JP2006340676A - Method for producing astaxanthin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing natural astaxanthin useful for the heightening of color of cultured fishes and shellfishes, and also utilized as a health food, more simply than the conventional method of production by using yeast, green algae or the like. <P>SOLUTION: This method for producing the astaxanthin by finding that microorganisms belonging to the genus Brevandimonas produces the astaxanthin in a high selectivity, is provided by culturing the bacteria belonging to the genus Brevandimonas and having a capacity of producing the astaxanthin, and collecting the astaxanthin from its cultured material. <P>COPYRIGHT: (C)2007,JPO&amp;INPIT

Description

  The present invention relates to a production method using astaxanthin microorganisms that are useful for coloring cultured seafood such as shrimps and red sea bream and also used as an antioxidant.

Conventionally, astaxanthin has been widely used for the coloring of cultured seafood such as salmon, shrimp and red sea bream, and as its production method, extraction from shellfish such as Antarctic krill, cultivation of a kind of yeast such as faffia Culture of a kind of green algae, Hematococcus, and organic synthesis are known. However, when using Antarctic krill, etc., a great deal of labor is required for its collection, extraction, separation from impurities such as lipids (Patent Document 1). Moreover, since the cell wall is strong in the culture | cultivation of yeast Phaffia, operation which decomposes | disassembles a cell wall at the time of extraction is needed, and it is difficult (patent documents 2, 3). In addition, the optical isomer of astaxanthin generally used for food applications is (3S, 3S '), whereas astaxanthin produced by Phaffia yeast is (3R, 3R'), and the steric structure is different ( Non-patent document 1). When cultivating the green alga Haematococcus, it is necessary to supply light that is indispensable for photosynthesis, and it is necessary to have facilities such as site conditions for collecting sunlight and a culture device for supplying artificial light (Patent Document 4). In addition, the purification process is complicated because it is difficult to separate chlorophyll from the mixture. Production methods for cultivating bacteria are described in terms of Agrobacterium, Paracoccus, etc., but all have low productivity, and there are many contaminants such as β-carotene and other precursors, making industrial implementation difficult. Yes (Patent Documents 5, 6, and 7). Although there is a report that the genus Brevundimonas produces astaxanthin, the selectivity of astaxanthin is low because it accumulates a considerable amount of hydroxyastaxanthin as a by-product (Non-patent Document 2, described as 9.9% of total carotenoids), and separation In addition, it is considered that the productivity of astaxanthin is extremely low. On the other hand, astaxanthin-producing bacteria have been reported by the new strain E-396 strain and mutant strain Y-1071. Astaxanthin selectivity is less than 50% (Patent Document 8), and a strain with higher astaxanthin selectivity is desired. ing. Moreover, in the organic synthesis method, considering that astaxanthin is used as a feed or food for seafood, problems such as by-products generated during the reaction remain. From the above, development of a simple production method of astaxanthin is desired.
Japanese Patent Laid-Open No. 02-255771 Japanese Patent Laid-Open No. 08-214870 Japanese Unexamined Patent Publication No. 08-000283 WO99 / 50384 pamphlet Japanese Patent Laid-Open No. 06-165683 Japanese Unexamined Patent Publication No. 07-184668 JP 07-079796 JP 2001-352995 Eric A. Johnson et al., “Astaxanthin from Microbial Sources”, Critical Reviews in Biotechnology, USA, CRC Press, 1991, Volume 11, Issue 4, p.297-329 Akihiro Yokoyama et al., “New trihydroxy-keto-carotenoids isolated from astaxanthin-producing marie bacterium”, Bioscience Biotechnology and Biochemistry, Japanese Society for Agricultural Chemistry, 1996, Vol. 60, No. 2, p200-2003

  An object of the present invention is to provide natural astaxanthin in a simpler and more efficient manner than conventional production methods using yeast or green algae.

The present inventors conducted extensive research on microorganisms that produce astaxanthin. As a result, they found that microorganisms belonging to the genus Brebandimonas produced astaxanthin with high selectivity, and completed the present invention. That is, the method for producing astaxanthin of the present invention is characterized by culturing Brevundimonas spp. Bacteria having an ability to produce astaxanthin in a medium and collecting astaxanthin from the culture.
1) A bacterium belonging to the genus Brevandimonas that has an astaxanthin selectivity such that the content of astaxanthin in the total carotenoids is higher than that of hydroxyastaxanthin and dihydroxyastaxanthin.
2) A bacterium belonging to the genus Brevundimonas having a production capacity such that the astaxanthin content in total carotenoids is 50% or more in bacteria producing carotenoids.
3) The Brevundimonas spp. Described in 1) or 2) above, wherein the Brevundimonas spp.
(1) Gram staining negative
(2) Fungal form: Spider
(3) Bacterial pigment: Orange
(4) Physiological properties: Catalase positive, oxidase positive, gelatin degradability positive,
Esculin hydrolysis positive
(5) Glucose, maltose and malic acid assimilation 4) Brevandymonas sp. FERM P-20516 strain.
5) Brevundimonas sp. FERM P-20515 strain.
6) A method for producing astaxanthin, comprising culturing the genus Brevundimonas as described in any one of 1) to 5) above in a medium and collecting astaxanthin from the culture.

  According to the present invention, astaxanthin obtained from a culture of Antarctic krill, yeast, or green algae can be easily obtained in a high yield by using a very general microorganism culture apparatus. Moreover, by using bacteria, it is possible to easily extract astaxanthin, and since the bacteria selectively produce astaxanthin, purification thereof becomes easy.

The present invention will be specifically described below. As an astaxanthin-producing strain, any strain can be used as long as it belongs to the genus Brebandimonas and selectively produces astaxanthin. Here, selectively producing means producing astaxanthin in a high yield from by-products such as hydroxyastaxanthin.
Moreover, in this invention, the strain which has the production capacity whose content of astaxanthin is 50% or more is used among the total carotenoids produced.
As a strain, for example, soil, seawater, lake water, etc. are collected from nature, suspended or diluted in sterilized water, physiological saline, etc., and then applied to an agar medium prepared in a petri dish, and the culture temperature is 20 ° C. Culturing is performed at -40 ° C, preferably 25 ° C-37 ° C, and a colony producing a red-yellow pigment is selected from the obtained colonies. The selected colony is further cultured in an agar medium or liquid medium, and then the cells are collected and the pigment is extracted with an organic solvent such as acetone or DMSO. A strain producing astaxanthin can be obtained by analyzing the extracted pigment by HPLC or the like. In addition, these strains selectively produce astaxanthin by artificial mutation methods such as ultraviolet irradiation, X-ray irradiation, mutagen treatment, naturally occurring mutant strains, and mutant strains by gene manipulation and cell fusion. Any of them can be used in the present invention.

  Specifically, strains belonging to the genus Brevandimonas are cultured in liquid culture such as bouillon medium at a culture temperature of 25 ° C. to 35 ° C. for 1 to 3 days. (N′-nitro-N-nitrosoguanidine)) After suspension in a solution of 50 to 500 mg / L, shake at 25 ° C. to 35 ° C. for 10 to 120 minutes. Thereafter, the cells are collected by centrifugation, washed with physiological saline or the like, applied to a normal agar medium or the like prepared in a petri dish with appropriate dilution, and cultured at 25 ° C. to 30 ° C. for 1 to 7 days. The obtained colonies are further cultured in an agar medium or a liquid medium, and then the cells are collected, and the pigment is extracted with an organic solvent such as acetone or DMSO. A strain that selectively produces astaxanthin is selected by analyzing the extracted dye by HPLC or the like.

In the present invention, a large number of screenings were performed from nature to obtain a strain that selectively produced astaxanthin, and a strain that also produced astaxanthin by induction of mutation.
Representative strains include the N5 strain and the M26G-4 strain, which is a mutant strain of the A1-8 strain.
The present inventors have identified strains N-5 and A1-8 isolated from nature based on the analysis of 16S rDNA and the following biochemical properties. As a result of homology search with respect to the international nucleotide sequence data bank (DDBJ), the N-5 strain 16S rDNA sequence was found to have a homology of 99.9% with respect to the reference strain Brevundimonas vesicularis LGM2350, and a homology of 99 with Brevundimonas nasdae GTC1042. It showed a high homology rate of 8%. From the biochemical properties, gelatin was not hydrolyzed and β-galactosidase reaction was positive, so it was close to B. nasdae, and it was difficult to determine which one it belongs to. On the other hand, as for the A1-8 strain, as a result of homology search with respect to the international nucleotide sequence data bank (DDBJ), the 16S rDNA sequence was 100% homologous to the reference strain Brerevundimonas vesicularis LGM2350, compared with Brerevundimonas nasdae GTC1042 and 99% homologous. It showed a high homology rate of 9%. From the biochemical properties, gelatin was not hydrolyzed and β-galactosidase reaction was positive. Therefore, it was close to B. nasdae, and it was difficult to determine which was the end. Therefore, it was found that all the strains belonged to the genus Brebandimonas, but the species was not yet identified, and it was considered appropriate to use Brevundimonas sp. These were deposited at the National Institute of Advanced Industrial Science and Technology, and were deposited under the accession numbers FERM P-20516 and FERM P-20515, respectively (consignment date April 26, 2005). The mycological properties of the N-5 strain and the A1-8 strain are as follows.

  In the production method of the present invention, the above microorganisms are cultured in a medium generally used for culturing microorganisms, and the produced astaxanthin is collected by a conventional method. First, the culture method is described. A normal culture method can be used for culturing Brevundimonas bacteria. As a medium, any medium can be used as long as it contains moderately assimilable carbon sources, nitrogen sources, inorganic substances, and necessary growth and production promoting substances. As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, lactose, xylose, arabinose, mannitol, molasses and the like are used alone or in combination. Furthermore, hydrocarbons, alcohols, organic acids, and the like are also used depending on the ability of assimilation of bacteria. As the nitrogen source, ammonium chloride, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soy flour, casamino acid and the like are used alone or in combination. In addition, salt, potassium chloride, magnesium sulfate, calcium carbonate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium chloride, manganese sulfate, zinc sulfate, copper sulfate and other inorganic salts and seawater are required. Add as appropriate. Furthermore, trace components that promote the growth of the bacteria used and the production of astaxanthin can be appropriately added.

  As the culture method, a liquid culture method, particularly a stirring culture method is most suitable. The culture temperature is suitably 20 to 40 ° C., particularly 25 to 37 ° C., and the pH of the medium during the culture can be maintained at 4 to 10, particularly 6 to 8 by adding aqueous ammonia or ammonium carbonate solution. desirable. When cultured for 1 to 7 days in liquid culture, the target substance astaxanthin is produced and accumulated in the cells. The culture is stopped when the production amount in the culture reaches the maximum.

  Isolation and purification of astaxanthin from the culture is performed according to a commonly used method for isolating and purifying a microbial metabolic product from the culture. For example, the culture is separated into a culture filtrate and cells by filtration, and the cells are extracted with an organic solvent (eg, hexane, benzene, chloroform, acetone, ether, ethyl acetate, etc.). Next, after concentration, astaxanthin is separated and purified by silica gel column chromatography, gel filtration (Sephadex LH-20) or the like. The trend of astaxanthin during culturing and purification operations can be traced using astaxanthin red color as a guide by thin layer chromatography.

  The carotenoid extraction method for quantitative analysis of carotenoids in the microbial cells is shown in [Extraction method for analyzing carotenoids] below.

  Astaxanthin and astaxanthin precursor can be identified and quantified by comparing the elution time with a commercial product by HPLC and by comparing the visible light absorption spectrum by a photodiode array detector. A specific method is shown in [HPLC analysis condition 1] below.

  Hydroxyastaxanthin can be identified by LC / MS analysis and by analyzing the visible light absorption spectrum of the HPLC separation peak. The LC / MS analysis can be performed under the conditions shown in (Table 1). A specific method of HPLC analysis is shown in [HPLC analysis condition 2] below.

  The stereoisomers and optical isomers of astaxanthin were identified by a chiral HPLC method (Enantiomer, Vol. 2, p17-25) by Saleh Abu-lafi et al. Specifically, it is shown in [HPLC analysis condition 3] below.

[Extraction method for analyzing carotenoids]
Take 2 ml of the culture solution, collect the cells by centrifugation, add 0.3 ml of DMSO to the obtained wet cells, and extract at 50 ° C. for 1 hour. Thereafter, 0.3 ml of a mixed solvent of acetonitrile / methanol / isopropyl alcohol (85/10/5) is added, and the supernatant is obtained as an extract by centrifugation.

[HPLC analysis condition 1]
Using CAPCELL PAC C18 (3 μm 4.6 mmφ × 35 mm Shiseido) as a separation column, an eluent of acetonitrile / methanol / isopropyl alcohol (85/10/5) is passed at a flow rate of 1.0 ml / min. For detection, a photodiode array detector is used, and the elution time and visible light absorption pattern of a peak obtained from a 478 nm chromatographic chart are measured, and identification and quantification can be performed by comparing with a commercially available reagent (FIG. 1). .

[HPLC analysis condition 2]
Using CAPCELL PAC C18 (3 μm 4.6 mmφ × 35 mm Shiseido) as a separation column, an acetonitrile / water (90/10) eluent is passed at a flow rate of 1.0 ml / min. Detection can be carried out using a photodiode array detector, and identification and quantification can be performed by measuring the elution time of the peak obtained from the 478 nm chromatograph and the visible light absorption pattern (FIG. 2).

[HPLC analysis condition 3]
Sumichiral OA-2000 is used as a separation column, and an eluent of n-heptane: methylene chloride: isopropyl alcohol (70/25/5) is passed at a flow rate of 1.0 ml / min. Detection uses a photodiode array detector to measure the 478 nm chromatograph and the visible light absorption pattern of the resulting peak.
The structural formulas of carotenoid and hydroxyastaxanthin used as standard products in the above HPLC are shown.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but it goes without saying that the scope of the present invention is not limited thereto.
[Example 1]
Brevandimonas N-5 strain (FERM P-20516) was transplanted into a slant medium of Nutrient agar (manufactured by Difco), cultured at 30 ° C. for 48 hours, and then Nutrient Broth (manufactured by Difco) in 100 ml portion in a 500 ml Erlenmeyer flask. Poured ears were inoculated into the medium that had been sterilized by autoclaving and cultured at 30 ° C. for 48 hours. After centrifuging the culture-terminated solution and collecting the cells, the cells were extracted according to the above [Extraction method for analyzing carotenoids]. It was confirmed that astaxanthin and hydroxyastaxanthin were produced by analyzing the extract with LC / MS (FIG. 3). LC / MS analysis conditions are shown in Table 1. Further, the extract was analyzed according to the above-mentioned [HPLC analysis condition 1] and [HPLC analysis condition 2]. Compared to commercially available astaxanthin and astaxanthin precursor, it was confirmed that the elution time and the visible light absorption spectrum coincided, and at the same time, astaxanthin, astaxanthin precursor and hydroxyastaxanthin were quantified. The results are shown in Table 2.

[Example 2]
In the same manner as in Example 1, 100 ml of the culture end solution in which the N-5 strain was cultured was centrifuged, and the collected cells were lyophilized. The pigment was extracted from the dried cells with 5 ml of ethyl acetate to obtain an extract. The extract was charged in a silica gel column (silica gel 60, 20 mm diameter x 200 mm height manufactured by Merck & Co., Inc.) filled with hexane as a solvent, washed with 50 ml hexane, and then eluted with hexane / acetone (60/40). The liquid was fractionated into 5-ml fractions and analyzed under [HPLC analysis condition 1] to obtain astaxanthin fractions. Further, by analyzing the astaxanthin fraction under [HPLC analysis condition 3], the three-dimensional structure of astaxanthin is (3S, 3'S) -3,3'-dihydroxy-β, β-carotene-4,4'-dione. It was confirmed (FIG. 4).

[Example 3]
Brevandimonas A1-8 strain is transplanted to a slant medium of Nutrient agar (Difco), and after culturing at 30 ° C for 48 hours, 100 ml of Nutrient Broth (Difco) is dispensed into a 500 ml Erlenmeyer flask and autoclaved. One white fungus ear was inoculated into the culture medium and cultured at 30 ° C. for 48 hours. The obtained culture end solution was centrifuged to obtain bacterial cells. The obtained microbial cells were suspended in 2 ml of 100 mM potassium phosphate buffer (pH 7) to a concentration of 10 ⁶ cells / mL, and NTG (N-methyl-N′-nitro-N-nitrosoguanidine) was further added at 250 mg / L. It added to the density | concentration, and the mutation process was performed by shaking at 30 degreeC for 1 hour. The bacterial cells were collected from this bacterial cell suspension by centrifugation, washed thoroughly with 100 mM potassium phosphate buffer (pH 7), diluted to 100 to 300 spores per plate in Nutrient agar medium, and applied. After culturing at 30 ° C. for 4 days, the obtained colonies were further transplanted into Nutrient broth, cultured at 30 ° C. for 48 hours, and then centrifuged to obtain bacterial cells. The obtained bacterial cells were freeze-dried, and the pigment was extracted with methanol, and then monohydroxyastaxanthin (Rf value 0.25) with TLC (silica gel 60, TLC aluminum plate (Merck), developing solvent hexane: acetone = 60: 40). ) And dihydroxyastaxanthin (Rf value 0.15) were eliminated, and the M26G-4 strain was obtained as a strain in which the astaxanthin (Rf value 0.4) spot became dark.

[Example 4]
When culturing in the same manner as in Example 1, the M26G-4 strain obtained in Example 3 was used as a strain and culture was performed. After centrifuging the culture end solution and collecting the bacterial cells, the cells were extracted according to the above [Extraction method for analyzing carotenoid], and then analyzed according to [HPLC analysis condition 1] [HPLC analysis condition 2]. Astaxanthin, astaxanthin precursor, and hydroxyastaxanthin were quantified. The results are shown in Table 2.

[Comparative example]
When culturing in the same manner as in Example 1, A1-8 strain, Brevundimonas aurantiaca (DMSZ4731), B. bacteroides (DMSZ4726), B. Subvibrioides (DMSZ4735), B. valiabilis (DMSZ7237), B. vesicularis (DMSZ7233) Or B. vesicularis (DMSZ7226) strain was used for cultivation. After centrifuging the culture-terminated solution and recovering the cells, extraction was performed according to the above [Extraction method for analyzing carotenoids] to obtain an extract. [HPLC analysis condition 1] Analysis was performed according to [HPLC analysis condition 2], and astaxanthin, astaxanthin precursor, and hydroxyastaxanthin were quantified. The results are shown in Table 2.
It can be seen that general Brevundimonas spp. Have lower astaxanthin selectivity during fermentation production than the strains of the present invention.

  The present invention relates to a method for producing astaxanthin. Astaxanthin is useful for coloring cultured seafood such as shrimp and red sea bream, and has recently been used as a health food.

HPLC analysis chart of astaxanthin and astaxanthin precursor by [HPLC analysis condition 1] HPLC analysis chart of astaxanthin and hydroxyastaxanthin by [HPLC analysis condition 2] LC / MS analysis chart of the extract in Example 1 HPLC analysis chart according to [HPLC analysis condition 3] of the extract in Example 1

Claims (6)

  A bacterium belonging to the genus Brevandimonas having an astaxanthin selectivity such that the content of astaxanthin in total carotenoids is higher than that of hydroxyastaxanthin and dihydroxyastaxanthin in the carotenoid-producing bacteria.   A bacterium belonging to the genus Brevundimonas having a production capacity such that the astaxanthin content in total carotenoids is 50% or more in bacteria producing carotenoids. Brevundimonas spp. According to claim 1 or 2, wherein the Brevundimonas spp.
(1) Gram staining negative
(2) Fungal form: Spider
(3) Bacterial pigment: Orange
(4) Physiological properties: Catalase positive, oxidase positive, gelatin degradability positive,
Esculin hydrolysis positive
(5) Glucose, maltose and malate utilization   Brevundimonas sp. FERM P-20516 strain.   Brevandimonas sp. FERM P-20515 strain.   A method for producing astaxanthin, comprising culturing the Brevundimonas bacterium or mutant thereof according to any one of claims 1 to 5 in a medium and collecting astaxanthin from the culture.

Images