Patent GB-814794-A (GB)
Title: A new antibiotic c-159 produced by fermentation
Filed: 1957-09-17
Abstract: A new antibiotic C159 is produced by cultivating a C159-producing strain of Streptomyces canus, e.g. Streptomyces canus ATCC 12646 and ATCC 12647 under aerobic conditions in an aqueous nutrient medium containing a source of carbon, nitrogen and inorganic salts.  The carbon source may be a sugar such as "Cerelose" (Registered Trade Mark), a polysaccharide or a glyceride oil.  Suitable nitrogen sources are ammonium sulphate, sodium nitrate, corn steep liquor, distillers' solubles, yeast and soybean meal (preferred).  Suitable mineral salts are potassium monohydrogen phosphate, sodium chloride and calcium carbonate. The temperature of cultivation is 27 DEG  to 30 DEG  C. and the duration 42 to 70 hours.  Antibiotic C159 is recovered from the clarified broth by (a) adsorption or (b) solvent extraction.  Adsorption may be on magnesium silicate (magnesol) or activated carbon at pH 8.5, followed by elution with 1 : 1 mixture of water and methanol or 3 : 1 mixture of n-butanol and water in the case of the magnesol, and 1 : 1 mixture of n-butanol and water acidified to pH 3.0 with hydrochloric acid in the case of the carbon. The eluate is concentrated at a ph of 6.5 to 7.5 by distillation in vacuo and then lyophilised or spray dried.  Alternatively, the antibiotic is recovered from the eluate concentrate by adding a nonsolvent such as Skellysolve B (hydrocarbon solvent).  Solvent extraction of the filtered broth is by means of n-butanol at pH 2.5, 6.0 or 9.0 but preferably 8.2 to 8.5, the antibiotic being recovered by lyophilisation or precipitation by means of a non-solvent.  The antibiotic may be further purified by dissolving in methanol, filtering and adding diethyl ether to precipitate the antibiotic. Alternatively, it may be purified by countercurrent distribution using as upper solvent phase a 1 : 1.5 mixture of n-butanol and methyl isobutyl ketone saturated with water and as lower aqueous phase water buffered with 0.02 molar phosphate at pH 6.0 and saturated with both n-butanol and methyl isobutyl ketone. Antibiotic C159 is effective in inhibiting the growth of Gram positive microorganisms; it is soluble in methanol and alkaline aqueous solutions, but insoluble in acetone, ether and butyl acetate and aqueous solutions of pH below 3.7; it contains carbon 58.7 per cent, hydrogen 7.4 per cent, nitrogen 9.9 per cent and oxygen 24.0 per cent (by difference); on hydrolysis (heat in sealed tube <PICT:0814794/IV (b)/1> with barium hydroxide at 150 DEG  C. for 1 1/2 hours or with 20 per cent hydrochloric acid for 5 hours) yields glycine, a -alanine, threonine and aspartic acid and no other amino acids; shows ultraviolet absorption maximum at 345 mm  (E 1 per cent=71 1 cm.) and shoulder at 260-280 mm ; infra-red absorption shows characteristic bands at 2.90, 3.38, 5.85, 6.05, 6.10, 6.55, 7.23, 7.50, 8.00 and 9.40 microns; it is an acid and is converted to the ammonium, amine and metal salts such as the sodium, potassium, calcium, magnesium and aluminium salts by adding the appropriate base, e.g. Ca(OH)2 or NaOH to an aqueous solution of the antibiotic.  It is used medicinally (see Group VI).  The antibiotic is distinguished from amphomycin. Specification 736,325 is referred to.ALSO:A pharmaceutical composition comprises a pharmaceutically acceptable carrier and at least one part per thousand of the antibiotic C159 which is effective against gram positive micro organisms.  The composition may be in dosage unit form. Specification 736,325, [Group IV (b)], is referred to.