JPH06165683A - Production of astaxanthin - Google Patents

Abstract

PURPOSE:To obtain astaxantin useful as an agent for the coloration of cultured fish, an antioxidant, a colorant, an anti-inflammatory agent, etc., by culturing an astaxanthin-producing bacterial strain belonging to the genus Flavobacterium, etc., in a medium and separating the compound from the cultured product. CONSTITUTION:Astaxanthin useful for the coloration of cultured fish such as shrimp and red sea bream and utilizable as food additives such as antioxidant and colorant or as pharmaceuticals such as anti-inflammatory agent can be produced by culturing a bacterial strain belonging to the genus Flavobacterium, Alcaligenes, Pseudomonas, Alteromonas, Hyphomonas or Caryophanon and capable of producing astaxanthin [e.g. Flavobacterium sp. N-81106 (FERM P-12782)] in a medium and separating the compound from the cultured product.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is useful for frying cultivated seafood such as shrimp and red sea bream, and is also used as a food as an antioxidant and a coloring agent, and as a medicine such as an anti-inflammatory agent. The present invention relates to a method for producing astaxanthin using a microorganism.

[0002]

BACKGROUND OF THE INVENTION Conventionally, astaxanthin is shrimp,
Widely used for the purpose of deep-fried farmed seafood such as red sea bream, as its production method, extracts from crustaceans such as Antarctic krill, cultures such as yeast a kind of huafia, green algae such as hematococcus. Production methods from cultures, organic synthesis methods and the like are known. However, when using crustaceans such as Antarctic krill, a great deal of labor is required for their collection, extraction, and separation from contaminants such as lipids. Further, in a culture product such as yeast one kind of sphere, a fatty acid ester is present as a by-product, which causes a problem in production. In a culture of hematococcus, a kind of green algae, it is necessary to supply light that is indispensable for photosynthesis at the time of cultivation, and it is necessary to provide a site condition for collecting sunlight and a culture device for supplying artificial light. Not only is equipment required, but separation from mixed chlorophyll is difficult and the purification process is complicated. Further, in the organic synthesis method, considering that astaxanthin is used as a feed for fish and shellfish or as a food additive, there remains a problem with by-products generated during the reaction. From the above, development of a simple production method of astaxanthin is desired.

[0003]

SUMMARY OF THE INVENTION It is an object of the present invention to provide natural astaxanthin more simply than conventional methods for producing yeast or green algae.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies on astaxanthin-producing microorganisms, and found that bacteria belonging to a specific genus produce astaxanthin, and completed the present invention. That is, the method for producing astaxanthin of the present invention is a flavobacterium (Fla
vobacterium), Alcaligenes, Pseudomonas, Alteromonas
A bacterium belonging to the genus omonas, the genus Hyphomonas or the genus Caryophanon and having the ability to produce astaxanthin is cultured in a medium, and astaxanthin is collected from the culture.

The present invention will be described in detail below. As the astaxanthin-producing strain, any strain can be used as long as it belongs to the above genus and has astaxanthin-producing ability. In addition, artificial mutation methods of these strains, for example, ultraviolet irradiation, X-ray irradiation, mutagen treatment, etc., or naturally occurring mutant strains, genetic manipulation,
Any mutant strain produced by cell fusion can be used in the present invention as long as it produces astaxanthin.

[0006] A representative strain is Flavobacterium sp. As a bacterium of the genus Flavobacterium.
N-81106 strain, Alcaligenes sp.PC-2 strain, Pseudomonas (Pseudom)
onas) bacterium, Pseudomonas sp. PC-3 strain, Alteromonas sp.
SD-402 strain, Hyp as Hypomonas sp.
The homonas sp. PC-4 strain and the Caryophanon sp. PC-5 strain can be mentioned as a bacterium belonging to the genus Caryophanon.

The mycological properties of Flavobacterium sp. N-81106 strain are shown below. (1) Morphology Shape / size of the bacterium: Rod-shaped, 0.9 μm × 1.2 μm Motility: Yes Flagella: With perilimes Cell polymorphism: No Spore formation: No Gram stain: Negative (2) Growth status in each medium Broth agar plating: non-diffusive, shiny, orange round colonies are formed. Broth agar slant culture: non-diffusive, shiny, grows in an orange band. Broth liquid culture: It grows uniformly on the whole medium and shows an orange color. Meat broth gelatin stab culture: Grows on the surface around the stab hole.

(3) Physiological properties Nitrate reduction: positive Denitrification reaction: negative Indole formation: negative Citric acid utilization: negative Pigment formation: lipophilic red-orange pigment urease activity: negative oxidase activity: positive catalase activity : Positive β-glucosidase activity (degradability of esculin): Positive β-galactosidase activity: Positive Growth range: pH 5-9, Temperature 10-40 ° C Attitude toward oxygen: Aerobic seawater resistance: Positive OF test: Oxidation of saccharides Assimilation ability: Positive: D-glucose, D-mannose, D-galactose, D-fructose, lactose, maltose, sucrose, glycogen, N-acetyl-D-glucosamine Negative: L-arabinose, D-mannitol, inositol, L -Rhamnose, D-sorbitol Assimilation ability of organic acids: positive: lactate negative: citrate, phosphorus , Gluconate, caprate, succinate, assimilating positive adipate other organics: inosine, uridine, glucose-1-phosphoric acid,
Glucose-6-phosphate negative: gelatin, L-arginine, DNA, casein

The mycological properties of Alcaligenes sp. PC-2 strain are shown below. (1) Morphology Shape / size of the bacterium: Rod-shaped, 0.4 μm × 1.7 μm Motility: Yes Flagella: With perilimes Cell polymorphism: None Spore formation: None Gram stain: Negative (2) Growth status in each medium Broth agar plating: non-diffusive, shiny, orange round colonies are formed. Broth agar slant culture: non-diffusive, shiny, grows in an orange band. Broth liquid culture: It grows uniformly on the whole medium and shows an orange color. Meat broth gelatin stab culture: Grows on the surface around the stab hole.

(3) Physiological properties Nitrate reduction: negative Denitrification reaction: negative Indole formation: negative Citric acid utilization: negative Pigment formation: lipophilic red-orange pigment urease activity: negative oxidase activity: positive catalase activity : Positive β-glucosidase activity (degradability of esculin): Positive β-galactosidase activity: Positive Growth range: pH 5-9, temperature 10-40 ° C Attitude toward oxygen: Aerobic seawater resistance: Positive OF test: Oxidation, fermentation None of these are shown. Assimilation ability of saccharides: Positive: D-glucose, maltose Negative: L-arabinose, D-mannose, D-mannitol, N-acetyl-D-glucosamine Assimilation ability of organic acids: Positive: Malate negative: Gluconate, Caprate, adipate, citrate, phenyl acetate Decomposition and assimilation ability of other organic substances Negative: Gelatin, L-arginine

The mycological properties of Pseudomonas sp. PC-3 strain are shown below. (1) Morphology Bacterial shape and size: Rod-shaped, 0.6 μm × 1.3 μm Motility: Yes Flagella: With polar hair Polymorphism of cells: None Spore formation: None Gram stain: Negative (2) Growth status in each medium Meat broth agar plating: hardly grows. Broth agar slope culture: Almost no growth. Broth liquid culture: hardly grows. Broth gelatin stab culture: hardly grows. Marine agar (Difco) plate culture: non-diffusive, glossy, orange round colonies are formed. Marine agar (manufactured by Difco) Slope culture: grows in a non-diffusive, glossy, orange band. Marine broth (manufactured by Difco) culture: Grow uniformly throughout the medium and show an orange color.

(3) Physiological properties Nitrate reduction: negative Denitrification reaction: negative Indole formation: negative Citric acid utilization: negative Pigment formation: lipophilic red-orange pigment urease activity: negative oxidase activity: positive catalase activity : Positive β-glucosidase activity (degradability of esculin): Positive β-galactosidase activity: Negative Growth range: pH 5-9, Temperature 10-40 ° C Attitude toward oxygen: Aerobic seawater resistance: Positive OF test: Oxidation, fermentation None of these are shown. Assimilation ability of sugars: Positive: D-glucose, maltose Negative: L-arabinose, D-mannose, D-mannitol, N-acetyl-D-glucosamine Assimilation ability of organic acids: Negative: gluconate, caprate, adipine Acid, malate, citrate, phenyl acetate Decomposition and assimilation ability of other organic substances Negative: Gelatin, L-arginine

The mycological properties of the Alteromonas sp. SD-402 strain are shown below. (1) Morphology Shape and size of bacteria: Rod-shaped, 0.4 μm × 1.6 μm Motility: Yes Flagella: With perilimes Cell polymorphism: None Spore formation: None Gram stain: Negative (2) Growth status in each medium Broth agar plating: non-diffusive, shiny, orange round colonies are formed. Broth agar slant culture: non-diffusive, shiny, grows in an orange band. Broth liquid culture: It grows uniformly on the whole medium and shows an orange color. Meat broth gelatin stab culture: Gelatin is liquefied and grows on the surface around the puncture hole.

(3) Physiological properties Nitrate reduction: Negative denitrification: Negative Indole formation: Negative Citric acid utilization: Negative Dye formation: Lipid-soluble red-orange pigment Urease activity: Negative Oxidase activity: Negative Catalase activity : Negative β-glucosidase activity (degradability of esculin): Positive β-galactosidase activity: Negative Growth range: pH 5-9, Temperature 10-40 ° C Attitude toward oxygen: Aerobic seawater resistance: Positive OF test: Oxidation, fermentation None of these are shown. Assimilation ability of saccharides: Positive: D-glucose, maltose Negative: L-arabinose, D-mannose, D-mannitol, N-acetyl-D-glucosamine Assimilation ability of organic acids: Positive: Malate negative: Gluconate, Caprate, adipate, citrate, phenyl acetate Decomposition and assimilation ability of other organic substances Positive: Gelatin Negative: L-arginine

The mycological properties of the Hyphomonas sp. PC-4 strain are shown below. (1) Morphology Shape / size of bacterium: sphere to short rod, 0.4 μm Motility: yes Flagella: with attached stalk Cell polymorphism: none Spore formation: none Gram stain: negative

(2) Growth status in each medium Meat agar plate culture: Almost no growth. Broth agar slope culture: Almost no growth. Broth liquid culture: hardly grows. Broth gelatin stab culture: hardly grows. Marine agar (manufactured by Difco): Non-diffusive, glossy, orange-colored amorphous colony is formed. Marine agar (manufactured by Difco) Slope culture: grows in a non-diffusive, glossy, orange band. Marine broth (manufactured by Difco) culture: Grow uniformly throughout the medium and show an orange color.

(3) Physiological properties Nitrate reduction: Negative denitrification: Negative Indole formation: Negative Citric acid utilization: Negative Pigment formation: Lipid-soluble red-orange pigment Urease activity: Negative Oxidase activity: Positive Catalase activity : Positive β-glucosidase activity (escrine degrading): Negative β-Galactosidase activity: Negative Growth range: pH 5-9, Temperature 10-40 ° C Attitude toward oxygen: Aerobic seawater tolerance: Positive OF test: Oxidation, fermentation None of these are shown. Assimilation ability of sugars: Negative: D-glucose, maltose, L-arabinose, D
-Mannose, D-mannitol, N-acetyl-D-
Glucosamine Anabolic ability of organic acids: Negative: Gluconate, caprate, adipate, malate, citrate, phenyl acetate Decomposition and assimilation of other organic substances Negative: Gelatin, L-arginine

The mycological properties of Caryophanon sp. PC-5 strain are shown below. (1) Morphology Shape / size of the bacterium: Rod-shaped, 0.5 μm × 2.5 μm Motility: Yes Flagella: With lateral hairs Cell polymorphism: None Spore formation: None Gram stain: Positive (2) Growth status in each medium Broth agar plating: non-diffusive, shiny, orange round colonies are formed. Broth agar slant culture: non-diffusive, shiny, grows in an orange band. Broth liquid culture: It grows uniformly on the whole medium and shows an orange color. Meat broth gelatin stab culture: Grows on the surface around the stab hole.

(3) Physiological properties Nitrate reduction: negative Denitrification reaction: negative Indole formation: negative Citric acid utilization: negative Pigment formation: lipophilic red-orange pigment urease activity: negative oxidase activity: positive catalase activity : Positive β-glucosidase activity (degradability of esculin): Positive β-galactosidase activity: Negative Growth range: pH 5-9, Temperature 10-40 ° C Attitude toward oxygen: Aerobic seawater resistance: Positive OF test: Oxidation, fermentation None of these are shown. Assimilation ability of saccharides: Positive: D-glucose, maltose Negative: L-arabinose, D-mannose, D-mannitol, N-acetyl-D-glucosamine Assimilation ability of organic acids: Positive: Malate negative: Gluconate, Caprate, adipate, citrate, phenylacetate Degradation and assimilation ability of other organic substances Negative: Gelatin, L-arginine The above-mentioned mycological properties were determined by the method of Shimizu et al. (Gen Kadota, Nobuo Taga). Ed., Marine Microbial Research, published by Japan Society for Publishing, 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.

Regarding the above-mentioned mycological properties, NR Krieg and J. G. Holt (J.
G. Holt), Bergey's Manual of Systemic Bacteriology
tematic Bacteriology)
It was appropriate to assign the -81106 strain to the genus Flavobacterium, but it was difficult to identify the species, and the N-81106 strain was assigned to Flavobacterium sp. N-81106.
(Microorganism Research Institute No. 12782 (FERM P-12782)) was deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology (Original deposit date: February 19, 1992).

As a result of conducting a similar search for the PC-2 strain, it was appropriate to assign the PC-2 strain to the genus Alcaligenes, but it is difficult to identify the species and PC-2 The strain was deposited as Alcaligenes sp. PC-2 (FERM P-13488) at the Institute of Biotechnology, Institute of Industrial Science and Technology (Original Deposit Date: March 2, 1993). As a result of conducting a similar search for the PC-3 strain,
It was appropriate to assign the PC-3 strain to the genus Pseudomonas, but it is difficult to identify the species,
The PC-3 strain was deposited as Pseudomonas sp. PC-3 (FERM P-13487) at the Institute of Biotechnology, Institute of Industrial Science and Technology (original deposit date: March 2, 1993).

The SD-402 strain was also searched in the same manner, resulting in SD-4.
Although it was appropriate to assign the 02 strain to the genus Alteromonas, it was difficult to identify the species and SD
-402 strain was deposited as Alteromonas sp. SD-402 (FERM P-13489) at the Institute of Biotechnology, Institute of Industrial Science and Technology (Original deposit date: March 2, 1993). As a result of conducting a similar search for the PC-4 strain, it was appropriate to assign the PC-4 strain to the genus Hyphomonas, but it was difficult to identify the species, and the PC-4 strain was identified as Hyphomonas. sp. PC-4 (FERM P-13486) was deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology (Original deposit date: March 2, 1993).

As a result of conducting a similar search for the PC-5 strain, it was appropriate to assign the PC-5 strain to the genus Caryophanon, but it is difficult to identify the species, and PC-5 The strain was deposited as Caryophanon sp. PC-5 (FERM P-13485) at the Institute of Biotechnology, Institute of Industrial Science and Technology (Original Deposit Date: March 2, 1993). In the production method of the present invention, the microorganism is cultured in a medium generally used for culturing the microorganism,
The astaxanthin produced is collected by a conventional method.

First, the culture method will be described. A usual culture method can be used for culturing the microorganism. As the medium, any synthetic medium or natural medium can be used as long as it is a medium containing an assimilable carbon source, a nitrogen source, an inorganic substance and necessary growth and production promoting substances. 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 a nitrogen source, ammonium chloride, ammonium nitrate,
Sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean powder, casamino acid, etc. may be used alone or in combination. In addition, inorganic salts such as sodium chloride, 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. Further, a metabolic map that is considered to be a precursor for astaxanthin biosynthesis (edited by the Japanese Biochemical Society, published by Tokyo Kagaku Dojin, 1980) 123-125 carotene or its precursor, echinenone, canthaxanthin, zeaxanthin, idoxanthin , 4-ketozeaxanthin, beta-carotenetriol, and other xanthophylls can be added. Furthermore, a trace amount component that promotes the growth of the used bacterium and the production of astaxanthin can be appropriately added.

As a culture method, a liquid culture method, particularly a deep agitation culture method is most suitable. Culture temperature is 16-40 ℃, especially
20 to 30 ° C. is suitable, and the pH of the culture medium during culture is preferably maintained at 4 to 10, particularly 6 to 8 by adding aqueous ammonia or ammonium carbonate solution. Usually 1 to 1 in liquid culture
When cultured for 7 days, the target substance, astaxanthin, is produced and accumulated in the cells. The culture is stopped when the maximum production in the culture is reached.

Isolation and purification of astaxanthin 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 cells by filtration or centrifugation, and the cells are treated with an organic solvent (for example, hexane, benzene, chloroform,
Acetone, ether, ethyl acetate, ethanol or a water-containing product of the above organic solvents). When astaxanthin is present in the culture filtrate, extraction with an organic solvent such as ethyl acetate or diethyl ether, or adsorption on an adsorption resin (Amberlite XAD-2, etc.), followed by extraction with an appropriate organic solvent It is possible to obtain Then, after concentrating the extract, astaxanthin is separated and purified by liquid chromatography using silica gel, chemically bonded silica gel, gel filtration agent and the like. The trend of astaxanthin during the culture and purification operations can be traced using the red color of astaxanthin by thin layer chromatography as a guide.

[0027]

The present invention will be described in more detail with reference to the following examples, but it goes without saying that the scope of the present invention is not limited to these examples. (Example 1) Flavobacterium sp. N-81106 as an inoculum
(Microtechnology Research Institute, No. 12782 (FERM P-12782)) was used. For pre-culture and main culture, a medium prepared by "MARINE BROTH" manufactured by DIFCO Co., Ltd. according to the method described in the manual was appropriately dispensed into a culture tank and used after sterilization. As a preculture, one platinum loop of the strain was inoculated into 150 ml of medium in a 500 ml Erlenmeyer flask,
Culture was carried out by shaking (100 rpm) at 48 ° C for 48 hours. The thus-obtained seed culture was transferred at a rate of 5% v / v to 3 L of a medium having the same composition as the above composition in a 10 L culture tank, and aerated and agitated at 25 ° C. (rotation speed 100 rpm, Main culture was performed at an aeration rate of 1 L / min. During the culture, the pH of the medium is not particularly controlled,
Cultured for 120 hours.

The culture solution was centrifuged (10,000 rpm) to obtain a bacterial cell fraction, 200 ml of acetone was added and stirred, the precipitate was filtered off, and the extract was dried to dryness. This is further silica gel column
(Silica gel 60 manufactured by Nacalai Tesque, Inc.) was used and developed with hexane: acetone = 7: 3. The eluted fraction A was concentrated to obtain 0.30 mg of astaxanthin. The astaxanthin thus obtained was in agreement with known ones in hydrogen nuclear magnetic resonance spectrum, visible absorption spectrum and mass spectrometry.

(Example 2) Alcaligenes sp.
PC-2 (FERM P-13488) was used. For pre-culture and main culture, a medium prepared by "MARINE BROTH" manufactured by DIFCO Co., Ltd. according to the method described in the manual was appropriately dispensed into a culture tank and used after sterilization.
As a preculture, 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) It culture | cultivated. The thus-obtained seed culture was transferred at a rate of 5% v / v to 3 L of a medium having the same composition as the above composition in a 10 L culture tank, and aerated and agitated at 25 ° C. (rotation speed 100 rpm, Main culture was performed with an aeration rate of 1 L / min. During the culturing, the pH of the medium was not particularly controlled, and the cells were cultivated for 120 hours.

The culture broth was centrifuged (10,000 rpm) to obtain a bacterial cell fraction, 200 ml of acetone was added and stirred, the precipitate was filtered off, and the extract was dried to dryness. This is further silica gel column
(Silica gel 60 manufactured by Nacalai Tesque, Inc.) was used and developed with hexane: acetone = 7: 3. The eluted fraction A was concentrated to obtain astaxanthin 0.16 mg. The astaxanthin thus obtained was in agreement with known ones in hydrogen nuclear magnetic resonance spectrum, visible absorption spectrum and mass spectrometry.

(Example 3) As an inoculum, Pseudomonas sp.
PC-3 (FERM P-13487) was used. For pre-culture and main culture, a medium prepared by "MARINE BROTH" manufactured by DIFCO Co., Ltd. according to the method described in the manual was appropriately dispensed into a culture tank and used after sterilization.
As a preculture, 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) It culture | cultivated. The thus-obtained seed culture was transferred at a rate of 5% v / v to 3 L of a medium having the same composition as the above composition in a 10 L culture tank, and aerated and agitated at 25 ° C. (rotation speed 100 rpm, Main culture was performed with an aeration rate of 1 L / min. During the culturing, the pH of the medium was not particularly controlled, and the cells were cultivated for 120 hours.

The culture solution was centrifuged (10,000 rpm) to obtain a bacterial cell fraction, 200 ml of acetone was added and stirred, the precipitate was filtered off, and the extract was dried to dryness. This is further silica gel column
(Silica gel 60 manufactured by Nacalai Tesque, Inc.) was used and developed with hexane: acetone = 7: 3. The eluted fraction A was concentrated to obtain astaxanthin 0.12 mg. The astaxanthin thus obtained was in agreement with known ones in hydrogen nuclear magnetic resonance spectrum, visible absorption spectrum and mass spectrometry.

(Example 4) As an inoculum, Alteromonas sp.
SD-402 (FERM P-13489) was used. For pre-culture and main culture, a medium prepared by "MARINE BROTH" manufactured by DIFCO Co., Ltd. according to the method described in the manual was appropriately dispensed into a culture tank and used after sterilization.
As a preculture, 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) It culture | cultivated. The thus-obtained seed culture was transferred at a rate of 5% v / v to 3 L of a medium having the same composition as the above composition in a 10 L culture tank, and aerated and agitated at 20 ° C. (rotation speed 100 rpm, Main culture was performed with an aeration rate of 1 L / min. During the culturing, the pH of the medium was not particularly controlled, and the cells were cultivated for 120 hours.

The culture broth was centrifuged (10,000 rpm) to obtain a bacterial cell fraction, 200 ml of acetone was added and stirred, the precipitate was filtered off, and the extract was dried to dryness. This is further silica gel column
(Silica gel 60 manufactured by Nacalai Tesque, Inc.) was used and developed with hexane: acetone = 7: 3. The eluted fraction A was concentrated to obtain 0.26 mg of astaxanthin. The astaxanthin thus obtained was in agreement with known ones in hydrogen nuclear magnetic resonance spectrum, visible absorption spectrum and mass spectrometry.

(Example 5) Hyphomonas sp.
PC-4 (FERM P-13486) was used. For pre-culture and main culture, a medium prepared by "MARINE BROTH" manufactured by DIFCO Co., Ltd. according to the method described in the manual was appropriately dispensed into a culture tank and used after sterilization.
As a preculture, one platinum loop of the strain was inoculated into 150 ml of medium in a 500 ml Erlenmeyer flask and shaken at 20 ° C for 48 hours.
(100 rpm) It culture | cultivated. The thus-obtained seed culture was transferred at a rate of 5% v / v to 3 L of a medium having the same composition as the above composition in a 10 L culture tank, and aerated and agitated at 25 ° C. (rotation speed 100 rpm, Main culture was performed with an aeration rate of 1 L / min. During the culturing, the pH of the medium was not particularly controlled, and the cells were cultivated for 120 hours.

The culture broth was centrifuged (10,000 rpm) to obtain a bacterial cell fraction, 200 ml of acetone was added and stirred, the precipitate was filtered off, and the extract was dried to dryness. This is further silica gel column
(Silica gel 60 manufactured by Nacalai Tesque, Inc.) was used and developed with hexane: acetone = 7: 3. The eluted fraction A was concentrated to obtain astaxanthin 0.24 mg. The astaxanthin thus obtained was in agreement with known ones in hydrogen nuclear magnetic resonance spectrum, visible absorption spectrum and mass spectrometry.

(Example 6) Caryophanon sp. As an inoculum
PC-5 (FERM P-13485) was used. For pre-culture and main culture, a medium prepared by "MARINE BROTH" manufactured by DIFCO Co., Ltd. according to the method described in the manual was appropriately dispensed into a culture tank and used after sterilization.
As a preculture, 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) It culture | cultivated. The thus-obtained seed culture was transferred at a rate of 5% v / v to 3 L of a medium having the same composition as the above composition in a 10 L culture tank, and aerated and agitated at 25 ° C. (rotation speed 100 rpm, Main culture was performed with an aeration rate of 1 L / min. During the culturing, the pH of the medium was not particularly controlled, and the cells were cultivated for 120 hours.

The culture solution was centrifuged (10,000 rpm) to obtain a bacterial cell fraction, 200 ml of acetone was added and stirred, the precipitate was filtered off, and the extract was dried to dryness. This is further silica gel column
(Silica gel 60 manufactured by Nacalai Tesque, Inc.) was used and developed with hexane: acetone = 7: 3. The eluted fraction A was concentrated to obtain astaxanthin 0.18 mg. The astaxanthin thus obtained was in agreement with known ones in hydrogen nuclear magnetic resonance spectrum, visible absorption spectrum and mass spectrometry.

[0039]

INDUSTRIAL APPLICABILITY According to the present invention, astaxanthin conventionally obtained from cultures of Antarctic krill, yeast and green algae can be easily obtained in a high yield with a very general culture device. In addition, there was no contamination of lipids, fatty acid esters, and chlorophyll peculiar to microalgae, and the purification process was greatly improved.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication (C12P 7/26 C12R 1:38) (C12P 7/26 C12R 1:01)

Claims (1)

[Claims] 1. Flavobacterium
Genus, Alcaligenes, Pseudomonas
(Pseudomonas) genus, Alteromonas (Alteromonas) genus,
The genus Hyphomonas or caryophanone
A method for producing astaxanthin, which comprises culturing a bacterium belonging to the genus anon) and having an ability to produce astaxanthin in a medium, and collecting astaxanthin from the culture.