JPWO2011052003A1 - Novel bacterial strain, culture, carotenoid pigment-containing composition, and method for producing carotenoid pigment - Google Patents

Abstract

The present invention provides a novel bacterium having a carotenoid pigment-producing ability and a method for producing a carotenoid pigment using the bacterium. The present invention relates to Sphingomonas sp. JPCC MB0017-6 strain (Accession No .: NITE BP-808); a culture obtained by culturing such a bacterial strain; a carotenoid pigment-containing composition isolated from such a culture. A method for producing a carotenoid pigment comprising culturing the bacterial strain.

Description

  The present invention relates to a novel mutant strain of a bacterium belonging to the genus Sphingomonas, a culture obtained using the mutant strain and a carotenoid pigment-containing composition, and a method for producing a carotenoid pigment using the mutant strain .

  Various carotenoid pigments such as astaxanthin and β-carotene have many physiological activities, and are useful as raw materials for supplement foods. On the other hand, due to the recent decrease in natural resources, the cultivation of these natural resources is expected, and in the production of artificial seeds and seedlings of fish and shellfish, there is a demand for feed that provides high feed effect and added value to the target species. . Under such circumstances, feed (colored fried feed) containing astaxanthin may be used to bring the color of cultured fish such as Thailand and salmon closer to natural ones. In addition, feeds containing pigments such as astaxanthin may be used for fish species such as Thailand and rainbow trout that require vivid body colors.

Astaxanthin as an additive to deep-fried feed is sold by Roche as calorie pink (synthetic astaxanthin). However, astaxanthin derived from nature has been attracting attention because of traceability problems and users' preference for natural products.
Astaxanthin is widely distributed in fish such as red sea bream and salmon; crustaceans such as crabs, shrimps and krill. Therefore, a method for extracting astaxanthin from crustaceans has been proposed as a method for obtaining naturally-derived astaxanthin. However, since the extraction efficiency is low, there is a problem that the extraction amount is small and the cost is high. In addition, due to the decrease in natural resources, there were commercial problems in terms of securing resources.

  On the other hand, as a microorganism producing astaxanthin, Haematococcus pluviaris, which is a green alga, is best known, and in addition, Xanthophyllomyces dendrous (formerly Phaffia rhodozyma), which is a red yeast, is known. It has been reported that the amount of astaxanthin produced by Haematococcus pluvialis is about 43 mg / g dry weight (see Non-Patent Document 1). In addition, it has been reported that the amount of astaxanthin produced by Xanthophyllomyces dendrous (formerly Phaffia rhodozyma) is about 0.4 mg / g dry weight (see Non-Patent Document 2).

  Thus, as a method of blending naturally derived astaxanthin into feed, a method of blending Hematococcus pluviaris or Xanthophyllomyces dendrhaus as it is has been proposed, but there are problems such as insufficient coloring. This was because the cell walls of Haematococcus plubialis and Xanthophyllomyces dendrous were thick, and the digestion / absorption rate in the target fish species was low. Therefore, a new microorganism having a high absorption rate is required.

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 yeast. It is. So far, in the production of astaxanthin by bacteria, 1.4 mg / g dry weight has been achieved with Escherichia coli gene recombinants (see Non-Patent Document 3). Moreover, in Bacillus firmus, the production amount of 0.05 mg / g dry weight was achieved (refer nonpatent literature 4). Furthermore, Paracoccus sp. In MBIC 1143, the production amount of 0.14 mg / g dry weight has been reported (see Non-Patent Document 5). However, production of astaxanthin by these gene recombinants has a problem in expression level and the like.
Therefore, bacteria that produce astaxanthin were searched, and bacteria belonging to the genus Flavobacterium, Alkaligenes, Pseudomonas, Alteromonas, Pipomonas, Caryophanone, Erytlobacter, Paracoccus have been reported so far. .

  In addition, the present inventors have reported that JPCMMB0017 strain (NITE P-48), which is a bacterium belonging to the genus Sphingomonas, has a carotenoid pigment producing ability (see Patent Document 1). ).

JP 2006-191919 A

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 Microbiology and Biotechnology, 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

However, bacteria having the ability to produce various carotenoid pigments including astaxanthin have not yet been fully searched, and development of a method for producing carotenoid pigments with higher production efficiency is strongly desired.
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a novel bacterium having a carotenoid pigment-producing ability and a method for producing a carotenoid pigment using the bacterium.

In order to solve the above problems, the present inventors have intensively studied and have completed the following invention.
(1) Sphingomonas sp. JPCC MB0017-6 strain (Accession number: NITE BP-808).
(2) A culture obtained by culturing the bacterial strain described in (1).
(3) A carotenoid pigment-containing composition separated from the culture according to (2).
(4) A method for producing a carotenoid pigment comprising the step of culturing the bacterial strain according to (1).

  ADVANTAGE OF THE INVENTION According to this invention, the novel bacterium which has a carotenoid pigment-producing ability, and the manufacturing method of the carotenoid pigment | dye using this bacterium are provided. Moreover, since the bacterium of the present invention has a high ability to produce carotenoid pigments, high-quality carotenoid pigments can be produced easily and in large quantities. As a result, an inexpensive carotenoid pigment can be provided.

It is the imaging data of the colonies of JPCC MB0017-6 strain and JPPCMB0017 strain.

The present invention will be described in detail below.
The Sphingomonas sp. JPCC MB0017-6 strain (Accession number: NITE BP-808) of the present invention is a variant of the Sphingomonas sp. JPCMBB strain. Hereinafter, in the present specification, “Sphingomonas sp. JPCMBB0017 strain” may be abbreviated as “wild strain”. “Sphingomonas sp. JPCC MB0017-6 strain (accession number: NITE BP-808)” is sometimes abbreviated as “mutant strain”.
In the present invention, the “carotenoid pigment” refers to a pigment having a polyene structure having 40 carbon atoms as a basic skeleton, and includes hydrocarbons such as carotene and lycopene, and alcohols such as xanthophyll, and β-carotene. Examples of various useful carotenoid pigments produced in the process of converting astaxanthin and β-carotene into astaxanthin.
More specifically, as carotenoid pigments, carotene, lycopene, astaxanthin, adonixanthin, zeaxanthin, adonilvin, canthaxanthin, cryptoxanthin, echinone, hydroechinone, lutein, fucoxanthin, anthaxanthin, violaxanthin, β- Examples include cryptoxanthin.

<Acquisition of mutant strain>
The mutant strain of the present invention was obtained by performing chemical mutation on the wild strain. More specifically, it is as follows.

(Acquisition of wild strains)
The wild strain was obtained by the present applicant as of December 3, 2004, from the Patent Microorganism Depositary Center for Product Evaluation Technology, Japan (2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan) -0818)) (deposit number: NITE P-48).
In addition, the present inventors have acquired a wild strain from the nature upon deposit, and the acquisition procedure is shown below.

  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 (manufactured by Senju Pharmaceutical).

  The stationary culture was performed at 25 ° C. for 7 to 10 days to obtain marine microorganisms forming orange and red colonies. In order to isolate these microorganisms, an operation of inoculating them in a liquid medium containing 5 ml of the above-mentioned components, growing them by shaking culture at 150 rpm, and applying them to an agar plate to form colonies. Repeated.

  Pigments and lipids were extracted from 0.3 mg of cell powder consisting of about 50 strains of marine bacteria with a solution of chloroform: methanol = 1: 1 and screened for astaxanthin and glycosphingolipids. Wild strains also have the ability to produce glycosphingolipids.

  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).

  The wild strain is an absolute aerobic gram-negative bacilli. From the phylogenetic analysis of 16S rDNA, this wild strain belongs to α-Proteobacteria, forms a cluster with the genus Sphingomonas, and also contains the sphingolipid characteristic of the genus Sphingomonas. Has been confirmed to be a bacterium.

(Chemical mutation)
The wild strain was subjected to chemical mutation using a DNA alkylating agent according to the following procedure.
First, wild strains were cultured in marine broth (hereinafter abbreviated as MB) 2216 medium (manufactured by Becton Dickinson) for 24 hours and collected by centrifugation.
Subsequently, 1 × 10 ⁹ cells of JPCMBB0017 strain was added at 30 ° C. for 30-60 in 1 ml of pH 7.0 15 mM phosphate buffer containing 1.0-5.0% ethyl methanesulfonate (EMS). Incubation for min and mutagenesis was performed.
Next, the obtained cells were washed twice with phosphate buffered saline (hereinafter abbreviated as PBS), and then reincubated with the MB2216 medium at 30 ° C. for 2 hours.
Next, after washing with PBS, the obtained bacterial cells were applied to an MB agar medium and incubated at 30 ° C. for 3 to 7 days.
As a result, mutant strains were obtained.

  Also, instead of 15 mM phosphate buffer pH 7.0 containing 1.0-5.0% ethyl methanesulfonate (EMS), 5.0-10% N-methyl-N′-nitro-N— Mutant strains were also obtained by the same procedure as above except that a pH 7.0 15 mM phosphate buffer containing nitrosoguanidine (NTG) was used.

<Properties of mutant strain>
The acquired mutants were all the same as the wild type except for the color of the colonies. More specifically, it is as follows.

(1) Morphological properties Cell shape: Aspergillus Cell size: (0.6-0.7) × (2.0-3.0 μm) (with extension type)
Presence of spores:-
Motility (flagellar state): −
Polymorphism:-

(2) Culture characteristics (2-1) Medium: Marine agar (Marine broth 2216 (Becton Dickinson) + 1.5% agar)
Temperature: 25 ° C
Pigment production: +
Color: Red Gloss: +
(2-2) Medium: Marine Broth 2216
Temperature: 25 ° C
Surface development:-
Medium turbidity: +
(2-3) Gelatin puncture culture Temperature: 25 ° C
Growth:-
Gelatin liquefaction:-
(2-4) Litmus milk Temperature: 25 ° C
Coagulation:-
Liquefaction:-

  Wild-type colonies are orange, whereas mutant-type colonies are red as described above, and mutants can be clearly distinguished from wild-types in terms of color tone. FIG. 1 shows imaging data of colonies of mutant and wild strains, respectively. In FIG. 1, the right side is a mutant colony, and the left side is a wild-type colony.

(3) Physiological properties Gram staining:-
Reduction of sulfate: +
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 ° C): + (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 production / gas production from saccharides L-arabinose:-/-
D-glucose:-/-
D-fructose:-/-
Maltose:-/-
Lactose:-/-
D-sorbitol:-/-
Inositol:-/-
D-xylose:-/-
D-Mannose:-/-
D-galactose:-/-
Circus:-/-
Trehalose:-/-
D-mannitol:-/-
Glycerin:-/-

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

(5) Assessability 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

(6) 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: +

<Base sequence of mutant 16S rDNA>
As a result of identifying the 16S rDNA base sequence of the mutant strain by a known method, it was as shown in SEQ ID NO: 1 and was the same as the wild strain.

<Mutant, culture, carotenoid pigment-containing composition>
As will be described later, the mutant strain of the present invention is a novel bacterium having a carotenoid pigment producing ability significantly higher than that of the wild strain under the same conditions. As described above, a bacterium having a high carotenoid pigment-producing ability has not been known so far in bacteria belonging to the genus Sphingomonas.
In addition, the mutant strain of the present invention is a bacterium belonging to the genus Sphingomonas, which has the ability to produce various carotenoid pigments and can produce a desired carotenoid pigment by adjusting the culture temperature. Has excellent properties not seen.

  The mutant strain of the present invention has an accession number of NITE BP-808 dated September 04, 2009, and is incorporated by the Incorporated Administrative Agency, National Institute of Technology and Evaluation of Microorganisms (2-5 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan) 8 (zip code 292-0818).

  The culture obtained by culturing the mutant strain contains the mutant strain having the produced carotenoid pigment. The culture may be used as it is for the intended purpose, or may be used after performing an arbitrary purification operation. For example, a composition containing a carotenoid pigment may be separated from a mutant after culturing, and the composition may be used for the intended use. Further, the composition is purified to improve purity. Carotenoid pigments may be used.

<Method for producing carotenoid pigment>
The method for producing a carotenoid pigment of the present invention includes a step of culturing the mutant strain of the present invention. The composition of the carotenoid pigment produced by the mutant strain can be adjusted by appropriately adjusting the culture temperature.
Mutant strains can be cultured in the same manner as for wild strains. Specifically, it is as follows.

The medium is not particularly limited as long as it contains essential components such as a carbon source, a nitrogen source, inorganic salts, and trace components necessary for growth.
Examples of the carbon source include sugars such as glucose and sucrose; alcohols such as ethanol and glycerol.
The addition ratio of the carbon source is preferably about 0.5 to 3.0% by mass although it depends on the type of the carbon source.

Examples of the nitrogen source include sodium nitrate, ammonium nitrate, ammonium sulfate, potassium nitrate, ammonium chloride, urea and the like.
Although the addition ratio of a nitrogen source is based also on the kind of nitrogen source, it is preferable that it is about 0.01%-0.1 mass%.

Examples of the inorganic salts include sodium chloride, sodium sulfate, calcium chloride, potassium chloride, sodium bicarbonate, potassium boride, strontium chloride, boric acid, sodium silicate, sodium fluoride, monopotassium phosphate, dipotassium phosphate, Examples thereof include monosodium phosphate, disodium phosphate, iron chloride, manganese chloride, manganese sulfate, magnesium chloride, and copper sulfate.
The addition ratio of the inorganic salt is preferably about 0.001% to 0.01% by mass, although it depends on the kind of the inorganic salt.

  Examples of the trace component include vitamins and trace metals.

The culture medium may contain optional components other than the essential components. Examples of optional components include yeast extract, peptone, and tryptone.
Although the addition ratio of an arbitrary component is based also on the kind of arbitrary component, it is preferable that it is about 0.01%-0.5 mass%.

  The pH of the medium is preferably 6.0 to 8.0.

  The culture temperature of the mutant strain can be arbitrarily selected within a range that does not hinder the growth of the mutant strain, but is preferably 20 to 45 ° C.

The production efficiency of the mutant carotenoid pigment varies depending on the culture temperature for each type of carotenoid pigment. That is, by appropriately adjusting the culture temperature of the mutant strain to a temperature suitable for the production of the desired carotenoid pigment, the production amount of the desired carotenoid pigment can be improved, and carotenoid pigments can be created separately.
When improving the production amount of astaxanthin, the culture temperature is preferably 20 to 30 ° C, more preferably 23 to 27 ° C.
When improving the production amount of echinenone, the culture temperature is preferably 25 to 40 ° C, more preferably 27 to 33 ° C.
When improving the production amount of adonixanthin, the culture temperature is preferably 25 to 40 ° C, more preferably 33 to 37 ° C.
When improving the production amount of zeaxanthin, the culture temperature is preferably 30 to 45 ° C, more preferably 33 to 37 ° C.
When improving the production amount of canthaxanthin, the culture temperature is preferably 25 to 45 ° C, more preferably 33 to 37 ° C.
When improving the production amount of β-cryptoxanthin, the culture temperature is preferably 35 to 45 ° C, more preferably 38 to 42 ° C.
When improving the production amount of β-carotene, the culture temperature is preferably 35 to 45 ° C, more preferably 38 to 42 ° C.

And the production amount of a desired carotenoid pigment | dye can be improved as follows by adjusting the culture | cultivation temperature of a mutant as mentioned above.
For example, in the case of astaxanthin, the astaxanthin content in the dried microbial cells of the mutant strain can be 0.04% by mass or more, and the astaxanthin content in all carotenoid pigments can be 30% by mass or more.
In the case of echinenone, the echinenone content in the dry cells of the mutant strain can be 0.03% by mass or more, and the echinenone content in all carotenoid pigments can be 11% by mass or more.
In the case of adonixanthin, the adonixanthin content in the dried cells of the mutant strain can be 0.04% by mass or more, and the adonixanthin content in all carotenoid pigments can be 6.5% by mass or more.
In the case of zeaxanthin, the zeaxanthin content in the dried cells of the mutant can be 0.1% by mass or more, and the zeaxanthin content in the total carotenoid pigment can be 19.5% by mass or more.
In the case of canthaxanthin, the canthaxanthin content in the dried microbial cells of the mutant strain can be 0.005% by mass or more, and the canthaxanthin content in all carotenoid pigments can be 1.5% by mass or more.
In the case of β-cryptoxanthin, the β-cryptoxanthin content in the dry cells of the mutant can be 0.05% by mass or more, and the β-cryptoxanthin content in all carotenoid pigments can be 6% by mass or more. .
In the case of β-carotene, the β-carotene content in the dried cells of the mutant strain can be 0.5 mass% or more, and the β-carotene content in the total carotenoid pigment can be 65 mass% or more.

  Thus, the carotenoid pigment-containing composition isolated from the mutant culture is a composition belonging to the genus Sphingomonas, including wild strains, obtained from a culture of bacteria having the ability to produce carotenoid pigments. The content of various carotenoid pigments is remarkably high. In addition, for example, the content of specific carotenoid pigments such as astaxanthin, zeaxanthin, and β-carotene can be greatly improved. Therefore, the carotenoid pigment-containing composition of the present invention is extremely useful.

The production amount (content) of the total carotenoid pigment in the mutant generally improves as the culture temperature increases.
For example, when the culture temperature is preferably 20 ° C. or higher, more preferably 23 ° C. or higher, the content of the total carotenoid pigment in the dried microbial cells of the mutant strain can be 0.12% by mass or higher.
Moreover, content of the total carotenoid pigment | dye in the dry microbial cell of a mutant can be made into 0.25 mass% or more by making culture | cultivation temperature preferably 25 degreeC or more, More preferably, 27 degreeC or more.
Moreover, content of the total carotenoid pigment | dye in the dry microbial cell of a mutant can be 0.55 mass% or more by making culture | cultivation temperature preferably 30 degreeC or more, More preferably, 33 degreeC or more.
Moreover, content of the total carotenoid pigment | dye in the dry microbial cell of a mutant can be 0.78 mass% or more by making culture | cultivation temperature preferably 35 degreeC or more, More preferably, 38 degreeC or more.

Although the culture time depends on the culture temperature, it is usually preferably 1 to 3 days.
The culture method may be appropriately selected depending on the type of medium, such as stationary culture, shaking culture, and stirring culture. And it is preferable to carry out aeration culture.

After culturing, the carotenoid pigment can be separated from the culture or from the sediment recovered by centrifugation of the culture.
For example, the culture or sediment is washed as necessary, and then subjected to freeze-drying or the like to obtain dry cells. And an organic solvent is added to the obtained dry microbial cell, carotenoid pigment | dye is extracted in this organic solvent, and this extraction operation is repeated in multiple times as needed. Subsequently, the carotenoid pigment is obtained by removing the solvent by a technique such as vacuum concentration.

The organic solvent used for extraction is not particularly limited as long as it can dissolve the carotenoid pigment, and any of a polar solvent and a nonpolar solvent can be used. Preferable examples include hydrocarbons such as hexane; ketones such as acetone; alcohols such as methanol, ethanol and 2-propanol; esters such as ethyl acetate; halogenated hydrocarbons such as dichloromethane and chloroform. Nitriles such as acetonitrile can be exemplified.
An organic solvent may be used individually by 1 type, and may use 2 or more types together. When two or more kinds are used in combination, the combination and ratio may be arbitrarily selected according to the purpose.
These organic solvents can also be used for the purification of carotenoid pigments.

  Carotenoid pigments can be identified, for example, by comparing their analytical data with analytical data of known compounds. Any analysis method may be used as long as it can acquire data related to the structure. For example, a commonly used analysis method such as HPLC, absorbance analysis, NMR, or LC / MS may be used. For example, if a calibration curve is prepared at the time of analysis by HPLC, the content of each component in the obtained composition can also be quantified.

  After the culture obtained by culturing the mutant strain, or the dried strain of the mutant strain, the frozen cell body, the solvent extract, or the crushed and crushed processed product is added to the zooplankton culture and preyed, Carotenoid pigments can also be produced by recovering zooplankton. According to this production method, a feed enriched with carotenoid pigments can also be provided.

  Examples of the zooplankton include rotifers, artemia, daphnia and the like. These zooplanktons are biologically enriched by preying on mutants, and the resulting zooplankton cultures have a much higher concentration of carotenoid pigments than those produced by conventional bacterial cultures. And its utility value is extremely high.

  A feed enriched with carotenoid pigments by zooplankton is suitable for use in farmed 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 produced by the above method is fed as a fried food, the concentrated carotenoid pigment can be fed, and the fish can be colored more efficiently than before.

  Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.

[Example 1]
<Production of carotenoid pigment using mutant strain (1)>
MB2216 (37.4 g), 3 g of glucose and 1 L of pure water were mixed to prepare a medium, and the medium was sterilized at 121 ° C. for 20 minutes in a 1 L media bottle.
Next, after cooling the medium to room temperature, 200 ml of this was dispensed into a 1 L Erlenmeyer flask in a clean bench, and 10 ml of the mutant strain previously cultured in a 10 ml L-shaped test tube was inoculated into the dispensed medium. did. The pH of the medium after inoculation was 7.6 to 7.8.
Subsequently, the mutant strain was cultured by shaking for 2 days under the conditions of a culture temperature of 30 ° C. and a stirring speed of 120 rpm using a constant temperature shake incubator bioshaker (trade name, manufactured by Taitec Co., Ltd.).
Subsequently, the culture was centrifuged to recover the mutant strain, freeze-dried, and then crushed in a mortar to obtain powdered dry cells.
And the carotenoid pigment | dye was extracted and isolate | separated from the obtained dried microbial cell using the mixed solvent of dichloromethane / methanol (3/1, volume ratio).
The separated carotenoid pigment was dissolved in methanol, and contaminants were removed from the obtained solution using a filter to prepare a sample for HPLC analysis. In addition, the separated carotenoid pigment was dissolved in acetone, and impurities were removed from the obtained solution using a filter to obtain a sample for absorption spectrum analysis. Then, HPLC analysis and absorption spectrum analysis were performed under the following conditions. Calculate the ratio of the content of each carotenoid pigment from the ratio of peak intensity (area value) in the profile of the carotenoid pigment at the time of HPLC analysis, and calculate the total carotenoid pigment content (total of carotenoid pigments) from the data at the time of absorption spectrum analysis. Content) and the content of each carotenoid pigment was calculated from these values.

(HPLC analysis conditions)
HPLC analyzer: LC-VP series (manufactured by Shimadzu Corporation)
Column: Reversed phase column Symmetry C18 (5 μm, 4.6 × 250 mm) (manufactured by Waters)
Column temperature: 40 ° C
Mobile phase: Mixed solvent of acetonitrile / methanol / 2-propanol (45/3/2, volume ratio) Mobile phase flow rate: 0.5 mL / min (absorption spectrum analysis)
Spectrophotometer: UV-2400 (manufactured by Shimadzu Corporation)
Cell: 1cm width

  As a result, the content of total carotenoid pigment in 1 g of dry cells was 1.246 mg (0.125% by mass), and the content of astaxanthin in 1 g of dry cells was 0.16 mg (0.016% by mass). Met. The content of astaxanthin in the total carotenoid pigment was 12.8% by mass.

[Comparative Example 1]
<Production of carotenoid pigment using wild type strain (1)>
A powdered dry cell was obtained in the same manner as in Example 1 except that the wild strain was used instead of the mutant strain, and the carotenoid pigment was separated.
As a result, the content of total carotenoid pigment in 1 g of the obtained dried cells was 0.162 mg (0.016% by mass), and the content of astaxanthin in 1 g of the dried cells was 0.01 mg (0.001 Mass%). The content of astaxanthin in the total carotenoid pigment was 6.2% by mass.

  As is clear from the results of Example 1 and Comparative Example 1, the content of total carotenoid pigment in the mutant strain (production amount of total carotenoid pigment) is about 8 times that of the wild strain, and the content of astaxanthin in the mutant strain is It was about 16 times the wild type.

[Example 2]
<Production of carotenoid pigment using mutant strain (2)>
Soy protein-derived peptide: High New R (trade name, manufactured by Fuji Oil Co., Ltd.) 10 g, yeast 1 g, iron iron citrate III 0.1 g, ammonium sulfate 0.0016 g, disodium hydrogen phosphate 0.008 g, glucose 3 g, artificial seawater 37 g Then, 4 L of a medium was prepared by mixing 1 L of pure water, and the medium was sterilized at 121 ° C. for 20 minutes in a 10 L container of a microorganism culture apparatus BML-10PI (trade name, manufactured by Able).
Next, the container was set in a reaction part of a microorganism culture apparatus, and the medium was cooled to room temperature, and then 200 ml of a mutant strain previously cultured in a 1 L Erlenmeyer flask in a clean bench was inoculated into the medium. The pH of the medium after inoculation was 7.6 to 7.8.
Subsequently, the mutant strain is shaken for 2 days under the conditions of a culture temperature of 25 ° C., a stirring speed of 200 rpm, and an aeration rate of 1 vvm (aeration rate (volume) per minute is 1 times the culture solution (4.2 L)). Cultured.
Subsequently, the culture was centrifuged to recover the mutant strain, freeze-dried, and then crushed in a mortar to obtain powdered dry cells.
And the carotenoid pigment | dye was extracted and isolate | separated from the obtained dried microbial cell by the method similar to Example 1, and content of each carotenoid pigment | dye was computed.
Table 1 shows the composition of the carotenoid pigment in 1 g of the obtained dried cells.
As is clear from Table 1, astaxanthin had the highest content among all carotenoid pigments.

[Example 3]
<Production of carotenoid pigment using mutant strain (3)>
The carotenoid pigment was separated and the content of each carotenoid pigment was calculated in the same manner as in Example 2 except that the culture temperature of the mutant strain was 30 ° C. instead of 25 ° C.
Table 2 shows the composition of the carotenoid pigment in 1 g of the obtained dried cells.
As is clear from Table 2, β-carotene had the highest content in all carotenoid pigments.

[Example 4]
<Production of carotenoid pigment using mutant strain (4)>
Carotenoid pigments were separated in the same manner as in Example 2 except that the culture temperature of the mutant strain was 35 ° C instead of 25 ° C, and the content of each carotenoid pigment was calculated.
Table 3 shows the composition of the carotenoid pigment in 1 g of the obtained dried cells.
As is clear from Table 3, β-carotene had the highest content in all carotenoid pigments.

[Example 5]
<Production of carotenoid pigment using mutant strain (5)>
Carotenoid pigments were separated and the content of each carotenoid pigment was calculated in the same manner as in Example 2 except that the culture temperature of the mutant strain was 40 ° C instead of 25 ° C.
Table 4 shows the composition of the carotenoid pigment in 1 g of the obtained dried cells.
As is clear from Table 4, β-carotene had the highest content among all carotenoid pigments.

[Comparative Example 2]
<Production of carotenoid pigment using wild type strain (2)>
Carotenoid pigments were separated and the content of each carotenoid pigment was calculated in the same manner as in Example 2 except that a wild strain was used instead of the mutant strain and the culture temperature was changed to 30 ° C. instead of 25 ° C.
Table 5 shows the composition of the carotenoid pigment in 1 g of the obtained dried cells.

As is clear from Table 5, in Comparative Example 2 in which the wild strain was cultured, production of astaxanthin, zeaxanthin, canthaxanthin, echinenone and β-carotene was not observed. And the content of the total carotenoid pigment was also very low.
On the other hand, in Example 3 where the mutant strain was cultured at the same culture temperature as in Comparative Example 2, the total carotenoid pigment content was about 39 times that in Comparative Example 2, as is apparent from Tables 2 and 5.
And the content of the carotenoid pigment was significantly higher in Example 3 than in Comparative Example 2 in all types. When compared with the carotenoid pigments produced in Comparative Example 2, adonixanthin was about 18 times, and β-cryptoxanthin was about 31 times.

Further, as is apparent from Tables 1 to 4, the content of each carotenoid pigment could be improved by adjusting the culture temperature of the mutant strain.
Specifically, astaxanthin is cultured at 25 ° C., echinenone is cultured at 30 ° C., adonixanthin, zeaxanthin and canthaxanthin are cultured at 35 ° C., and β-cryptoxanthin and β-carotene are cultured at 40 ° C. , Each had the highest content.
Thus, the desired carotenoid pigment was efficiently obtained by appropriately adjusting the culture temperature.

  Furthermore, as is clear from Examples 1 to 5, when a carotenoid pigment was produced using a mutant strain, the ability to produce carotenoid pigment did not decrease even when the mutant strain was cultured in large quantities, and the production of carotenoid pigment was carried out. It was confirmed that the efficiency was extremely high.

  Since the present invention can be used for the production of health food ingredients and feed additives, it is extremely useful industrially.

NITE BP-808

[0001]
Technical field [0001]
The present invention relates to a novel mutant strain of a bacterium belonging to the genus Altererislobacter, a culture obtained using the mutant strain and a carotenoid pigment-containing composition, and a method for producing a carotenoid pigment using the mutant strain.
Background art [0002]
Various carotenoid pigments such as astaxanthin and β-carotene have many physiological activities, and are useful as raw materials for supplement foods. On the other hand, due to the recent decrease in natural resources, the cultivation of these natural resources is expected, and in the production of artificial seeds and seedlings of fish and shellfish, there is a demand for feed that provides high feed effect and added value to the target species. . Under such circumstances, feed (colored fried feed) containing astaxanthin may be used to bring the color of cultured fish such as Thailand and salmon closer to natural ones. In addition, feeds containing pigments such as astaxanthin may be used for fish species such as Thailand and rainbow trout that require vivid body colors.
[0003]
Astaxanthin as an additive to deep-fried feed is sold by Roche as calorie pink (synthetic astaxanthin). However, astaxanthin derived from nature has been attracting attention because of traceability problems and users' preference for natural products.
Astaxanthin is widely distributed in fish such as red sea bream and salmon; crustaceans such as crabs, shrimps and krill. Therefore, a method for extracting astaxanthin from crustaceans has been proposed as a method for obtaining naturally-derived astaxanthin. However, because extraction efficiency is low, the amount of extraction is small and the cost is high.

[0003]
43, the production amount of 0.14 mg / g dry weight has been reported (see Non-Patent Document 5). However, production of astaxanthin by these gene recombinants has a problem in expression level and the like.
Therefore, bacteria that produce astaxanthin were searched, and bacteria belonging to the genus Flavobacterium, Alkaligenes, Pseudomonas, Alteromonas, Pipomonas, Caryophanone, Erytlobacter, Paracoccus have been reported so far. .
[0007]
In addition, the present inventors have reported that JPCMMB0017 strain (NITE P-48), which is a bacterium belonging to the genus Sphingomonas, has a carotenoid pigment producing ability (see Patent Document 1). ). Further analysis revealed that JPCMB0017 strain (NITE P-48) belongs to the genus Alterellislobacter, not Sphingomonas.
Prior Art Literature Patent Literature [0008]
Patent Document 1: Non-Patent Document [0009] of JP-A-2006-191919
Non-Patent Document 1: Lee, Y.-K. and Soh, C.-W., “Journal of Phycolology”, USA, 1991, Vol. 27, No. 3, p. 575-577
Non-Patent Document 2: Flores-Cotera, LB et al., Applied Microbiology and Biotechnology (Appl. Microbiol. Biotechnol.), USA, 2001, Vol. 55 , No. 2, p. 341-347
Non-Patent Document 3: Wang et al. (Wang, C.-W. et al.), Biotechnology and Bioengineering (Biotechnol. Bioeng.), USA, 1999, Vol. 62, No. 3, p. 235-241
Non-Patent Document 4: Yokoyama et al., Bioscience, Biotechnology and Biochemistry (Bioscience, Biotechnology, and Biochemistry)

[0004]
y), USA, 1994, Vol. 58, No. 10, p. 1842-1844
Non-Patent Document 5: Pane, L. et al., Journal of Biology Research (J. Biol. Res.), USA, 1996, Vol. 22, p. 303-308
SUMMARY OF THE INVENTION Problems to be Solved by the Invention [0010]
However, bacteria having the ability to produce various carotenoid pigments including astaxanthin have not yet been fully searched, and development of a method for producing carotenoid pigments with higher production efficiency is strongly desired.
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a novel bacterium having a carotenoid pigment-producing ability and a method for producing a carotenoid pigment using the bacterium.
Means for Solving the Problems [0011]
In order to solve the above problems, the present inventors have intensively studied and have completed the following invention.
(1) Alter Ellis Lobacter genus JPCC MB0017-6 strain (Accession number: NITE BP-808).
(2) A culture obtained by culturing the bacterial strain described in (1).
(3) A carotenoid pigment-containing composition separated from the culture according to (2).
(4) A method for producing a carotenoid pigment comprising the step of culturing the bacterial strain according to (1).
Effects of the Invention [0012]
ADVANTAGE OF THE INVENTION According to this invention, the novel bacterium which has a carotenoid pigment-producing ability, and the manufacturing method of the carotenoid pigment | dye using this bacterium are provided. Moreover, since the bacterium of the present invention has a high ability to produce carotenoid pigments, high-quality carotenoid pigments can be produced easily and in large quantities. As a result, an inexpensive carotenoid pigment can be provided.

[0005]
BRIEF DESCRIPTION OF THE DRAWINGS [0013]
[FIG. 1] Imaging data of colonies of JPCC MB0017-6 strain and JPCCMB0017 strain.
MODE FOR CARRYING OUT THE INVENTION [0014]
The present invention will be described in detail below.
The Alter erythrobacter genus JPCC MB0017-6 strain (Accession number: NITE BP-808) of the present invention is a mutant of the Alter erythrobacter genus JPCMBB0017 strain. Hereinafter, in the present specification, the “Alter Ellis Lobacter genus JPCMBB0017 strain” may be abbreviated as “wild strain”. In addition, “Alter Ellis Lobacter genus JPCC MB0017-6 strain (accession number: NITE BP-808)” may be abbreviated as “mutant strain”.
In the present invention, the “carotenoid pigment” refers to a pigment having a polyene structure having 40 carbon atoms as a basic skeleton, and includes hydrocarbons such as carotene and lycopene, and alcohols such as xanthophyll, and β-carotene. Examples of various useful carotenoid pigments produced in the process of converting astaxanthin and β-carotene into astaxanthin.
More specifically, as carotenoid pigments, carotene, lycopene, astaxanthin, adonixanthin, zeaxanthin, adonilvin, canthaxanthin, cryptoxanthin, echinone, hydroechinone, lutein, fucoxanthin, anthaxanthin, violaxanthin, β- Examples include cryptoxanthin.
[0015]
<Acquisition of mutant strain>
The mutant strain of the present invention was obtained by performing chemical mutation on the wild strain. More specifically, it is as follows.
[0016]
(Acquisition of wild strains)
Wild strains are independent administrative corporations dated December 3, 2004 by the applicant.

[0006]
It is deposited at the Product Evaluation Technology Infrastructure Organization Patent Microorganism Deposit Center (2-5-8 Kazusa-Kamashita, Kisarazu City, Chiba Prefecture, Japan, postal code 292-0818) (Accession Number: NITE P-48).
In addition, the present inventors have acquired a wild strain from the nature upon deposit, and the acquisition procedure is shown below.
[0017]
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 (manufactured by Senju Pharmaceutical).
[0018]
The stationary culture was performed at 25 ° C. for 7 to 10 days to obtain marine microorganisms forming orange and red colonies. In order to isolate these microorganisms, an operation of inoculating them in a liquid medium containing 5 ml of the above-mentioned components, growing them by shaking culture at 150 rpm, and applying them to an agar plate to form colonies. Repeated.
[0019]
Pigments and lipids were extracted from 0.3 mg of cell powder consisting of about 50 strains of marine bacteria with a solution of chloroform: methanol = 1: 1 and screened for astaxanthin and glycosphingolipids. Wild strains also have the ability to produce glycosphingolipids.
[0020]
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).
[0021]
The wild strain is an absolute aerobic gram-negative bacilli. From the phylogenetic analysis of 16S rDNA, this wild strain belongs to α-Proteobacteria, forms a cluster with Altera erythrobacter genus, and also contains sphingolipid, which is characteristic of Alter erythrobacter genus. It has been confirmed that it is a bacterium belonging to the genus Erytlobacter.

[0013]
It is a novel bacterium with significantly higher productivity than the wild type. Thus, a bacterium having a high carotenoid pigment-producing ability has not been known so far in bacteria belonging to the genus Alterellislobacter.
In addition, the mutant strain of the present invention has the ability to produce various carotenoid pigments, and can control the culture temperature to produce the desired carotenoid pigments separately. It has excellent properties not found in
[0036]
The mutant strain of the present invention has an accession number of NITE BP-808 dated September 04, 2009, and is incorporated by the Incorporated Administrative Agency, National Institute of Technology and Evaluation of Microorganisms (2-5 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan) 8 (zip code 292-0818).
[0037]
The culture obtained by culturing the mutant strain contains the mutant strain having the produced carotenoid pigment. The culture may be used as it is for the intended purpose, or may be used after performing an arbitrary purification operation. For example, a composition containing a carotenoid pigment may be separated from the mutant after culturing, and the composition may be used for the intended use. Further, the composition is purified to improve purity. Carotenoid pigments may be used.
[0038]
<Method for producing carotenoid pigment>
The method for producing a carotenoid pigment of the present invention includes a step of culturing the mutant strain of the present invention. The composition of the carotenoid pigment produced by the mutant strain can be adjusted by appropriately adjusting the culture temperature.
Mutant strains can be cultured in the same manner as for wild strains. Specifically, it is as follows.
[0039]
The medium is not particularly limited as long as it contains essential components such as a carbon source, a nitrogen source, inorganic salts, and trace components necessary for growth.
Examples of the carbon source include sugars such as glucose and sucrose; alcohols such as ethanol and glycerol.
The addition ratio of the carbon source depends on the type of the carbon source, but is generally 0.5 to 3.0 mass.

[0016]
In the case of β-cryptoxanthin, the β-cryptoxanthin content in the dry cells of the mutant can be 0.05% by mass or more, and the β-cryptoxanthin content in all carotenoid pigments can be 6% by mass or more. .
In the case of β-carotene, the β-carotene content in the dried cells of the mutant strain can be 0.5 mass% or more, and the β-carotene content in the total carotenoid pigment can be 65 mass% or more.
[0048]
As described above, the carotenoid pigment-containing composition isolated from the mutant culture is a composition belonging to the genus Altera erythrobacter including wild strains, and obtained from a culture of bacteria having carotenoid pigment-producing ability. The content of various carotenoid pigments is remarkably higher than the product. In addition, for example, the content of specific carotenoid pigments such as astaxanthin, zeaxanthin, and β-carotene can be greatly improved. Therefore, the carotenoid pigment-containing composition of the present invention is extremely useful.
[0049]
The production amount (content) of the total carotenoid pigment in the mutant generally improves as the culture temperature increases.
For example, when the culture temperature is preferably 20 ° C. or higher, more preferably 23 ° C. or higher, the content of the total carotenoid pigment in the dried microbial cells of the mutant strain can be 0.12% by mass or higher.
Moreover, content of the total carotenoid pigment | dye in the dry microbial cell of a mutant can be made into 0.25 mass% or more by making culture | cultivation temperature preferably 25 degreeC or more, More preferably, 27 degreeC or more.
Moreover, content of the total carotenoid pigment | dye in the dry microbial cell of a mutant can be 0.55 mass% or more by making culture | cultivation temperature preferably 30 degreeC or more, More preferably, 33 degreeC or more.
Moreover, content of the total carotenoid pigment | dye in the dry microbial cell of a mutant can be 0.78 mass% or more by making culture | cultivation temperature preferably 35 degreeC or more, More preferably, 38 degreeC or more.
[0050]
The culture time is usually 1 to 3 days, although it depends on the culture temperature.

[0025]
[Table 5]
[0073]
As is clear from Table 5, in Comparative Example 2 in which the wild strain was cultured, production of astaxanthin, zeaxanthin, canthaxanthin, echinenone and β-carotene was not observed. And the content of the total carotenoid pigment was also very low.
On the other hand, in Example 3 where the mutant strain was cultured at the same culture temperature as in Comparative Example 2, the total carotenoid pigment content was about 39 times that in Comparative Example 2, as is apparent from Tables 2 and 5.
And the content of the carotenoid pigment was significantly higher in Example 3 than in Comparative Example 2 in all types. When compared with the carotenoid pigments produced in Comparative Example 2, adonixanthin was about 18 times, and β-cryptoxanthin was about 31 times.
[0074]
Further, as is apparent from Tables 1 to 4, the content of each carotenoid pigment could be improved by adjusting the culture temperature of the mutant strain.
Specifically, astaxanthin is cultured at 25 ° C., echinenone is cultured at 30 ° C., adonixanthin, zeaxanthin and canthaxanthin are cultured at 35 ° C., and β-cryptoxanthin and β-carotene are cultured at 40 ° C. , Each had the highest content.

Claims (4)

  Sphingomonas sp. JPCC MB0017-6 strain (Accession number: NITE BP-808).   A culture obtained by culturing the bacterial strain according to claim 1.   A carotenoid pigment-containing composition separated from the culture according to claim 2.   The manufacturing method of the carotenoid pigment | dye which has the process of culturing the bacterial strain of Claim 1.