JP2003052357A - Marine microorganism, and method for manufacturing carotenoid pigment and/or higher unsaturated fatty acid by using the microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new microorganism having high productivity of a carotenoid substance and a higher unsaturated fatty acid, which are nutritional supplements, and a method for manufacturing the carotenoid substance and the higher unsaturated fatty acid in high yields by using the microorganism. SOLUTION: KH 105 strain (FERM P-18431) of the genus Schizochytrium. A method comprising culturing the strain KH 105 in a medium and collecting a carotenoid substance and/or a higher unsaturated fatty acid from the culture products.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel marine microorganism and a method for producing a carotenoid pigment and / or a polyunsaturated fatty acid using this microorganism.

[0002]

2. Description of the Related Art Carotenoid (or carotenoid) type substances are a general term for pigments which exhibit a wide distribution throughout the animal and plant kingdom and which exhibit a yellow color, a reddish color or a reddish color. For example,
Green and yellow vegetables such as carrots, tomatoes, strawberries, spinach,
That is a fat-soluble pigment that gives off the color of the vegetable. This pigment group is roughly classified into a hydrocarbon carotenoid "carotene (or carotene)" (which does not contain oxygen) and xanthophyll (which contains oxygen).
The former is represented by α-carotene, β-carotene, lycopene and the like, and the latter is represented by lutein, cryptoxanthin and zeaxanthin. It has been said for a long time that these pigments are good for the body, but recent studies have made them even clearer. It is becoming more and more demonstrated that "antioxidant nutrients" are important in most of the fields of the current research "to live long and healthy". One of the most important nutrients among them is the carotenoid group, which has been confirmed to have more than 600 kinds. It is said that ingestion of these carotenoids as a carotenoid group is more effective in preventing aging than ingestion of one type. That is, carotenoids are said to be stored in different body locations and perform some protective function at that location (for example, the retina of the eye stores lutein and plays a role in preventing retinal oxidation). Therefore, it is said that it is preferable to take in a complex form rather than taking only one kind of carotenoid such as β-carotene.

Conventionally, more than 600 different bacteria, yeasts, fungi, and plants having carotenogenic ability are known, but only two of them, β-carotene and astaxanthin, are commercially available from microorganisms. It is only produced. Astaxanthin is produced in a paffia strain created by classical mutation.

[0004] Carotenoids are synthesized by an isoprene basic biosynthetic pathway that is partially common with isoprenoids such as sterols, hopanol, and dolichol. Hydroxymethylglutaryl CoA (HMG-CoA) is converted to C ₅ isopentenylpyrroline (IPP) via mevalonic acid
P is dimethylallyl pyrophosphate (DMAPP) by isomerization reaction
Is converted to. Further, DMAPP is sequentially condensed with C ₅ IPP to give C ₁₀ geranylpyrophosphate (GPP), C 10
_The number of carbon atoms is increased by 5, such as ₁₅ farnesyl pyrophosphate (FPP) and C ₂₀ geranylgeranyl pyrophosphate (GGPP).

A biosynthetic pathway specific to carotenoids is GG
It branches from the isoprene-based biosynthetic pathway in PP. That is, two molecules of GGPP are condensed to synthesize the first carotenoid, colorless phytoene. Phytoene is converted into lycopene by a desaturation reaction, and lycopene is further converted into β-carotene by a cyclization reaction. Then, a hydroxyl group or a keto group is introduced into β-carotene, and zeaxanthin (zeaxa
Various xanthophylls such as nthin and astaxanthin are synthesized.

[0006] The knowledge of genes responsible for carotenoid biosynthesis has exploded in the last 10 years. For example,
The carotenoid biosynthetic genes of the plant resident non-photosynthetic bacterium Erwinia uredovora are cloned into Escherichia coli using the yellow color as an index, and various combinations of these genes are introduced into microorganisms such as Escherichia coli and expressed, thereby expressing these genes. The function of the gene has been clarified. As a result, it has become possible to obtain a gene group necessary for biosynthesizing phytoene, lycopene, β-carotene, zeaxanthin, and zeaxanthin-β-D-diglucoside (Norihiko Misawa, “Carotenoids clarified at the gene level. "Biosynthetic pathway" protein nucleic acid enzyme, 41, 337-346,199
6). However, these methods have not been put to practical use because the operations are still complicated.

As a specific method for producing carotenoids from plants currently in practical use, a raw material containing a yellow pigment such as citrus is soaked in hot water and dehydrated by hot water temperature to produce a carotenoid-containing raw material. Method of recovering, deoiling, drying and crushing (Japanese Patent Publication No. 4-38388), and further crushing carotenoid-containing natural products such as pericarp in water with biopolymer degrading enzyme to complete the enzymatic reaction. A method for producing a carotenoid powder pigment by solid-liquid separation by centrifugation at a certain time, separating the water-soluble component and the solid component, and drying and pulverizing the obtained solid (Japanese Patent Laid-Open No. 62-190090).
Etc. have been reported.

On the other hand, docosahexaenoic acid (DHA) is a kind of essential fatty acid, but it is a substance that is not produced in the human body and must be ingested from outside the body. This DHA is a polyunsaturated fatty acid (PUFA) that is abundant in phospholipids such as synaptic membranes and photoreceptor membranes of the cerebral cortex and retina, and is considered to play an important role in learning ability and visual function. In addition, anti-cancer, hypolipidemic, anti-thrombotic, anti-inflammatory and anti-allergic actions have been reported, and while their diverse physiological functions have been noted, foods intended to enhance and regulate nutrition by ingestion from outside the body.・ Demand for feed additives is increasing.
However, its source is mainly orbital fat of skipjack and tuna, and although improvements have been made in the production process of high-purity DHA, further cost reduction and a new source for that are required. Also, in skipjack and tuna,
It is not produced in the body, but is merely accumulated in the body by ingesting what is produced by the microorganisms in seawater. On the other hand, most recently, the genes of all enzymes involved in the biosynthesis of γ-linolenic acid (GLA) and eicosapentaenoic acid (EPA), which are intermediates, have been isolated, and transgenic plants containing them have been isolated. Creation has become a reality. However, a few more steps are required to reach DHA, and the enzyme involved is unidentified. Thus, in the production of fats and oils by gene recombination, various unsaturated fatty acids are targeted, and rapid technological progress has been made, especially in the United States, but DHA production is still unclear.

As a source of DHA other than fish oil, a production method using microorganisms is considered. Examples of microorganisms that produce DHA-containing fats and oils include Vibrio marinus (ATCC 15381), a bacterium isolated from the deep sea, Vibrio spp. Trust Chitrium (T
Thraustochytrium aureum (ATCC34304), Japonochytrium sp. (ATCC 28207), a microalgae, Cyclotella cry
ptica) is known, and the production of DHA-containing fats and oils by a culture method using these microorganisms has been studied (PK
Bajapai, P.Bajapai and OPWard, J.Am.Oil Chem.So
c., 68: 509 (1991) and JP-A-1-199588).

Thraustochytrium Aureum (Thraus
For tochytrium aureum) (ATCC 34304), for example,
Journal of Fermentation and Bioengineering Vol.81,
No. 1,76-78 (1996) has a detailed report on DHA production by this microorganism. However, according to the method using these microorganisms, the amount of DHA-containing oil / fat produced per liter of medium is as low as about 100 to 700 mg. Therefore, the amount of DHA produced per liter of culture medium is as low as several tens to 500 mg.

Among these microorganisms, the labyrinthine Schizochytrium (Schizochytrium) has recently been mentioned as a particularly excellent organism.
A new species belonging to the genus Strium, Schizochytrium SR21 strain has been attracting attention. Journal of American Oil Ch
emists' Society Vol.73, No.11, 1421-1426 has a detailed report on the production of docosapentaenoic acid (DPA) and DHA by this microorganism.

Initially, this SR21 strain was considered to be a microorganism of the genus Thraustochytrium.
However, as a result of detailed examination of its mycological properties, it was found that this strain is a microorganism recognized as a new species of the genus Schizochytrium.

According to the overview of the prior art as described above, carotenoid substances by marine microorganisms, and
Utilizing the production capacity of DHA, carotenoids and D
Although it is possible to produce HA, it has not reached the commercial level due to the uneven distribution of the substances produced by these microorganisms and the low yield.

[0014]

As described above, an industrial production method of carotenoid pigment and DHA using a microorganism having the ability to produce carotenoid pigment and DHA has not been established yet. In addition, carotenoid pigments and DH
A microorganism that has a high productivity of A and can be used for industrial production is not yet known.

The invention of this application has been made in view of the above-mentioned conventional techniques, and an object thereof is to provide a microorganism having a high ability to produce carotenoid substances and highly unsaturated fatty acids.

Further, the invention of this application is to provide a method for producing a carotenoid-based substance and a polyunsaturated fatty acid in a higher yield than in the prior art using the above-mentioned microorganism and without requiring complicated steps. I am trying.

[0017]

Means for Solving the Problems The inventors of the present application
Up to now, it has been possible to provide a next-generation DHA supply source by expressing mainly in the microorganism itself having DHA productivity, and the genes of the enzymes that act in the DHA biosynthesis system in microorganisms such as yeast or plants. Although we have carried out the research aimed at, we have focused our attention on labyrinthuloid microorganisms, which are eukaryotic microorganisms with high DHA productivity, as a microbial source for this purpose. This microbial group is basically marine, acts as a decomposer of leaf litter, and is considered to be the primary producer of PUFA that accumulates in marine fish. However, in terms of the classification of microorganisms, it does not belong to algae or fungi, and its ecology is hardly known. Also,
It cannot be said that systematic search, classification, and analysis for the purpose of application of PUFA such as DHA to production have been sufficient. Therefore, the inventors first analyzed the lipid production characteristics of several hundred DHA-producing bacteria isolated from seawater coastal areas. Among them, we have found an excellent strain that exhibits excellent growth and lipid productivity, as well as pigment productivity. Furthermore, we have clarified in detail the optimal culture conditions for the strain, and as a new dye and DHA supply source. The present invention has been completed and the present invention has been completed.

[0018] Therefore, as an invention for solving the above-mentioned problems, this application provides a novel marine microorganism, Schizochytrium sp. Strain KH105 (FERM P-18431).

The invention of this application also relates to the above-mentioned microorganism KH105.
A method for producing a carotenoid substance and / or a polyunsaturated fatty acid, which comprises culturing a strain (FERM P-18431) in a medium and collecting a carotenoid pigment and / or a polyunsaturated fatty acid from the culture. provide.

According to the method of the present invention, a carotenoid pigment and a polyunsaturated fatty acid can be produced with high efficiency. Moreover, a carotenoid pigment and a polyunsaturated fatty acid can be simultaneously produced with high efficiency. In particular,
Polyunsaturated fatty acids extracted from microorganisms are extremely susceptible to oxidation, which is a cause of deterioration in the quality of oils and fats containing them. Since carotenoid pigments have antioxidant effects, simultaneous production of both from a single microorganism is extremely effective in suppressing the oxidation of highly unsaturated fatty acids.

In the invention of this production method, the concentration of seawater in the medium is 50% or more, the carbon source in the medium is one or more of glucose, glycerol and sodium acetate, and the nitrogen source in the medium is , Peptone, sodium nitrate, and potassium nitrate are preferably one or more.

The Schizochytrium of the present invention (Schizoc
The KH105 strain of the genus hytrium (FERM P-18431) is classified into the genus Schizochytrium from the viewpoint of morphology, but this classification is not necessarily definitive and there are many undetermined parts.

The embodiments of the invention of this application will be described in detail below.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION The KH105 strain of the present invention is a marine microorganism isolated from seawater and highly produces carotenoid substances and highly unsaturated fatty acids such as DHA. This K
The medium used for culturing the H105 strain may be of any composition as long as the strain can grow well, and it is different for the bacterial cell growth and the substance production.
Different types of media may be used. A liquid medium or a solid medium can be used as the medium, and as the medium components, in addition to the carbon source, a nitrogen source and inorganic ions may be contained. The concentration of seawater in the medium is preferably 50% or more.

There are no particular restrictions on the carbon source in the medium for growing the bacterial cells, and known carbon sources can be used, but lower fatty acids such as acetic acid, malic acid, succinic acid, propionic acid, and barmitic acid, starch, Sucrose and the like can be preferably used. These carbon sources can be added to the medium alone or in combination. On the other hand, it is preferable to add one or more of glucose, glycerol and sodium acetate as a carbon source to the medium for producing the carotenoid substance and / or the polyunsaturated fatty acid. Further, it is preferable that the concentration of the carbon source is 6-12%.

As the nitrogen source, inorganic nitrogen compounds such as ammonium sulfate and ammonium nitrate, and organic nitrogen sources such as peptone and yeast extract can be used. It is preferable to add one or more of peptone, sodium nitrate, and potassium nitrate to the medium for producing carotenoid substances and / or highly unsaturated fatty acids.

Further, examples of the inorganic ion include phosphate ion, magnesium ion, calcium ion, potassium ion, iron ion, copper ion, manganese ion and the like, and these inorganic ions are contained in the medium in the form of inorganic salt or the like. May be.

As a method of culturing the microorganism of the present invention, a method of culturing with a liquid medium such as a shaking culturing method and a method of culturing with a solid medium such as an agar medium can be used. When producing carotenoid substances and / or highly unsaturated fatty acids, the culture temperature is, for example, 20 to 35 ° C, preferably 25 to 30 ° C.
The culturing time can be determined according to the amount of cells, the amount of substance produced, etc., but 0.5 day to 5 days, more preferably about 1 day to 3 days is suitable.

After such culture, the carotenoid substance and / or polyunsaturated fatty acid can be isolated and purified from the culture by known means. For example, salting out or ammonium sulfate precipitation method, dialysis, centrifugation, ultrafiltration, gel filtration,
Examples include SDS-PAGE, isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography and the like.

The method of screening the KH105 strain of the present invention, the mycological properties, etc. will be described in detail below. 1. Method 1.1 Screening for DHA-producing bacteria A suitable amount of pine pollen is floated on the surface of a seawater sample diluted with sterilized water and left standing for 2 to 4 days, and the pine pollen is agar with a platinum loop (3% glucose, 0.1% peptone). , 0.05% yeast extract, 50% artificial seawater (ASW), 2% agar) and spread. After culturing at 28 ° C. for several days, the appeared colonies were spread on a new agar medium and further cultivated for purification. The colonies grown on the agar medium were cut out together with the agar and dried overnight in an open chamber, and then the lipids of the bacterial cells were extracted and the fatty acid composition was measured (Aki et al. (1998) Lipi.
d Composition of Newly Isolated Polyunsaturated Fa
tty Acid Producing fungus, Achlya sp.ma-2801, J Fer
ment Bioeng 86: 504-507). 1.2 Culture and lipid analysis Liquid cultures are performed in 200 ml baffle flasks containing 50 ml medium.
It was carried out by shaking culture at 28 ° C. for 2 to 7 days. When adding fats and oils such as fatty acids, 1% of tergitol NP-40
Were added and cultured. After collecting cells by centrifugation, wet cells or dried cells were homogenized in the presence of glass beads in a chloroform-methanol mixture (2: 1, v / v), filtered, washed with water, dehydrated, and concentrated. The total lipids of the cells were used and weighed. Equal amount of 10 in 1% chloroform solution of lipid
% Hydrochloric acid-methanol was added and the mixture was heated at 60 ° C. for 3 hours to methylate the fatty acid, and the hexane extract was measured for the fatty acid composition by capillary gas chromatography (GC). The fatty acid content was calculated using arachidic acid or nonadecanoic acid as an internal standard.
Identification of fatty acid molecular species was performed by GC-mass spectrometry analysis. The chloroform phase obtained above was concentrated to an appropriate amount for analysis of the dye components. Perform thin layer chromatography (silica gel 60: Merck) (TLC) in the dark,
The pigment component was separated. Developing solvent is acetone-hexane
(3: 7, v / v) was used. The quantification of the dye component was performed using a densitometer. 1.3 Molecular phylogenetic analysis Bacteria were collected by centrifugation and lysed (8M urea, 2%
SDS, 0.15% NaCl, 1 mM EDTA, 100 mM Tris-HCl, pH 7.
5) and liquid nitrogen (Murray and Thom
pson (1980) Rapid isolation of high molecular weigh
t plant DNA.Nucleic Acid Res 8: 4321-4325). Total genomic DNA was phenol-chloroform method (Sambrook et al. (19
89) Analysis and Cloning of eukaryotic genomic DN
A, In: Nolan C, Ferguson M (eds) Molecular Cloning: A
Laboratory Manual, ed 2, Cold Spring Harbor Laborato
ry Press, Cold Spring Harbor, pp9-31) and used as a template for PCR reaction. 18S ribosomal RNA (r
For amplification of (RNA) gene, oligo DNA primer 18sl (5'-CCA
ACCTGGTTGATCCTGCCAGTA-3 ': SEQ ID NO: 1) and 18s12 (5'
-CCTTGTTACGACTTCACCTTCCTCT-3 ': SEQ ID NO: 2) was used
(Honda et al. (1999) Molecular phylogeny of labyri
nthulids and thrsustochytrids based on the sequenc
ing of 18S ribosomal RNA gene. J Eukaryot Microbio
l 46: 637-647). PCR reaction solution (50 μl) is 5 μl 10 x PCR b
uffer, 20 mM dNTPs, 0.5 μM each primer, 2 units Taq
Contains pclymerase, 2 μg genomic DNA. After denaturing at 95 ° C. for 5 minutes, the reaction of 95 ° C. × 30 sec, 55 ° C. × 1 min, 72 ° C. × 30 sec was repeated 30 times, and finally the extension reaction was performed at 72 ° C. for 10 min. The DNA nucleotide sequence of the obtained gene fragment was analyzed using a PCR amplification product or a plasmid obtained by inserting it into pGEM-T Easy Vector (Promega) as a template and using a Gene Rapids sequencer (Amersham Pharmacia Blotech.). Cafeteria roen for molecular phylogenetic analysis and creation of phylogenetic trees
bergensis and Haematococcus pluvialis as an outgroup distance method (Saitou and Nei (1987) The neighbo
r-joining method: a new method for reconstructing p
hylogenetic trees. Mol Biol Evol 4: 406-425). 2. Isolation of DH-producing strain KH105 strain Seawater samples were collected at 50 points on the Seto Inland Sea coast centering on Hiroshima Bay and Kure Bay and 10 points on the mangrove community on the coast of Iriomote Island, and fishing using pine pollen was performed. About 500 species of bacteria were isolated and purified by the bait method. As a result of examining the fatty acid composition in the total lipids in the cells of each of these strains by GC analysis, 25 kinds of DHA contained 30% or more in the total fatty acids,
It was found that three kinds contained eicosapentaenoic acid (EPA) in an amount of 15% or more, and three kinds contained arachidonic acid (AA) in an amount of 25% or more, respectively. Among them, a significant amount of DHA
Attention was paid to the KH105 strain which produced n-6 docosapentaenoic acid (DPA) and showed high proliferation (Fig. 1). In flask-level liquid culture, PUFA composition in total lipid was DHA.
It was 27.9% and DPA 8.5% (Table 1). In addition, it was speculated that the cells were orange or orange-yellow and accumulated carotenoid-like pigment in the cells. However, n- found in many algal lipids that are pigment-producing and have photosynthetic systems
3 Octadecatetraenoic acid (C18: 4n-3) was not detected.

The KH105 strain isolated as described above was used in 2001
On July 25, 2013, the patent was deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depository Center, with the deposit number FERM P-18431.

[0032]

[Table 1]

3. Species identification of KH105 strain In order to perform a molecular phylogenetic analysis based on the DNA base sequence of the 18S rRNA gene, the gene of KH105 strain was first amplified by PCR, and its DNA base sequence was analyzed using the PCR product as a template. However, it could not be decoded because the signals from the sequencer overlapped in some areas. This indicates that a plurality of DNA fragments differing in sequence only partially exist in the PCR product used as the template. Although the 18S RAN gene exists in multiple copies on the genome, since such diversity has not been reported, it was decided to analyze it in further detail. Ten clones were randomly selected from the clones obtained by cloning the PCR product, and the DNA base sequence was determined for each clone. As shown in FIG. 2, five different sequences ranging from 21 to 23 bp were found. Interestingly,
This region was discovered by Honda et al. Thraustochytri
d Phylogenetic group (TPG) -specific insertion sequence (Honda
It coincided with the site of et al. (1999): supra. TPGs are newly grouped by the presence of this specific sequence Thra
It is a taxon that contains some labyrinthula marine microorganisms such as Ustochytrium and Schizochytrium. The TPG-specific sequence is located at region 8 helix 46 in 18S rRNA (Neefs et al. (1993) Compilation of small rib
Nucleic Acids Res 2
1: 3025-3049), the sequence differs depending on the species and its function is not clear. Therefore, although the significance of the diversity found in the KH105 strain is unknown, it may be used as a molecular marker for this microorganism and its related microorganisms. At least due to the presence of this characteristic sequence, KH105
It was strongly suggested that the strain is contained in Labyrinthula such as Thraustochytrium and Schizochytrium.

Then, when the molecular phylogenetic analysis of the KH105 strain was carried out using the labyrinthula microbial group as a control, it was found that it was the closest relative to S. limacinum SR21 as shown in the phylogenetic tree of FIG. In fact, the fatty acid composition of KH105 strain (Table 1) and the assimilability of carbon expression in the medium (described later) were very similar to those of SR21 strain (Yokochi et al. (1998) Optimizat
ion of docosahexaenoic acid production by Schizoch
ytrium limacinum SR21. Appl Microbio Biotechnol 4
9: 72-76). Furthermore, when the life cycle of the KH105 strain was observed under a microscope, continuous mitosis of vegetative cells peculiar to the genus Schizochytrium was observed (Fig. 1) (Honda et al. (1998) Schizoc
hytnum hmacmum sp.nov., a new thraustochyirid from
a mangrove area in the west Pacific Ocean. Mycol R
es 102-439-448). From these facts, the KH105 strain is Schizo
It was concluded that it is included in the chytrium genus. But S.limacinu
18Sr RNA gene of mSR21 strain and KH105 strain was 86.3% (1544 bp
However, since the SR21 strain does not produce pigment, it cannot be concluded that it is the same species. In fact, regarding the group of microorganisms belonging to the Labyrinthula, the conventional classification and nomenclature based on morphology and the classification by molecular phylogenetic analysis do not necessarily match. It is considered that this is because the labyrinthula microorganisms have various life cycles and the morphology changes remarkably depending on the culture conditions. Therefore, morphological observation under unified conditions is required in the future. In addition, reorganization that adds molecular phylogenetic analysis to the amino acid sequence of proteins such as EF1-α may be effective (Roger e
t al. (1999) An evaluation of elongation factor 1
alpha as a phylogenetic marker for eukaryotes. Mol
Biol Evol 16: 218-233).

Hereinafter, the invention of this application will be described in more detail and specifically with reference to Examples, but the invention of this application is not limited by the following examples.

[0036]

Examples Example 1: Acquisition of carotenoid pigments from KH105 strain Carotenoid pigments were collected from KH105 strain by the following method.

Glucose (or galactose, fructose, mannose, arabinose, glycerol) 3
%, Yeast extract 0.5%, peptone 1.5%, artificial seawater 50%, 50 ml of a medium was placed in a 200 ml Erlenmeyer flask and sterilized at 120 ° C. for 20 minutes.

The Schizochytrium sp. Strain KH105 was inoculated on an agar medium containing 0.85% of corn meal agar, 0.75% of bactoagar and 50% of artificial seawater to form colonies. Take one platinum loop, inoculate in 50 ml medium, and use a reciprocal shaker (160 rp).
The cells were cultured at 28 ° C. for 2 days.

After culturing, cells were collected by centrifugation (3000 rpm, 10 min) and washed twice with distilled water. 30 ml of acetone / methanol (7: 3) was added, and homogenized (10000 rpm, 5 min) under ice-cooling together with approximately the same amount of glass beads. After dehydration by adding an appropriate amount of anhydrous sodium sulfate, the mixture was evaporated to dryness at room temperature under a nitrogen stream. This was stored at -20 ° C protected from light.

The concentrated solution of carotenoid dye in acetone was spotted on a silica gel thin layer plate (silica gel 60) and subjected to chromatographic development under light-shielding conditions using acetone / hexane (3: 7) as a developing solvent. At this time, ethoxyquin was added to the solvent as an antioxidant at 7.5 mg / ml.

13 silica gel 60 for column chromatography
It was activated by heat treatment at 0 ° C. for 3 hours. This was suspended in chloroform and packed in a 20 cm high column. After washing the column sequentially with chloroform and hexane, a carotenoid dye sample was applied.

As the elution solvent, hexane, hexane / acetone (50: 1), the same (20: 1) and the same (10:
1), the same (4: 1), and stepwise elution with acetone. The visible part was fractionated by looking at the color of the band, and the other part was fractionated with time. The above operation was performed under the shaded condition as much as possible.

The absorbance of the hexane solution of the dye component at 400 to 500 nm was measured with a spectrophotometer. Further, based on the absorbance at 450 nm, the β-carotene concentration was calculated from the extinction coefficient.

E (1%) / (1cm) = 2592 FIG. 4 shows the results of thin layer chromatographic analysis of the extracted components.

As a result, β-carotene, astaxanthin and astaxanthin fatty acid ester were confirmed as pigment components derived from the KH105 strain.

FIG. 5 shows the results of thin layer chromatographic analysis of the fractionated dye components.

FIG. 6 shows the absorption characteristics of the fractionated dye components (Fr.1, Fr.2 and Fr.5) shown in FIG.

Β-carotene was fractionated as a single dye component, and exhibited absorption maximums at 420 nm, 450 nm and 475 nm. About 1 to 5 mg of β-carotene was produced per liter of medium. From the spot intensity in the thin layer chromatographic analysis, about 2 to 10 mg of astaxanthin and its fatty acid ester were produced per liter of the medium. Example 2: KH
Acquisition of carotenoid pigment and DHA from strain 105 The carotenoid pigment and highly unsaturated fatty acids such as DHA were simultaneously obtained from strain KH105 by the method described below.

The medium, inoculation, and culture were the same as in Example 1. After culturing, the cells were collected by centrifugation (3000 rpm, 10 min) and washed twice with distilled water. 30 ml of chloroform / methanol (2: 1) was added, and the mixture was homogenized with water in an amount of glass beads under water cooling (10000 rpm, 5 min). After centrifugation (3000 rpm, 10 min), the chloroform layer was taken and evaporated to dryness at room temperature under a nitrogen stream. This was stored at -20 ° C protected from light.

Thin layer chromatographic analysis of carotenoid dye was carried out in the same manner as in Example 1.

The lipid was hydrolyzed and methyl-esterified by adding an equal amount of 10% hydrochloric acid / methanol solution and heating at 60 ° C. for 3 hours. After evaporating to dryness at 60 ° C. under a nitrogen stream, hexane and distilled water (1 ml each) were added, and the mixture was vigorously stirred and then centrifuged (3000 rpm, 3 min). The hexane layer was taken, and again evaporated to dryness at 60 ° C under a nitrogen stream. It was dissolved in 50 μl of hexane and the fatty acid composition was analyzed by gas chromatography.

The results of thin layer chromatographic analysis of carotenoid dyes were the same as those shown in FIG. 4 in Example 1.

The results of fatty acid composition analysis are shown in Table 1.

As described above, from the chloroform-extracted lipid components derived from the KH105 strain, carotenoid pigments containing β-carotene and astaxanthin, DHA (27.9%) and DPA (8.
Polyunsaturated fatty acids containing 5%) could be extracted simultaneously. Example 3: Acquisition of highly unsaturated fatty acid containing DHA from KH105 By the method shown below, a large amount of highly unsaturated fatty acid containing DHA was produced using KH105 strain.

3 liters of a medium (pH 7.0) containing 9% glucose, 1% yeast extract, 2% peptone and 50% artificial seawater was added.
Place in a liter jar fermenter tank, 120 ° C for 20
Sterilized for a minute.

The Schizochytrium sp. Strain KH105 was inoculated on an agar medium containing 0.85% cornmeal agar, 0.76% bactoagar and 50% artificial seawater to form colonies. Take one platinum loop, inoculate in 50 ml medium, and use a reciprocal shaker (160 rp).
The cells were cultured at 28 ° C. for 2 days.

This was inoculated into a 2 liter medium and cultured for 7 days at 28 ° C., a stirring speed of 500 rpm, and an aeration rate of 5 liter / min.

10 ml of the culture solution was collected during and after the culture, and the cells were collected by centrifugation (3000 rpm, 10 min).
It was dried at 105 ° C for several hours. By measuring this weight, the amount of bacterial cells was calculated. After finely crushing the dry cells,
10 ml of chloroform / methanol (2: 1) was added, and the mixture was homogenized (10000 rpm, 5 min) under ice cooling. Centrifuge (3000r
After that, the chloroform layer was removed and evaporated to dryness at room temperature under a nitrogen stream.

An arachidic acid standard substance was used as an internal standard.
μg equivalent amount was added. This was stored at -20 ° C protected from light.

An equal amount of a 10% hydrochloric acid / methanol solution was added to the extracted lipid, and the mixture was heated at 60 ° C. for 3 hours for hydrolysis and methyl esterification. After evaporating to dryness at 60 ° C. under a nitrogen stream, hexane and distilled water (1 ml each) were added, and the mixture was vigorously stirred and then centrifuged (3000 rpm, 3 min). The hexane layer was taken, and again evaporated to dryness at 60 ° C under a nitrogen stream. 50
After dissolving in μl of hexane, fatty acid composition analysis by gas chromatography was performed. The total lipid amount and each fatty acid amount were calculated by comparison with the internal standard substance.

FIG. 7 shows the results of jar fermenter culture of KH105 strain.

By jar fermenter culture of the KH105 strain, 17 g / liter of dry cells, 9.9 g / liter of total fatty acids and 3.4 g / liter of DHA were obtained as maximum values at the time of 83 hours of culture. Example 4: Examination of DHA production conditions by KH105 strain KH105 strain was used as a basic medium (glucose 3%, yeast extract 0.5.
%, Peptone 1%, artificial seawater 50%), flask culture (50 ml) at 28 ° C. reached a stationary phase in 36-48 hr,
It was found that DHA production did not increase after that. When the artificial seawater concentration was 50-100%, the cell growth and the DHA production reached the maximum (Fig. 8), so it was decided to set the artificial seawater concentration to 50% as described above. When the assimilation and DHA production of the KH105 strain were examined for several types of carbon sources and fats and oils, the maximum was obtained when glucose was used,
Glycerol and sodium acetate have also been found to be suitable (Table 2).

[0063]

[Table 2]

As compared with them, a lower value was obtained with soluble starch which is a disaccharide such as sucrose or maltose or a polysaccharide. This tendency for carbon source assimilation is similar to that of the SR21 strain (Honda et al. (1998) Schiz
ochytnum hmacmum sp. nov., a new thraustochyirid fr
om a mangrove area in the west Pacific Ocean. Myco
l Res 102: 439-448). Next, as a result of examining the nitrogen source,
It was found that peptone was the most suitable (Table 2). In the case of yeast extract, the lipid accumulation was low, but the proliferation and DHA content were high, so we decided to use them in combination. Preliminary experiments revealed that a suitable ratio of peptone and yeast extract was 2: 1.

When the glucose concentration was examined,
6-12% yielded almost the same DHA productivity, but 9%
Was highest (1.1 g DHA / L; Fig. 9). Also, even at 15%
It was found to be an interesting strain showing a DHA productivity of about 0.8 g / L and resistant to a high concentration medium. Next, when various nitrogen source concentrations were compared, the highest DHA production was shown when 2% peptone and 1% yeast extract were used (1.2 g-DHA / L; FIG. 10). At this time, biomass (biomass) was 11.4 g / L, total fatty acid content was 5.2 g / L, and DHA in total fatty acid was
The content was 23.1%. Therefore, at least at the flask level, the optimal medium composition for DHA production by the KH105 strain is glucose 9%, peptone 2%, yeast extract 1
%, Artificial seawater 50%.

As described above, the KH105 strain showed a maximum DHA productivity of 1.2 g / L after 2 days of culture at the flask level.
This value corresponds to the productivity (0.7-0.7) reported in the analysis under almost the same conditions for the SR21 strain, which is currently considered to have the highest DHA productivity.
1.5g / L; (Yokochi et al. (1998) Optimization of do
cosahexaenoic acid production by Schizochytrium li
macinum SR21, Appl Microbiol Biotechnol 49; 72-76)
Can be said to be a promising strain for industrial use. Further, it has been suggested that the aeration rate (dissolved oxygen content) is an important factor at the preliminary experiment level, and it is considered that this point also needs to be strictly controlled when culturing on a large scale in the future. .

[0067]

INDUSTRIAL APPLICABILITY As described in detail above, the invention of this application provides a novel microorganism which is excellent in proliferative properties and oil and fat accumulation properties, and has a high ability to produce carotenoid substances and docosahexaenoic acid (DHA). By using this microorganism, it is possible to efficiently produce carotenoid substances and fats and oils having a high DHA content, which are useful in the fields of foods and pharmaceuticals, at high yields that have never been achieved. The substance produced by the method according to the present invention has a great effect even if it is used as a food or a medicine as it is as a mixture. However, the carotenoid substance and the DHA-containing oil and fat obtained by the production method of the present invention contain icosapentaenoic acid (C20 in Table 1). : 5n-3) The low content ratio makes it easy to separate and purify DHA from fats and oils. Therefore, DHA can be efficiently produced from the DHA-containing oil and fat. That is, according to the present invention, in particular, carotenoid substances and DHA-containing fats and oils by the culture method, which replace the DHA-containing fats and oils separated and purified from the carotenoid-based substances and fish oils that are currently classified and purified from citrus, and which contain icosapentaenoic acid, An excellent method for producing a fat and oil containing DHA having an extremely low ratio is provided.

[0068]

[Sequence list] SEQUENCE LISTING <110> Japan Science and Technology Corporation <120> A marine bacterium and a method for producing carotenoid substance      and / or poly unsaturated fatty acids by using the bacterium <130> NP01274 <160> <210> 1 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Synthesized oligonucleotide <400> 1 ccaacctggt tgatcctgcc agta 24 <210> 2 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Synthesized oligonucleotide <400> 2 ccttgttacg acttcacctt cctct 25

[Brief description of drawings]

FIG. 1 shows a micrograph of Schizochytrium KH105 strain.

Figure 2: Schizochytrium KH105 strain 18S
Thraustochytrid phyloge DNA sequence of rRNA gene
It is shown in comparison with the insertion sequence specific to the netic group (TPG).

[Fig. 3] KH10 with a labyrinthula microbial group as a control
The phylogenetic tree resulting from the molecular phylogenetic analysis of 5 strains is shown.

FIG. 4 shows the results of thin layer chromatographic analysis of KH105 strain dye components.

FIG. 5 shows the results of thin layer chromatographic analysis of the dye components fractionated in FIG.

FIG. 6 shows the absorption characteristics of the dyes fractionated in FIG.

FIG. 7 shows the results of jar fermenter culture of KH105 strain.

FIG. 8 shows the effect of artificial seawater concentration (50-100%) on DHA production by the KH105 strain.

FIG. 9 shows the effect of glucose concentration on DHA production by strain KH105.

FIG. 10 shows the effect of nitrogen source concentration on DHA production by KH105 strain.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ identification code FI theme code (reference) C12R 1: 645) C12R 1: 645 (C12P 7/64 C12R 1: 645) (C12P 23/00 C12R 1: 645) (72) Inventor, Seiko Shigeta 2-9-13, Oki-Shioya, Ono-cho, Saiki-gun, Hiroshima Prefecture (72) Shoji Kawamoto, 8-3-3, Saijo Chuo, Higashi-Hiroshima City, Hiroshima Prefecture (reference) 4B064 AD88 AH01 CA05 CC03 CD02 CD09 CD20 CD21 CD30 DA10 DA20 4B065 AA58X AC08 BA22 BB02 BB06 BB08 BB12 BB15 BB19 BB22 BB24 BB40 BC01 CA05 CA13

Claims (8)

[Claims] 1. A schizochytrium genus KH10.
5 shares (FERM P-18431). 2. A Schizochytrium genus KH10.
A method for producing a carotenoid substance, which comprises culturing 5 strains (FERM P-18431) in a medium and collecting a carotenoid pigment from the culture. 3. A Schizochytrium genus KH10.
A method for producing a carotenoid substance and a polyunsaturated fatty acid, which comprises culturing 5 strains (FERM P-18431) in a medium and collecting the carotenoid substance and the polyunsaturated fatty acid from the culture. 4. A schizochytrium genus KH10.
A method for producing a highly unsaturated fatty acid, which comprises culturing 5 strains (FERM P-18431) in a medium and collecting the highly unsaturated fatty acid from the culture. 5. The method according to claim 2, wherein the concentration of seawater in the medium is 50% or more. 6. The carbon source in the medium is one or more of glucose, glycerol and sodium acetate.
One of the manufacturing method of. 7. The carbon source concentration is 6-12%.
Manufacturing method. 8. The method according to claim 2, wherein the nitrogen source in the medium is at least one of peptone, sodium nitrate and potassium nitrate.