JP2012139166A - New microorganism and production method of carotenoid using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microorganism storable of much more carotenoid or astaxanthin than conventionally reported microorganisms, in consideration of industrial carotenoid or astaxanthin production.SOLUTION: There is provided a carotenoid-productive genus Paracoccus microorganism producible of carotenoid of ≥17 mg per 1g of dried fungus body.

Description

  The present invention relates to a carotenoid-producing Paracoccus microorganism capable of producing a carotenoid of 17 mg or more per 1 g of dry cell weight.

  Astaxanthin is used for coloring when marine products such as trout, red sea bream, and salmon are cultivated. In recent years, its antioxidant activity has attracted attention, and it has also been applied to health foods such as cosmetics and supplements. ing. Astaxanthin can be obtained by extraction from crustaceans such as krill, but there is a problem in the stability of supply, and in addition, it requires a step of crushing the cell wall at the time of extraction, so the production method becomes complicated and the production cost is kept low Is difficult.

  On the other hand, a method for producing astaxanthin by culturing a marine Agrobacterium genus (later categorized as a bacterium belonging to the genus Paracoccus, strain N-81106 has been reported (see, for example, Patent Document 1). According to the method, it is possible to easily extract astaxanthin simply by mixing and stirring an organic solvent such as acetone after culturing the microorganism, and it is possible to simplify the manufacturing method and keep the manufacturing cost low. However, the amount of astaxanthin accumulated by microorganisms contained in 1 liter of medium (hereinafter referred to as “the amount of astaxanthin accumulated by microorganisms per liter of medium”) even if the amount of microbial cells reached the upper limit due to its growth. Etc.), and further improvement has been sought after. 06 strains of mutant breeding strains were reported, and the total amount of carotenoids per liter of culture medium was 400 mg, and the amount of astaxanthin contained in the microorganism TSN18E7 strain (Patent Document 2), the total amount of carotenoids per liter of culture medium was 720 mg. Microorganism TSTT052 accumulating 250 mg in the amount of astaxanthin in it (consigned as FERM P-20690 to the National Institute of Advanced Industrial Science and Technology (AIST) on October 18, 2005. (Deposited as FERM BP-10754 for the original deposit on the day) (Patent Document 3).

Japanese Patent Laid-Open No. 7-184668 JP-A-2005-58216 JP 2007-181449 A

  The mutant breeding strain of the N-81106 strain reported by the present applicant is a microorganism that can accumulate higher carotenoids per liter of culture medium than the N-81106 strain. However, considering the industrial production of carotenoids or astaxanthin, the creation of microorganisms capable of accumulating a larger amount of carotenoids has been an issue.

  Accordingly, an object of the present invention is to provide a microorganism capable of accumulating a larger amount of carotenoid or astaxanthin than conventionally reported microorganisms in consideration of industrial production of carotenoid or astaxanthin.

  As a result of intensive studies on the above problems, the present inventors have completed the present invention. That is, the present invention is a carotenoid-producing Paracoccus microorganism capable of producing 17 mg or more of carotenoid per gram of dry cell weight. Moreover, this invention is a manufacturing method of astaxanthin characterized by culture | cultivating this microorganism and collect | recovering astaxanthin from a microbial cell or a culture solution. The present invention will be described in detail below.

  The carotenoid-producing Paracoccus microorganism capable of producing 17 mg or more of carotenoid per 1 g of dry cell weight of the present invention can be obtained by breeding Paracoccus bacteria having carotenoid productivity. Suitable as the Paracoccus bacterium used for breeding can be exemplified by the Paracoccus bacterium TSTT052 strain found by the present applicant and having a certain level of carotenoid and astaxanthin productivity. In addition, breeding a carotenoid-producing Paracoccus genus strain TSN18E7 (Japanese Patent Laid-Open No. 2005-58216) can also be exemplified. Furthermore, a marine Agrobacterium microorganism (later reclassified as a microorganism belonging to the genus Paracoccus) N-81106 strain can be exemplified. This is because the above-described TSTT052 strain is a strain obtained by breeding the TSN18E7 strain, and the TSN18E7 strain is a strain obtained by breeding the N-81106 strain. The N-81106 strain contains astaxanthin, β-carotene, β-cryptoxanthin, 3-hydroxyechinenone, canthaxanthin, 3′-hydroxyechinenone, cis-adonixanthin, adonilvin, adonixanthin in the cell. It is known to accumulate various carotenoids such as.

  Specific contents of breeding include a method of selecting excellent microorganisms derived from natural mutation, as well as carotenoids after accelerating mutation by treating cells with mutagens and ultraviolet rays. Examples thereof include a method for selecting microorganisms with improved astaxanthin productivity and accumulation, and a method for cell fusion of microorganisms having different properties obtained by the above method. Among them, a method using a mutagen is preferable as a method for obtaining a useful microorganism in a short period of time. Specifically, a compound such as N-methyl-N′-nitro-N-nitrosoguanidine or ethyl methanesulfonate is used as a mutagen. The method to be used can be exemplified. More specifically, an example of breeding using the TSTT052 strain will be described. The cells of the TSTT052 strain obtained by culturing in advance are suspended in an aqueous solution of the mutagen as described above and contacted for a certain period of time, followed by centrifugation. The cells are collected by such a method to remove the mutagen. Thereafter, the cells are cultured on a plate medium and colonies of excellent microorganisms are selected. Colonies are selected by selecting and isolating dark colonies unique to astaxanthin-producing bacteria, performing liquid culture, then extracting carotenoids from the cells and analyzing their accumulation and astaxanthin accumulation by HPLC, etc. For example, it is possible to narrow down microorganisms having improved productivity.

  The productivity and accumulation of carotenoids and astaxanthin can be evaluated by the amount of carotenoids and the like that can be recovered from 1 liter (L) of the medium and the production amount per dry cell weight. For example, when a solid medium is used, an arbitrary colony may be picked up and evaluated by quantifying the growth ability and the amount of carotenoids produced after liquid culture. The TSN47R3 strain found by the present applicant through breeding using the TSTT052 strain (deposited as FERM AP-22034 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on December 15, 2010) Carotenoids of 17 mg or more per 1 g of body weight can be produced, and 60% by weight or more thereof is astaxanthin (accumulating 1100 mg or more of astaxanthin per liter of medium), a carotenoid-producing Paracoccus microorganism. For reference, the amount of dry cells per liter of the medium varies depending on the culture conditions and the like, but is 100 g or more in Examples described later.

  The method for producing carotenoids or astaxanthin of the present invention comprises culturing a microorganism obtained by breeding as described above. As the culture conditions, generally known conditions can be used. In the method for producing carotenoids and the like of the present invention, the culture is preferably performed in a culture solution. For example, the culture temperature is set to 10 to 35 ° C., the pH of the medium is set to a range of 6 to 9, and the culture is performed for 20 to 200 hours. Regarding the culture temperature, the temperature may be changed at each stage by distinguishing between the initial stage, the middle stage, and the late stage.

  The medium components of the nutrient medium used for the culture include molasses, glucose, fructose, maltose, sucrose, starch, lactose, glycerol, acetic acid and the like for the carbon source, and corn steep liquor, peptone, yeast extract for the nitrogen source, Natural ingredients such as meat extract and soybean meal, ammonium salts such as ammonium acetate, ammonium chloride and ammonium sulfate, and amino acids such as glutamic acid, aspartic acid and glycine, inorganic salts include monosodium phosphate, disodium phosphate, Phosphate and sodium chloride such as monopotassium phosphate and dipotassium phosphate include metal chloride, magnesium chloride, magnesium sulfate, ferrous sulfate, ferric sulfate, ferrous chloride, ferric chloride, Iron citrate, ammonium iron sulfate, calcium chloride dihydrate, calcium sulfate, zinc sulfate, chloride Lead, copper sulfate, copper chloride, manganese sulfate, manganese chloride, and yeast extract and biotin, nicotinic acid, thiamine, riboflavin, inositol, pyridoxine, or the like can be used as vitamins.

  Suitable culture conditions using the nutrient medium as described above are a culture temperature of 20 to 30 ° C., a pH of about 7.0 to 7.6, and a culture time of 60 to 180 hours. The sugar concentration in the medium is preferably maintained at a low concentration and not depleted, and it is preferably fed using a high-concentration sugar solution such as glucose. When the microorganism of the present invention is cultured, carotenoids containing astaxanthin are produced and accumulated in the cells or in the culture solution. The extraction of carotenoids is not particularly limited as long as carotenoids and astaxanthin are stably and efficiently recovered from the cells or the culture solution. For example, methanol, ethanol, isopropyl alcohol, acetone, methyl ethyl ketone, methyl isobutyl ketone, dichloromethane, chloroform, dimethyl Examples include extraction with an extraction solvent such as formamide and dimethyl sulfoxide, and supercritical fluid extraction. Among them, an organic solvent using acetone is preferable. The extracted carotenoid can be separated and purified with high purity using liquid chromatography or the like. Examples of the separation principle of liquid chromatography include ion exchange, hydrophobic interaction, molecular sieving and the like. Preferred are reverse phase chromatography and normal phase chromatography. Further, according to high performance liquid chromatography, the extracted carotenoid can be quantified.

  When extracting from the microbial cells, for example, after completion of the culture, the microbial cells are separated from the medium using a method such as centrifugation, decantation, or filtration, and water is added to a viscosity that is easy to use to form a slurry. good. Later, the prepared slurry is homogenized using, for example, a crusher using glass beads or zirconia beads or a high-pressure homogenizer, and is preferably dried by a spray drying method and subjected to extraction. Here, an antioxidant such as ascorbic acid may be added to the slurry in order to prevent the decomposition of astaxanthin.

  In addition, the microorganisms of this invention can also be added as it is to the feed of marine products, such as cultured fish, without extracting carotenoid and astaxanthin, for example. In addition, the residue of microorganisms substantially free of carotenoids and astaxanthin after the extraction operation as described above can also be used as an ideal source of protein and vitamins for raising poultry and the like. .

  The microorganism of the present invention makes it possible to efficiently produce carotenoids, particularly astaxanthin, useful as cosmetics, health foods or food colorings.

It is the figure which showed the time-dependent change of the astaxanthin production amount and the total carotenoid production amount in the fermenter culture | cultivation using TSN47R3 strain | stump | stock. It is a HPLC chart which shows the carotenoid production pattern at the time of 170-hour culture | cultivation.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by these Examples.

Example 1 Mutation Introduction and Production of Excellent Strains The TSTT052 strain was inoculated into 3 mL of the medium shown in Table 1, and cultured overnight in a test tube at 25 ° C. and 150 rpm. 1 mL of this culture solution was transferred to a 1.5 mL Eppendorf tube, and the cells were collected by centrifugation at 15000 rpm for 5 minutes. This microbial cell was suspended in 1 mL of 0.1 M potassium phosphate buffer (hereinafter referred to as Buffer A) at pH 7.0, and then 3 mg / mL N-methyl-N′-nitro-N-nitrosoguanidine (hereinafter referred to as NTG). And 10 μL of an aqueous solution was added and allowed to stand for 60 minutes. Thereafter, the supernatant was removed by centrifugation, and the operation of resuspending in buffer A was repeated twice to remove NTG. Further, the cells were suspended in 1 mL of the medium shown in Table 1, and cultured with shaking in a test tube at 25 ° C. and 150 rpm for 5 hours. The obtained culture broth was appropriately diluted and applied to a plate medium having the composition shown in Table 1 which was solidified by adding 15 g / L of agar, followed by stationary culture at 25 ° C. for 1 week. Among the grown colonies, strong red ones were selected and used for evaluation of mutant strains by flask culture.

Example 2 Evaluation of Mutant Strains Mutant strains selected in Example 1 were inoculated into 3 mL of the medium shown in Table 1, and cultured overnight in a test tube at 25 ° C. and 150 rpm. Next, 0.5 mL of this culture solution was inoculated into 60 mL of the medium shown in Table 1 placed in a 100 mL baffled Erlenmeyer flask and cultured with shaking at 25 ° C. and 120 rpm for 6 days. During the culture, the culture solution was appropriately extracted, and turbidity (OD660 nm), residual glucose concentration, medium pH, and carotenoid production were analyzed over time. Carotenoid production was quantified as follows. First, 1 mL of the culture solution was transferred to a 1.5 mL Eppendorf tube, and the cells were collected by centrifugation at 15000 rpm for 5 minutes. The cells were suspended in 20 μL of pure water, and then 480 μL of dimethylformamide and 500 μL of acetone were sequentially added and shaken to extract carotenoids. After removal of the extraction residue by centrifugation at 15,000 rpm for 5 minutes, various types of high-performance liquid chromatography (hereinafter referred to as HPLC) using a commercially available liquid chromatography column (TSKgel-ODS80TM column, manufactured by Tosoh Corporation) are used. Carotenoids were quantified.

  Carotenoids were separated using a 5:95 mixed solvent of pure water and methanol as the A liquid, and a 7: 3 mixed solvent of methanol and tetrahydrofuran as the B liquid, and the A liquid was columned for 5 minutes at a flow rate of 1 mL / min. Then, linear concentration gradient elution from solution A to solution B was carried out for 5 minutes at the same flow rate, and the solution B was further passed for 5 minutes. The astaxanthin concentration was monitored by measuring the absorbance at 470 nm, and the concentration was calculated from a calibration curve prepared with known concentrations of astaxanthin reagent (Alexis / Cosmo Bio).

  The carotenoid productivity of the mutant strain was evaluated according to the above method, and a new mutant strain TSN47R3 was obtained from the strains having improved productivity over the parent strain TSTT052. Table 2 shows the amount of carotenoid produced at the end of the culture of the TSN47R3 strain and the TSTT052 strain, which is the strain before mutation introduction. As shown in Table 2, it can be seen that the production of carotenoid and astaxanthin is improved in the TSN47R3 strain as compared with the TSTT052 strain which is the parent strain.

Example 3 Cultivation of new microorganisms in fermentor and quantification of carotenoids TSN47R3 strain was inoculated into 60 mL of the medium shown in Table 1, and cultured overnight in a 100 mL baffled Erlenmeyer flask at 25 ° C. and 120 rpm overnight. Cultured. Next, 3 mL of this culture solution was inoculated into 100 mL of the medium shown in Table 3 placed in a 500 mL baffled Erlenmeyer flask, and cultured overnight at 25 ° C. and 120 rpm for pre-culture. Next, 1.8 L of the medium shown in Table 4 was placed in a 3.0 L fermentor (Sakura Seiki Co., Ltd., TFLC-3) and sterilized at 121 ° C. for 20 minutes. And cultured for about 170 hours. The operation of the fermenter was as follows. First, the culture temperature of the fermenter was 22 ° C., the pH was 7.0 to 7.2, and the pH was adjusted using a 10% aqueous ammonia solution. Moreover, the stirring speed of the fermenter was set to 516 rpm at the start of the culture, and the stirring speed was gradually increased as the culture progressed, and increased to 594 rpm during the 117 hours of culture. The shortage of carbon source generated during the culturing process was compensated for by appropriately adding 70% glucose. The glucose concentration was adjusted to be 0.5 g / L to 33 g / L by starting the culture at 10 g / L and starting the addition of glucose 18 hours after the culture. The addition of glucose was terminated when 117 hours had elapsed from the start of the culture, and 25 mL of an aqueous solution of 0.2 M ferrous sulfate heptahydrate was added to the culture solution.

  FIG. 1 shows changes over time in OD660 nm, astaxanthin production and total carotenoid production. As shown in FIG. 1, the production amounts of astaxanthin and total carotenoids increase with the lapse of culture time, and the amounts of each carotenoid at 170 hours of culture are quantified as shown in Table 5. Moreover, the HPLC chart in that case is shown in FIG. As shown in FIG. 1, the production amounts of carotenoid and astaxanthin increase with the lapse of the culture time, and the value of OD660 exceeds 490 at about 140 hours after the start of the culture. The dry cell weight at this time was about 123 g per liter of medium, the carotenoid production was about 1665 mg per liter of medium, of which the astaxanthin production was about 694 mg per liter of medium. Astaxanthin production reaches a maximum at about 168 hours after the start of culture. At this time, the value of OD660 is about 440, the dry weight of the cells is about 109 g per liter of the medium, the carotenoid production is about 1864 mg per liter of the medium, of which the astaxanthin production is about 1118 mg About 10 mg / g body dry weight). It can be seen that by this experimental operation, about 1100 mg of astaxanthin and about 1850 mg of carotenoid are produced per liter of the medium.

Claims (5)

  A carotenoid-producing Paracoccus microorganism capable of producing 17 mg or more of carotenoid per gram of dry cell weight.   The microorganism according to claim 1, wherein the microorganism produces carotenoids containing 60% by weight of astaxanthin in the carotenoids containing astaxanthin.   The microorganism according to claim 1 or 2, wherein the microorganism is capable of growing at least 100 g per liter of the medium by dry weight of the cells. The microorganism according to claim 1 or 2, wherein the microorganism is a carotenoid-producing Paracoccus genus strain TSN47R3 (FERM P-22034).     A method for producing astaxanthin, comprising culturing a carotenoid-producing microorganism of the genus Paracoccus that can produce 17 mg or more of carotenoid per gram of dry cell body and recovering astaxanthin from the cell body.

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