Patent GB-1034944-A (GB)
Title: Production of ª‰-carotene
IPC: C12P23/00
Filed: 1964-08-04
Abstract: Mated (+) and (-) strains of species of the family Choanephoraceae are grown in submerged culture in an aqueous medium containing sources of carbon, nitrogen, thiamin and mineral salts to produce a new substance designated "factor-b ". The cultivation is carried out at from 24 DEG  to 35 DEG C. at a pH of from 5.5 to 7 for from 3 to 10 days, preferably in the presence of stated proportions of b -ionone, 2,2,6-trimethylcyclohexanone, orange peel extract, or a higher aliphatic hydrocarbon such as dodecane, tetradecane, hexadecane or kerosene. The mated (+) and (-) strains may be of the species Blakeslea trispora, Choanephora conjuncta, Choanephora cucurbitarum and Choanephora circinans. The factor-b  produced is extracted in the form of the free acid by a water-immiscible organic solvent and may be converted into an alkali metal (or ammonium), salt or into a methyl ester by reaction with an alkali metal (or ammonium) hydroxide or bicarbonate or esterification with diazomethane, respectively.ALSO:A microbiological process of producing b -carotene comprises growing in submerged culture a (-) strain of a species of the family Choanephoraceae in the presence of a new compound designated "factor b ," in an aqueous medium containing sources of carbon, nitrogen, thiamin and mineral salts.  The cultivation is carried out at from 20 DEG  to 35 DEG  C. at a pH of from 5.5 to 7.5 for from 3 to 12 days, preferably in the presence of stated proportions of b -ionone, 2,2,6-trimethyl cyclohexanone, kerosene or orange peel extract.  The following species are preferred: Blakeslea trispora, Choanephora cucurbitarum, Choanephora circinans and Choanephora conjuncta.  The factor-b  may be added to the culture medium as such, as a solution in an organic solvent or as an aqueous solution of an alkali metal or ammonium salt thereof, or the culture broth containing factor-b  obtained from the fermentation process for the preparation of the factor-b  itself may be used.  The factor-b is added at the beginning of, or during, the fermentation at a concentration of from 50 to 500 g  per c.c. of culture medium. An example (Example 15) is directed to the cultivation of Blakeslea trispora on a medium of pH 5.9-6.1 consisting of distiller's solubles, starch, yeast extract, cotton seed oil and tap water, and including also 1.5% by volume of kerosene.  After 51 hours of fermentation, 0.3 c.c. of a b -ionone solution in 20% kerosene and 14% of a filtered broth containing the factor b , obtained as described in Example 5, are added so that the end-concentration of the factor-b  in the culture is 280 g /c.c.  After 6 days of fermentation the production of b -carotene amounts to 970 g /c.c. (The same fermentation carried out without adding the factor-b yields 46 g /c.c. of b -carotene.)  When the fermentation is over, the mycelium is separated from the culture broth by filtration, and is vacuum dried and then extracted with petroleum ether.  The b -carotene contained in the extracts is determined spectrophotometrically.  The factor-b  which is produced by growing mated (+) and (-) strains of species of the family Choanephoraceae is a hydroxy carboxylic acid containing a conjugate system of a keto-group and three ethylenic bonds, is soluble in chlorine - containing aliphatic hydrocarbons, lower aliphatic alcohols, aromatic hydrocarbons, diethyl ether, ethyl acetate and acetone, under U.V. irradiation shows in chloroform an absorption maximum at 325 mm  with two shoulders at 350 mm  and at 310 mm  and in ethanol an absorption maximum of 325 mm  and a shoulder at 233 mm , forms a sodium salt which is soluble in water, acetone and lower aliphatic alcohols and under the U.V. irradiation shows in water at pH7 an absorption maximum at 335 mm  and a shoulder at 230-235 mm , and forms on esterification with diazomethane a methyl ester from which there are obtained by chromatography on silica gel and elution with carbon tetrachloride containing 0.5-5% of acetone, three fractions, viz. esters b 3, b 2 and b 1. Ester-b 3 of factor-b  has the empirical formula C19H26- 28O4, contains a carboxyl group, a a , b -unsaturated keto-group, a secondary alcohol group and three conjugated double bonds, has an Rf value of 0.36 (silica gel thin layer chromatography and elution with benzene-gasoline-ethyl acetate (7: 1.4)) and on hydrogenation in the presence of a catalyst such as palladium on charcoal, gives a compound having the formula C19H32O4 boiling at 170-180 DEG  C. at 0.02 mm. Hg.  Ester-b 3 yields a crystalline semicarbazone derivative of the formula C20H35O4N3 and melting at 112-113 DEG  C., an on complete reduction yields the methyl ester of the corresponding saturated acid having the formula C19H36O2 (as a mixture of two diastereoisomers) and boiling at 115 DEG  C. at 0.02 mm. Hg; spectral characteristics of all these compounds and of esters b 2 and b 1 are disclosed. Ester-b 2 has the same carbon skeleton as that of ester-b 3 and on complete reduction yields a mixture of two saturated diastereoisomer esters of the formula C19H36O2.  Esters b 2 and b 1 have Rf values of 0.36 and 0.68, respectively.