JPH07265088A - Method for producing carotene and xanthophyll compounds - Google Patents

Abstract

PURPOSE:To easily obtain beta-carotene or a xanthophyll compound in high yields, the beta-carotene or the xanthophyll compound having been produced by organic synthesis methods or extracted and purified from yeasts, green algae, blue-green algae, etc. CONSTITUTION:A method for producing beta-carotene or a xanthophyll compound (excluding astaxanthin) comprises culturing a microorganism belonging to the genus Agrobacterium and having a beta-carotene or xanthophyll-producing ability in a medium and subsequently collecting the beta-carotene or xanthophyll from the culture product.

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 β-carotene and xanthophylls using microorganisms, which are useful as color improvers for ornamental fish and cultured fish, antioxidants and the like.

[0002]

2. Description of the Related Art Conventionally, in order to enhance the commercial value of cultured fish and shellfish such as shrimp and red sea bream and appreciation fish such as goldfish and Nishikigoi, the color tone of the fish has been increased by people involved in the aquaculture industry and the feed manufacturing industry. Research on improvement has been advanced.
It has been reported that the color tone of these seafood is derived from β-carotene and xanthophylls.

On the other hand, as methods for producing β-carotene and xanthophylls, organic synthesis, yeast, and extraction from cultures of green algae and cyanobacteria have been reported. However, in the case of organic synthesis, there are problems in complexity of synthesis reaction and removal of reaction by-products, and in yeast and green algae, culture conditions, extraction conditions, chlorophyll and lipid removal.

[0004]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a method for easily producing β-carotene and xanthophylls, which has been a labor intensive work for obtaining the same.

[0005]

[Means for Solving the Problems] The inventors of the present invention have conducted diligent research on microorganisms that produce β-carotene or xanthophylls. As a result, microorganisms belonging to the genus Agrobacterium produce β-carotene and xanthophylls. This has led to the completion of the present invention. That is, the present invention is a β-carotene characterized by culturing a microorganism belonging to the genus Agrobacterium having the ability to produce β-carotene or xanthophylls in a medium, and collecting β-carotene or xanthophylls from the culture. Alternatively, it is a method for producing xanthophylls.

However, within the scope of the present invention, Japanese Patent Application No. 5-33
The method for producing astaxanthin using a bacterium of the genus Agrobacterium described in 3101 is not included. Less than,
The present invention will be described in detail. In the present invention, the xanthophylls refer to a group of carotenoids containing oxygen in the form of a hydroxyl group, a carbonyl group, an epoxide and the like, and examples thereof include echinenone, canthaxanthin, phenicoxanquine and 3
-Hydroxyequinone, 3'-hydroxyequinone, β-cryptoxanthin, zeaxanthin, 4-
It refers to ketozeaxanthin, astaxanthin and the like.

As a typical strain used in the present invention, N-81106 strain isolated from seawater in the waters of the Kerama Islands, Okinawa can be mentioned. The mycological properties of this N-81106 strain are shown below. (1) Gram stain Negative (2) Morphology Shape / size of fungus: Aggressive, 0.9 μm × 1.2 μm Motility: Yes Flagella: Peripheral hair (3) Cell pigment: Orange (4) Physiological properties Oxetase : Positive Glucose degrading: Positive Gelatin degrading: Negative DNA degrading: Negative (5) OF test Oxidation (6) G + C content 67.4% (7) Ubiquinone type Quinone Q-10 Shimizu et al.'S method [Gen Kadota, Nobuo Taga: Marine Microbial Research, Academic Publishing Center
pp.229 (1985)]. The morphological examination was performed by using an optical microscope, and particularly by using a scanning electron microscope for the morphology of the spore surface.

[0008] Regarding the above-mentioned mycological properties, N. Krieg and J. J. Holt (J.
G. Holt) edited by Bergey's Manual of System Bacteriology
Search based on (atic Bacteriology), N-811
It is appropriate to assign the 06 strain to Agrobacterium aurantiacus, and it was identified as Agrobacterium aurantiacus. Then, this N-81106 strain was designated as Agrobacterium aurantiacus sp. Nov N-81106.
Strain (Agrobacterium aurantiacus sp. Nov N-81106) FERM P-14023
No. (Deposit date: December 16, 1993).

As the strain used in the present invention, any strain can be used as long as it belongs to the genus Agrobacterium and has the ability to produce β-carotene or xanthophylls. Further, a method for artificially mutating a microorganism belonging to the genus Agrobacterium, for example, ultraviolet irradiation, X-ray irradiation, treatment with a mutagen or a naturally occurring mutant strain, or a genetically engineered cell fusion mutant strain, β-carotene or xanthophyll Any of these can be used in the present invention.

In the production method of the present invention, the above-mentioned microorganism is cultivated in a medium generally used for culturing microorganisms, and β-carotene or xanthophylls produced are collected by a conventional method. For culturing microorganisms belonging to the genus Agrobacterium, ordinary culture methods can be used. As a medium, assimilable carbon source, nitrogen source, minerals and necessary growth,
If it is a medium containing a production promoting substance in a suitable amount, a synthetic medium,
Any natural medium can be used. As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol, molasses, etc. may be used alone or in combination. Further, depending on the assimilation ability of the bacterium, hydrocarbons, alcohols, organic acids and the like are also used. As the nitrogen source, ammonium chloride, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steve liquor, soybean powder, casamino acid, etc. may be used alone or in combination. In addition, inorganic salts such as table salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate, copper sulfate and seawater or natural seawater. Inorganic salts present therein are added as needed. In addition, a metabolic map that is considered to be a precursor in β-carotene biosynthesis (edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin, 1980) 123-
The precursor described on page 125 and trace components that promote the production of β-carotene or xanthophylls can be appropriately added.

As a culture method, a liquid culture method, particularly a deep agitation culture method is most suitable. Culture temperature is 16-4
The temperature is preferably 0 ° C., particularly 20 to 30 ° C., and the pH of the medium during culture is preferably maintained at 4 to 10, particularly 6 to 8 by adding aqueous ammonia or ammonium carbonate solution. When liquid culture is generally performed for 1 to 7 days, β-carotene or xanthophylls are produced and accumulated in the cells. The culture is stopped when the maximum production in the culture is reached.

Isolation and purification of β-carotene or xanthophylls from the culture is carried out according to a conventional method for isolating and purifying microbial metabolites from the culture. For example, the culture is separated into a culture filtrate and bacterial cells by filtration or centrifugation, and the bacterial cells are treated with an organic solvent (for example, hexane, benzene, chloroform, acetone, ether, ethyl acetate,
Extract with ethanol, methanol and the above hydrous substances. When β-carotene or xanthophylls are present in the culture filtrate, extraction with an organic solvent such as ethyl acetate or diethyl ether or adsorption type resin (Amber
After adsorption to liteXAD-2, etc., it is possible to obtain the extract in a suitable organic solvent. Then, the extract is concentrated, and β-carotene or xanthophylls are separated and purified by liquid chromatography using silica gel, chemically bonded silica gel, gel filtration agent or the like. The trend of β-carotene or xanthophylls during the culture and purification operations can be traced by thin layer chromatography using the yellow, orange and red colors characteristic of β-carotene and xanthophylls as a guide.

[0013]

EXAMPLES The present invention will be described below with reference to examples, but it goes without saying that the present invention is not limited thereto. [Example 1] As an inoculum, Agrobacterium aurantiacus sp. Nov N-81106 strain (FERM P-14023) was used. For pre-culture and main culture, "MARINE BROT manufactured by DIFCO
A medium prepared by "H" according to the method described in the manual was appropriately dispensed into a culture tank and sterilized before use. As a pre-culture, one platinum loop of the strain was inoculated into 150 ml of medium in a 500 ml Erlenmeyer flask and shaken at 25 ° C. for 48 hours (100 rpm).
Cultured. The seed culture obtained in this way was added to 10 L
1. In 6 L of medium having the same composition as the above composition in the volume culture tank.
The cells were transferred at a rate of 5% v / v, and main culture was carried out at 25 ° C by an aeration and stirring method (rotation speed 100 rpm, aeration amount 500 ml / min). During the culture, the pH of the medium was not particularly controlled, and the culture was performed for 96 hours.

Centrifugation of the culture solution (10,000 rpm)
The bacterial cell fraction was obtained by adding 200 ml of acetone and stirring, the precipitate was filtered off, and the extract was concentrated to 20 ml.
200m each of ethyl acetate and distilled water was added to the obtained concentrated liquid.
l was added, and the ethyl acetate fraction was obtained with a separating funnel and concentrated.
Dried up. The dried solid was further fractionated with benzene: ethyl acetate = 85: 15 using a silica gel column (Silica gel 80, manufactured by Nacalai Tesque, Inc.).

Fraction 1 (0.6 mg) was obtained.
R, visible light absorption spectrometry, and mass spectrometry were in agreement with known β-carotene. Fraction 2 yielded 0.4 mg, hydrogen nucleus N
Consistent with known β-cryptoxanthin in MR, visible light absorption spectrometry, and mass spectrometry. Fraction 3 was obtained in an amount of 0.5 mg and was consistent with known 3-hydroxyequinone in hydrogen nucleus NMR, visible light absorption spectrometry and mass spectrometry.

Fraction 4 was obtained in an amount of 0.4 mg.
In R, visible light absorption spectrometry, and mass spectrometry, it was in agreement with the known 3'-hydroxyequinone. Fraction 5 is 0.7 mg
The obtained product was in agreement with known astaxanthin in hydrogen nucleus NMR, visible light absorption spectrometry, and mass spectrometry. Fraction 6 is 2.
1 mg was obtained, which was consistent with known 4-ketozeaxanthin in hydrogen nucleus NMR, visible light absorption spectrometry, and mass spectrometry.

Fraction 7 was obtained in an amount of 0.5 mg.
R, visible light absorption spectrometry, and mass spectrometry agreed with known zeaxanthin. [Example 2] Agrobacterium aurantiacus sp. Nov N-81106 strain (FERM P-14023) was used as an inoculum. For pre-culture and main culture, "MARINE BROT manufactured by DIFCO
A medium prepared by "H" according to the method described in the manual was appropriately dispensed into a culture tank and sterilized before use. One platinum loop of the strain was inoculated into 300 ml of medium in a 500 ml Erlenmeyer flask, the pH of the medium was not particularly controlled, and 72 mL at 25 ° C. on a 9 L scale.
Culture was performed with shaking (100 rpm).

Centrifuge the culture (10,000 rpm)
The bacterial cell fraction was obtained by adding 200 ml of acetone and stirring, the precipitate was filtered off, and the extract was concentrated to 20 ml.
200m each of ethyl acetate and distilled water was added to the obtained concentrated liquid.
l was added, and the ethyl acetate fraction was obtained with a separating funnel and concentrated.
Dried up. The dried solid was further fractionated with benzene: ethyl acetate = 85: 15 using a silica gel column (Silica gel 80, manufactured by Nacalai Tesque, Inc.).

Fraction 1 was obtained in an amount of 0.9 mg.
R, visible light absorption spectrometry, and mass spectrometry were in agreement with known β-carotene. Fraction 2 yielded 1.1 mg, hydrogen nucleus N
Consistent with known echinenone in MR, visible light absorption spectrometry, and mass spectrometry. Fraction 3 was obtained at 0.7 mg, hydrogen nucleus N
It was in agreement with known canthaxanthin in MR, visible light absorption spectrometry, and mass spectrometry.

Fraction 4 was obtained in an amount of 0.3 mg.
R, visible light absorption spectrometry, and mass spectrometry were in agreement with known phenicoxane.

[0021]

According to the present invention, conventional organic synthesis and
The β-carotene and xanthophylls that have been extracted and purified from yeast, green algae, cyanobacteria, etc. can be easily obtained in high yield. Further, by-products in the synthetic reaction, lipids such as yeast, green algae and cyanobacteria, chlorophyll, etc. are not mixed, and the purification process is greatly improved.

Claims (1)

[Claims] 1. A method of culturing a microorganism belonging to the genus Agrobacterium having the ability to produce β-carotene or xanthophylls in a medium and collecting β-carotene or xanthophylls (excluding astaxanthin) from the culture. A method for producing a characteristic β-carotene or xanthophylls (excluding astaxanthin).