Abstract:
A process for production of canthaxanthin comprising the steps of culturing a microorganism capable of producing canthaxanthin and belonging to the genus Corynebacterium, such as Corynebacterium sp. SQH 348 (FERM BP-4284), and recovering canthaxanthin from the culture.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a process for production of canthaxanthin which is one of carotenoid pigments. Canthaxanthin produced by the present process is useful as a natural red pigment in foddor additives, food additives, cosmetics and the like. 
     2. Related Art 
     It is known that canthaxanthin is present in a mushroom (Botanical Gazette 112, 228-232, 1950), fishes, and crustaceans (Carotenoids of Aquatic Organisms, Nippon Suisan Gakukai, 1978). In addition as microorganisms producing canthaxanthin, microorganisms belonging to the genus Brevibacterium (Applied and Environmental Microbiology, 55(10), 2505, 1989), and microorganisms belonging to the genus Rhodococcus (Japanese Unexamined Patent Publication (Kokai) No. 2-138996) are known. Moreover, canthaxanthin can be chemically synthesized by oxidation of B-carotene (J. Amer. Chem. Soc., 78, 1427 (1956)) and synthesis from novel 3-oxo-C 15  phosphonium salt (Paure Appl. Chem. 51, 875 (1979)). 
     SUMMARY OF THE INVENTION 
     The conventional processes, however, have various drawbacks; for example, extraction from natural products is expensive, raw materials are not stably available, productivity by microorganisms is low, and products are accompanied with a lot of impurity. It is problemable rouse chemically synthesized canthaxanthin due to safety problems. 
     The present invention provides a simple process for production of canthaxanthin having high purity and being safe. 
     The present inventors carried out various attempts to develop a process for production of canthaxanthin using microorganisms. As a result the present inventors found that microorganisms belonging to the genus Corynebacterium accumulate a high concentration of canthaxanthin in their cells, and completed the present invention. The present invention relates to a process for production of canthaxanthin comprising culturing a microorganism having an ability to produce canthaxanthin and belonging to the genus Corynebacterium, and extracting and purifying canthaxanthin accumulated in the cells to obtain canthaxanthin. 
    
    
     BRIEF EXPLANATION OF DRAWINGS 
     FIG. 1 represents an infrared absorption spectrum of canthaxanthin produced according to the present invention. 
     FIG. 2 represents a result of mass analysis of canthaxanthin produced according to the present invention. 
     FIG. 3 represents a  13  C nuclear magnetic resonance of canthaxanthin produced according to the present invention. 
    
    
     DETAILED DESCRIPTION 
     According to the present invention, any microorganism belonging to the genus Corynebacterium and producing canthaxanthin can be used. As an example, Corynebacterium sp. SQH 348 isolated by the present inventors can be mentioned. This strain was deposited to National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305 Japan, on April 27, 1993 as FERM BP-4284. 
     This strain has the following taxonomical properties. 
     
         ______________________________________(a) Morphology    Bouillon liquid medium(1)    Shape and size of cell  Rod, 0.8 to 1.0 × 1.0 to 1.5 μm(2)    Polymorphism  Present(3)    Motility      None(4)    Spore formation                None(5)    Gram stain    Positive(b) Cultural properties on medium(1)    Bouillon agar plate  State of growth                Abundant  Color of colony                Orange  Shape of colony                Circle (smooth)  Gloss of colony                Present  Diffusible pigment                None(c) Physiological properties(1)    Reduction of nitrate                     -(2)    Oxidase            -(3)    Catalase           +(4)    Range for growth  Growth at pH 6.0   -  Growth at pH 7.0   +  Growth at pH 12.0  +(5)    Behavior toward oxygen                     aerobic(6)    Liquefaction of gelatin                     -(7)    Decomposition of esculin                     -(8)    Decomposition of hippuric acid                     -(9)    Decomposition of casein                     -(10)   Decomposition of urea                     +(11)   Methyl red test    -(12)   Glycolate test     -                     (acetyl type)(13)   Diamino acid of cell wall  meso-diaminopimelic acid(14)   Sugar composition of cell wall  Arabinose          +  Galactose          +(15)   Quinone type       MK-8 (H.sub.2)(16)   GC content         69 mol %(17)   Formation of acid from carbohydrates(1)       Arabinose        -(2)       Galactose        -(3)       Xylose           -(4)       Glucose          -(5)       Salicin          -(6)       Sucrose          -(7)       Starch           -(8)       Dextrin          -(9)       Trehalose        -(10)      Fructose         -(11)      Maltose          -(12)      Mannose          -(13)      Lactose          -(14)      Raffinose        -(15)      Rhamnose         -______________________________________ 
    
     As a result, the SQH 348 strain was identified as a microorganism belonging to the genus Corynebacterium and designated as Corynebacterium sp. SQH 348. 
     According to the present invention, microorganisms other than the strain SQH 348 can be used. Microorganisms which can be used in the present invention can be selected from microorganisms belonging to the genus Corynebacterium. For example, microorganisms belonging to Corynebacterium are obtained from depository institutes such as ATCC, NRRL, FRI etc. Next they are cultured in a medium such as that described in Table 1, and inoculated into a production medium such as that described in Example 1, and the culture is assaied for canthaxanthin according to the procedure described in Exmple 1. Microorganisms which produce canthaxanthin is selected and used for the present invention. 
     Medium for production of canthaxanthin using the present microorganisms is, for example, as follow. Namely, it contains a carbon source, a nitrogen source and inorganic salts necessary for the growth of producer microorganisms, as well as if necessary special required substances (for example, vitamines, amino acids, nucleic acids etc.). As the carbon sourses, sugars such as glucose, fructose, trehalose, mannose etc., organic acids such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid; alcohols such as ethanol, propanol, butanol, pentanol, hexanol, isobutanol; hydrocarbons such as linear hydrocarbons having 11 to 20 carbon atoms; branched hydrocarbons such as squalene; oil or fat such as rape oil, soybean oil, olive oil, corn oil, linseed oil, and the like are mentioned. Amount of the carbon source added varies according to the kind of the carbon source, and usually 1 to 100 g, preferably 2 to 50 g per 1 l medium. 
     As the nitrogen sourses, for example, potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, urea etc. are used alone or in combination. Amount of the nitrogen source added varies according to the kind of the nitrogen source, and usually 0.1 to 10 g, and preferably 1 to 3 g per 1 l medium. 
     As the inorganic salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric sulfate, ferrous sulfate, ferric chloride, ferrous chloride, calcium chloride, calcium carbonate, sodium carbonate etc. may be used alone or in combination. Amount of inorganic acid varies according to the kind of the inorganic salt, and usually 0.001 to 10 g per 1 l medium. 
     As special required substances, vitamines, nucleic acids, yeast extract, peptone, meat extract, malt extract, corn steep liquor, dried yeast etc. may be used alone or in combination. 
     Amount of the special required substance used varies according to the kind of the substance, and usually 0.2 g to 200 g, and preferably 3 to 100 g per 1 l medium. A pH value of a medium is adjusted to pH 2 to 12, preferably 6 to 10. Culturing is carried out at temperature of 15° to 80° C., and preferably 25° to 40° C., usually for 1 to 20 days, and preferably 2 to 8 days, under an aerobic condition provided by shaking or aeration/agitation. 
     Finally the present compound is isolated and purified from the culture. Namely, microbial cells are separated from the culture by a conventional means such as centrification or filtration, and the cells are subjected to an extraction with a solvent. As a solvent for the extraction, any substance in which the present compound is soluble can be used. For example, organic solvents such as acetone, chloroform, dichloromethane, hexane, cyclohexane, ethanol, benzene, carbon disulfide, diethyl ether etc. are used, and preferably chloroform, dichloromethane, acetone or ethanol is used. The purification can be carried out by conventional procedures such as absorption, elution, dissolving and the like, alone or preferably in combination. 
     Canthaxanthin produced by a microorganism of the present invention is characterized by that it contains a high ratio of all-trans canthaxanthin; a ratio of all-trans:cis is 95:5 to 98:2. The all-trans canthaxanthin is natural type product, and the present microorganisms are advantageous in that they produce the natural type canthaxanthin. If necessary, the cis type canthaxanthin can be synthesized from the all-trans type canthaxanthin, while the all-trans type canthaxanthin cannot be prepared from the cis type canthaxanthin. 
     The present producer microorganisms are characterized in that they produce canthaxanthin in a wide range of pH value. Namely, they can produce canthaxanthin under an alkaline side pH condition (pH 7 to 10), and therefore suitable for the production of canthaxanthin which is instable under an acidic condition. 
     An infrared absorption spectrum of canthaxanthin produced according to the present invention is shown in FIG. 1, a mass spectrum thereof is shown in FIG. 2, and  13  C nuclear resonance spectrum thereof is shown in FIG. 3. 
     EXAMPLES 
     Now, the present invention is explained in detail by Examples, but the scope of the present invention should not be restricted to the Examples. 
     Example 1 
     First, 10 ml of a medium having a composition shown in Table 1 was put into a test tube having a diameter of mm, and was autoclaved at 121° C. for 15 minutes. 
     
                       TABLE 1______________________________________  Glucose        10 g/L  Polypepton     5 g/L  Yeast extract  5 g/L  KH.sub.2 PO.sub.4                 1 g/L  MgSO.sub.4.7H.sub.2 O                 0.2 g/L  pH 8.0 (adjusted with Na.sub.2 CO.sub.3)______________________________________ 
    
     Said medium was inoculated with a piece of cells of SQH 348 strain (FERM BP-4284), and culturing was carried out at 30° C. for 3 days with shaking to prepare an inoculum culture. The inoculum culture was inoculated into a production medium in an amount of 2% by weight inoculum per 100% by weight of the production medium, and culturing was carried out at 30° C. for 8 days with shaking. 50 ml of the production medium was contained in a 500 ml volume Sakaguchi flask, and the production medium contained 2 g/l of a carbon source selected from the group consisting of glucuse, fructose, ethanol, propanol, butanol, and squalene, as well as the components shown in Table 2. 
     
                       TABLE 2______________________________________Component             Amount______________________________________Yeast extract         0.2 g/LNH.sub.4 NO.sub.3     2.5 g/LKH.sub.2 PO.sub.4     1.5 g/LNa.sub.2 HPO.sub.4    1.5 g/LMgSO.sub.4.7H.sub.2 O 0.5 g/LFeSO.sub.4.7H.sub.2 O 0.01 g/LCaCl.sub.2.2H.sub.2 O 0.01 g/LpH 8.0 (adjusted with Na.sub.2 CO.sub.3)______________________________________ 
    
     The cultured medium was centrifuged to obtain microbial cells, and the cells obtained from 10 ml of the cultured medium was extracted with 10 ml of acetone, and 10 ml of hexane and 10 ml of 0.85% Sodium Chloride were added to the extract, and the mixture was stirred. The upper layer was separated and the solvent was distilled off at 35° C. under a reduced pressure. An amount of canthaxanthin in the pigment extract was analysed by high performance liquid chromatography. A result is shown in Table 3. The method for analysis by high performance liquid chromatography is described in Applied and Environmental Microbiology, 55(12), p 3065 (1989). Namely, a ZORBAX ODS (Du pont, 4.6 mm I.D.×250 mm column) was used, and elution was carried out with a mixed solvent of methanol/acetonitrile/dichloromethane (5:4:1). Canthaxanthin was ditected by absorption at 470 nm, and quantitated from a ratio of the peak areas for a sample tested and a standard canthaxanthin in a high performance liquid chromatography. In addition, a ratio of canthaxanthin among the other pigments was calculated from a ratio of an area of a peak of canthaxanthin and a total area of peaks of other pigments. In addition, a ratio of all-trans: cis of the canthaxanthin isomers was calculated from a ratio of areas of peaks of the isomers. 
     
                       TABLE 3______________________________________                   Ratio of     Canthaxanthin canthaxanthin                              Ratio of     produced      in pigments                              all-trans:Carbon source     (mg/L)        (%)        cis______________________________________Glucose   0.34          98.5       96:4Fructose  0.32          99.0       96:4Ethanol   0.57          98.9       96:4Propanol  0.64          99.0       96:4Butanol   0.48          98.9       96:4Squalene  0.21          99.0       96:4______________________________________ 
    
     Example 2 
     First, 10 ml of a medium having a composition shown in Table 1 was put into a test tube having a diameter of mm, and autoclaved at 121° C. for 15 minutes. The medium was inoculated with a piece of cells of SQH 348 strain (FERM BP-4284), and culturing was carried out at 30° C. for 3 days with shaking to prepare a inoculum culture. The inoculum culture was inoculated into a production medium in an amount of 2% by weight inoculum culture per 100% by weight of the production medium, and culturing was carried out at 30° C. for 5 days with shaking. 50 ml of the production medium was included in a 500 ml volume Sakaguchi flask, and the production medium contained 10 g/l of a carbon source selected from the group consisting of rape oil, olive oil, corn oil, linseed oil and soybean oil as well as the component shown in Table 1. An extraction and quantification of canthaxanthin were carried out as described in Example 1. A result is shown in Table 4. 
     
                       TABLE 4______________________________________       Canthaxanthin producedPlant oil   (mg/L)______________________________________Rape oil    3.9Olive oil   4.5Corn oil    4.8Linseed oil 1.6Soybean oil 5.7______________________________________ 
    
     Example 3 
     First, 10 ml of a medium having a composition shown in Table 1 was put into a test tube having a diameter of 18 mm, and autoclaved at 121° C. for 15 minutes. The medium was inoculated with a piece of cells of SQH 348 strain (FERM BP-4284), and culturing was carried out at 30° C. for 3 days to prepare an inoculum culture. The inoculum culture was inoculated into a production medium in an amount of 2% by weight per 100% by weight of the production medium, and culturing was carried out at 30° C. for 7 days with shaking. 50 ml of the production medium was included in a 500 ml valve Sakaguchi flask. The production medium had a composition shown in Table 2, except that it further contained 10 g/l glucose, 30 g/l yeast extract and 5 ml/l soybean oil, but did not contain NH 4  NO 3 . Extraction and quantification of canthaxanthin were carried out according to the same procedure as described in Example 1. Amount of canthaxanthin produced was 14.1 mg/l. 
     Example 4 
     First, 10 ml of a medium having a composition shown in Table 1 was put into a test tube having a diameter of 18 mm, and autoclaved at 121° C. for 15 minutes. The pH value of the medium was adjusted to pH 10.0 with a sterilized 20% Na 2  CO 3  aqueous solution, inoculated with a piece of cells of SQH 348 strain (FERM BP-4284), and culturing was carried out at 30° C. for 3 days to prepare a inoculum culture. The inoculum culture was inoculated into a production medium in an amount of 2% by weight inoculum culture per 100% by weight of the production culture, and culturing was carried out at 30° C. for 7 days with shaking. The production medium had a composition shown in Table 1, except that pH value was 10.0. Extraction and quantificaiton of canthaxanthin were carried out according to the same procedure as described in Example 1. Amount of canthaxanthin produced was 0.50 mg/l. 
     Example 5 
     First, 200 ml of a medium having a composition shown in Table 1 was put into a one liter Sakaguchi flask, and autoclaved at 121° C. for 15 minutes. The medium was inoculated with SQH 348 strain (FERM BP-4284), and culturing was carried out at 30° C. for 3 days. 2.4 l of this culture was inoculated into 25 l of a production medium in a 50 liter fermenter, having a composition shown in Table 1 except that it further contained 0.3 ml/l of Nissan Disfoam BC-51Y (Nippon Yushi) as a antifoaming agent, and culturing was carried out at 30° C., 300 rpm, 1.0 vvm, for 188 hours. 
     Extraction and quantification of canthaxanthin were carried out according to the procedure as described in Example 1. Amount of canthaxanthin produced was 2.0 mg/l. 21.4 kg of the culture was centrifuged to obtain 298 g of wet cells, which were then homogeneously mixed with 500 ml of chloroform. The mixture was centrifuged to separate and recover the aqueous lower layer. The aqueous layer was extracted twice with chloroform to obtain 1.5 l of an extract containing canthaxanthin. The extract was evaporated off under a reduced pressure, and the concentrated extract containing canthaxanthin was adsorbed on a silica gel column. 
     Canthaxanthin was eluted with a mixed solvent of hexane/ethyl acetate (9:1), and solvent was evaporated off from the elute. The extract was dissolved in a small amount of chloroform, and ethanol was dropwise added to the solution so as to crystallize canthaxanthin. 7.5 mg of crystallized canthaxanthin was obtained. Canthaxanthin thus obtained was identical with authentic canthaxanthin in an infrared absorption spectrum, mass spectrum,  13  C nuclear magnetic resonance spectrum and absorption spectrum. 
     Example 6 
     First, 100 ml of a medium having a composition shown in Table 1 was put into a 500 ml Sakaguchi flask, and autoclaved at 121° C. for 15 minutes. The medium was inoculated with SQH 348 strain (FERM BP-4284), and culturing was carried out at 30° C. for 3 days to prepare an inoculum culture. 100 ml of the culture was inoculated into 1 l of a medium having a composition shown in Table 1 in a 2.5 l fermenter, and culturing was carried out at 30° C., 500 rpm and 1.0 vvm, for 67 hours. Extraction and quantification of canthaxanthin were carried out according to the same procedure as described in Example 1. Amount of canthaxanthin produced was 3.4 mg/l. 
     Example 7 
     First, 100 ml of a medium having a composition shown in Table 1 was put into a 500 ml Sakaguchi flask, and autoclaved at 121° C. for 15 minutes. The medium was inoculated with SQH 348 strain (FERM BP-4284) and culturing was carried out at 30° C. for 3 days with shaking to prepare an inoculum culture. 100 ml of the culture was inoculated into 1 l of a production medium in a 2.5 l fermenter, and culturing was carried out at 30° C., 500 rpm and 1.0 vvm, for 44 hours in an aerobic condition. The production medium has a composition shown in Table 1, except that it contained 10 ml/l of ethanol in place of glucose. Extraction and quantificaiton of canthaxanthin were carried out according to a procedure as described n Example 1. Amount of canthaxanthin produced was 2.4 mg/l. 
     Example 8 
     First 100 ml of a medium having a composition shown in Table 2, except that it contained 20 g/l glucose, 20 g/l yeast extract and 5 g/l of soybean oil but did not contain NH 4  NO 3 , was autoclaved at 121° C. for 15 minutes. The medium was inoculated with SQH 348 strain (FERM BR-4284) and culturing was carried out at 30° C. for 3 days with shaking to prepare an inoculum culture. 100 ml of the culture was inoculated into 1.25 l of a production medium in a 2.5 l fermenter, and culturing was carried out at 30° C., 500 rpm mand 1.0 vvm, for 163 hours in an aerobic condition. The production medium had a composition shown in Table 2, except that it contained 10 g/l glucose, 30 g/l yeast extract, and 5 ml/l soybean oil and does not added NH 4  NO 3 . During the culturing at 50, 66, 74 and 90 hours from the inoculation, 12 g of glucose was added to maintain the presence of glucose. In addition at 42, 66 and 90 hours from the inoculation, 3 g/l of soybean oil was added. Extraction and quantification of canthaxanthin were carried out according to the same procedure as described in Example 1. Amount of canthaxanthin produced was 19.5 mg/l. 
     Example 9 
     First, 100 ml of a medium having a composition shown in Table 2 (except that it contained 20 g/l glucose, 20 g/l yeast extract and 5 g/l soybean oil, but does not contain added NH 4  NO 3 ) was put into a 500 ml Sakaguchi flask, and autoclaved at 121° C. for 15 minutes. The medium was inoculated with SQH 348 strain (FERM BP-4284), and culturing was carried out at 30° C. for 3 days to prepare an inoculum culture. 100 ml of this culture was inoculated into 1.2 l of a production medium in a 2.5 liter fermenter, and culturing was carried out at 30° C., 800 rpm and 1.0 vvm, for 141 hours. The production medium had a composition shown in Table 2, except that it contained 10 ml/l ethanol, 30 g/l yeast extract and 5 ml/l soybean oil, but does not contain added NH 4  NO.sub. 3. Total 19 ml of ethanol was periodically added so as to maintain the presence of ethanol. When foam was formed on the medium, soybean oil was fed with a pump cooperating with a foam detecting electrode. Total amount of soybean oil fed was 43 ml. Extraction and quantification of canthaxanthin were carried out according to the same procedure as described in Example 1. Amount of canthaxanthin produced was 13.6 mg/l. 
     Example 10 
     First, 100 ml of a medium having a composition shown in Table 1 was put into a 500 ml Sakaguchi flask, and autoclaved at 121° C. for 15 minutes. The medium was inoculated with SQH 348 strain (FERM BP-4284) and culturing was carried out at 30° C. for 3 days to prepare an inoculum culture. 100 ml of the culture was inoculated into 1.2 l of a production medium in a 2.5 liter fermenter, and culturing was carried out at 30° C., 500 rpm and 1.0 vvm for 158 hours. The production culture had a composition shown in Table 1 except that it contained 10 ml/l proponol, 40 g/l yeast extract, and 0.01 ml/l Nissan Disfoam BC-51Y, but did not contain added NH 4  NO 3 . Extraction and quantification of canthaxanthin were carried out according to the same procedure as described in Example 1. Amount of canthaxanthin produced was 7.2 mg/l. 
     This application claims priority from Japanese Patent Application Serial No. 5-140874, which is incorporated herein by reference in its entirety.