Patent GB-715804-A (GB)
Title: Process for the production of vitamin substances by fermentation
IPC: C12P19/42
Filed: 1949-07-11
Abstract: A vitamin product suitable for the enrichment of animal feeds deficient in "animal protein factor" (APF) is manufactured by fermenting an aqueous nutrient medium under submerged conditions by means of an LLD-activity-producing micro-organism selected from the genera of Fungi consisting of Myxomycetes, Schizomycetes and Eumycetes, for a sufficient period of time to produce a fermentation product having an LLD activity, based on the solid content thereof, of at least 20 LLD units per mg., and recovering the APF-active vitamin product from the broth by removing the bulk of the water therefrom or by treating it with adsorbent material. In the latter case the adsorbate may be dried at temperatures below those destructive of Vitamin B12, or the active material may be eluted with a solvent and the eluate subjected to chromatographic fractionation to recover Vitamin B12, ultimately in crystalline form.  When the micro-organism employed is a streptomycin-producing strain of Streptomyces griseus, the broth may first be treated with an ion-exchange resin to adsorb the streptomycin therefrom.  The fermentation is preferably effected under aerated conditions in an aqueous nutrient medium containing a source of assimilable carbon, a source of assimilable nitrogen and inorganic salts.  In examples: (1) an aqueous medium containing beef extract, tryptic digest of casein, sodium chloride, ferrous sulphate, cobaltous nitrate and soy bean oil is fermented under aerated conditions for 48 hours at 28 DEG  C. with a grisein-producing strain of S. griseus, then acidified, filtered, made slightly alkaline and again filtered, and the filtrate worked up as follows: (a) evaporated to 6 per cent of its original volume and then spray-dried; (b) treated with activated carbon and the adsorbate filtered off and part of it dried in a vacuum oven at 45 DEG  C.; (c) the rest of the adsorbate from (b) is eluted by stirring with 25 per cent pyridine and the eluate partially evaporated at reduced pressure below 35 DEG  C. and finally freeze-dried; (d) the product of (c) is extracted with methanol, the extract passed through a column of activated alumina which is then eluted with methanol, the most active fractions of eluate are concentrated, and crude Vitamin B12 is precipitated with acetone, purified by reprecipitation from ethanol by addition of acetone and finally crystallized from aqueous acetone; (2) a medium as in (1) but omitting ferrous sulphate and cobaltous nitrate is similarly fermented and worked up to the carbon adsorption stage, and the adsorbate is eluted with 25 per cent a -picoline, the eluate partially evaporated as in (1) (c), the concentrate is diluted with methanol and filtered to remove precipitated inert material, and the active material is precipitated with ether; (3) an aqueous medium containing soy bean meal, dextrose, distillers' solubles, sodium chloride and cobaltous ions is fermented under submerged aerated conditions for 2 days at 27 DEG  C. with a streptomycin-producing strain of S. griseus, the broth is acidified and filtered, the filtrate is passed through a column of carboxylic acid-type ion exchange resin to adsorb streptomycin, and the effluent is worked up as follows: (a) treated with activated carbon and the adsorbate washed and dried; (b) acidified with phosphoric acid and treated with fuller's earth, and the adsorbate washed and dried; (c) evaporated to 15 per cent of its original volume and then spray-dried; (4) a medium containing tryptic digest of casein, beef extract and cobaltous nitrate is fermented with S. roseochromogenus, and the p  broth, after filtration, is evaporated under reduced pressure and finally freeze-dried, or is treated with activated carbon, the adsorbate eluted with a mixture of ethanol, pyridine and water, and the eluate evaporated under reduced pressure and then freeze-dried; (5) a medium as in (4) is fermented with S. albidoflavis, the broth is acidified, filtered, neutralized and treated with activated carbon, and the adsorbate is dried; (6) the medium in (1) is replaced by one containing Pfeiffer's dried yeast and cobalt nitrate; (7) the beef extract and tryptic digest of casein in (1) are replaced by one of the following: dried yeast, protein digested tankage, "Quaker" oats, "Armour" blood meal (the words "Quaker" and "Armour" are Registered Trade Marks), SMA fish meal, salmon waste meal, protein meat and bone scraps, Anglo beef extract; (8) a medium containing brain-heart infusion, agar and cobalt nitrate is fermented with Clostridium tetanomorphum, C. cochlearium, C. flabelliferum or C. butyricum and the broth is treated as in (1).  Specifications 623,082, 639,943 and 640,950 are referred to.ALSO:Animal food comprising a nutrient material deficient in "animal protein factor" (APF) is rendered dietically adequate in respect to said factor by the addition of a vitamin product obtained by fermenting an aqueous nutrient medium under submerged conditions by means of an LLD-activity-producing micro-organism selected from the genera of Fungi consisting of Myxomycetes, Schizomycetes and Eumycetes, for a sufficient period of time to produce a fermentation product having an LLD activity, based on the solid content thereof, of at least 20 LLD units per mg., and recovering the APF-active vitamin product from the broth by removing the bulk of the water therefrom or by treating it with adsorbent material.  In the latter case, the dried adsorbate, an eluate therefrom evaporated to dryness, or crystalline vitamin B12 prepared from such an eluate, may be added to the animal food.  In examples, various forms of the APF-active vitamin product, and, for comparison, crude liver extract, are added to chick foods consisting of soya bean meal, dextrose, cellulose, wheat germ oil, calcium gluconate, glycine, DL-methionine, calcium carbonate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, magnesium sulphate, calcium hydrogen phosphate, ferric citrate, manganese sulphate, potassium iodide, copper sulphate, zinc chloride, L-arginine, L-cysteine, choline, inositol, p-aminobenzoic acid, niacin, calcium pantothenate, riboflavin, thiamin, pyridoxine, pteroylglutamic acid, menadione, biotin, and A and D powder. Specifications 623,082, 639,943 and 640,950, [all in Group IV(b)], are referred to.