Patent GB-707332-A (GB)
Title: Antibiotics and methods for obtaining same
Filed: 1952-03-11
Abstract: Antibiotic C and antibiotic D are produced by cultivating the micro-organism Streptomyces plicatus, which is described, in a nutrient medium under aerobic conditions at a temperature of 20-40 DEG  C. for 2 to 15 days.  It is preferred to use an aqueous nutrient medium of initial pH 6-9, especially pH 7.3-7.7, a temperature of 22-29 DEG  C., submerged culture, and aeration with air or oxygen. The medium contains (a) assimilable carbon, e.g. glycerol, arabinose, fructose, galactose, lactose, maltose, rhamnose, xylose, mannitol, rhamnitol, starch, dextrin, fermentation and distillers slops, corn steep water solids, corn steep liquor, and fermentation residues, e.g. of butanol-acetone; (b) proteinaceous material, e.g. beef extract, acid hydrolysed casein, beef peptone, soybean oil meal, dried fermentation residues; water-soluble, defatted and partially hydrolysed soybean meal; amino acids, urea and purines; (c) mineral salts, e.g. sodium chloride, calcium carbonate, dipotassium hydrogen phosphate and mono-potassium hydrogen phosphate; (d) vitamins and growth stimulants.  Anti-foaming agent, e.g. lard or mineral oils containing mono- and di-glycerides may be used.  The antibiotics are isolated from (a) liquid media by acidifying to pH 2-4 and filtering off the mycelium, and (b) solid media by extracting with a solvent such as water.  A crude concentrate is prepared from the liquids so produced by (a) extraction with a water-immiscible alcohol, e.g. n-butanol, or (b) adsorption on activated carbon or an ion-exchange resin, and elution with acidified methanol or aqueous ammonia.  The concentrate is purified by dissolving in (a) methanol and adding a nonsolvent such as ether, or (b) water, adding a dye, e.g. p-hydroxy azo benzene p-sulphonic acid, and regenerating the antibiotics from the dye complex.  Antibiotic C is obtained by dissolving the purified crude concentrate in water and (a) passing the solution down a column of ion-exchange resin on the base cycle and thereafter concentrating the effluent (b) adding aqueous ammonia or potassium, acetate to precipitate the antibiotic.  Antibiotic D is obtained by dissolving the purified crude concentrate in a mixture of ethyl acetate, water and acetic acid, passing the solution down a column of cellulose, eluting with water and freeze drying the eluate.  Antibiotic C is basic, crystalline, contains C, H, O and N only; has melting-point of 182-184 DEG  C., molecular weight 552 in camphor solution; [a ]26D 180.9 in methanol; contains an arylamino grouping; shows characteristic ultra-violet and infra-red absorption spectra; soluble in dilute mineral acid and aliphatic alcohols of 1 to 4 carbon atoms, moderately soluble in water, insoluble in ether; precipitated from aqueous solution by p-hydroxyazobenzene p-sulphonic acid, picric acid, Orange II and other dyes; has Rf value; active against Mycobacterium tuberculosis var. hominis.  Antibiotic D is basic, contains elements C, H, O and N only; shows characteristic ultra-violet and infra-red absorption phenomena; soluble in water and methanol, less so in ethanol, insoluble in ether; precipitated from aqueous solutions by means of p-hydroxyazobenzene p-sulphonic acid, picric acid, Orange II and other dyes; has Rf value; active against M. tuberculosis var. hominis.