Patent GB-775115-A (GB)
Title: Production of tetracycline by fermentation
IPC: C12P29/00
Filed: 1954-07-26
Abstract: Tetracycline is produced by aerobically fermenting an aqueous nutrient medium containing assimilable sources of carbon and nitrogen and mineral substances with a micro-organism of the genus Streptomyces which tends to produce chlortetracycline in the presence of chloride ions, the chloride ion content of the nutrient medium being controlled so that formation of the chlortetracycline is restricted and tetracycline is formed as the predominating antibiotic.  The preferred medium contains less than 1 p.p.m. of available chloride ions.  Streptomyces aureofaciens and reactants thereof are suitable micro-organisms. The fermentation is submerged aerobic, the temperature 25 DEG  to 35 DEG  C., and preferably 30-33 DEG  C., and the pH 3.5-8.5 and preferably 7.0.  The medium contains (a) a source of assimilable carbon, e.g. sucrose, starch and dextrin; (b) a source of assimilable nitrogen, e.g. corn steep liquor, amino acids such as d,l-alanine, l-arginine, d,l-methionine, l-glutamic acid, l-histidine, d,l-beta-asparagine, and ammonium salts, e.g. ammonium sulphate and ammonium phosphate; (c) trace elements, e.g. copper, zinc, manganese, iron and chromium.  Buffers, e.g. calcium carbonate and antifoaming agents, may also be present.  In another medium there is used corn steep liquor which has been deionized by treatment with ion-exchangers.  Tetracycline may be isolated from the fermentation broth by acidifying to pH 3 or less, filtering and (a) adding an alkaline earth metal salt at pH 6.5 and extracting the precipitate so formed with acidified water or with a water-miscible solvent such as an alcohol, alkoxy-alkanol or ketone; (b) extracting with a water-immiscible organic solvent, e.g. n-butanol, chloroform and ethyl acetate, if desired, in the presence of a water-soluble inorganic or organic salting out agent; (c) adsorption on a synthetic resin, e.g. Amberlite IRC 50 in the hydrogen cycle and elution with acidified aqueous methanol; or (d) adjusting to pH 8-11 and adding a quaternary ammonium salt especially an alkyl trimethylammonium chloride or a dialkyl dimethylammonium chloride wherein the alkyl group contains 8 to 18 carbon atoms.  The quaternary ammonium salt is purified by treatment with water and chloroform and extraction of the chloroform layer with aqueous acid of pH 1 to 2.5, the extract then being adjusted to pH 3 to 7.  Tetracycline has the formula C22H24N2O8 <FORM:0775115/IV(b)/1> It is amphoteric forming salts with inorganic and organic acids, e.g. hydrochloride, hydrobromide, sulphate, borate, nitrate, phosphate, ascorbate, citrate, succinate, acetate and sulphamate; with metals such as potassium, lithium, calcium, barium, strontium, magnesium; and with amines such as ethylene diamine, triethylamine and piperidine.  It forms complexes with sodium borate.  Forms yellow, tubular crystals of trihydrate, [a ]29D=-234 DEG  (0.82 per cent in methanol), melting-point 160-165 DEG C.  Soluble in methanol, 2-methoxyethanol, ethanol, n-butanol; almost insoluble in ether, benzene and petroleum ether.  Infrared maxima at 3300-3400 cm.-1, 1655, 1613, 1580, 1228, 1178, 950, 862, 785, 722 and 703 cm.-1.  Ultra-violet E1 %1 cm. at 269 mu=271 E1 %1 cm. at 365 mu=272.  Distinguished from chlorotetracycline and oxytetracycline also by Craig countercurrent distribution technique.  Tetracycline gives a violet colour with concentrated sulphuric acid and a deep yellow with Ehrlich reagent (p-dimethylaminobenzaldehyde in hydrochloric acid).  Tetracycline is of low toxicity and is effective against both gram positive and gram negative micro-organisms.  It may be administered to animals or humans in the conventional forms or it may be incorporated in animal feed.ALSO:Tetracycline (see Group IV(b)) is added directly to animal feeds in the form of a fermentation broth, a clarified fermentation broth, or a concentrate of a clarified broth in order to stimulate growth or control disease. The filtered broth may be added to drinking water.ALSO:Tetracycline (see Group IV(b)) is administered to humans and animals in dosage forms appropriate to the desired route of administration.