JP2007151475A - New microorganism and method for producing zeaxanthin using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microorganism capable of selectively producing zeaxanthin in large amounts and to provide a method for producing zeaxanthin by the microorganism. <P>SOLUTION: The zeaxanthin-producing microorganism is selected from a variant obtained by breeding a bacterium of the genus Paracoccus producing carotenoids in which zeaxanthin occupies ≥85 wt.% based on total amount of carotenoids, in carotenoids containing β-carotene, β-cryptoxanthin, echinenone, canthaxanthin, 3-hydroxyechinenone, 3'-hydroxyechinenone, zeaxanthin, phoenicoxanthin, adonixanthin and astaxanthin produced. The method for producing zeaxanthin comprises recovering zeaxanthin from a bacterium cell or a culture solution using the microorganism. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel microorganism used in the production of zeaxanthin useful as a feed additive, food coloring, functional food, antioxidant, and a method for producing zeaxanthin using the same.

  Zeaxanthin, a kind of carotenoid, is used to color cultured ayu and improve egg yolk quality. In recent years, zeaxanthin and its structural isomer lutein are known to reduce the risk of developing diseases such as age-related macular degeneration and cataracts, and the demand for supplements such as health foods is expanding. . Zeaxanthin is naturally contained mainly in green-yellow vegetables such as spinach, corn seeds, foods such as egg yolk, and yellow flowers such as marigold, but these are low in content and are suitable for mass production of zeaxanthin by the extraction method. No.

  Conventionally, zeaxanthin has been produced by mass culture of spirulina, a kind of microalgae. However, photosynthetic bacteria generally have several problems in fermentation production, such as slow growth rate and the necessity of supplying light for photosynthesis. Moreover, since it contains a large amount of fat-soluble chlorophyll as in zeaxanthin, separation from zeaxanthin is difficult. As a solution to these problems, production studies using Artenomonas bacteria (see, for example, Patent Document 1), Flexibacter bacteria (see, for example, Patent Document 2), Paracoccus bacteria, and the like have been conducted. Can be easily extracted without. However, the amount of zeaxanthin produced by these bacteria is as low as 1 to 4 mg / L, and further strain breeding is necessary for industrialization. Breeding of strains to improve zeaxanthin production has also been studied, and mutations are usually made using ultraviolet radiation or mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine and ethyl methanesulfonate. After the original treatment, single colonies are isolated and high production strains are selected based on the color of the colonies. However, since zeaxanthin is a yellow pigment, it is difficult to distinguish the shade of the color, so that efficient breeding has conventionally been difficult.

  In order to solve such a breeding problem, first, a Paracoccus bacterium was bred that has a colony color ranging from orange to red and that can easily distinguish the shade of the color, and that can easily obtain a high carotenoid-producing strain. This strain is obtained by chemically modifying β-carotene as shown in FIG. 1, and β-cryptoxanthin, echinenone, canthaxanthin, 3-hydroxyechinone, 3′-hydroxyechinenone, zeaxanthin, phenicoxanthine, ad It is known to produce astaxanthin via nixanthin or the like (see, for example, Non-Patent Document 1). Subsequently, the carotenoid high-producing strain was further bred to obtain a strain that selectively produces and accumulates zeaxanthin, an intermediate metabolite, by changing the activity balance and metabolic balance of enzymes related to this metabolism. It came to be completed.

  The Paracoccus bacterium N-81106 used as a zeaxanthin-producing bacterium is known to produce carotenoids such as adonixanthin, astaxanthin, or glycosides thereof, but most of the metabolic intermediate zeaxanthin is Does not accumulate. Further, the total production amount of carotenoids is about several milligrams per liter of the medium, which is not always a satisfactory carotenoid productivity.

  This Paracoccus bacterium N-81106 strain was later re-identified as a Paracoccus bacterium as a result of sequence analysis of the 16S ribosomal RNA gene. It is also registered as MBIC01143 at the Marine Biotechnology Research Institute, and a summary of information regarding its properties can be obtained from the National Institute of Genetics, Japan DNA Data Bank (DDBJ) or the NIH Database (NCBI) (for example, Non-Patent Documents 2 and 3).

  Breeding with this N-81106 strain as a parent strain, TSN18E7 strain (Accession No. FERM P-19746) has been reported which produces 220 mg of astaxanthin per liter of medium and 400 mg of total carotenoid to date (for example, Patent Document 4). reference).

  Research has also been conducted on zeaxanthin production by a new genus of carotenoid-producing bacteria (see, for example, Patent Document 3), achieving a zeaxanthin production of 19.7 mg / L per culture. However, the selectivity of zeaxanthin in all carotenoids is as low as 34.3%, and further improvement in productivity is necessary to obtain only zeaxanthin with high purity and efficiency.

Japanese Patent Laid-Open No. 5-49497 JP-A-5-328978 JP 2005-87097 A JP-A-2005-58216 Journal of Biotechnology, 59, 169-181, 1998 National Institute of Genetics Japan DNA Data Bank Home Page <URL: http: // www. ddbj. nig. ac. jp /> US National Institute of Health, National Center of Biotechnology Information Homepage <URL: http: // www. ncbi. nlm. nih. gov />

  An object of this invention is to provide the novel microorganisms which can produce zeaxanthin selectively and in large quantities, and the manufacturing method of zeaxanthin by the said microorganisms.

  As a result of intensive studies on the above problems, the present inventors have reached the present invention. That is, the present invention is a bacterium belonging to the genus Paracoccus having an improved zeaxanthin productivity in which zeaxanthin, which is an intermediate metabolite, is well accumulated by mutating the carotenoid-producing paracoccus genus N-81106 strain. The present invention also provides a method for producing zeaxanthin, which comprises culturing such bacteria and recovering zeaxanthin from the bacterial cells or the culture solution.

  Therefore, the present invention provides a zeaxanthin-producing microorganism selected from mutant strains obtained by breeding carotenoid-producing Paracoccus bacteria, such as the TSTT002 strain deposited under the accession number FERM P-20695 at the Patent Organism Depositary. It is.

  Further, the present invention is a microorganism that produces 14.9 mg or more of zeaxanthin per liter of culture medium, and further produced β-carotene, β-cryptoxanthin, echinone, canthaxanthin, 3-hydroxyequinone, 3′-hydroxy In carotenoids containing echinenone, zeaxanthin, phenicoxanthin, adonixanthin and astaxanthin, zeaxanthin is a microorganism that accounts for 85% by weight or more of the total amount of the carotenoids.

  Moreover, this invention is a manufacturing method of zeaxanthin which culture | cultivates said microorganisms and collects zeaxanthin from the microbial cell or culture solution after culture | cultivation. The present invention will be described in detail below.

  The microorganism of the present invention is a novel microorganism induced by breeding of the Paracoccus bacterium N-81106, and is selected from mutant strains obtained by breeding of a carotenoid-producing Paracoccus bacterium, such as the TSTT002 strain. It is a zeaxanthin-producing microorganism.

  The Paracoccus genus N-81106 strain is a microorganism discovered by Marine Biotechnology Research Institute, Inc. and is published in the company's Culture Collection (MBIC). The N-81106 strain is known to accumulate astaxanthin, adonixanthin, or glycosides thereof in cells at a high content, but zeaxanthin is quickly used as an intermediate metabolite to various carotenoids that are the next metabolites. Because it is converted, it hardly accumulates in the cell.

  The microorganism of the present invention is induced by breeding of Paracoccus genus N-81106. As a breeding method, in addition to a method of selecting excellent strains derived from natural mutation, mutagen and ultraviolet rays are used. Various methods such as selecting strains with improved productivity after accelerating mutation by treating cells with, and fusing strains with different properties obtained by the above methods The method can be performed. In particular, methods using mutagens can induce mutations, and methods for obtaining useful strains in a short time are known. Methods for inducing these mutations are well known to those skilled in the art. As mutagens, compounds such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and ethyl methanesulfonate (EMS) can be used.

  For example, the cells of Paracoccus genus N-81106 obtained by culturing in advance are suspended in an aqueous solution of these compounds. After standing for a certain period of time, the cells are collected by a method such as centrifugation, and the mutagen is removed and cultured on a plate medium. Next, a colony having a changed color tone is selected. Colonies can be selected by arbitrarily selecting a large number of colonies, performing liquid culture after separation, extracting carotenoids from the collected cells with an appropriate solvent, and measuring the absorbance at around 470 nm of the extract.

  In this way, the primary selection is performed, and then the composition of the extract is analyzed by HPLC or the like to obtain a strain having improved carotenoid productivity or a specific carotenoid composition. For example, it is a strain that selectively synthesizes zeaxanthin, canthaxanthin, β-carotene, lycopene and the like.

  More specifically, the microorganism of the present invention is a microorganism that produces 14.9 mg or more of zeaxanthin per liter of the medium, and the produced β-carotene, β-cryptoxanthin, echinone, canthaxanthin, 3-hydroxyequine. Among the carotenoids including non-3′-hydroxyechinone, zeaxanthin, phenicoxanthin, adonixanthin and astaxanthin, zeaxanthin is a microorganism that accounts for 85% by weight or more of the total amount of the carotenoids.

  The reason why the microorganism of the present invention can efficiently produce specific carotenoids is due to reduced activity or inactivation of enzymes responsible for carotenoid metabolism, although the details of the mechanism are unknown. The TSTT002 strain that accumulates zeaxanthin is considered to have a significantly reduced or inactivated activity of β-carotene ketating enzyme (an enzyme that ketolates the 4th and 4 ′ positions of β-carotene).

  As the medium used in the present invention, any medium can be used as long as bacteria can grow and produce carotenoids, and the carbon source is molasses, glucose, fructose, maltose, sucrose, starch, lactose, glycerol. Acetic acid, etc., nitrogen sources such as corn steep liquor, peptone, yeast extract, meat extract, soybean cake, ammonium salts such as ammonium acetate, ammonium chloride, ammonium sulfate, glutamic acid, aspartic acid, glycine, etc. Amino acids include inorganic salts such as monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, and sodium chloride, and metal ions include magnesium chloride, magnesium sulfate, and sulfate. 1 iron, ferric sulfate, ferrous chloride, ferric chloride, iron citrate, ammonium sulfate Mu iron, calcium chloride dihydrate, calcium sulfate, zinc sulfate, zinc chloride, copper sulfate, copper chloride, manganese sulfate, manganese chloride, and other vitamins such as yeast extract, biotin, nicotinic acid, thiamine, riboflavin, inositol , Pyridoxine and the like can be used.

  The culture conditions in the present invention are not particularly limited as long as bacteria can grow and produce carotenoids, but the culture temperature is preferably 15 to 35 ° C., and the pH of the culture solution is preferably 6 to 9, The time is preferably 24 to 200 hours.

  The present invention is a method for producing zeaxanthin, in which the above microorganism is cultured, and zeaxanthin is collected from the cultured cells or the culture solution. In order to collect zeaxanthin, zeaxanthin-accumulating microorganisms are collected from the culture solution, What is necessary is just to extract with a suitable organic solvent etc.

  As the organic solvent, methanol, ethanol, isopropyl alcohol, acetone, methyl ethyl ketone, methyl isobutyl ketone, dichloromethane, chloroform, dimethylformamide, dimethyl sulfoxide and the like are preferable. Acetone is preferred. Furthermore, it is also possible to separate with high purity using liquid chromatography or the like. Examples of the separation principle of liquid chromatography include ion exchange, hydrophobic interaction, separation sieve and the like. Preferred are reverse phase chromatography and normal phase chromatography.

  According to the present invention, it is possible to efficiently produce zeaxanthin useful as a feed additive, food coloring, functional food, and antioxidant.

Hereinafter, although it demonstrates still in detail using an Example, this invention is not limited to these.

Example 1
Acquisition of strains TSN18E7 (FERM P-19746) strain obtained from Paracoccus genus N-81106 through several mutation breeding is various astaxanthin, adonixanthin, phenicoxanthine, canthaxanthin, echinone, β-carotene, etc. However, the zeaxanthin-selective highly synthetic bacterium TSTT002 strain used in the present invention was obtained by mutation modification of the TSN18E7 strain.

  Specifically, 5 ml of the medium shown in Table 1 was placed in a test tube and sterilized, and then the TSN18E7 strain was inoculated and cultured with shaking at 25 ° C. and 150 rpm for 1 day. 1 ml of this culture solution was transferred to a 1.5 ml Eppendorf tube, and the cells were collected by centrifugation at 15,000 rpm for 10 minutes. This microbial cell was suspended in 1 ml of 0.1 M potassium phosphate buffer (buffer A) at pH 7.0, and then 3 mg / ml N-methyl-N′-nitro-N-nitrosoguanidine (hereinafter referred to as NTG). (Abbreviated) 10 μl of aqueous solution was added and allowed to stand for 30-60 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 0.5 ml buffer A, inoculated into 3 ml of the medium shown in Table 1, and cultured for 4 to 5 hours. The obtained culture broth was appropriately diluted, applied on a plate medium having the composition shown in Table 2, and kept at 25 ° C. for 5 days. Yellow colonies were preferentially selected and grown from the grown colonies, and cultured with shaking in a medium having the composition shown in Table 1 at 25 ° C. and 100 rpm for 7 days. The culture broth was analyzed and a strain with improved zeaxanthin productivity was selected.

  Carotenoids were identified and quantified as follows. First, 1 ml of the culture solution was placed in a 1.5 ml Eppendorf tube and centrifuged at 15,000 rpm for 5 minutes to obtain a cell pellet. The cells were suspended in 20 μl of pure water, and then 200 μl of dimethylformamide and 500 μl of acetone were added and shaken to extract carotenoids. After the residue was removed by centrifugation at 15,000 rpm for 5 minutes, various carotenoids were quantified by high performance liquid chromatography (hereinafter abbreviated as HPLC) using a TSKgel-ODS80TM column (manufactured by Tosoh Corporation).

  Carotenoids were separated using a 5:95 mixed solvent of pure water and methyl alcohol as liquid A, and a 7: 3 mixed solvent of methyl alcohol and tetrahydrofuran as liquid B, and liquid A was columned for 5 minutes at a flow rate of 1 ml / min. Then, the solution was passed from a 100% solution A to 100% solution B at a linear flow rate gradient for 5 minutes at the same flow rate, and the solution B was further passed for 5 minutes. Component assignment of carotenoids was performed by comparison of HPLC retention times with commercially available reagents. By the above method, zeaxanthin is detected at a retention time of 9.5 to 10.0 minutes.

  Through the above operation, a bacterial TSTT002 strain that selectively produces zeaxanthin at high yield was obtained.

(Example 2)
Cultivation of new microorganisms and determination of carotenoids in flasks Inoculate 50 ml of a glycerol stock solution of TSTT002 in 5 ml of a sterilized medium having the composition shown in Table 1 at 25 ° C for 1 day at a shaking speed of 150 revolutions per minute. Culture was performed.

  Next, 60 ml of a sterilized medium having the composition shown in Table 1 was placed in a 100 ml baffled Erlenmeyer flask and sterilized at 121 ° C. for 20 minutes. Then, 2 ml of the above culture solution was inoculated, and at 25 ° C. for 7 days every minute. Culturing was performed at a shaking speed of 100 revolutions.

  The turbidity (OD 660 nm) of the culture solution at the end of the culture was 3.6. When carotenoids were extracted from the cells after completion of the culture and the amount of carotenoids was quantified by HPLC, 14.9 mg of zeaxanthin was produced per liter of the medium. Moreover, the total amount of carotenoids including β-carotene and β-cryptoxanthin was 17.5 mg. Therefore, the ratio of zeaxanthin to the total amount of carotenoid was 85% or more. The carotenoid production pattern (HPLC chart) at this time is shown in FIG.

  Moreover, when the TSN18E7 strain, which is the parent strain of the TSTT002 strain, is cultured by the same culture method, astaxanthin, adonixanthin, phenicoxanthine, canthaxanthin and echinone are accumulated, and zeaxanthin is not accumulated. The carotenoid production pattern at this time is shown in FIG. 3 (HPLC chart). In addition, Table 3 shows quantitative values for each component.

It is a pathway diagram starting from phytoene, chemically modifying β-carotene, and finally producing astaxanthin. It is a HPLC chart of carotenoids extracted from TSTT002 strain, and in the figure, the X axis (horizontal axis) indicates retention time (unit is minutes), and the Y axis (vertical axis) is HPLC peak intensity (unit is mV (arbitrary intensity)). ). It is a HPLC chart of carotenoids extracted from TSN18E7 strain, in which X-axis (horizontal axis) indicates retention time (unit: minutes), Y-axis (vertical axis) HPLC peak intensity (unit: mV (arbitrary intensity)) ).

Explanation of symbols

1: Zeaxanthin peak 2: Astaxanthin

Claims (5)

A zeaxanthin-producing microorganism selected from mutants obtained by breeding carotenoid-producing Paracoccus bacteria. The zeaxanthin-producing microorganism according to claim 1, wherein the microorganism is a carotenoid-producing Paracoccus genus strain TSTT002 (Accession No. FERM P-20695). The microorganism according to claim 1 or 2, wherein 14.9 mg or more of zeaxanthin is produced per liter of the medium. Among the carotenoids produced including β-carotene, β-cryptoxanthin, echinenone, canthaxanthin, 3-hydroxyechinenone, 3'-hydroxyechinenone, zeaxanthin, phenicoxanthine, adonixanthin and astaxanthin, zeaxanthin The microorganism according to any one of claims 1 to 3, wherein the amount of the microorganism is 85% by weight or more of the total amount of the carotenoids. A method for producing zeaxanthin, comprising culturing the microorganism according to any one of claims 1 to 4 and recovering zeaxanthin from the cultured cells or culture solution.

Images