JPH06237787A - Production of canthaxanthin - Google Patents

Abstract

PURPOSE:To industrially and efficiently produce canthaxanthin at a low cost. CONSTITUTION:This method for producing canthaxanthin comprises culturing a microorganism, belonging to the genus Phaffia and having the ability to produce the canthaxanthin in a culture medium, producing and accumulating the canthaxanthin in the resultant culture and collecting the prepared canthaxanthin from the culture.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing canthaxanthin which is a kind of natural carotenoid. As a natural red pigment, canthaxanthin is useful for improving the color tone of cosmetics, eggs and farmed fish.

[0002]

BACKGROUND ART Canthaxanthin is known to exist in crustaceans and red fish. However, its accumulated amount is very small, and its existence is only confirmed. As a microorganism that produces canthaxanthin, Micrococcus ( Mi
Microorganisms belonging to the genus crococcus [Jonrnal of Bacteriology, 96, 23
4 pages, 1968], Brevibacte
Microorganisms belonging to the genus rium (Bulletin of the Chemical Society of Japan
Chemical Society of Japan) Volume 44, Page 481, 1971
Year], Rhodococcus (Rhodococcus) microorganism belonging to the genus (JP-A-2-138996) are known. However,
There is no known microorganism belonging to the genus Phaffia and capable of producing canthaxanthin.

[0003]

The amount of naturally occurring canthaxanthin accumulated is very small, and it is not efficient to extract and separate it from crustaceans and red fish. An object of the present invention is to provide an industrially inexpensive and efficient method for producing canthaxanthin.

[0004]

According to the present invention, a microorganism belonging to the genus Phaffia and having a canthaxanthin-producing ability is cultivated in a medium, and canthaxanthin is produced and accumulated in the culture. It is possible to provide a method for producing canthaxanthin, which comprises collecting

The present invention will be described in detail below. As the microorganism used in the present invention, a wild strain belonging to the genus Phaffia, as long as it has a canthaxanthin-producing ability,
Any mutant strain that has been mutated by a normal mutagenesis treatment can also be used. The wild-type strain, Phaffia rhodozyma (Phaffia rhodozyma) ATCC24202 (below, ATCC
24202 shares. ) Can be given.

Examples of the mutant strains include microorganisms belonging to the genus Phaffia, resistant to citronellol or primaquine, and capable of producing canthaxanthin. In the present specification, “having resistance to a drug” means that it can grow even in the presence of a drug (herein, referred to as citronellol or primaquine) at a concentration at which the parent strain inhibits growth.

[0007] Such a mutant strain is a canthaxanthin-producing bacterium belonging to the genus Phaffia (for example, ATCC24202 strain).
By applying ordinary mutagenesis means (for example, ultraviolet irradiation, treatment with a mutation-inducing agent such as ethyl methanesulfonate or N-methyl-N′-nitro-N-nitrosoguanidine) to give resistance to citronellol or primaquine. Obtainable.

Specifically, Phaffia rhodozyma H-
8264 (hereinafter referred to as H-8264 strain) and Phaffia rhodozyma H-8434 (hereinafter referred to as H-8434 strain). In the present specification, the H-8264 strain has a citronellol content of 220 μg / ml, which is not viable in the parent strain,
The H-8434 strain can grow to 3.9 g / l of primaquine.

The specific method for obtaining the mutant strain will be described below. ATCC24202 strain ethyl methanesulfonate 0.02
After treatment with ml / ml at 22 ℃ for 60 minutes, the parent strain ATCC
24202 Minimal agar plate containing 20 μg / ml of citronellol that inhibits growth (glucose 20 g / ml
l, Yeast Nitrogen Base (7 g / l, agar 20 g / l, manufactured by Difco). After culturing at 22 ° C. for 7 to 10 days, among the mutant strains that grew, a strain having a higher canthaxanthin-producing ability than the parent strain in the liquid culture test in a 250 ml Erlenmeyer flask was selected. H-8264 strain was obtained as a particularly excellent strain.

Further, H-8434 strain was obtained as a strain having an excellent canthaxanthin producing ability, in the same manner as described above except that primaquine (3.9 g / l) was used instead of citronellol. H-8264 is effective as of May 18, 1991, H-8434 is effective as of March 1, 1992.
Based on the Budapest Treaty on 3rd, the Institute of Biotechnology, Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology No. 3404 (FERM B
(P-3404), and has been deposited as Micro Engineering Research Article No. 3800 (FERM BP-3800).

The H-8264 strain and the H-8434 strain are described as astaxanthin-producing bacteria in the international application JP92 / 00722 filed on June 4, 1992 by the present applicant. The resistance of these mutants was confirmed as follows. When the mutant strains (H-8264 and H-8434 strains) and the parent strain (ATCC24202 strain) were applied to the minimal agar plate medium containing the additives at the concentrations shown in Tables 1 and 2, and cultured at 22 ° C. Was examined. The results are shown in Tables 1 and 2.

[0012]

[Table 1]

[0013]

[Table 2]

Cultivation of the microorganism used in the present invention may be carried out aerobically in a medium containing a carbon source, a nitrogen source, an inorganic salt, a growth factor and the like according to a usual method for culturing a microorganism. Examples of the carbon source include glucose, fructose, sucrose, molasses and the like. Examples of nitrogen sources include ammonia, ammonium sulfate, peptone, yeast extract, corn steep liquor and the like. Further, examples of the inorganic salt include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, calcium chloride and the like.
Examples of growth factors include vitamins, amino acids, nucleic acid-related substances and the like.

Culturing is carried out at a temperature of 15 to 35 ° C., preferably 20 to 35 ° C.
It is carried out at 25 ° C and pH of 3 to 8, preferably 4 to 6, and is usually completed in 2 to 7 days. The pH of the medium is adjusted with calcium carbonate, an inorganic or organic acid, an alkaline solution, ammonia, a pH buffer solution (eg potassium hydrogen phthalate) or the like. After completion of the culture, the cells are separated from the culture broth, crushed, and then canthaxanthin is extracted from the crushed cells with a solvent and treated by silica gel chromatography.

Examples of the present invention will be shown below.

[0017]

【Example】

Example 1 ATCC24202 strain, H-8264 strain, and H-8434 strain were inoculated into 8 ml of a seed medium having the following composition in a test tube, and then cultured at 22 ° C for 3 days. Seed medium composition: glucose 10 g / l, yeast extract 3
g / l, peptone 5 g / l, meat extract 3 g / l, potassium hydrogen phthalate 20.4 g / l (pH 5.0, KO
(Adjust by H and sterilize under pressure with steam at 120 ° C for 20 minutes.) 3 ml of the obtained seed culture was inoculated into 30 ml of a production medium having the following composition in a 250 ml Erlenmeyer flask at 22 ° C.
The cells were cultured for 4 days. Production medium composition: glucose 30 g / l, ammonium sulfate 2 g / l, yeast extract 2 g / l, KH ₂ PO _{4
1g / l, MgSO 4 · 7H} 2 O 0.5g / l, CaC
_{l 2 · 2H 2 O 0.1g /} l, potassium hydrogen phthalate
20.4 g / l (pH 5.0, adjusted with KOH, 2 at 120 ° C)
As a result, the production amount of canthaxanthin in the culture solution was 0.02 mg / l, 0.9 mg / l and 1.1 mg / l, respectively.

Example 2 Each of the H-8264 strain and the H-8434 strain was placed in a 2 liter Erlenmeyer flask and the seed medium 300 shown in Example 1 was used.
After inoculating the cells in ml, the cells were cultured at 22 ° C for 3 days. 300 ml of the obtained seed culture solution was inoculated into 3 liters of a medium obtained by removing potassium hydrogen phthalate from the medium shown in Example 1 in a 5 liter culture tank, and then cultured at 22 ° C. for 48 hours (rotation speed 60
(0 rpm, aeration rate 3 l / min). The pH during the culture was adjusted to 5 with aqueous ammonia. As a result, the amount of canthaxanthin produced in the culture was 1.9 mg / l and 2.5 mg / l, respectively.
It was l. After the culture was completed, the cells of the culture solution of the H-8434 strain were collected by centrifugation, and the cells were disrupted with a glass bead cell homogenizer. Canthaxanthin was extracted from the disrupted cells using a mixed solution of chloroform and methanol. The extract was concentrated under reduced pressure, and the residue was subjected to silica gel chromatography (elution solvent hexane: ethyl acetate = 10:
Purification according to 1) yielded 4.5 mg canthaxanthin. The canthaxanthin obtained in Examples 1 and 2 was confirmed by the fact that the absorption spectrum in the ultraviolet-visible region was in agreement with the absorption spectrum of a standard canthaxanthin (manufactured by Funakoshi).

[0019]

Industrial Applicability According to the present invention, canthaxanthin can be industrially produced at low cost and efficiently.

Claims (1)

[Claims] 1. A method of culturing in a medium a microorganism belonging to the genus Phaffia and capable of producing canthaxanthin, producing and accumulating canthaxanthin in the culture, and collecting canthaxanthin from the culture. And a method for producing canthaxanthin.