Patent GB-619572-A (GB)
Title: Improvements in and relating to the production of an antibiotic
Filed: 1946-12-21
Abstract: An antibiotic is obtained by making a culture of the species of bacteria known as Bacillus aerosporus Greer or B. polymyxa (Prazmowski) Migula, removing the bacterial cells from the metabolism product (e.g. by filtration or centrifuging), adsorbing the antibiotic on activated charcoal under neutral conditions (pH 6-8) and eluting it with a solvent therefor.  The culturing of the bacillus (of which strains shown by biological assay to give a high yield of the antibiotic are preferably selected) is effected aerobically (in shallow layers or in deeper vessels with artificial aeration) on the usual culture media, the best results being obtained with an aqueous medium (pH about 7.4) containing about 10 per cent by volume of nutrient broth with the addition of small amounts (e.g. 3 per cent and 0.6 per cent respectively) of glucose and diammonium hydrogen phosphate and a trace (e.g. 0.002 per cent) of manganese sulphate.  The adsorption of the antibiotic on charcoal is preferably preceded by a preliminary treatment at pH 2.0-2.5 with an activated charcoal which under such conditions adsorbs colouring matter and impurities but not the antibiotic.  Elution, preferably preceded by washing with dilute hydrochloric acid (pH 2.0-2.5) or water, or both, may be effected with aqueous acetone (about 40 per cent concentration) acidified (pH about 2.5) with sulphuric acid, and the sulphate of the antibiotic may be precipitated from the eluate by adding acetone until the concentration thereof is about 75 per cent and cooling (to about 4 DEG  C.), and may be purified by dissolving in water, neutralizing (pH 7) with alkali, filtering and freeze drying.  Alternatively the eluate is adjusted to pH 3, the acetone distilled off under reduced pressure, the antibiotic (sulphate) again adsorbed on charcoal from the aqueous solution and eluted as before and the eluate neutralized with calcium carbonate, filtered and freeze dried; or, after evaporation of the acetone, picric acid may be added to precipitate the picrate of the antibiotic.  The sulphate may be converted by treatment with hydrochloric acid or calcium chloride, to the hydrochloride, which may be further purified by dissolving it in methanol and reprecipitating it with ether. Alternatively the hydrochloride may be obtained directly by washing the charcoal on which the antibiotic is adsorbed with neutral ethanol (twice), eluting with dry methanolic hydrochloric acid (approximately decinormal) and precipitating with ether.  Purification of the sulphate, hydrochloride or picrate may also be effected by conversion to the helianthate by dissolving in aqueous methanol and adding a saturated solution of methyl orange, preferably such amount that about 80 per cent of the antibiotic activity is associated with the precipitate.  The bacterial cells after separation from the metabolism fluid may be subjected to autolysis, e.g. by suspension in sterile saline solution (preferably with the addition of 1 per cent of toluene) and incubation at about 37 DEG  C. for about 48 hours, and the resulting supernatant fluid added to or worked up in the same way as the metabolism fluid.ALSO:In the production of an antibiotic, a culture of the species of bacteria known as Bacillus aerosporous Greer or B. polymyxa (Praymowski) Migula is made on the usual culture media, preferably an aqueous medium (pH about 7.4) containing about 10 per cent by volume of nutrient broth with the addition of small amounts (e.g. 3 per cent and 0.6 per cent respectively) of glucose and diammonium hydrogen phosphate and a race (e.g. 0.002 per cent) of manganese sulphate.  The culturing (for which strains of the bacillus shown by biological assay to give a high yield of antibiotic are preferably selected) is effected aerobically, e.g. at 22-28 DEG  C., in shallow layers (for 3-8 days) or in deeper vessels with artificial aeration for 20 - 24 hours).  The metabolism fluid is separated from the bacteria (e.g. by filtration or centrifuging, if desired with the addition of 0.4 per cent of chloroform as preservative) and is worked up to recover the antibiotic, whilst the residual bacteria may be subjected to autolysis, e.g. by suspension in sterile saline solution (preferably with the addition of 1 per cent of toluene) and incubation at about 37 DEG  C. for about 48 hours, and the resulting supernatant fluid may be added to or used in the same way as the metabolism fluid.