oil or other suitable commercially available defoamers, light be kept within the desired limits.

If the medium contains appreciable amounts of available chlorine ions, some chlorotetracycline will be formed concurrently with tetracycline. In order to achieve the highest possible ratio of tetracycline to chlortetracycline in the end product obtained in the process according to the invention, a nutrient medium is therefore preferably used which contains only 17 parts / 1 million chlorine ions or less. The fermentation is preferably performed until at least 500 t £ g tetracycline e j ml fermentation liquor were formed.

For example, a synthetic, practically chloride-free medium has been developed that is easy to manufacture, inexpensive, has a constant composition, and produces outstanding yields. This medium contains sucrose as a carbohydrate, ammonium sulfate as a nitrogen source, potassium phosphate as an inorganic salt, magnesium sulfate, calcium carbonate, sodium citrate and acetic acid for buffering and small amounts of copper sulfate, zinc sulfate and manganese sulfate as sources of trace elements. Virtually chloride-free types of these substances are selected, and distilled water is used so that this medium does not contain more than 1 part / million chloride.

The composition of this synthetic medium is as follows: Sucrose ................ 30 g / 1 (N HJ, S 0. ............... 8.1 g / 1 Na, C, H, 0, - 5 H, 0 ....... 1.0 g / 1 MgS04 - 7 H20 ........... 0.25 g11 K, HPO . ................. 0.57 g / 1 CaCO . ................... 1.0 g / 1 CH, COOH ............... 0.1 ini CUS04 5 11.0 ............ 3 part effilillion ZnS04 7 H, 0 ............ 50 parts / million MnS04 4 H, 0 ........... 2.5 Teüe / .YLglion The inoculum for fermentation is prepared from the growth of slant cultures inoculated with Streptomyces aureofaciens. A medium suitable for slant culture is Waksman agar with the following composition: Glucose .................. 10 gil Peptone ................... 5 gll KH, PO . ................. 1 g / 1 M9S04 - 7 H, 0 ........... 0.5 g / 1 Agar ..................... 20 g / 1 The slant culture is transferred to shake flasks, which are used as laboratory fermentation vessels on a small scale or to produce inoculum for larger fermentations.

In tank fermentation on an industrial scale, the inclined culture is used to inseminate a suitable liquid medium in a shake flask which is shaken on a shaking machine with back and forth movement at temperatures in the range from 26 to 35 ° C, preferably at 30 to 33 3 C, is shaken. Typically, a second shake flask stage is used in conjunction with submerged tank fermentation to increase the volume of the shaken inoculum to 1.5 to 2.5 O1 the volume of liquid in the tank. The reaction of the medium in the shake flask is initially in the range of pil 6.5 to 7.5. As soon as growth occurs, a steady fall in pH is observed, with values as low as 3.8 . However, a physiological age indicated by a p11 value of 5.0 appears to be the most favorable time for the transfer of the vaccine material.

The inoculum in the shaker, which is 1.5 to 2.5 of the volume of liquid in the tank, is aseptically transferred to the fermentation vessel and cultured for about 2 to 3 days with continuous agitation and aeration. Air velocities equal to 0.5 to 1.5 volumes of fresh air per volume of liquid medium can be used, depending on the size of the fermentation vessel. The foaming of the broth can be controlled by sterile addition of a defoaming agent such as lard oil containing 2 % octadecanol.

Recovery and Purification The recovery of tetracycline from your fermentation medium can be done in a number of ways. For example, the fermented Material can be added directly to animal feed, thereby stimulating growth and / or created a particularly suitable product for the prevention of diseases will. If the mycelium and insolubles are undesirable, that can Fermentation medium with or without acidification filtered off and the clear liquid be added to drinking water or animal feed for the same purposes. If so desired the liquid containing the tetracycline can be under reduced pressure or by Spray drying concentrate and the antibiotic in raw egg form to stimulate growth or used to control disease treatment in humans, animals and poultry will.

If the tetracycline is to be obtained in a more purified state, it can be isolated from the fermentation medium by various methods, which include one or more of the following steps in different order: adsorption, elution, solvent extractions with or without a carrier, crystallization, precipitation in the form of insoluble salts, for example of the calcium, barium, strontium or magnesium salt, at pn values above about 6.5 or as insoluble salts with sulfates or sulfonic acids of the wetting agent or azo dye type; or it can be solvent extracted with carriers such as sulfates or sulfonates of the surfactant or azo dye type. The tetracycline can be separated from the raw mash by acidification to a pH of about 3 or less. The tetracycline goes into the aqueous layer and the insoluble solids can be separated and removed. At a pH-value above about 6 5 tetracycline can be precipitated as Erdalkaiiniederschlag in aqueous solutions. This precipitate can be extracted with acidified water or with organic solvents such as alcohols, alkoxyalkanols or ketones. The extraction can be carried out under acidic, neutral or basic conditions. In the same way, the aqueous solution containing the tetracycline can be extracted by means of organic solvents which are immiscible with the aqueous layer, for example with lower molecular weight alcohols, alkoxyalkanols, esters and ketones which are immiscible with the aqueous layer. Of known solvents such as butanol, chloroform and ethyl acetate, butanol is preferred for tetracycline extraction. Certain alcohols, such as isopropanol, which are miscible with water but immiscible with a salt solution, can also be used well. The addition of a water-soluble salt insoluble in the organic layer, such as an ammonium or amine salt or alkali halides and sulfates, is beneficial in the extraction.

The tetracycline can be obtained from aqueous solutions by adding water-soluble ones Salts such as ammonium or amine salts or alkali halides and sulfates precipitated will. The salting out can be done with a small amount of an organic solvent get supported.

If chlortetracycline is present in the fermentation mash, so it goes through these process steps together with the tetracycline, chlortetracycline can be degraded by keeping the mixture under such conditions it becomes that the tetracycline is the more stable, or the chlortetracycline can become tetracycline be reduced.

Furthermore, a novel recovery method has been developed which is extremely effective both in terms of obtaining a product free of inactive impurities and in terms of obtaining a product which does not contain any other antibiotic substances which are simultaneously formed in the medium. This recovery method yields a crystallized product which has a high degree of purity in these two aspects. Its main features are the use of quaternary ammonium salts which selectively precipitate tetracycline from the broth at an alkaline pH (pH 8 to 11) as the quaternary ammonium salts of tetracycline. Such a salt, after being filtered off, is then slurried with a small amount of water and a relatively large amount of chloroform, whereby the quaternary ammonium salt of tetracycline is dissolved in the chloroform phase of the slurry. The water and chloroform phases are then preferably separated to remove any impurities dissolved in water. The tetracycline is then extracted with an aqueous acidic solution at a p11 of about 1 to 2.5 to form the acid salt of tetracycline which goes into solution in the aqueous phase. After the pH has been increased to a range from 3 to 7 , it is precipitated from this solution as a crystallized product, with precipitation starting at the lower pH.

The quaternary ammonium salts particularly useful in this recovery method are alkyltrimethylammonium chlorides and dialkyldimethylammonium chlorides in which the alkyl group contains from 8 to 18 carbon atoms, inclusive.

The following examples are intended to explain the invention in more detail without restricting it. Examples 1 to 4 explain the production of the antibiotic substance according to the invention by fermentation under submerged aerobic conditions; in the case of fermentation in shake flasks on a smaller scale, the same media can be used as in fermentation in tanks on an industrial scale. Examples 5 to 11 explain the extraction and purification.

Example 1 A vaccine medium (50 ml in 250 ml Erlenmeyer flasks) containing the following ingredients was prepared: Sucrose ....................... 30.0 g / 1 Sodium citrate .................... 1.0 g / 1 (N HJ, SO. ...................... 3.3 g / 1 MgS0, - 7H, 0 ................... 0.25 gll KH, PO . ........................ 0.10 g / 1 K, HPO . ........................ 0.10 g / 1 Caco . .......................... 1.0 g / 1 Acetic acid ....................... 0.4 ml Sov grits ......................... 0.5 g Trace element solution ............. 1.0 ml The volume was then made up to 11 with distilled water.

The trace element solution had the following composition: CuS0, - 511.0 ................. 0.3 g '7100 ml ZiiSC, 7H #, 0 ................. 5.0 MnSO, 4H, 0 ................ 0.25 (The pil value was adjusted to 6.2 with NaOH.) The materials were added in the order given and placed in an autoclave. After cooling, the flasks were inoculated from a slant culture of the UV-8 culture and shaken for 48 hours at 30 to 33 ° C. on a shaker with to-and- fro movement (97 periods / .#, linute).

The following medium was prepared to generate tetracycline: Sucrose ................ 30 g / 1 distilled water. MgSO, -7H, 0 ............ 0.25 KH, PO . ................. 0.15 CaCO . ................... 1.5 Citric acid ............. 11.5 NH, OH .................. 10 ml Trace element solution ...... 1.0 ml (as described above). 50 ml of the medium was placed in each 250 ml flask and, after sterilization, 2.5 ml of vaccine was aseptically transferred to each flask. The original pH ranged from 6.0 to 7.0. After 3 days of incubation on a rotating shaker at 250 rpm at 30 ° C , an average value of 610 tg was obtained in ten individual shake flasks for the pure material with E. coli, Iml against a tetracycline standard of 1000 tg / ml. The sample against the tetracycline standard was 610 µg / ml. At a chloride content of 0.6 parts / million, the total effective amount of chlorotetracycline formed was 8 tg / ml, or about 1.4 "/, of total antibiotic.

Example 2 The following example relates to the recovery of tetracycline from the fermentation medium. Around 600 l of the broth with an activity of 68 tg / ml, essentially prepared according to Example 2, were mixed with 5600 g of oxalic acid in order to make the antibiotic soluble. The pu value was adjusted to 3.5 with 4600 ml of concentrated ammonium hydroxide and the mixture was filtered with filter aid in order to remove the alycel and the precipitated calcium oxalate. The filtered broth were added to 120 1 of a 50 / solution of N- (lauroyl colaminoformyl-methyl) pyridinium chloride in ethyl acetate, then the mixture 2200 ml of 500 sodium sodium hydroxide to pH 8.5 was. "Adjusted. The The mixture was stirred for 20 minutes and then allowed to settle.

The 82 L ethyl acetate layer containing the antibiotic was separated and extracted three times using 1500 ml of water. The pi value was adjusted to 1.7 each time with hydrochloric acid. Sulfuric acid can be used. The extracts were combined and adjusted to p.1 7.8 with 6.5 ml of 4N ammonium hydroxide, the relatively insoluble isoelectric material being formed. The mixture was filtered off and the cake dried in a vacuum desiccator. 21 g of crude material with an activity of 417 tg / nig were obtained. Countercurrent distribution of the material indicated that about 75 % of the antibiotic present was tetracycline.

Example 3 A fermentation medium containing more than 130 .mu.g! Ml of tetracycline was acidified to pH 3.5 with concentrated sulfuric acid to solubilize the antibiotic and filtered off after adding a filter aid. The volume of the broth filtered off was around 40660 1. This broth was mixed with 2140 ml of a mixture of alkyl trimethylammonium chloride and dialkyl dimethylammonium chloride in isopropanol, the alkyl groups being composed of 901 /, hexadecyl, 601, octadecyl and 3 9 1 4 0 /, Octadecenyl are added and the pH is brought to a value of 10.0 by adding 6,100 ml / 150 l) /, aqueous sodium hydroxide. The alcohol makes up about 50,070 of the preparation. After the addition, the pu value was adjusted to 8 to 11 in order to form the quaternary ammonium salts of tetracycline which precipitate in this p1 range. After adding filter aid, the broth was filtered off and the solids thus obtained were washed with water. About four fifths of this gewascbenen filter cake was slurried with about 45.6 1 methanol as solvent and concentrated hydrochloric acid in an amount sufficient to bring the pH to a value of 1.8, whereby the solubility of tetracycline is increased. After 15 minutes of stirring, the mixture was filtered and the solids washed with methanol until - they were substantially colorless. The total volume of extract and washing liquids was 92 1. The extract combined with the washing liquids was brought to a pH of 6.8 with 10N aqueous sodium hydroxide, whereby the relatively insoluble free base was formed, which precipitated. The solids were filtered off and air dried. 1545 g of crude amorphous isoelectric tetracycline trihydrate with an activity of 296 yg / nig were obtained. Example 4 2140m1 of the above mixture were added to around 406601 of filtered fermentation broth at p113.5 and the pH was brought to a value of 10 . Filterbilfe was added and the mixture was filtered off and the solids washed with water. A 1 kg aliquot of the washed filter cake was slurried with 3300 ml of chloroform and the pi adjusted to 10.5. This pH can range from 8 to 11 . After stirring for 15 minutes, the solids were filtered off and reslurried twice in succession with a mixture of 150 ml of water and 3300 ml of chloroform. The combined chloroform extracts containing the antibiotic, which had been separated off from the aqueous phase, were then stirred with 600 ml of 0.4N sulfuric acid and the pH was adjusted to a value of 2 with 4N sulfuric acid. After stirring for 5 minutes, the aqueous phase was separated off and filtered off. A 340 ml aliquot of this acid concentrate was adjusted to pH 5.3 and left in the refrigerator overnight. The crystals were filtered off, washed with ice water and dried in vacuo over anhydrous calcium chloride. 19.5 g of substance with an activity of 629 tg / ing were obtained. Chlortetracycline was not present in detectable amounts.

This recovery method is preferred because it is an effective method and a crystallized material is recovered. The filter cake, which comprises the precipitated quaternary ammonium salts of tetracycline, is extracted with chloroform which is moist because of its own water content or the water content of the filter cake or because water has been added. When using chloroform, lower yields are achieved. The filter cake is preferably repeatedly extracted and the aqueous phase discarded. The chloroform extract is acidified with an inorganic acid to a pil value, preferably in the range from about 1 to 2.5 , in order to convert the tetracycline salts into the corresponding acid salts of the tetracycline, which are water-soluble. Before or during the addition of the acid, water is added in order to form two phases, the tetracycline acid salts being in the aqueous phase. The aqueous phase is separated from the chloroform phase and the pH increased to a value of about 3 to 7 to form the relatively insoluble isoelectric material. Relatively low pH values are preferred because purer crystals are obtained. The isoelectric material separates out on standing in the form of trihydrate crystals, which can easily be separated off by filtration and, if desired, washed.

Example 5 Two columns, each containing 50 g Carboxvi-cation-exchange resin in the hydrogen state entbi # Iten, were buffered to p, 1 7 by minute through each of 11 of 1 N-sodium acetate-acetic acid buffer at a rate of 10 ml '/ was passed through. 10 l of the filtered broth with an activity of -11 ygiml was adjusted to pH 7 with 4N aqueous sodium hydroxide and passed through one of the buffered exchange resin columns at a rate of 5 ml / minute. The following fractions of the escaping liquid were collected: 0 to 2 1 activity 13.3 # Lg / rnl 2 to 5 1 activity 8.7 # tg / ml 5 to 7 1 activity 15.1 tg / ml 7 to 10 1 activity 25.0 # tg / III1 The liquid emerging from this column was passed through the second buffered column at the same rate of 5 minutes. Fractions of the liquid emerging from the second column were collected for testing for their activity. 0 to 2 1 activity ............. 16.9 pg / M1 2 to 4 1 activity ............. 12.4 # Lg / ml 4 to 6 1 activity ............. 7.5 # ig / M1 6 to 10 1 activity ............. 7.2 #Lgiiml 11 1N hydrochloric acid in 600% strength methanol was passed through the first column at a rate of 10 ml / minute. The eluate 0 /, containing 74 of the activity of the filtered broth, was concentrated in vacuo to about one third of its volume, whereby the pli to 2.5 fell. The pH was brought to 1.8 by the addition of a filter filter to remove the solids that had separated out during the concentration. The filtered solids were washed with water and the filtrate combined with washing liquids was brought to pH 7.8 by adding 4N sodium hydroxide. The solids which separated out were washed and then dried in vacuo. 1.375 g of a product with an activity of 78 # tg / ing were obtained. Example 6 Tetracycline can be precipitated from its aqueous solutions at alkaline pH values by adding a mixture of barium chloride and magnesium chloride. For example, when 15 g Bariumehlorid dihydrate to bring and 2 g Magnesiumehlorid hexahydrate to a solution of 3 g of crude tetracycline in water at 11 p, 1 2.5, and finally aqueous sodium hydroxide was added to the pH to a value of 8.5 , all activity was found in the precipitate, which is separated by filtration from the liquid containing the impurities. After slurrying the precipitate with water and adjusting the pli value to 1.5 with sulfuric acid and filtering off the insoluble barium sulfate from the mixture, the resulting aqueous extract contained practically all of the originally present microbiological activity. This procedure can be used to concentrate solutions and broths containing tetracycline and to remove impurities.

Example 7 By dissolving crude amorphous tetracycline in water, crystals of the free base tetracycline were formed. This crude material had an activity of about 427 pg / mg. The aqueous solution was adjusted to pl. 3 by adding hydrochloric acid. The solution was then saturated with n-butanol and then subjected to countercurrent distribution with nine separating funnels, using equal volumes of n-butanol saturated with water, the pH of which had been adjusted to a value of 3 with dilute hydrochloric acid. After distribution, the middle tubes 4, 5, 6 and 7 were combined and the pI was brought to a value of 7 by adding dilute sodium hydroxide solution. The mixture was concentrated in vacuo at 40 to 50 ° C. The crystals which formed on cooling were filtered off and dried over calcium chloride in a vacuum desiccator. The crystallized trihydrate thus obtained had an activity of 780 μg / mg.

Example 8 Spores of Streptomyces aureofaciens strain S-77 grown on an agar slant are collected by washing with water. An amount of the suspension containing about 1,000,000,000 spores is used to inoculate a medium in a 500 ml Erlenmeyer flask, the volume of which is 100 ml and which is composed as follows: g / 1 distilled water. Corn steeple solids ........ » 20 Calcium carbonate ................ 6.25 Sucrose ..................... 30 Ammonium sulfate ............... 2 The medium is sterilized prior to inoculation by 20-minute treatment in Autoldav at 120 'C. The vaccine is grown on an orbital shaker (180 revolutions / minute) at 26 ° C for 24 hours.

A fermentation medium is produced with the following composition: Quantity / liter aqua dest. Starch ......................... 50.0 g Corn steeple solids ......... 4.0 g Calcium carbonate ................ 7.5 g Ammonium sulfate ............... 7.0 g MgS0.711.0 .................. 1.5 g FeS04'711.0 ..... ............. 16.0 mg MnS04 * 411.0 ................. 50.0 mg ZnSO, .711.0 ................. 100.0 mg CoC1, - 6H, 0 ................... 5.0 mg H3P0, ......................... 0.4 g KISO .......................... 0.5 g Soybean flour ................ 10.0 g Lard oil ...................... 2.00 /, The medium containing 36 parts / million chlorine ions is adjusted to pH 5.85 by adding 100% sulfuric acid and kept in the autoclave at 120 ° C. for 20 minutes.

1 ml of the vaccine prepared as described above is used to inoculate 25 ml of fermentation medium in a 250 ml Erlenmeyer flask. The fermentation is carried out on a rotary shaker (180 revolutions / minute) at 26 ° C. After 144 hours, the test of the fermentation mash shows a content of 3635 y tetracycline and 380 y chlorotetracycline in the ml of fermentation liquid.

Testing the activity of tetracycline To test the activity of tetracycline, use can be made of E. coli 9637 and Difco nutrient agar at pH 6.0 to determine concentrations in the range from 15 to 100 %. S. Lutea 9341 with Difco Penassay Seed Agar at pl, 6.0 represents a more sensitive organism and can be used to determine concentrations in the range from about 5 to 50 tg / ml. In all cases, the dilutions were made in 0.1 ni potassium phosphate buffer at pl, 6.0 and applied in volumes of 0.1 ml to S and S-No.420E paper disks of 12.5 mm. All plates were incubated at 30 ° C. for 18 hours and the zones of inhibition were measured on a Quebec colony counter. The results can be read from a standard curve made on the same day. A multiple dose method can also be used. A sample of purified tetracycline was assigned an arbitrarily chosen value of 1000 # tg / rag, based on anhydrous material. All values given were calculated against this standard, unless otherwise stated.

Toxicity of Tetracycline Tetracycline shows little toxicity when administered to animals. The following Table IV shows the relative amounts of tetracycline, chlortetracycline and oxytetracycline necessary to kill mice in the manner indicated after administration of a single dose. It can be seen that the toxicity of tetracycline is comparable to that of chlortetracycline and oxytetracycline. Table IV Chlorine oxv Type of tetracycline tetracycline tetrac-vclin administration -g / kg mg / kg rag / kg Intravenous ...... LD "145 LD" 140 Intraperitoneally . . LDJ55 LD5,365 LD5,280 Subeutane ....... LDJ00 LD "400 LD2,340 Oral ........... LD "4250 - LD2.3750 Pneumoniae administration of tetracycline animal protection tests with mice pneumoniae D., X. and Spyogenes were infested show that tetracycline g after subcutaneous or oral Verabreichun has an activity which is comparable to that of oxytetracycline and chlortetracycline.

Tetracycline is produced in a purity greater than 9511/0 been with negligible levels of other antibiotic substances. Another form of the product contains a significant amount of others that are more compatible with it Antibiotics, however, not more than half that either added to the preparation were formed or simultaneously in the fermentation broth and at the recovery stage were also crystallized out, for example when using those described above Butanol or emulsion extraction. These products can be used in humans and animals in the same way as with known broad spectrum antibiotics in conventional ones Dosage forms appropriate for the desired route of administration, administered.

Claims (2)

PATENT CLAIMS 1. A process for the production of tetracycline by fermentation technology, characterized in that an aqueous nutrient medium containing a carbon, nitrogen and mineral salt source is fermented by means of an organism of the species Streptomyces aureofaciens or a natural or induced mutant thereof under aerobic conditions , the chlorine ion content of the nutrient medium having a value at which the formation of chlortetracycline is limited with the organism used and the respective constituents of the medium and a higher proportion of tetracycline than of chlortetracycline is obtained at the end of fermentation. 2. The method according to claim 1, characterized in that the fermentation is carried out within a temperature range of 25 to 35 'C and within a pl, -Beieichs of 5.0 to 8.5 . 3. The method according to claim 1, characterized in that the chlorine ion content of the medium is 17 parts / million parts of medium, preferably below 17 parts / million parts of medium. 4. The method according to claim 1, characterized in that the tetracycline formed is precipitated selectively from the fermentation liquid at an alkaline pH value with a quaternary ammonium salt. 5. The method according to claim 1, characterized in that the antibiotic formed together with the mycelium and other solids contained in the fermentation mash at a pu-M, ert of w:, at least 6 as an insoluble or complex compound with calcium, barium , Strontium and / or magnesium ions are separated and, if necessary, the solution obtained by extraction of the separated solids is treated again at a pn value of at least 6 with calcium, barium, strontium and / or magnesium ions. 6. The method according to claim 1, characterized in that the pH value of the aqueous solution obtained after separation of the mycelium and the solids contained in the fermentation mash in the presence of calcium, barium, strontium and / or magnesium ions to a value between 6 and 10 adjusted and the formed insoluble or complex compound of the antibiotic is separated. When the registration was announced, 3 priority documents were displayed.