Patent GB-793797-A (GB)
Title: Improvements in or relating to antibiotics
Inventor: WHITFIELD JEAN
Filed: 1955-03-25
Abstract: <PICT:0793797/IV (b)/1> A new antibiotic designated E.129 is produced by culturing under aerobic conditions an antibiotic E129-producing-strain of Streptomyces E.129 nov. spec., especially Streptomyces ostreogriseus NCIB No. 8792 or NRRL No. 2558 in a nutrient medium containing an assimilable source of carbon, nitrogen and nutrient salts.  It is preferred to use submerged aerobic fermentation for 16 to 28 hours at 28 DEG  C.  Specified sources of (a) nitrogen are ammonium sulphate and phosphate, soya bean meal and flour, maize corn meal, oat meal, corn steep liquor, distiller's solubles, casein hydrolysate and enzyme digest, peptone, tyrosine; (b) carbon are dextrin, starch and sugars such as dextrose, maltose, sucrose and glucose; (c) nutrient salts are sodium and magnesium chlorides, potassium hydrogen phosphate, calcium carbonate, calcium lactate, magnesium, zinc, ammonium and ferrous sulphates. In addition to the nutrient salts, a preferred medium contains 0.4 to 0.9, and preferably 0.7, per cent corn steep liquor (as solids) and 1.5 per cent carbohydrate especially glucose, as such, or with the further addition of 1 to 5, and especially 2.5, per cent oatmeal.  Improved yields are obtained when the medium contains 30 to 135, and preferably 20 to 115 p.p.m. of a soluble salt such as ferrous sulphate, ferrous ammonium sulphate, ferric citrate and ferric chloride.  Antibiotic E.129 is recovered from the filtered broth by (a) solvent extraction, or (b) adsorption.  Extraction is at pH 7 to 9.5 with a water-immiscible solvent such as benzene, ethyl acetate, butyl acetate, chloroform or n-butanol.  The solution so obtained is preferably washed with aqueous sodium bicarbonate, hydrochloric acid and water, dried over magnesium sulphate and the solvent removed. Alternatively, the solid obtained by removing the solvent without the above washing steps, is purified by treating with a petroleum hydrocarbon solvent such as light petroleum in order to remove impurities.  Adsorption is an acidwashed charcoal or on magnesium trisilicate, followed by elution with a solvent such as n-butanol or aqueous acetone. Antibiotic E.129 is a broad spectrum antibiotic, containing 64 per cent carbon, 7 per cent hydrogen and 8 per cent nitrogen; it is readily soluble in methanol, ethanol, butanol, acetone, amyl acetate, chloroform and benzene, less soluble in ether and water and insoluble in petroleum; it is a neutral substance which does not form salts with either the common acids or common bases; it is stable over pH 2 to 10 at room temperatures, less so at higher temperatures; it melts at 121 DEG  to 134 DEG  C.; it exhibits no characteristic ultra-violet absorption spectrum in ethanol; it has an infra-red absorption spectrum with characteristic absorption bands at 810 cms-1, 1058 cms-1, 1022 cms-1 and 1575 cms-1, the characteristic relative intensites of these maxima being I810/I1022 greater than 0.5, I810/I1575 greater than 0.4, I1058/I1022 greater than 0.95 and I1058/I1575 greater than 0.7.  R values on paper chromatogram: (1) Rf = 0.64 in 1 per cent glacial acetic acid; (2) Rf = 0.65 in 3 per cent ammonium chloride solution; (3) Rf = 0.78 in methanol/acetone/water (19 : 6 : 78). Antibiotic E.129 is distinguished from streptogramin and erythromycin.  Specification 776,035 is referred to.