Patent GB-687661-A (GB)
Title: Method of preparing vitamin b
IPC: C12P19/42
Filed: 1949-09-15
Abstract: Vitamin B12 is produced by growing a streptomycin-producing organism in contact with a liquid nutrient medium suitable for the production of streptomycin, which includes as a source of nitrogenous and growth-promoting substances at least one member of the group consisting of the meals, flours, meal-infusions and flour-infusions of beans, peanuts, cottonseed and linseed and to which a non-toxic cobalt salt has been added in non-toxic concentration, and recovering vitamin B12 from the culture.  The nutrient medium should contain also a carbohydrate assimilable by the mould, and salt if not already present in sufficient quantity.  After filtering the culture, vitamin B12 may be recovered from the filtrate as the main product, or the streptomycin may first be removed from the filtrate, which is then intimately contacted with an adsorbent or organic solvent for vitamin B12, this latter procedure being applicable also when no cobalt salt has been added to the nutrient medium.  Vitamin B12 is also recoverable from the waste mycelium, whilst, when the streptomycin is recovered from the filtrate by adsorption on activated charcoal and elution with aqueous acid, some vitamin B12 is also adsorbed but is not eluted by the aqueous acid.  The charcoal may be re-used in the process, thus accumulating more vitamin B12, and may then be dried to provide a vitamin B12 containing product, or the vitamin B12 may be eluted with a solvent therefor.  The vitamin B12 obtained may be further purified by precipitation of impurities with absolute alcohol, by adsorption on and elution from activated charcoal, by extraction from the impure material with phenol, or by any combination of these treatments.  In examples: (1) an aqueous medium containing soybean meal and dextrose is fermented with Streptomyces griseus, then acidified, filtered, and adjusted to pH 7.5 with dilute caustic soda, whereupon streptomycin and some vitamin B12 are adsorbed on activated charcoal, and, after filtration, vitamin B12 is extracted from the aqueous medium with phenol, the phenol extracts are mixed with ether and extracted with water and the aqueous extracts are evaporated; (2) alternatively the second filtrate in (1) is treated with activated charcoal at pH6-8 to adsorb vitamin B12, which is eluted with (a) phenol, or (b) absolute ethanol, or, after slurrying with water, adjusting to pH6 and filtering, with (c) aqueous ethanol, or (d) aqueous acetone; (3) cobalt chloride is added to the nutrient medium in (1), the product being (a) worked up as in (1) or (2), or (b) treated with charcoal as in (1), the streptomycin being eluted with dilute nitric acid and precipitated therefrom by the process of Specification 660,583, and the charcoal being successively re-used until it contains sufficient vitamin B12 to be useful as a supplement to animal foods, or to make recovery by elution as in (1) or (2) worth while; vitamin B12 is also recovered from the mycelium-containing solids by slurrying with water, filtering and evaporating the filtrate.  Specification 613,469 also is referred to.  The Specification as open to inspection under Sect. 91 comprises the use of any suitable liquid nutrient medium with any method of recovering vitamin B12 from the culture, and includes additional examples.  This subject-matter does not appear in the Specification as accepted.  Reference has been directed by the Comptroller to Specification 695,273.ALSO:A vitamin B12-containing product useful as a supplement in animal feeding, e.g. as an addition to vegetable protein diets, is obtained by growing Streptomyces grisens in contact with a liquid nutrient medium suitable for the production of streptomycin, which includes as a source of nitrogenous and growth-promoting substances at least one member of the group consisting of the meals, flours, meal-infusions and flour-infusions of beans, peanuts, cottonseed and linseed and to which a non-toxic cobalt salt has been added in non-toxic concentration, filtering the culture, treating the filtrate with activated charcoal to adsorb the streptomycin and some vitamin B12, eluting the adsorbed streptomycin with aqueous acid and drying the waste charcoal, preferably after re-use to increase the vitamin B12 content.  In an example the culture medium contains soybean meal and cobalt chloride, the elution of streptomycin is effected with dilute nitric acid, and the charcoal is re-used at least twice.  Specifications 613,469 and 660,583, [both in Group IV(b)], are referred to.  Reference has been directed by the Comptroller to Specification 695,273.ALSO:Vitamin B12 and concentrates thereof, obtained from cultures of streptomycin-producing organisms, may be used in therapeutic preparations.  In an example, the product obtained from a filtered Streptomyces griseus broth by adjusting the pH to 6-8, adsorbing the vitamin B12 on activated charcoal, slurrying the adsorbate in water and adjusting the pH to 6.0 with caustic soda, filtering, eluting with aqueous acetone and concentrating the eluate, is made into a sterile, highly concentrated, aqueous solution, preserved with phenol and put up in rubber capped vials for intramuscular injection. Specifications 613,469 and 660,583, [both in Group IV (b)], are referred to.  Reference has been directed by the Comptroller to Specification 695,273.