Patent GB-817730-A (GB)
Title: Fermentation of a tetracycline-producing-streptomyces
Filed: 1956-02-03
Abstract: The antibiotics tetracycline and P.A.121 (also known as antibiotic P.A. 371 or quinocycline), are produced by cultivating in a nutrient medium under submerged aerobic conditions a tetracycline-producing-strain of Streptomyces fuscopaciens nov. spec. especially Streptomyces fuscofaciens ATCC 12061, or a mutant thereof. The medium contains a source of carbon, nitrogen, inorganic salts and growth stimulants.  Specified carbon sources are molasses, glucose, starch and glycerol.  Nitrogen sources are soybean meal, wheat gluten, peanut meal, hydrolysate of casein, corn steep liquor, distiller's solubles, yeast extract and cottonseed meal.  Inorganic salts are sodium chloride, sodium nitrate, potassium phosphate and magnesium sulphate.  Media of low chloride ion content produce increase yields of tetracycline.  The temperature of cultivation is 25 DEG  to 30 DEG  C. and preferably 28 DEG  C. and the duration 2 to 6 days.  The crude broth or the dried whole or filtered broth may be used as an animal feed supplement or plant growth stimulant. Recovery of tetracycline is by the methods known to be used for the recovery of tetracycline, namely (a) precipitation as a complex with one or more polyvalent metals of Group II of the periodic table, e.g. barium, calcium, magnesium, strontium, beryllium, mercury, and cadmium which are added as water-soluble salts and the pH is adjusted to 7.5 to 9.0. The complex may be used as such or treated to recover the tetracycline by e.g. addition of an acid forming insoluble salts with the metal of the complex; (b) extraction at pH 1 to 2.5 or 7.5 to 10 with a water - immiscible organic solvent, e.g. butanol, if desired, after the addition of a sequestering agent for polyvalent ions such as ethylene diamine tetraacetic acid; (c) precipitation at a pH of 8 to 9.5 as a complex with an organic base such as a C8 to C18 alkyl trimethyl ammonium halide, preferably after the addition of a sequestering agent; (d) precipitation as a complex with an aryl azo sulphonic acid dye; (e) adsorption at pH 3 to 8 on ion exchange resins, activated carbon and earths.  The above methods of isolation, in general, yield mixtures of antibiotics.  Antibiotic P.A. 121 may initially be extracted from the filtered fermentation liquor at pH 6 to 8 by means of water-immiscible organic solvents, e.g. aromatic hydrocarbons, esters, ketones, alcohols and halogenated hydrocarbons, e.g. ethyl acetate, methyl isobutyl ketone, propyl acetate, chloroform and benzene.  P.A.121 is recovered by removing the solvent or adding a non-solvent, e.g. hexane or petroleum ether to the extract.  Tetracycline may then be recovered by one of the methods described above.  The crude broth may be used for the stimulation of plant growth or may be mixed with animal feed (see Group I). Specifications 769,239 and 817,729 are referred to.ALSO:An animal feed has incorporated therein the crude fermentation broth formed by cultivating streptomyces purcofaciens ATCC 12061, or a mutant thereof, in a nutrient medium (see Group IV(b)).  The broth contains the antibiotics tetracycline and antibiotic PA 121 (also known as antibiotic PA 371 or quinocycline).  Specifications 769,239 and 817,729 [both in Group IV(b)] are referred to.