JP2006191919A - Method for producing carotenoid pigment, sphingoglycolipid, ubiquinone q-10 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel microorganism which simultaneously produces carotenoid pigments and sphingoglycolipids and furthermore produces ubiquinone Q-10, and a method for producing the pigment, glycolipid and ubiquinone Q-10 using the microorganism. <P>SOLUTION: The novel Sphingomonus, JPCCMB0017, has production ability of both carotenoid pigments and sphingoglycolipids, and furthermore has production ability of ubiquinone Q-10. The carotenoid pigments, sphingoglycolipids and ubiquinone Q-10 can be produced by culturing the bacterial strain. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a novel microorganism that simultaneously produces a carotenoid pigment and a glycosphingolipid, a novel microorganism that produces ubiquinone Q-10 (also known as coenzyme Q-10), the pigment using these microorganisms, and the glycolipid. And a method for producing the ubiquinone Q-10.

  Astaxanthin, a carotenoid pigment, is widely distributed in fishes such as red sea bream and salmon, and crustaceans such as crabs and shrimps. As an example of the production of microorganisms, the green alga Heamatococcus pluvialis is the most famous. In addition, red yeast Xanthophyllomyces dendrohous (former Phaffia rhodozyma) is mentioned. It is known that the amount of astaxanthin produced by Heamatococcus pluvialis is about 43 mg / g dry weight (Non-patent Document 1). Moreover, in red yeast Xanthophyllomyces dendrohous (former Phaffia rhodozyma), the production amount of 0.4 mg / g dry weight is reported (nonpatent literature 2).

  Prokaryotic bacteria are one of the most promising microorganisms in the production of useful substances because they have a wide substrate utilization capacity, show a high growth rate in simple culture, and do not have a thick cell wall like the yeast described above. It is. In astaxanthin production in bacteria, 1.4 mg / g dry weight has been achieved by the Escherichia coli gene recombinant (Non-patent Document 3). Moreover, in the method using Bacillus firmus, 0.05 mg / g dry weight has been achieved (Non-patent Document 4). Furthermore, 0.14 mg / g dry weight has been reported in the marine bacterium Paracoccus sp. MBIC1143 (Non-patent Document 5). However, the production of astaxanthin by these genetic recombinants is currently problematic in terms of expression level.

  On the other hand, in the production of astaxanthin by separation from natural products, extraction from krill and crustaceans has a problem that the extraction amount is low and the cost is high due to low extraction efficiency. There is also a commercial problem in terms of securing resources due to the decrease in natural resources.

  Astaxanthin has many physiological activities and is sold as a supplement food. Moreover, in the cultured fish such as Thailand and salmon, a feed (colored fried feed) containing astaxanthin is used in order to bring the color tone closer to a natural one.

  Due to the recent decrease in natural resources, production of natural resources by aquaculture is expected. In the artificial seed production of seafood, there is a demand for feed that adds high feed effects and added value to the target fish species. Feeds containing pigments such as astaxanthin are used for fish species that require vivid colors such as Thailand and rainbow trout.

  Astaxanthin as a fried food additive is sold by Roche as calorie pink (synthetic astaxanthin), but the natural product astaxanthin is attracting attention due to traceability problems and natural product orientation. Up to now, astaxanthin, a natural product for deep-fried color, has used the green alga Heamatococcus pluvialis and the red yeast Xanthophyllomyces dendrohous, but has problems such as insufficient coloring. The green alga Heamatococcus pluvialis and the red yeast Xanthophyllomyces dendrohous have a thick cell wall and a low digestion and absorption rate of the target fish species. For this reason, a new microorganism having a high absorption rate is required.

  Bacteria are characterized by a thin cell wall and rapid growth. Thus, bacteria producing astaxanthin have been searched, and the genus Flavobacterium, Alkaligenes, Pseudomonas, Alteromonas, Pipomonas, Caryophanone, Erytlobacter and Paracoccus are already known.

  Examples of humectant substances produced by microorganisms include various substances such as polysaccharides, glycerol, hyaluronic acid, and ceramide. Bacteria belonging to the genus Sphingomonas are known to produce glycosphingolipids with high moisture retention. Glycosphingolipids are shown in Patent Document 1, Patent Document 2, Patent Document 3, and Patent Document 4 to have a moisture retention effect. Furthermore, Patent Document 4 discloses a cosmetic containing a glycosphingolipid as a constituent component.

  Glycosphingolipids are present throughout the family Sphingomonas, and include sphingomonas genus, blastmonas genus, erythrobacter genus, erythromicbium genus, erythromonas genus, porphylobacter genus, lysomonas genus, zynomonas genus and the like.

  In general, glycosphingolipids are generally obtained by culturing sphingomonadaceae bacteria and extracting them with a specific solvent. Many sphingomonads contain pigments, but if the pigments to be produced are highly functional, such as astaxanthin, which has an antioxidant action, it is possible to provide a moisturizer having an antioxidant action as a single microorganism. Can be provided.

  Ubiquinone Q-10 (CoQ-10) is the official name 2,3-dimethoxy-5-methyl-6-buoprenyl-1,4 benzoquinone, but this substance is widely distributed in animals, plants and microorganisms ( Non-patent document 7). Ubiquinone Q-10 has a pharmacological effect on congestive heart failure, ischemic heart disease, and essential hypertension, and is used clinically. Furthermore, an antioxidant action and a fat burning action are known, and in recent years, utilization to health foods, supplements, and quasi drugs is progressing. Ubiquinone Q-10 is produced by fermentation using microorganisms such as yeast and photosynthetic bacteria containing a large amount of ubiquinone Q-10. There are also methods using fungi and basidiomycetes.

When producing ubiquinone Q-10 by fermentation, the microorganisms to be used add nutrients (glucose, alkane, methanol, etc.) necessary for ubiquinone production to the medium, grow the microorganisms, and produce the ubiquinones in the microorganisms. Q-10 is extracted with an organic solvent or the like. Microorganisms that can be used for fermentation include yeast: Aureobasidium, Brettanomyces, Bullera, Candida, Cryptococcus, Leucosporidium, Oosporidium, Rhodotorula, Rhodosporidium, Schizosaccharomyces, Sporobolomyces, Torulopsis, Tremella, Tricosporon, Sporidiolus, Breconos Geotrichum, Monascus, Paecilomyces, Sporotrichum, Tilletiopsis, Basidiomycetes: Ustilago, Bacteria: Escherichia, Acetobacter, Agrobacterium, Corynebacterium, Erytrobacter, Flavobacterium, Methylobacter, Microcyslus, Phyllobacterium, Protaminobacter, R
Japanese Unexamined Patent Publication No. 1-242690 Japanese Patent Laid-Open No. 2-48520 JP-A-4-159203 Japanese Patent Laid-Open No. 6-172833 JP-A-5-333101 Japanese Patent Laid-Open No. 5-70334 Japanese Patent Laid-Open No. 6-2017878 JP-A-6-152240 Japanese Patent Laid-Open No. 11-279337 Japanese Patent Laid-Open No. 11-42082 Lee, Y.-K. and Soh, C.-W., "Journal of Phycolology", USA, 1991, Vol. 27, No. 3, p. .575-577 Flores-Cotera, LB et al., Applied Microbiol. Biotechnol., USA, 2001, Vol. 55, No. 2, p.341 -347 Wang, C.-W. et al., Biotechnol. Bioeng., USA, 1999, Vol. 62, No. 3, p.235-241 Yokoyama et al., Bioscience, Biotechnology, and Biochemistry, USA, 1994, Vol. 58, No. 10, p.1842-1844 Pane, L. et al., Journal of Biology Research (J. Biol. Res.), USA, 1996, Vol. 22, p. 303-308 Kawahara et. Al., FEBS letter, Netherlands, 1991, 292, 107, p.107-110 Ernster, L and Dallner, G, “Biochem. Biophys. Acta”, USA, 1995, 1271, p.195-204

  The present invention does not have a thick cell wall such as yeast and algae, and can produce both substances such as astaxanthin and glycosphingolipid, and microorganisms capable of producing ubiquinone Q-10. The issue is to produce.

  Another object of the present invention is to provide a method for easily and efficiently producing the substance by culturing these microorganisms, and the substance produced by these methods. According to the present invention, compared to conventional carotenoid-containing feeds and the like, it is expected that it will be possible to provide a more concentrated carotenoid and provide an efficient food additive for coloring. It is expected.

  In order to solve the above problems, the present invention has the following configuration. That is, the present invention provides a novel microorganism according to the first invention, which has a carotenoid pigment-producing ability and a glycosphingolipid-producing ability, and the base sequence of 16S rDNA is the sequence described in SEQ ID NO: 1. The bacterial strain JPCCMB0017 is provided.

According to the second aspect of the present invention, in the second invention, the base sequence of 16S rDNA has at least 96% homology with the sequence shown in SEQ ID NO: 1, and has carotenoid pigment production ability and glycosphingolipid production ability. Bacterial strains are also provided.
In the second invention of the present invention, as long as the properties of the invention are satisfied, it can be a strain that is a gene recombinant.

  In the third invention, the present invention provides the bacterial strain described in the first invention or the second invention, wherein the carotenoid pigment is astaxanthin.

  According to the fourth aspect of the present invention, there is provided a bacterial culture obtained by culturing the bacterial strain according to any one of the first to third aspects.

The present invention provides, in its fifth invention, a method for producing carotenoid pigments and / or glycosphingolipids, comprising a culturing step of culturing the bacterial strain of any one of the first to third inventions. .
In the sixth aspect of the present invention, the step of separating the carotenoid pigment and / or glycosphingolipid from the culture obtained in the step of culturing can be further included after the step of culturing in the fifth aspect of the present invention.
In the seventh invention, the present invention provides a composition containing a carotenoid pigment and / or glycosphingolipid produced by the production method of the fifth or sixth invention.

  In the eighth invention, the present invention relates to a carotenoid-producing bacterial strain, a glycosphingolipid-producing bacterial strain, a culture of the bacterial strain described in any one of the first to third inventions, or a bacterial strain of these bacterial strains. A carotenoid pigment comprising: a step of adding a dried microbial cell, a frozen microbial cell, a solvent extract, or a pulverized / broken product to a culture solution of zooplankton; and a step of appropriately recovering the zooplankton from the solution. And / or a method for producing glycosphingolipids.

  In the ninth invention, the present invention relates to a carotenoid-producing bacterial strain, a glycosphingolipid-producing bacterial strain, a culture of the bacterial strain described in any one of the first to third inventions, or a bacterial strain of these bacterial strains. Provided is a feed obtained by feeding zooplankton to dry cells, frozen cells, solvent extracts, or crushed and crushed products.

  In the tenth aspect of the present invention, the present invention provides Sphingomonas sp. JPCCMB0017, which has the ability to produce ubiquinone Q-10 and whose base sequence of 16S rDNA is the sequence set forth in SEQ ID NO: 1.

  In the eleventh invention, the present invention relates to a bacterial strain of the genus Sphingomonas, wherein the base sequence of 16S rDNA has at least 96% homology with the sequence of SEQ ID NO: 1 and has the ability to produce ubiquinone Q-10. I will provide a.

  The twelfth aspect of the present invention provides a bacterial culture obtained by culturing the bacterial strain according to the tenth or eleventh aspect.

  The thirteenth aspect of the present invention provides a method for producing ubiquinone Q-10, comprising a culture step of culturing the bacterial strain of the tenth or eleventh aspect.

  In the fourteenth aspect of the present invention, after the culturing step in the thirteenth aspect, the present invention can further include a step of separating ubiquinone Q-10 from the culture obtained in the culturing step.

  In the fifteenth aspect of the present invention, there is provided a composition comprising a carotenoid pigment and / or glycosphingolipid produced by the method of the thirteenth aspect, and ubiquinone Q-10.

  When the novel bacterium of the present invention and its culture are used, it is possible to simultaneously produce carotenoid pigments such as astaxanthin, which is a useful pigment, and glycosphingolipids having a moisturizing effect, and further to produce ubiquinone Q-10. Is possible. Furthermore, according to the method for producing carotenoid pigments and / or glycosphingolipids of the present invention, it is possible to efficiently produce carotenoid pigments such as astaxanthin, which is a useful pigment, and coloring that contains pigments such as astaxanthin Feeds such as feeds can be efficiently prepared. Moreover, according to the production method of ubiquinone Q-10 of the present invention, it is possible to efficiently produce a composition containing ubiquinone Q-10 and ubiquinone Q-10.

Hereinafter, an optimal embodiment of the present invention will be described in detail.
In the practice of the present invention, a sphingomonas bacterium strain JPCCMB0017, which has carotenoid pigment production ability, glycosphingolipid production ability, and ubiquinone Q-10 production ability, and the base sequence of 16S rDNA is the sequence described in SEQ ID NO: 1. Can be used. As for this strain, the applicant filed a deposit application with the Patent Microorganisms Depositary Center of National Institute of Technology and Evaluation on December 3, 2004. (Receipt number NITE AP-48).

The properties of this strain were as follows.
-Morphological properties Cell shape: Neisseria gonorrhoeae Cell size: 0.6-0.7 × 2.0-3.0 μm (extended)
Presence of spores:-
Motility (flagellar state):-
Polymorphism:-

・ Cultural properties:
Medium: Marine Agar (Marine Broth 2216 (Becton Dickinson) + 1.5% agar)
Temperature: 25 ° C
Pigment production: +
Color: Orange Gloss: +
Medium: Marine Broth 2216
Temperature: 25 ° C
Surface development:-
Medium turbidity: +
Gelatin puncture culture Temperature: 25 ° C
growth:-
Gelatin liquefaction:-
Litmus milk Temperature: 25 ° C
coagulation:-
Liquefaction:-

・ Physiological properties Gram staining:-
Sulfate reduction: +
Denitrification reaction:-
MR test:-
VP test: +
Indole production:-
Production of hydrogen sulfide:-
Hydrolysis of starch:-
Use of citric acid (Koser):-
Use of citric acid (Christensen):-
Use of inorganic nitrogen source (nitrate):-
Use of inorganic nitrogen source (ammonium salt):-
Urease:-
Catalase: +
Oxidase: +
Growth (pH 5):-
Growth (pH 8): +
Growth (pH 9): +
Growth (20 ℃): + (weak)
Growth (25 ° C): +
Growth (30 ° C): +
Growth (37 ° C): + (weak)
Growth (salt concentration 0%):-
Growth (salt concentration 1%): + (weak)
Growth (salt concentration 2%): +
Growth (salt concentration 3%): +
Growth (salt concentration 7%): + (weak)
Growth (salt concentration 10%):-
Anaerobic growth:-
O / F test (oxidation / fermentation):-/-
Acid / gas production from sugars:
L-arabinose:-/-
D-glucose:-/-
D-fructose:-/-
Maltose:-/-
Lactose:-/-
D-sorbitol:-/-
Inositol:-/-
D-xylose:-/-
D-Mannose:-/-
D-galactose:-/-
Circus:-/-
Trehalose:-/-
D-mannitol:-/-
Glycerin:-/-

Other physiological properties β-galactosidase activity: +
Alkinin dihydrase activity:-
Lysine decarboxylase activity:-
Tryptophan deaminase activity:-
Gelatinase activity:-
Esculin degradation activity: + (weak)
Hippuric acid degradation activity:-
Malonic acid availability:-
Ornithine decarboxylation:-
Phenylalanine deamination reaction:-
Coagulase activity:-
Hemolytic:-
Phosphatase activity: +
Lipase activity: +
Lecithinase activity:-
Cytochrome oxidase activity: +

・ Utilization test Glucose: Negative
L-arabinose: negative
D-Mannose: Negative
D-mannitol: negative
N-acetyl-D-glucosamine: negative maltose: negative potassium gluconate: negative n-capric acid: negative adipic acid: negative d1-malic acid: negative sodium citrate: negative phenyl acetate: negative

Chemical taxonomic properties DNA base composition (GC content): 59.1 mol%
Cellular lipid analysis Major quinone: Ubiquinone Q-10
fatty acid:
10: 0 3OH 0.26%
12: 0 2OH 0.13%
12: 0 3OH 0.30%
14: 0 0.17%
13: 0 2OH 0.24%
15: 0 1.32%
14: 0 2OH 3.05%
16: 0 5.82%
15: 0 2OH 2.58%
17: 0 ISO 0.08%
17: 1 w8c 2.83%
17: 1 w6c 2.95%
17: 0 1.39%
16: 1 2OH 0.09%
16: 0 2OH 1.40%
18: 1 w7c 71.90%
18: 1 w5c 0.39%
18: 0 0.20%
17: 0 ISO 3OH 1.61%
18: 1 2OH 0.24%
19: 0 10 methyl 0.42%
Species with similar fatty acids: Sphingomonas paucimobilis
Similarity (SI): 0.267
Bacteriochlorophyll production (anaerobic): not growing Bacteriochlorophyll production (aerobic): negative Presence of sphingolipid: +
Salt (NaCl) requirement in growth: +

  In the present invention, a bacterial strain of the genus Sphingomonas having a base sequence of 16S rDNA having at least 96% homology with the sequence shown in SEQ ID NO: 1 can be used. If 16SrDNA exhibits at least 96% homology with the sequence set forth in SEQ ID NO: 1, it is considered that the 16SrDNA is a strain of the same genus and the same species as those having the sequence set forth in SEQ ID NO: 1. Like the JPCCMB0017 strain, it is considered to have the ability to simultaneously produce carotenoid pigments and glycosphingolipids and the ability to produce ubiquinone Q-10.

  An example of producing astaxanthin using these strains is shown below. The medium for culturing the strain of the present invention is not particularly limited as long as the production microorganism contains carbon and nitrogen sources, inorganic salts and trace elements (vitamins and trace metals) necessary for growth. More specifically, examples of the carbon source include saccharides such as glucose and sucrose, and alcohols such as ethanol and glycerol. The addition ratio depends on the carbon source to be added, but can be about 0.5 to 3.0%.

  The nitrogen source can be any of nitrate nitrogen such as sodium nitrate, ammonium nitrate, ammonium sulfate, potassium nitrate, ammonium chloride, urea, and ammonia nitrogen. The addition ratio depends on the nitrogen source to be added, but can be about 0.01% to 0.1%.

  Inorganic salts include sodium chloride, sodium sulfate, calcium chloride, potassium chloride, sodium bicarbonate, potassium boride, strontium chloride, boric acid, sodium silicate, sodium fluoride, 1 and 2 potassium phosphate, 1 phosphate and Trace metal elements such as sodium, iron chloride, manganese chloride, manganese sulfate, magnesium chloride, and copper sulfate can be used. The addition ratio depends on the kind of inorganic salt to be added, but can be about 0.001% to 0.01%.

  As a special necessary substance, yeast extract, peptone, tryptone and the like can be used. The addition ratio depends on the substance to be added, but can be about 0.01% to 0.5%. It is desirable to adjust the pH of the medium to 6.0 to 8.0. Culture conditions can be in the temperature range of 25-30 ° C., and usually shake culture and aeration culture for 2 to 3 days.

  Finally, astaxanthin is obtained from the cultured strain. That is, it is extracted with an organic solvent directly from a strain culture or from a precipitate collected by centrifugation. As the solvent used here, any solvent can be used as long as it is a solvent in which astaxanthin is sufficiently dissolved or a mixed solution of solvents.

  As the solvent to be used, nonpolar solvents such as acetone, methanol and ethyl acetate having high polarity to hexane, dichloromethane and chloroform can be used alone, or a mixture thereof can be used. For purification, these solvents can be combined and elution and dissolution can be repeated.

  Next, specific examples for producing glycosphingolipid using the strain of the present invention are shown below. The medium is not particularly limited as long as it contains carbon and nitrogen sources, inorganic salts and trace elements (vitamins and trace metals) necessary for the growth of the producing bacteria, as in the case of producing astaxanthin.

  The culture conditions can also be essentially the same as in the production of astaxanthin.

  Finally, glycosphingolipid is obtained from the cultured microorganism. Extract with organic solvent directly from cultured microorganisms or from centrifugally collected precipitates. As the solvent used here, any solvent can be used as long as it is a solvent in which the glycosphingolipid is sufficiently dissolved or a mixed solution of the solvents.

  As the solvent to be used, a nonpolar solvent such as acetone, alcohol or chloroform having high polarity can be used alone, or a mixture with water can be used. Purification is carried out by combining these solvents and repeating elution and dissolution. It is also possible to use the method described in Non-Patent Document 6.

  In the present invention, as described above, the carotenoid pigment and the glycosphingolipid can be separately extracted and used, but can also be separated as a mixed composition. Such a composition is appropriately mixed with other ingredients and applied to skin compositions, cosmetic soaps and facial cleansers, cosmetics such as UV absorbers and moisturizing creams, and pharmaceuticals such as anti-atopic drugs. It is also possible to use an external composition for skin.

  Next, specific examples for producing ubiquinone Q-10 using the strain of the present invention are shown below. As in the case of astaxanthin production and carotenoid pigment production, the medium is not particularly limited as long as it contains carbon and nitrogen sources, inorganic salts and trace elements (vitamins and trace metals) necessary for the growth of the producing bacteria.

  The culture conditions can also be essentially the same as in the case of astaxanthin production and carotenoid pigment production.

  Finally, ubiquinone Q-10 is obtained from the cultured microorganism. Extract with organic solvent directly from cultured microorganisms or from centrifugally collected precipitates. As the solvent used here, any solvent can be used as long as it is a solvent in which ubiquinone Q-10 is sufficiently dissolved or a mixed solution of solvents.

  As the solvent to be used, a nonpolar solvent such as acetone, alcohol or chloroform having high polarity can be used alone, or a mixture with water can be used. Purification is carried out by combining these solvents and repeating elution and dissolution.

  In the present invention, not only the culture of the above-mentioned novel bacterial strain, but also other carotenoid-producing bacterial strains, cultures of glycosphingolipid-producing bacterial strains, or dried cells, frozen cells of these bacterial strains, It is possible to produce carotenoid pigments and / or glycosphingolipids by adding a solvent extract or a pulverized / crushed product to a culture of zooplankton and feeding on it. According to this production method, it is also possible to provide a feed enriched with carotenoid pigments.

  Examples of the zooplankton used in the feed of the present invention and the carotenoid pigment and / or glycosphingolipid production method of the present invention include rotifers, artemia and daphnia. These zooplankton are biologically enriched by feeding on carotenoid-producing bacterial strains and glycosphingolipid-producing bacterial strains, and the resulting zooplankton cultures are produced by conventional bacterial strain cultures. Compared to carotenoid pigments and glycosphingolipids, they are much more concentrated and their utility value is high.

  The feed produced by the method for producing carotenoid pigments and / or glycosphingolipids of the present invention using concentration by zooplankton can be suitably used in cultured fish. Some juveniles feed on crustacean plankton containing carotenoid pigments, and it is known that the color of fish is affected by this feeding. Therefore, when these fry are cultivated, if the feed of the present invention is fed as a coloring feed, it becomes possible to feed carotenoid pigments concentrated by zooplankton, which is more efficient than before. The fish can be deep-fried.

  EXAMPLES Hereinafter, although an Example and a comparative example are given and this invention is demonstrated in detail, this invention is not limited to these Examples.

Acquisition of Astaxanthin and Glycosphingolipid Simultaneously Producing Microorganisms We tried to acquire microorganisms that produce astaxanthin and glycosphingolipid from marine environment and sea sand, mud and water collected from mangrove forests. A sample of 100 μl was applied to an agar plate prepared by adding 5.0 g / l of yeast extract, 1.0 g / l of peptone, and 5.0 g / l of glucose to artificial seawater (Senju Pharmaceutical).

  Ground culture was performed for 7 to 10 days at 25 ° C., and marine microorganisms forming orange and red colonies were obtained. In order to turn these microorganisms into single bacteria, inoculate them in a liquid medium containing 5 ml of the above ingredients, grow them by shaking culture (150 rpm), apply them to an agar plate and repeat colony formation. Bacterialized.

  The pigment and lipid were extracted from 0.3 mg of cell powder composed of about 50 strains of marine bacteria with a chloroform-methanol (1: 1) solution, and astaxanthin and glycosphingolipid were screened.

  The presence or absence of astaxanthin and glycosphingolipid was evaluated using TLC (Thin Layer Chromatography). As a result, a strain producing only astaxanthin and glycosphingolipid was identified and deposited (JPCCMB0017).

  Strain JPCCMB0017 was an absolute aerobic gram-negative bacilli. From the phylogenetic analysis of 16S rDNA, it was found that this strain belongs to α-Proteobacteria and forms a cluster with the genus Sphingomonas. JPCCMB0017 was confirmed to be a new bacterium belonging to the genus Sphingomonas since it also contains sphingolipid, which is a characteristic of the genus Sphingomonas.

Separation of Astaxanthin from JPCCMB0017 Strain Culture A marine broth (Difco) medium containing all nutrients necessary for growth of the strain JPCCMB0017 was prepared and sterilized at 121 ° C. for 15 minutes. After collecting 250 ml of the medium in a 500 ml Erlenmeyer flask, this strain was inoculated and cultured at 25 ° C. for 3 days under the condition of 150 rpm. This culture solution was centrifuged and freeze-dried to obtain 0.6 g of dried cells. After the pigment was extracted from this cell with a dichloromethane: methanol (3: 1) solution, the silica gel was suspended in a chloroform: methanol 15: 1 moving bed, and the pigment was collected using an open column (caliber 30 mm, total length 600 mm). Separation.

  The pigment extracted from this strain was divided into 8 fractions (FIG. 1). As a result of fractionating each fraction and analyzing by HPLC, the presence of a substance showing the same spectrum as that of known astaxanthin was confirmed for fraction 2. Furthermore, the analysis using LC / MS was consistent with the known one.

Acquisition of glycosphingolipids The strain JPCCMB0017 was inoculated into 250 ml of marine broth medium and cultured at 25 ° C. and 150 rpm for 3 days. After centrifugation, 0.5 g of dried cells were obtained by lyophilization. To this dried cell freeze-dried cell, chloroform-methanol (2: 1, v / v, CM) was added and stirred to extract glycosphingolipid. This operation was repeated twice. Further, chloroform-methanol (1: 3) was added to the extraction residue, and the mixture was further extracted by refluxing at 80 ° C. for 1 hour. This operation was repeated twice.
All of the obtained extracts were mixed and dried by a rotary evaporator. 0.1 M NaOH was added to the obtained solid content, and hydrolysis was performed in a water bath at 100 ° C. for 30 minutes. Further, the solution was neutralized with 1 M HCl, dialyzed and lyophilized. The precipitate was resuspended in chloroform-methanol (2: 1), and the glycosphingolipid was purified using a silica gel column packed with chloroform. 1.6 mg of glycosphingolipid was obtained from JPCCMB0017 strain.
The purified glycosphingolipid of JPCCMB0017 strain was confirmed using a silica gel plate. Analysis was performed using a developing solvent chloroform: methanol: water (65: 16: 2). The detection was performed visually using a color reagent containing anisaldehyde (anisaldehyde: sulfuric acid: ethanol (1: 1: 18)), and as a result, glycosphingolipids having glucose in the sugar chain were detected.

Concentration of astaxanthin by zooplankton JPCCMB0017 strain was inoculated into 250 ml of marine broth medium and cultured at 25 ° C. and 150 rpm for 3 days. 1 L of a culture solution containing bacterial cells was prepared. The zooplankton rotifer and Artemia were grown by aeration at 27 ° C. The grown rotifer and artemia were added to the culture solution of the strain JPCCMB0017, and the strain was preyed on by the rotifer and artemia under shaking culture at 150 rpm.

  After 5 hours, rotifer and artemia zooplankton that had sufficiently incorporated the strain were strained with a rough mesh and washed with artificial seawater. The washed rotifer and artemia were collected by centrifugation and freeze-dried. Astaxanthin contained in rotifer and artemia was quantified using about 0.1 mg of dried cells.

  As a result, it was found that both rotifer and artemia zooplankton contained about 1% by weight of astaxanthin per dry cell. This ratio is about 10 times the ratio of astaxanthin normally contained in bacteria itself. Therefore, in the method of the present invention, it was found that by using the concentration by zooplankton, a product with a high concentration can be obtained as compared with the case of directly using a conventional producer of carotenoid pigments.

Production of ubiquinone Q-10 using JPCCMB0017 Microbial culture containing yeast extract 1.0 g / l, peptone 5.0 g / l, glucose 5 g / l using commercially available reagents (Senju Pharmaceutical Co., Ltd.) that can easily produce artificial seawater A medium was prepared. After autoclave sterilization, JPCCMB0017 strain was inoculated into 250 ml of this medium and cultured for 3 days under conditions of 150 rpm and 25 ° C.
Three days later, the cells were collected by centrifugal collection, and 0.5 g of cell powder was obtained by freeze-drying.
Ubiquinone Q-10 was extracted from the microbial cells using 30 ml of a methanol: chloroform (1: 2) solution of 0.5 g of the microbial powder obtained from the culture. This operation was repeated three times, and the entire extraction solvent was dried by a rotary evaporator to obtain 50 mg powder. Acetone was added to this powder to purify ubiquinone Q-10. After filtration, ubiquinone Q-10 was quantified using HPLC (column: Inartsil ODS-3) from the acetone solution.
As a result, it was confirmed from the standard calibration curve that JPCCMB0017 produced 0.3 mg of ubiquinone Q-10 per g bacterial cells.

  The novel bacterial strain of the present invention is capable of simultaneously producing useful pigments such as astaxanthin and / or water-retaining glycosphingolipid, and further capable of producing ubiquinone Q-10. In the method for producing carotenoid pigments and / or glycosphingolipids of the present invention, the use of bacteria that do not have a thick cell wall as in the conventional production of pigments and the like, and the predation of bacterial cells by zooplankton By performing biological concentration, more concentrated pigments and glycosphingolipids can be obtained, and these can be used as feed additives for farmed fish.

It is a figure which shows a fraction (1-8) at the time of isolate | separating astaxanthin from a culture of a strain JPCCMB0017 using an open column.

Explanation of symbols

  1-8 ... Dye fraction eluted from open column

Claims (15)

  Sphingomonas bacterium strain JPCCMB0017, which has carotenoid pigment production ability and glycosphingolipid production ability, and the base sequence of 16S rDNA is the sequence described in SEQ ID NO: 1.   A bacterial strain of the genus Sphingomonas, wherein the base sequence of 16S rDNA has at least 96% homology with the sequence set forth in SEQ ID NO: 1, and has carotenoid pigment production ability and glycosphingolipid production ability.   The bacterial strain according to claim 1 or 2, wherein the carotenoid pigment is astaxanthin.   A bacterial culture obtained by culturing the bacterial strain according to any one of claims 1 to 3.   A method for producing carotenoid pigments and / or glycosphingolipids, comprising a culture step of culturing the bacterial strain according to any one of claims 1 to 3.   The method for producing carotenoid pigments and / or glycosphingolipids according to claim 5, further comprising the step of separating the carotenoid pigment and / or glycosphingolipid from the culture obtained in the culture step after the culture step.   A composition containing a carotenoid pigment and / or a glycosphingolipid produced by the production method according to claim 5 or 6. A carotenoid-producing bacterial strain, a glycosphingolipid-producing bacterial strain, or a culture of the bacterial strain according to any one of claims 1 to 3, or a dried cell, frozen cell, solvent extract of these bacterial strains, Or a step of adding the pulverized / crushed product to the culture solution of zooplankton;
Feeding the zooplankton with the bacterial strain culture, fungus body, extract, or crushed and crushed product; and optionally collecting the zooplankton from the solution;
A method for producing a carotenoid pigment and / or a glycosphingolipid.   A carotenoid-producing bacterial strain, a glycosphingolipid-producing bacterial strain, or a culture of the bacterial strain according to any one of claims 1 to 3, or a dried cell, frozen cell, solvent extract of these bacterial strains, Alternatively, feed that is obtained by precipitating crushed and crushed products to zooplankton.   Sphingomonas bacterium strain JPCMB0017, which has the ability to produce ubiquinone Q-10 and whose base sequence of 16S rDNA is the sequence set forth in SEQ ID NO: 1.   A bacterial strain of the genus Sphingomonas, wherein the base sequence of 16S rDNA has at least 96% homology with the sequence shown in SEQ ID NO: 1 and has the ability to produce ubiquinone Q-10.   A bacterial culture obtained by culturing the bacterial strain according to claim 10 or 11.   The production method of ubiquinone Q-10 including the culture | cultivation process which culture | cultivates the bacterial strain of Claim 10 or 11.   The method for producing ubiquinone Q-10 according to claim 13, further comprising a step of separating ubiquinone Q-10 from the culture obtained in the culturing step after the culturing step. A composition comprising a carotenoid pigment and / or glycosphingolipid and ubiquinone Q-10 produced by the method of claim 13.

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