JP6132905B2 - Method for producing zeaxanthin and bacterial culture method - Google Patents

Description

  The present invention relates to an efficient method for culturing a zeaxanthin-producing microorganism under pressure conditions and an efficient method for producing zeaxanthin using the same.

  Zeaxanthin is contained in various plants such as corn and added to feed as a natural yellow pigment, and is known for its use in improving the color of egg yolk, meat and epidermis in poultry such as chickens and as a colorant for foods. Moreover, it has a strong antioxidant action (Non-patent document 1), and an antitumor effect has been reported (Non-patent document 2). Zeaxanthin is known to exist in the retina and lens together with lutein and is involved in maintaining eye health (Non-patent Document 3). From these physiological effects, zeaxanthin is useful as a health food material, cosmetic material and pharmaceutical material.

  Conventionally, as a method for producing zeaxanthin, a chemical synthesis method using an optically active hydroxyketone obtained by asymmetric reduction of oxoisophorone as a raw material (Non-Patent Document 4), a method of extracting from plant raw materials such as corn seed and marigold (Non-patent document 5, Patent document 1), Spirulina algae (patent document 2), Nannochloris microalgae (patent document 3), Flexibacter bacteria (patent document 4), Alteromonas bacteria (patent document 5) In addition, production methods using microorganisms such as Flavobacterium bacteria (Non-patent Document 6), bacteria Agrobacterium aurantiacum (Non-patent Document 7), and Paracoccus bacteria (Patent Documents 6-9) have been known.

  However, chemical synthesis methods use organic solvents, and thus have problems in terms of safety and recent natural product orientation. Also, the zeaxanthin content in plants is very low, and zeaxanthin production by conventional microorganisms was not sufficiently high, so zeaxanthin can be obtained efficiently by the method using plants and microorganisms. It was not practical.

  In order to solve these problems, the present inventors have so far developed a Paracoccus microorganism having a high ability to produce zeaxanthin (hereinafter referred to as “zeaxanthin-producing microorganism”) and uses the microorganism to improve efficiency. It has been reported that zeaxanthin can be produced specifically (Patent Document 10).

JP-A-8-092205 Japanese Patent Laid-Open No. 10-155430 Japanese Unexamined Patent Publication No. 7-59558 Japanese Patent Laid-Open No. 5-328978 JP-A-5-49497 Japanese Unexamined Patent Publication No. 7-79796 JP-A-8-9964 U.S. Pat.No. 5,607,839 U.S. Pat.No. 5,858,761 JP 2005-87097 A

Fisheries Science, 62 (1), 134-137, 1996 Biol. Pharm. Bull., 18 (2), 227-233, 1995 FOOD Style 21, 3 (3), 50-53, 1999 Pure Appl. Chem., 63 (1), 45, 1991 Biological pigment, Asakura Shoten, 1974 publication Carotenoids, in Microbial Technology, 2nd edn, Vol.1, 529-544, New York: Academic Press FEMS Microbiology Letters, 128, 139-144, 1995

  For the purpose of producing zeaxanthin on a commercial scale, the inventors of the present invention used the zeaxanthin-producing microorganism in a large culture tank (for example, a height of 10 m), or in a culture tank lacking oxygen supply capacity. In order to increase the supply amount, when the amount of liquid to be applied is reduced (for example, 5 m in height) and the air phase is further pressurized, the microorganisms located at the bottom of the culture tank are It has been found that the zeaxanthin production ability and the growth ability of the microorganisms are significantly reduced due to the combined pressure of the gas phase.

  Therefore, the present invention provides a zeaxanthin-producing ability and a proliferating ability under a pressure condition that the zeaxanthin-producing microorganism can undergo in culture in a large-scale culture tank on a commercial scale without significantly reducing the zeaxanthin-producing ability and the proliferating ability of the microorganism. It is an object of the present invention to provide a method for cultivating a zeaxanthin-producing microorganism and a method for producing zeaxanthin using the culture method.

  As a result of diligent research to solve the above problems, the inventors of the present invention used a chelating agent having an action of chelating iron element and solubilizing in the medium, for example, citric acid or a salt thereof, By cultivating a zeaxanthin-producing microorganism, the inventors have found that zeaxanthin-producing ability and proliferation ability can be maintained under a predetermined pressure condition without significantly reducing the zeaxanthin-producing ability and proliferation ability of the microorganism, and the present invention has been completed. I came to let you.

  That is, the present invention has the following features.

[1] A method for producing zeaxanthin using a zeaxanthin-producing microorganism belonging to the genus Paracoccus,
(i) culturing the microorganism under an average pressure condition of 0.008 MPa or more and 0.08 MPa or less in a medium containing a chelating agent; and
(ii) recovering the produced zeaxanthin from the culture,
Including the above method.

[2] The method according to [1], wherein the step (ii) includes a step of obtaining a composition containing zeaxanthin by removing medium components and moisture from the culture and drying the culture.

[3] The method according to [2], further comprising a step of purifying zeaxanthin from a composition containing zeaxanthin.

[4] The method according to any one of [1] to [3], wherein the chelating agent is citric acid or a salt thereof.

[5] The method according to [4], wherein the medium contains citric acid or a salt thereof at a concentration of less than 14 g / L.

[6] The method according to [5], wherein the medium contains citric acid or a salt thereof at a concentration of 0.5 g / L or more and 10 g / L or less.

[7] The method according to any one of [1] to [6], wherein the microorganism is cultured under an average pressure condition of 0.02 MPa or more and 0.08 MPa or less.

[8] Improving the ability of zeaxanthin to be produced under pressure conditions of 0.008 MPa or more and 0.08 MPa or less, including culturing zeaxanthin producing microorganisms belonging to the genus Paracoccus in a medium containing a chelating agent How to make.

[9] The method according to [8], wherein the chelating agent is citric acid or a salt thereof.

[10] The method according to [9], wherein the medium contains citric acid or a salt thereof at a concentration of less than 14 g / L.

[11] The method according to any one of [8] to [10], wherein the pressure condition is 0.02 MPa or more and 0.08 MPa or less.

[12] A method for culturing a microorganism under pressure of 0.008 MPa or more and 0.08 MPa or less, comprising culturing a zeaxanthin-producing microorganism belonging to the genus Paracoccus in a medium containing a chelating agent.

[13] The method according to [12], wherein the chelating agent is citric acid or a salt thereof.

[14] The method according to [13], wherein the medium contains citric acid or a salt thereof at a concentration of less than 14 g / L.

[15] The method according to any one of [12] to [14], wherein the pressure condition is 0.02 MPa or more and 0.08 MPa or less.

[16] A composition containing zeaxanthin produced by the method of [2].

[17] Zeaxanthin produced by the method of [3].

  This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2013-073833, which is the basis of the priority of the present application.

  According to the present invention, zeaxanthin-producing ability and proliferation can be achieved without significantly reducing the zeaxanthin-producing ability and proliferation ability of the microorganism even under pressure conditions that the zeaxanthin-producing microorganism can undergo in culture in a large-scale culture tank on a commercial scale. It is possible to provide a method for cultivating a zeaxanthin-producing microorganism and a method for producing zeaxanthin using the culturing method that make it possible to maintain the performance.

FIG. 1 is a schematic diagram showing a biosynthetic pathway of carotenoids of Paracoccus microorganisms. FIG. 2 shows the measurement results of the zeaxanthin concentration (solid line) and the microorganism concentration (dotted line) in a culture solution obtained by culturing zeaxanthin-producing microorganisms under different pressure conditions. The zeaxanthin concentration in the culture solution is shown as a relative value with the zeaxanthin concentration in the culture solution obtained under a pressure condition of 0.003 MPa as 100%. Fig. 3 shows the measurement results of zeaxanthin concentration (solid line) and microorganism concentration (dotted line) in the culture solution obtained by culturing zeaxanthin-producing microorganisms in a medium with different amounts of sodium citrate added under a pressure of 0.05 MPa. Show. The zeaxanthin concentration in the culture solution is shown as a relative value with the zeaxanthin concentration in the culture solution obtained under the condition of 0 g / L added sodium citrate as 100%. Fig. 4 shows the measurement of zeaxanthin concentration (solid line) and microorganism concentration (dotted line) in the culture solution obtained by culturing zeaxanthin-producing microorganisms in a medium supplemented with 2.8 g / L sodium citrate under different pressure conditions. Results are shown. The zeaxanthin concentration in the culture solution is shown as a relative value with the zeaxanthin concentration in the culture solution obtained under a pressure condition of 0.003 MPa in a medium without addition of sodium citrate being 100%. FIG. 5 shows the measurement results of various ion concentrations in the supernatants of media with different amounts of sodium citrate added. (Round) Phosphate ion; (Triangle) Potassium ion; (Batsu) Magnesium ion; (Rhombus) Calcium ion; (Square) Iron ion.

1. Zeaxanthin-producing microorganism The zeaxanthin-producing microorganism in the present invention is a microorganism belonging to the genus Paracoccus, and has a high ratio of zeaxanthin to the total amount of carotenoid produced.

  A zeaxanthin-producing microorganism can be obtained based on a known method (for example, see JP-A-2005-87097). That is, astaxanthin-producing microorganisms belonging to the genus Paracoccus (hereinafter referred to as “astaxanthin-producing microorganisms”) are mutagenized to induce mutation, and the ratio of zeaxanthin produced to the total carotenoid production (mass%) ) Can be obtained by selecting and obtaining a mutant strain higher than that of the parent strain astaxanthin-producing microorganism.

  Astaxanthin-producing microorganisms include astaxanthin-producing bacteria in which the base sequence of DNA corresponding to 16S ribosomal RNA is substantially homologous to the base sequence described in SEQ ID NO: 1. Examples of such astaxanthin-producing microorganisms include E-396 strain (FERM BP-4283) and A-581-1 strain (FERM BP-4671), and these mutant strains and related species thereof. The E-396 strain (FERM BP-4283) and the A-581-1 strain (FERM BP-4671) are registered with the National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center) It is deposited on April 27, 1993 and May 20, 1994, respectively, with the Patent Biological Depositary Center (National Institute of Technology and Evaluation).

  Mutation treatments for astaxanthin-producing microorganisms include, for example, chemical methods such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS), ultraviolet irradiation, X-ray irradiation, and the like. Physical methods, biological methods such as genetic recombination, transposon, and the like can be used. This mutation treatment may be performed once, or, for example, a mutation of an astaxanthin-producing microorganism may be obtained by this mutation treatment, and the mutation treatment may be further performed twice or more.

  Among mutant mutants of astaxanthin-producing microorganisms that have been mutated, select mutants in which the ratio (mass%) of zeaxanthin produced to the total carotenoid production is higher than that of the parent strain, and select zeaxanthin-producing microorganisms. Can be obtained. When colonies are formed on a solid medium, the colonies of zeaxanthin-producing microorganisms exhibit a yellow to orange color, and can be easily distinguished from the red to red-orange colonies of astaxanthin-producing microorganisms. A zeaxanthin-producing microorganism (mutant strain) can be selected. The mutant colonies selected in this manner are cultured by a conventional method, and after completion of the culture, carotenoid compounds contained in the culture solution of each mutant strain can be obtained by, for example, high performance liquid chromatography, thin layer chromatography, paper chromatography. Analyze using etc. Thereby, a mutant strain with a high ratio of zeaxanthin to the total carotenoid amount can be selected and obtained. The total amount of carotenoid referred to here is the total amount of carotenoid compounds such as astaxanthin, canthaxanthin, adonixanthin, β-carotene, echinone, zeaxanthin, β-cryptoxanthin, 3-hydroxyechinenone, astereudenone, and adonilbin. Say. In addition, astaxanthin biosynthesis in an astaxanthin-producing microorganism is assumed to be carried out with β-carotene as the upstream and the 6-membered rings at both ends modified by ketozyme and hydroxylase, respectively (see FIG. 1). If the ketozyme is completely or incompletely lost here, the reaction indicated by the arrow “keto” in FIG. 1 does not occur or decreases, thereby “keto”. Echinenone, canthaxanthin, 3-hydroxyechinenone, astereudenone, adonilvin, adonixanthin and astaxanthin, which are products of the above, are not produced or the production amount is reduced. Therefore, another effective means for selecting zeaxanthin-producing microorganisms from mutant strains is the total carotenoids of echinenone, canthaxanthin, 3-hydroxyechinenone, asteroidenone, adonilbin, adonixanthin and astaxanthin. A method of selecting on the basis of the low ratio of each to the amount can be used. Selection can be made on the basis that the ratio of each of the aforementioned compounds to the total carotenoid is less than 10% by mass, more preferably less than 5% by mass, and even more preferably less than 1% by mass.

2. Medium The medium used in the present invention contains a chelating agent. The “chelating agent” is not particularly limited as long as it has a function of chelating iron elements and solubilizing them in the medium. Known chelating agents can be used, for example, citric acid, malic acid, succinic acid, fumaric acid, tartaric acid, acetic acid, gluconic acid, phytic acid, polyaminophosphoric acid, lactic acid, alkyl diphosphones. Acid, ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid, 1,2-diaminocyclohexanetetraacetic acid, hydroxyethylethylenediaminetriacetic acid, hydroxyethyliminodiacetic acid, iminodiacetic acid, diethylenetriaminepentaacetic acid, dihydroxyethylglycine, diaminopropanetetraacetic acid , Nitrilotris (methylenephosphonic acid), ethylenediaminetetrakis (methylenephosphonic acid), and salts thereof. Examples of the salt include water-soluble salts, such as alkali metal salts such as sodium salts and potassium salts, ammonium salts, and the like. The chelating agent is preferably citric acid or citrate. By including such a chelating agent in the culture medium, the zeaxanthin producing ability and the proliferating ability of the zeaxanthin producing microorganism can be improved even under pressure conditions that inhibit zeaxanthin production by the zeaxanthin producing microorganism and the growth of the microorganism. The addition amount of these chelating agents added to the medium varies depending on the type of chelating agent used and the ability to chelate iron elements, but even under pressure conditions that inhibit zeaxanthin production by the zeaxanthin-producing microorganism and the growth of the microorganism, It can adjust suitably in the range which improves the zeaxanthin production ability and growth ability of a zeaxanthin production microorganism. If the chelating agent used is citric acid or a salt thereof, the amount is less than 14 g, preferably 10.0 g or less, more preferably 8.4 g or less per liter of medium. The lower limit of the amount of citric acid or a salt thereof is not particularly limited, but if it is too small, a sufficient chelating effect cannot be obtained, so 0.5 g or more, preferably 1.0 g or more, more preferably 1.5 g or more, per liter of medium. More preferably, it is 2.8 g or more. Therefore, the amount of citric acid or a salt thereof added is preferably 0.5 g or more and 10.0 g or less, more preferably 2.8 g or more and 8.4 g or less per liter of medium.

  The medium further contains a carbon source, a nitrogen source, an inorganic salt, and special requirement substances (for example, vitamins, amino acids, nucleic acids, etc.) necessary for the growth of the zeaxanthin-producing microorganism. Carbon sources include sugars such as glucose, sucrose, fructose, trehalose, mannose, mannitol, maltose, organic acids such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid, ethanol, propanol, butanol, pen Examples include alcohols such as butanol, hexanol, and isobutanol. The addition ratio varies depending on the type of carbon source, but is usually 1 g to 100 g, preferably 2 g to 50 g, per liter of the medium. As the nitrogen source, for example, one or more of potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, urea and the like are used. The addition ratio varies depending on the type of nitrogen source, but is usually 0.1 g to 20 g, preferably 1 g to 10 g, per 1 L of the medium. Inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, manganese sulfate, manganese chloride, zinc sulfate, zinc chloride, copper sulfate, and chloride. One or more of calcium, calcium carbonate, sodium carbonate and the like are used. Although an addition ratio changes with kinds of inorganic salt, it is 0.1 mg-10 g normally with respect to 1 L of culture media. Special requirements include vitamins, nucleic acids, yeast extract, peptone, meat extract, malt extract, corn steep liquor, dry yeast, soybean meal, soybean oil, olive oil, corn oil, linseed oil, etc. The above is used. The addition ratio varies depending on the type of special requirement substance, but is usually 0.01 mg to 100 g with respect to 1 L of the medium. The pH of the medium is adjusted to 2-12, preferably 6-9.

3. Culturing method The present invention is a culture of a zeaxanthin-producing microorganism, 0.008 MPa or more and 0.08 MPa or less, or 0.008 MPa or more and less than 0.08 MPa, preferably 0.02 MPa or more and 0.08 MPa or less, or 0.02 MPa or more with respect to the zeaxanthin producing microorganism. In addition, the present invention can be applied to a culture method having a situation in which a pressure of less than 0.08 MPa, more preferably 0.05 MPa is applied. However, when a large culture tank is used, the pressure in the liquid phase portion (water pressure) varies depending on the depth, and a difference occurs between the pressure condition at the upper part of the medium and the pressure condition at the lower part of the medium. Therefore, the pressure value when a large culture tank is used can be an average pressure value applied to the culture system (liquid phase part). For example, when exemplifying a general culture method using a large culture tank (height 10 M), when a culture medium is filled to a depth of 6 M in the culture tank and a pressure of 0.02 MPa is applied to the gas phase, The average pressure value applied to the culture system can be determined as 0.02 MPa (gas phase part) +0.03 MPa (medium water depth median water pressure of 3 M).

  When the microorganism is cultured in a normal medium under a condition where a pressure of 0.008 MPa or more and 0.08 MPa or less is applied to the zeaxanthin-producing microorganism, the zeaxanthin production ability and the growth ability of the microorganism are significantly reduced. On the other hand, by culturing using the above-mentioned medium, the zeaxanthin-producing ability of the zeaxanthin-producing microorganism is not significantly reduced even under the above pressure conditions, and the production ability can be maintained. In addition, by culturing a zeaxanthin-producing microorganism using the medium, it is possible to promote the growth of the zeaxanthin-producing microorganism, and the ability of the microorganism to grow does not significantly decrease even under the pressure condition. That is, by culturing a zeaxanthin-producing microorganism using the above medium, zeaxanthin can be produced and produced with high efficiency even under the above pressure conditions.

  Culturing can be performed at a temperature of 10 to 70 ° C., preferably 20 to 35 ° C., usually for 1 to 20 days, preferably 2 to 9 days by shaking culture or aeration and agitation culture.

4). Recovery of zeaxanthin The zeaxanthin produced in the present invention can be recovered based on a known technique. That is, after completion of the culture, a culture precipitate containing zeaxanthin can be obtained by removing medium components and water from the culture solution by filtration, centrifugation, or the like. If necessary, the culture precipitate can be further dried by spray drying, drum drying, freeze drying, or the like to obtain a dried precipitate containing zeaxanthin.

  Next, zeaxanthin is extracted from the culture precipitate or the precipitate dried using a water-soluble organic solvent. The water-soluble organic solvent is not particularly limited, and for example, tetrahydrofuran, pyridine, dioxane, cyclohexanone, cyclohexanol, methanol, ethanol, isopropanol, acetone, ethyl methyl ketone, dimethylformamide, dimethyl sulfoxide, and the like can be used. These may be used alone, in combination of two or more, or in combination with water. An extract containing zeaxanthin can be obtained by removing the solvent from the obtained water-soluble organic solvent extract by concentration under reduced pressure or the like. The obtained extract may be subjected to a deodorizing treatment if necessary, or may be suspended in a vegetable oil.

  Furthermore, zeaxanthin can be further purified by adding a nonpolar organic solvent and water to the water-soluble organic solvent extract and performing liquid-liquid extraction. As the nonpolar solvent, for example, hexane, heptane, cyclohexane, benzene, toluene, xylene, petroleum ether, naphtha, kerosene and the like can be used. After the nonpolar solvent phase obtained after the liquid-liquid extraction is separated and recovered, high-purity zeaxanthin can be precipitated by concentration, addition of water, addition of nonpolar solvent or cooling. The steps of concentration, addition of water, addition of a nonpolar solvent and cooling may be carried out independently, but they can also be carried out in combination as necessary. Since the obtained precipitate is easily recovered by filtration, it can be washed with a solvent or the like, if necessary, and dried to obtain a highly purified zeaxanthin extract and purified product.

  Select and use the necessary product forms as feed formulation, food material, cosmetic material and pharmaceutical material from among the culture precipitate, precipitate dried product, extract, extract and purified product containing zeaxanthin obtained by the above method. can do.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not limited to these.

[Seed flask medium]
A seed flask medium having the following composition was used.

  Sucrose 30 g / L; corn steep liquor 30 g / L; potassium dihydrogen phosphate 1.5 g / L; dipotassium hydrogen phosphate anhydrate 3 g / L; calcium chloride dihydrate 5.0 g / L; magnesium sulfate 7 water Japanese product 12 g / L; Iron sulfate heptahydrate 0.3 g / L, pH 7.2, 100 ml was placed in a 500 ml Erlenmeyer flask with a cotton plug, autoclaved at 121 ° C. for 20 minutes, and used in the following examples. It was.

[Basic medium]
A basic medium having the following composition was used.

  Glucose 30 g / L; Corn steep liquor 30 g / L; Ammonium sulfate 5 g / L; Potassium dihydrogen phosphate 2.25 g / L; Disodium hydrogen phosphate dodecahydrate 5.7 g / L; Calcium chloride dihydrate 1 g / L Magnesium sulfate heptahydrate 5 g / L; iron sulfate heptahydrate 5 g / L; sodium L-glutamate monohydrate 6 g / L; alcohol defoamer 0.5 g / L.

  2.0 L of the basic medium was placed in a 5 L fermentor, a predetermined amount of sodium citrate was added as necessary, and autoclaved at 121 ° C. for 30 minutes, and used in the following examples.

[Zeaxanthin-producing microorganism]
As the zeaxanthin-producing microorganism, a mutant obtained by the above method from an astaxanthin-producing microorganism belonging to the genus Paracoccus was used.

[Culture method]
The microorganism was inoculated with one platinum ear in the above seed flask medium, cultured at 28 ° C. for 2 days at 100 rpm, and then inoculated with 80 mL of the culture into a fermentor containing a basic medium.

  Under predetermined pressure conditions and predetermined sodium citrate addition conditions, aerobic culture at 28 ° C. and aeration volume of 1 vvm was performed for 140 hours. The pH was continuously controlled with 15% aqueous ammonia so that the pH was maintained at 7.2 during the culture. Glucose was added in an amount of 50 g on the first day, the second day, the third day, and the fourth day so as not to be depleted. Further, the minimum stirring rotation speed was 500 rpm, and the dissolved oxygen concentration in the culture medium in the middle of the culture was maintained at 2.4 ppm. By detecting foaming with a bubble sensor, an alcohol-based antifoaming agent was automatically added, or an antifoaming agent was continuously added as necessary to suppress foaming.

[Control of pressure conditions]
The pressure was controlled by manually adjusting the back pressure valve.

  In addition, the value of the pressure condition described in the following Examples shows the average pressure value concerning a culture system (liquid phase part).

[Measurement of zeaxanthin concentration]
Quantification of zeaxanthin was performed as follows using high performance liquid chromatography (HPLC).

  Two analytical columns, Inertsil SIL-100A, 5 μm (φ4.6 × 250 mm) (manufactured by GL Sciences) were connected. Elution was carried out by flowing 1.0 mL / min of a mobile phase n-hexane / tetrahydrofuran / methanol mixture (40: 20: 1) at a constant temperature near room temperature. In the measurement, the sample was dissolved in tetrahydrofuran and 20 μL of a 100-fold diluted solution in the mobile phase was used as the injection amount, and the column eluent was detected at a wavelength of 470 nm. Further, zeaxanthin (Cat. No. 0307 S) manufactured by EXTRASYNTHESE was used as a standard for quantification. The zeaxanthin concentration of the standard solution was set using the following formula after measuring the absorbance at 453 nm (A) of the standard solution and the area percentage% (B) of the zeaxanthin peak when HPLC analysis was performed under the above conditions. It was.

Zeaxanthin concentration (mg / L) = A ÷ 2327 × B × 100
[Measurement of bacterial cell concentration]
At a wavelength of 610 nm, the absorbance (OD ₆₁₀ ) of the culture solution after completion of the culture was measured using a spectrophotometer, and this value was taken as the cell concentration of the culture solution.

(Example 1) Effect of pressure on zeaxanthin-producing microorganism The above culturing method using a basal medium not added with sodium citrate under pressure conditions of 0.003 MPa, 0.025 MPa, 0.05 MPa, 0.075 MPa, or 0.1 MPa. Incubated at

  After completion of the culture, the culture solution was collected, and the zeaxanthin concentration and the microorganism concentration in the culture solution were measured.

  The results are shown in FIG. The zeaxanthin concentration in the culture solution is shown as a relative value with the zeaxanthin concentration in the culture solution obtained under a pressure condition of 0.003 MPa as 100%. During the culture, the zeaxanthin concentration of the culture broth was decreased by applying pressure, and the lowest value was shown when 0.05 MPa was applied. The microbial concentration in the culture showed the same tendency.

(Example 2) Effect of citrate on zeaxanthin-producing microorganisms under pressure conditions Sodium citrate was added at 0 g / L, 2.8 g / L, 8.4 g / L, 14 g / Culture was carried out by the above culture method using a basic medium prepared by adding L or a concentration of 21 g / L.

  After completion of the culture, the culture solution was collected, and the zeaxanthin concentration and the microorganism concentration in the culture solution were measured.

  The results are shown in FIG. The zeaxanthin concentration in the culture solution is shown as a relative value with the zeaxanthin concentration in the culture solution obtained under the condition of 0 g / L added sodium citrate as 100%. It was shown that the zeaxanthin concentration in the culture broth was improved by adding sodium citrate to the medium even in the culture under a pressure of 0.05 MPa. The microbial concentration in the culture showed the same tendency. In particular, by adding sodium citrate so as to have a concentration of 2.8 g / L to 8.4 g / L in the medium, both the zeaxanthin concentration and the microorganism concentration showed high values.

(Example 3) Effect of pressure on zeaxanthin-producing microorganism in the presence of citrate zeaxanthin-producing microorganism was added at a pressure of 0.003 MPa, 0.02 MPa, 0.04 MPa, 0.06 MPa, 0.08 MPa, or 0.1 MPa. Using a basic medium prepared by adding to a concentration of g / L, culturing was performed by the above culture method.

  After completion of the culture, the culture solution was collected, and the zeaxanthin concentration and the microorganism concentration in the culture solution were measured.

The results are shown in FIG.

  The zeaxanthin concentration in the culture broth is shown as a relative value in which the zeaxanthin concentration in the culture broth obtained under the pressure condition of 0.003 MPa in the medium without addition of sodium citrate of Example 1 is 100%. By adding sodium citrate to the medium, the concentration of zeaxanthin in the culture can be greatly reduced not only under the pressure of 0.05 MPa but also under the pressure of 0.02 MPa to 0.08 MPa required on a commercial scale. There was no. However, as is clear from the results of Example 1 and FIG. 2, when the pressure condition is smaller than 0.008 MPa and larger than 0.08 MPa, the culture solution is obtained by adding sodium citrate to the medium. Zeaxanthin concentration was shown to decrease. On the other hand, by adding sodium citrate to the culture medium, the microbial concentration in the culture solution was not limited to 0.05 MPa pressure condition and was not greatly reduced under other pressure conditions. Moreover, it was shown that by adding sodium citrate to the medium, the microorganism concentration in the culture solution can be improved regardless of the pressure condition (see FIG. 2).

(Example 4) Change in ion concentration in culture supernatant by addition of citrate Sodium citrate was added to a concentration of 0 g / L, 2.8 g / L, 8.4 g / L, or 14 g / L, respectively. The basal medium prepared in this manner was centrifuged to recover the supernatant. Subsequently, the concentration of calcium ions, iron ions, potassium ions, magnesium ions, and phosphate ions in each of the obtained supernatants was measured. Each cation concentration was measured by ICP emission spectrometry, and phosphate ions were measured by molybdenum blue absorbance method.

  The results are shown in FIG. It was shown that the amount of phosphate ion, calcium ion and iron ion in the culture supernatant increased depending on the amount of sodium citrate added. In particular, iron ions were below the limit of detection in the culture supernatant without sodium citrate, but about 80 ppm in the culture supernatant with sodium citrate added to a concentration of 14 g / L. Increased to.

  In the carotenoid biosynthetic pathway of Paracoccus microorganisms shown in Fig. 1, it has been clarified that iron ion is required for the hydroxylation reaction involved in the synthesis of zeaxanthin (Fraser, PD et al., In Vitro Characterization of Astaxanthin Biosynthetic Enzymes, J. Biol. Chem., 1997, 272, 6128-6135). As one factor of the increase in the amount of zeaxanthin produced by the addition of sodium citrate shown in the above examples, iron element is eluted as iron ions in the medium by the chelating action of the added sodium citrate, and this is used as the zeaxanthin producing microorganism Is suggested to be available.

  According to the present invention, it is possible to maintain high zeaxanthin-producing ability and proliferative ability of zeaxanthin-producing microorganisms even in culture in a large-scale culture tank on a commercial scale, and efficiently produce a large-scale zeaxanthin on a commercial scale. be able to.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (15)

A method for producing zeaxanthin using a zeaxanthin-producing microorganism belonging to the genus Paracoccus,
(i) culturing the microorganism under an average pressure condition of 0.008 MPa or more and 0.08 MPa or less in a medium containing a chelating agent to produce zeaxanthin;
(ii) recovering the produced zeaxanthin from the culture,
Including the above method.   The method according to claim 1, wherein step (ii) comprises a step of obtaining a composition containing zeaxanthin by removing medium components and water from the culture and drying the culture.   Furthermore, the method of Claim 2 including the process of refine | purifying zeaxanthin from the composition containing zeaxanthin.   The method according to any one of claims 1 to 3, wherein the chelating agent is citric acid or a salt thereof.   The method according to claim 4, wherein the medium contains citric acid or a salt thereof at a concentration of less than 14 g / L.   The method according to claim 5, wherein the medium contains citric acid or a salt thereof at a concentration of 0.5 g / L or more and 10 g / L or less.   The method according to any one of claims 1 to 6, wherein the microorganism is cultured under an average pressure condition of 0.02 MPa or more and 0.08 MPa or less.   A method for improving the zeaxanthin producing ability of a microorganism under a pressure condition of 0.008 MPa or more and 0.08 MPa or less, comprising culturing a zeaxanthin producing microorganism belonging to the genus Paracoccus in a medium containing a chelating agent.   The method according to claim 8, wherein the chelating agent is citric acid or a salt thereof.   The method according to claim 9, wherein the medium contains citric acid or a salt thereof at a concentration of less than 14 g / L.   The method according to any one of claims 8 to 10, wherein the pressure condition is 0.02 MPa or more and 0.08 MPa or less.   A method of culturing a microorganism under a pressure condition of 0.008 MPa or more and 0.08 MPa or less, comprising culturing a zeaxanthin-producing microorganism belonging to the genus Paracoccus in a medium containing a chelating agent.   The method according to claim 12, wherein the chelating agent is citric acid or a salt thereof.   The method according to claim 13, wherein the medium contains citric acid or a salt thereof at a concentration of less than 14 g / L.   The method according to any one of claims 12 to 14, wherein the pressure condition is 0.02 MPa or more and 0.08 MPa or less.

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