JP2012139164A - Method for producing carotenoid by using microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing carotenoids using microorganisms, particularly carotenes such as lycopene.
The above-mentioned problems are solved by culturing a microorganism having a carotenoid-producing ability using a medium to which a calcium compound is added so as to have a final concentration of 3.6 mM or more.
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Description

  The present invention relates to a method for producing carotenoids using microorganisms, particularly carotenes such as lycopene.

  Carotenoids are red, orange, and yellow pigments produced by animals, plants, microorganisms, etc., and are useful as natural food pigments and feeds. In recent years, carotenoids have a carcinogenic inhibitory effect due to their excellent antioxidant activity. It is attracting attention and is used in health foods such as supplements.

  Carotenoids are roughly classified into two types, xanthophylls containing oxygen atoms and carotenes containing no oxygen atoms. The former includes astaxanthin, lutein, β-cryptoxanthin, zeaxanthin, etc., and the latter includes lycopene, β-carotene, ζ-carotene, phytofluene, α-carotene, phytoene, γ-carotene, neurosporene and the like.

  Many carotenoids are extracted from plants. For example, lycopene is extracted from tomato fruit and the like, and lutein is extracted from marigold petals and the like. However, the amount of carotenoids that can be extracted from plants is small, and a large amount of plant raw materials is required to meet the increasing demand in recent years. However, because the growth of plants depends on the weather, etc., it is a stable and inexpensive carotenoid. Is difficult to supply. Therefore, production of carotenoids using microorganisms has been proposed in order to supply carotenoids more efficiently, stably and inexpensively.

  As a method for producing carotenoids using microorganisms, for example, a method for producing lycopene using algae (patent document 1) and fungi (patent document 2) has been proposed. Further, a method using a lycopene-producing bacterium derived from the marine Agrobacterium genus (later reclassified to the genus Paracoccus) bacterium N-81106 (Japanese Patent Laid-Open No. 7-184668), TSTT003 strain (October 25, 2005) A method using the National Institute of Advanced Industrial Science and Technology, the Patent Biological Depositary Center (FERM P-20696) has also been proposed (Japanese Patent Application Laid-Open No. 2003-151867).

JP-A-9-313167 JP 2003-304895 A JP 2007-151474 A

  Among the methods described above, a method using a lycopene-producing bacterium derived from marine Agrobacterium is an excellent method for producing a proposed carotenoid. However, such gram-negative microorganisms have a problem that the produced carotenoid accumulates in the cell membrane and impairs the growth. This is because carotenoids have high hydrophobicity, so that accumulation reduces the fluidity of the cell membrane and suppresses the growth of microorganisms. In particular, carotenes that do not contain oxygen atoms such as lycopene and β-carotene have higher hydrophobicity than xanthophylls such as astaxanthin and lutein that contain oxygen atoms. As a result, efficient production of carotenoids becomes difficult.

  Then, this invention is providing the method of manufacturing carotenoid, especially lycopene more efficiently.

  In view of the above problems, the present inventors have intensively studied and, as a result, have completed the present invention capable of achieving the above object. That is, the present invention is a carotenoid production method capable of efficiently producing carotenoids, in particular lycopene, using a medium to which a calcium compound is added so that its final concentration is 3.6 mM or more. And culturing a microorganism having an ability to produce carotenoids. Hereinafter, the present invention will be described in detail.

  The present invention alleviates the growth inhibition of microorganisms due to the accumulation of carotenoids by utilizing calcium, which has the property of changing the structure of the cell membrane of microorganisms, particularly Gram-negative bacteria. In the present invention, a medium containing calcium at a final concentration of 3.6 mM or more, preferably 3.6 mM to 7 mM, more preferably 5 mM to 7 mM is used. To obtain such a medium, calcium chloride, calcium sulfate, A widely available calcium compound such as calcium carbonate, calcium hydroxide, calcium phosphate, or calcium acetate may be added to the medium. Calcium may be hydrated or anhydrous, and the amount added to the medium may be as described above at the final concentration. This is because if the final concentration of calcium to be added is less than 3.6 mM, the effect of the present invention is diminished, and conversely, even if it is added so as to be 7 mM or more, the effect of adding calcium reaches its peak. Strictly speaking, calcium ions affect changes in the cell membrane structure of microorganisms, but considering the high ionization tendency of calcium, it is not possible to add a calcium compound with a final concentration of 3.6 mmol or more to the medium. This is synonymous with setting the calcium ion in the medium to 3.6 mmol or more.

  As medium components other than calcium, medium components conventionally used when producing carotenoids using microorganisms can be used without particular limitation, and in the present invention, in addition to the addition of calcium described above There is no need to add special ingredients. Specifically, examples of medium components other than calcium include, for example, molasses, glucose, fructose, maltose, sucrose, starch, lactose, glycerol or acetic acid as a carbon source, and corn steep liquor, peptone as a nitrogen source. Yeast extract, meat extract, soybean cake, ammonium acetate, ammonium chloride, ammonium sulfate, glutamic acid, aspartic acid or glycine as inorganic salts such as monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate Phosphate, sodium chloride, etc. of magnesium chloride, magnesium sulfate, ferrous sulfate, ferric sulfate, ferrous chloride, ferric chloride, iron citrate, ammonium iron sulfate, zinc sulfate, chloride Zinc, copper sulfate, copper chloride, manganese sulfate, manganese chloride, etc. Yeast extract and biotin as glutamic acids, nicotinic acid, thiamine, riboflavin, may be used to inositol or pyridoxine like. In addition, the hydrate or anhydride of the compound illustrated above can be used for a culture medium component without a restriction | limiting.

  The microorganism to be cultured in the present invention is not limited as long as it has a carotenoid producing ability, but is particularly effective against a microorganism having a property of accumulating the produced carotenoid in its cell membrane. As such microorganisms, gram-negative bacteria can be exemplified, but bacteria, particularly bacteria of the genus Paracoccus are preferable. Among the Paracoccus bacteria, the N-81106 strain has a good carotenoid producing ability and is particularly preferable as a microorganism used in the present invention.

  In the present invention, in addition to the naturally occurring microorganism such as the N-81106 strain described above, a modified microorganism whose carotenoid production ability has been improved by genetic modification can also be used. Such modified microorganisms have acquired or originally possessed carotenoid-producing ability that was not originally possessed by naturally occurring microorganisms by natural mutation, or by artificial mutagenesis by mutagen, ultraviolet rays, etc. It is a microorganism with enhanced carotenoid production ability. In the present invention, it is also possible to use a genetically modified microorganism such as introducing a gene related to carotenoid production.

  As an example of the above-described modified microorganism, the TUG1C11 strain (Japanese Patent Laid-Open No. 2005-58216), the TSN18E7 strain (Japanese Patent Laid-Open No. 2005-58216), and TSTT031 that have been artificially mutated to the N-81106 strain to enhance the carotenoid production ability Stock (Japanese Patent Laid-Open No. 2007-181449), TSTT052 strain (October 18, 1999, the National Institute of Advanced Industrial Science and Technology (AIST), Patent Biological Depositary Deposited under the deposit number FERM P-20690, and then the original deposit BP- TSTT003 strain (consigned as FERM P-20696 at the National Institute of Advanced Industrial Science and Technology (AIST) on October 25, 2005) or TSLGO3-2 strain (Heisei 22) December 15, 2015 National Institute of Advanced Industrial Science and Technology Can be exemplified place are accession an accession number FERM P-22035 in International Patent Organism Depositary).

  Moreover, as an example of the above-mentioned genetically modified microorganism, a recombinant microorganism obtained by further performing genetic recombination operations on the N-81106 strain, the TSN18E7 strain, the TSTT031 strain, or the TSTT052 strain can be exemplified (Japanese Patent Application No. 2005-315070). Issue).

  Carotenoids can be efficiently produced by culturing the microorganisms described above in the medium described above. The operation for culturing microorganisms is not particularly limited in the present invention, but an example will be described. For example, at a culture temperature in the range of 15 ° C. to 35 ° C., preferably 22 ° C. to 30 ° C., the pH of the medium is from pH 6 to It is possible to exemplify culturing for 20 hours to 200 hours, preferably 60 hours to 180 hours while maintaining pH 9, preferably pH 6.5 to pH 8.0. The exemplified culture temperature and pH can be changed so as to be appropriate for each stage in the initial stage, the middle stage and the late stage of the culture. The sugar concentration in the medium is preferably maintained at a low concentration and not depleted by feeding a sugar solution such as high concentration glucose.

  When the microorganism used in the present invention is cultured, carotenoid is produced in the microorganism, and the carotenoid is accumulated in the microorganism, particularly in its cell membrane or released outside the microorganism, and accumulated in the culture solution. A conventionally used method can also be used for recovering the carotenoid thus produced. As such a method, for example, a method of extracting a carotenoid produced using an organic solvent can be exemplified. Specific examples of the organic solvent include methanol, ethanol, isopropyl alcohol, acetone, methyl ethyl ketone, methyl isobutyl ketone, dichloromethane, chloroform, dimethylformamide, and dimethyl sulfoxide. These organic solvents can be used alone or in admixture of two or more. The carotenoid recovered in this manner can be highly purified as necessary. As a purification method, when carotenoids accumulate in the cell membrane of microorganisms, the microorganisms are first separated from the medium by centrifugation, decantation, or filtration, and then added to form a slurry, such as glass beads or zirconia beads. It is better to homogenize using a crusher or high-pressure homogenizer. Here, in particular, in order to prevent decomposition of lycopene, it is preferable to add an antioxidant to the slurry. Examples of the antioxidant include reducing agents such as ascorbic acid.

  Thereafter, it can be purified by liquid chromatography such as ion exchange chromatography, hydrophobic chromatography or gel filtration chromatography, among which reverse phase chromatography or normal phase chromatography is preferred. When purifying carotenoids by liquid chromatography, a fraction having an absorption peak from 420 nm to 510 nm may be obtained, and a fraction having an absorption peak from 450 nm to 490 nm may be obtained to achieve a higher degree of purification.

  Carotenoid productivity of microorganisms capable of producing carotenoids can be improved by a very simple operation of culturing microorganisms capable of producing carotenoids in a medium supplemented with calcium. Since this operation can be carried out simply by adding a certain amount of calcium compound to the medium, the practitioner does not require skill, and the time required for the implementation is extremely short.

It is a HPLC chart which shows a carotenoid production pattern when TSLGO2-23 strain | stump | stock is culture | cultivated by the culture medium which added 1.2 mmol of calcium chloride. It is a HPLC chart which shows a carotenoid production pattern when TSLGO2-23 stock | strain is culture | cultivated by the culture medium which added 3.6 mmol calcium chloride. It is a HPLC chart which shows a carotenoid production pattern when TSLGO2-23 strain | stump | stock is culture | cultivated by the culture medium which added 7.2 mmol of calcium chloride.

  EXAMPLES Hereinafter, examples will be described to describe the present invention in more detail, but the present invention is not limited to these examples.

  Example 1 Preparation of lycopene production mutant

  The TSTT052 strain was inoculated into 3 mL of the medium shown in Table 1, and cultured with shaking overnight at 25 ° C. and 150 rpm in a test tube. 1 mL of this culture solution was transferred to a 1.5 mL Eppendorf tube, and microorganisms were collected by centrifugation at 15,000 rpm for 5 minutes. This microorganism is 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 “buffer A”). 10 μL of an aqueous solution (referred to as “NTG”) was added and allowed to stand for 30 to 60 minutes. Thereafter, the supernatant was removed by centrifugation, and the operation of resuspending in buffer A was repeated twice to remove NTG. Furthermore, this microorganism was suspended in 1 mL of the medium shown in Table 1, and cultured in a test tube with shaking at 25 ° C. and 150 rpm for 4 to 5 hours. The obtained culture broth was appropriately diluted and applied to a plate medium having the composition shown in Table 2, followed by static culture at 25 ° C. for 1 week. Among the colonies that grew, colonies exhibiting a pink color peculiar to lycopene-producing bacteria were selected and subjected to evaluation of mutant strains by flask culture.

  The selected mutant strain was inoculated into 3 mL of the medium shown in Table 1, and cultured in a test tube with shaking at 25 ° C. and 150 rpm overnight. 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 cultivation, the culture solution was appropriately removed, and turbidity (OD 660 nm), glucose concentration, and carotenoid production were analyzed over time.

  Carotenoid production was quantified as follows. First, 0.8 mL of the culture solution was transferred to a 1.5 mL Eppendorf tube, and microorganisms were collected by centrifugation at 15,000 rpm for 5 minutes. This microorganism was suspended in 50 μL of pure water, and then 550 μL of dimethylformamide and 1000 μL of acetone were sequentially added and shaken to extract carotenoids. After removing the extraction residue by centrifugation at 15,000 rpm for 20 minutes, the obtained extraction supernatant was subjected to high-performance liquid chromatography using reverse phase chromatography (trade name, TSKgel-ODS80TM column, manufactured by Tosoh Corporation) ( (Hereinafter referred to as “HPLC”). 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 carotenoid concentration was determined by monitoring the absorbance at 470 nm and calculating the concentration from a calibration curve prepared with a known concentration of lycopene reagent (manufactured by Wako Pure Chemical Industries, Ltd.).

  According to the above method, the lycopene productivity of the mutant strain was evaluated, and a new mutant strain 2D038 that selectively produced lycopene was obtained.

実施例２ ２Ｄ０３８株への変異導入によるリコペン高生産菌の作製
実施例１と同様に、２Ｄ０３８株を表１に示す培地３ｍＬに植菌し、試験管中、２５℃、１５０ｒｐｍで１晩振とう培養を行った。この培養液のうち１ｍＬを１．５ｍＬエッペンドルフチューブに移し、１５，０００ｒｐｍ、５分間の遠心分離により微生物を回収した。

Example 2 Production of a lycopene high-producing bacterium by introducing a mutation into the 2D038 strain In the same manner as in Example 1, the 2D038 strain was inoculated into 3 mL of the medium shown in Table 1 and shaken overnight at 25 ° C. and 150 rpm in a test tube. Culture was performed. 1 mL of this culture solution was transferred to a 1.5 mL Eppendorf tube, and microorganisms were collected by centrifugation at 15,000 rpm for 5 minutes.

  This microorganism was suspended in 1 mL of Buffer A, then 10 μL of 3 mg / mL NTG aqueous solution was added, and the mixture was allowed to stand for 30 to 60 minutes. Thereafter, the supernatant was removed by centrifugation, and the operation of resuspending in buffer A was repeated twice to remove NTG. Furthermore, this microorganism was suspended in 1 mL of the medium shown in Table 1, and cultured in a test tube with shaking at 25 ° C. and 150 rpm for 4 to 5 hours. The obtained culture broth was appropriately diluted and applied to a plate medium having the composition shown in Table 2, followed by static culture at 25 ° C. for 1 week. Among the grown colonies, those with strong pink were selected and used for evaluation of mutant strains by flask culture.

  In the same manner as in Example 2, the selected mutant strain was inoculated into 3 mL of the medium shown in Table 1, and cultured in a test tube with shaking at 25 ° C. and 150 rpm overnight. Next, 1 mL of this culture solution was inoculated into 60 mL of the medium shown in Table 1 placed in a 100 mL baffled conical flask, and cultured with shaking at 25 ° C. and 120 rpm for 5 days. During the cultivation, the culture solution was appropriately removed, and turbidity (OD 660 nm), glucose concentration, and carotenoid production were analyzed over time.

  Carotenoid production was quantified as follows. First, 0.2 mL of the culture solution was transferred to a 2.0 mL Eppendorf tube, and microorganisms were collected by centrifugation at 15,000 rpm for 5 minutes. The microorganism was 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 the extraction residue was removed by centrifugation at 15,000 rpm for 20 minutes, carotenoids were quantified by HPLC analysis according to the method described in Example 2.

  According to the above method, the proliferation and lycopene productivity of the mutant strain were evaluated, and a mutant TSLGO2-23 strain having lycopene productivity equivalent to 2D038 strain and improved proliferation was obtained. Table 4 shows the maximum values of turbidity and lycopene production.

実施例３ リコペン生産変異株の評価
実施例２で示した変異株２Ｄ０３８株及びＴＳＬＧＯ２−２３株を表１に示す培地３ｍＬに植菌し、試験管中、２５℃、１５０ｒｐｍで１晩振とう培養した。次いでこの培養液１ｍＬを、１００ｍＬ容バッフル付三角フラスコに入れた表３に示す培地２０ｍＬへ植菌し、２５℃、１２０ｒｐｍで４日間振とう培養を行った。培養中は培養液を適宜抜き取り、濁度（ＯＤ６６０ｎｍ）、グルコース濃度、ｐＨ、及びカロテノイド生産量を経時的に分析した。カロテノイド生産量の定量は以下のように行った。まず培養液０．１ｍＬを２．０ｍＬ容エッペンドルフチューブに移し、１５，０００ｒｐｍ、５分間の遠心分離により微生物を回収した。この微生物を２０μＬの純水に懸濁し、次いで４８０μＬのジメチルフォルムアミド、及び５００μＬのアセトンを順次加え振とうすることでカロテノイドを抽出した。抽出残渣を１５，０００ｒｐｍ、２０分間の遠心分離により除去した後、実施例２に記載の方法に従ってＨＰＬＣ分析によりカロテノイドを定量した。

Example 3 Evaluation of Lycopene Production Mutant Strains The mutant 2D038 strain and TSLGO2-23 strain shown in Example 2 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. did. Next, 1 mL of this culture solution was inoculated into 20 mL of the medium shown in Table 3 placed in a 100 mL baffled conical flask, and cultured with shaking at 25 ° C. and 120 rpm for 4 days. During the culture, the culture solution was appropriately extracted, and the turbidity (OD660 nm), glucose concentration, pH, and carotenoid production amount were analyzed over time. Carotenoid production was quantified as follows. First, 0.1 mL of the culture solution was transferred to a 2.0 mL Eppendorf tube, and microorganisms were collected by centrifugation at 15,000 rpm for 5 minutes. This microorganism was 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 the extraction residue was removed by centrifugation at 15,000 rpm for 20 minutes, carotenoids were quantified by HPLC analysis according to the method described in Example 2.

  According to the above-mentioned method, the growth property and lycopene productivity of the mutant strain were evaluated. As Table 5 shows the results of turbidity (OD660 nm), lycopene production, and glucose consumption, TSLGO2-23 was superior to the 2D038 strain in growth and the same results as in Example 3 were obtained. The lycopene production amount was 120 mg / L, which was higher than that in Example 3.

実施例４ リコペン生産変異株のカルシウムによる影響
ＴＳＬＧＯ２−２３株を表１に示す培地３ｍＬに植菌し、試験管中、２５℃、１５０ｒｐｍで１晩振とう培養した。次いでこの培養液１ｍＬを、１００ｍＬ容バッフル付三角フラスコに入れた表３に示す培地のうち塩化カルシウム濃度を３．６ｍＭ、７．２ｍＭ、１０．８ｍＭに変更した組成の培地２０ｍＬへ植菌し、２５℃、１２０ｒｐｍで４日間振とう培養を行った。培養終了後、濁度（ＯＤ６６０ｎｍ）、グルコース濃度、及びカロテノイド生産量を測定した。カロテノイド生産量の定量は以下のように行った。まず培養液０．１ｍＬを２．０ｍＬ容エッペンドルフチューブに移し、１５，０００ｒｐｍ、５分間の遠心分離により微生物を回収した。この微生物を２０μＬの純水に懸濁し、次いで４８０μＬのジメチルフォルムアミド、及び５００μＬのアセトンを順次加え振とうすることでカロテノイドを抽出した。抽出残渣を１５，０００ｒｐｍ、２０分間の遠心分離により除去した後、実施例２に記載の方法に従ってＨＰＬＣ分析によりカロテノイドを定量した。

Example 4 Effect of calcium on lycopene production mutant strain TSLGO2-23 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. Next, 1 mL of the culture solution was inoculated into 20 mL of a medium having a composition in which the calcium chloride concentration was changed to 3.6 mM, 7.2 mM, or 10.8 mM in the medium shown in Table 3 placed in a 100 mL baffled conical flask. The shaking culture was performed at 25 ° C. and 120 rpm for 4 days. After completion of the culture, turbidity (OD660 nm), glucose concentration, and carotenoid production were measured. Carotenoid production was quantified as follows. First, 0.1 mL of the culture solution was transferred to a 2.0 mL Eppendorf tube, and microorganisms were collected by centrifugation at 15,000 rpm for 5 minutes. This microorganism was 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 the extraction residue was removed by centrifugation at 15,000 rpm for 20 minutes, carotenoids were quantified by HPLC analysis according to the method described in Example 2.

  According to the above-mentioned method, the growth property and lycopene productivity of the mutant strain were evaluated. Table 6 shows the turbidity (OD 660 nm), lycopene production, and glucose consumption at the end of the culture. As shown in Table 6, turbidity and lycopene production were significantly increased by increasing the concentration of calcium chloride to 3.6 mmol or more.

Claims (6)

A method for producing carotenoids, comprising culturing a microorganism having an ability to produce carotenoids using a medium to which a calcium compound is added so that the final concentration thereof is 3.6 mM or more. The method for producing carotenoid according to claim 1, wherein the carotenoid is a carotenes containing no oxygen atom. A carotenoid production method, wherein the carotenoid according to claim 1 is lycopene. The method for producing carotenoid according to any one of claims 1 to 3, wherein the microorganism is a microorganism that accumulates the produced carotenoid in a cell membrane. The method for producing carotenoid according to claim 4, wherein the microorganism is a bacterium belonging to the genus Paracoccus. The carotenoid production method according to claim 5, wherein the Paracoccus bacterium is N-81106 strain or a mutant thereof.

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