JP2002291464A - New arachidonic acid and carotenoid producing microorganism, method for producing the same, and method for producing the arachidonic acid and carotenoid by using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide microorganisms capable of effectively producing carotenoids and arachidonic acid in large amounts from easily available raw materials, without using fine algae which are conventionally required for producing the carotenoids, nor using fisheyes and specific bacteria which are conventionally required for producing the arachidonic acid. SOLUTION: This microorganisms consist of Labyrinthla sp. CHN-1 belonging to Labyrinthula genus, produce the carotenoids so that their cells are colored into red, and produce the arachidonic acid in a culture medium to which no phosphorus is added.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel microorganism belonging to the genus Labyrinthula, a method for producing the same, and a method for producing arachidonic acid or a carotenoid using the novel microorganism.

[0002]

2. Description of the Related Art Arachidonic acid is present in the cell membrane and endoplasmic reticulum membrane of animals in a state bound to the 2-position of the glycerol skeleton of phospholipids, which enhances the fluidity of biological membranes and enhances the activity of prostaglandins, tropoxane and leukotriene. Although it is a physiologically important compound such as a precursor, it is not synthesized in the animal body and must be supplied from the outside as an essential fatty acid.

[0003] On the other hand, carotenoids are pigments widely distributed in the animal and plant kingdoms, and include vitamin A in animals.
It is important as a nutrient. As described above, arachidonic acid and carotenoids are attracting attention from the viewpoint of nutritional physiology.In recent years, arachidonic acid and carotenoids have been found to be effective in controlling aging, detoxifying and preventing cancer, and the demand has rapidly increased. ing.

[0004] By the way, arachidonic acid and carotenoids are extracted from organisms such as carrots and algae. To grow these organisms efficiently, it is necessary to use special nutrients having high nutritional value. The use of commonly used nutrients such as sugars, peptones, and phosphorus cannot be grown in large quantities, so that the cost is unavoidable.

[0005]

DISCLOSURE OF THE INVENTION The present invention is easy to obtain without the use of fish eyes and special bacteria required for the production of microalgae and arachidonic acid, which have been required for the production of carotenoids. The purpose is to produce carotenoids and arachidonic acid efficiently and in large amounts from various raw materials.

[0006]

Means for Solving the Problems The present inventors have intensively studied a method for producing a useful substance using a marine microorganism, and have first studied Labyrinthul.
a) Labyrinthura sp. S3-2 (Labyrinthulasp. S3) which is one of microorganisms belonging to the genus
-2), a method for producing long-chain highly unsaturated fatty acids mainly comprising docosahexaenoic acid and docosapentaenoic acid was proposed (Japanese Patent Application No. 2000-94441). Among the microorganisms belonging to the present invention, carotenoids and carotenoids can be obtained by using a novel microorganism that differs from the previous ones in that it ingests carotenoids, colors it red, and produces higher unsaturated fatty acids containing arachidonic acid as a main component. The inventors have found that arachidonic acid can be produced efficiently, and based on this finding, have accomplished the present invention.

[0007] That is, the present invention relates to a labyrinthula (L
Labyrinthura sp. CHN-1 is a microorganism belonging to the genus abyrinthula, which is characterized by producing carotenoids and coloring the cells red and producing arachidonic acid in a medium without phosphorus.
(Labyrinthula sp. CHN-1), in which pine pollen is suspended in seawater collected from the Seto Inland Sea, and microorganisms adsorbed on the surface are inoculated into a nutrient medium and cultured, and Labyrinthura sp. CHN-
1. A method for producing arachidonic acid or carotenoid, comprising culturing Labyrinthura sp. CHN-1 in a phosphorus-free medium containing saccharides and extracting arachidonic acid or carotenoid from the culture solution. Is provided. The microorganism of the present invention, Labyrinthura sp. CHN-1 (Labyrint)
hula sp. CHN-1) has accession number FERM
P-18262 has been deposited with the National Institute of Advanced Industrial Science and Technology (AIST).

[0008]

DETAILED DESCRIPTION OF THE INVENTION The microorganism of the genus Labyrinthura to which the microorganism of the present invention belongs is easily distinguished from other microorganisms by its mycological properties. That is, the vegetative cells of the Labyrinthula microorganism are characteristic spindle-shaped, and gliding through a protoplasmic network. The two upper taxonomic Labyrinthurae are composed of Labyrinthurae and Thraustochytrium, the Labyrinthurae is composed of Labyrinthula 1 and the Thraustochytrium family is Thraustochytrium, Schizochytrium and the like. Seven genera have been reported (Handbook of Proctista,
David Porter, 1990).

Also, fungi of the Thraustochytrium family are:
The vegetative cells are spherical or ovoid, and are easily distinguished from Labyrinthula, which has a characteristic spindle shape. These Labyrinthura microorganisms were previously classified in the kingdom Fungi,
Known as so-called marine bacteria, which are widely present in the marine environment and multiply by heterotrophs, the microorganisms of the Thraustochytrium family are classified into oomycetes, and the microorganisms of the Labyrinthura family are classified into slime molds. However, these microorganisms of the Labyrinthula generally have a life cycle through zoospores having flagella of two different lengths and morphologies, and based on the zoospore structure or gene phylogenetic analysis,
In recent years, it has been incorporated into the unequal hair gate of the yellow plant kingdom.

[0010] Labyrinthura SPCHN of the present invention
-1 is a microorganism consisting of a single cell of 10 to 30 μm that grows in the ocean and can be easily collected from the sea near Japan, for example, the seawater of the Seto Inland Sea. Collection of the drug substance
For example, the method can be carried out by suspending pine pollen in seawater and leaving it to stand for 1 to 10 days, separating pine pollen, and collecting microorganisms attached to the surface of the pollen.

[0011] In this case, as the pine, Korean pine (Pine koraiensis), Himekomatsu (P. pentaphylla), slash pine,
Daisho (P. palastris), Scots pine (P. Sylvestris), loblolly pine, red pine (P. densiflora), Japanese black pine and the like are used.

[0012] Next, the raw material thus collected is purely cultured in a saccharide medium, that is, a culture solution rich in saccharides and peptone (for example, a medium containing 50 g of glucose and 1 g of peptone in 1 liter of seawater). . This pure culture is preferably performed under aeration and stirring conditions while continuously irradiating light at a constant temperature.

Thus, when the growth of Labyrinthura reaches the maximum concentration (10 g / L or more), the Labyrinthura microorganisms are precipitated and concentrated from the culture by centrifugation, and only the Labyrinthura microorganisms are recovered. . As a result of the analysis, it was found that the microorganism thus obtained contained a considerable amount of docosahexaenoic acid (DHA) together with a large amount of arachidonic acid and carotenoid. At this time, as the carotenoid to be produced, for example, astaxanthin and carotene can be mentioned.

Labyrinthura SPCHN of the present invention
-1 produces such carotenoids and is colored red by possessing them in cells, but the known rabirintula microorganisms do not have such ability.

Labyrinthula is a microorganism that grows on glucose, light energy, and inorganic phosphorus. In order to maximize its production function, the illuminance as light energy, temperature, and elemental composition in the medium are appropriate. It is necessary to adjust.

For example, the conditions of pure culture are as follows: air stirring, light irradiation of 1,000 lux or more, and temperature of 20 to 3 times.
It is 0 ° C, preferably around 28 ° C. The source of phosphorus in the medium is preferably KH ₂ PO ₄ , and its amount is 0.1
A range of 3 g / liter is preferred. The range in which Labyrinthula can grow (1 liter of seawater contains 6 glucose
0 g, 1 g of peptone, 1 g of KH ₂ PO ₄ ), when the growth stops, the concentration of nutrient elements in the medium is increased to a higher concentration so that the production of DHA, arachidonic acid and carotenoids can be reduced. Can be enhanced.

Further, as described above, when the stimulation of Labyrinthula is increased by changing the culture conditions, particularly the phosphorus composition in the medium, the production of DHA, arachidonic acid and carotenoid can be further increased.

Therefore, in order to produce DHA, arachidonic acid and carotenoid in high yield, it is necessary to keep the growth environment conditions of Labyrinthula at an extreme state and to maximize the production function of DHA, arachidonic acid and carotenoid. Is advantageous. It is effective to add, for example, phosphorus to the initial medium.

That is, when an aqueous solution containing phosphorus is added to the medium, the phosphorus in the aqueous solution is taken up by the cells of Labyrinthula, which is an adsorbent, and the content of DHA, arachidonic acid, and carotenoids is 1 g of microorganisms (dry matter). DHA, arachidonic acid and carotenoids increase to as high as 300 mg. If the culture medium is changed to a phosphorus-free culture at that time, the content of DHA, arachidonic acid, and carotenoids is increased to 380 mg / g of microbial organism (dry matter) because there is no phosphorus taken up by the cells of Labyrinthura.

When arachidonic acid and a carotenoid are produced by the method of the present invention, the culture is first carried out in a medium to which phosphorus has been added, and after the phosphorus has been consumed, phosphorus is not supplemented. It is preferable to culture under addition conditions. By culturing in a phosphorus-free medium in this way, the yield of carotenoid and arachidonic acid are increased by 1.5 times or more and 20 times or more, respectively, as compared with the case where phosphorus is added as usual. The DHA yield drops to less than 1/5.

Next, in order to efficiently extract DHA, arachidonic acid and carotenoids from the thus obtained DHA, arachidonic acid and carotenoid-rich Labyrinthula microorganisms, a chloroform-methyl alcohol solution is used. Is preferred.

In this manner, the produced DHA, arachidonic acid and carotenoid are separated from the microorganism. Then, the solution is then passed through a column packed with silica gel, and developed with a mixed solution of methyl alcohol, whereby 100
% Yield of DHA, arachidonic acid and carotenoids can be recovered. Usually, a mixed solvent of methyl alcohol having a volume of 90 to 95% by volume of methyl alcohol and the remainder of chloroform is used.

[0023]

Next, the present invention will be described in more detail by way of examples, which should not be construed as limiting the present invention.

Example 1 Labyrinthura sp. CHN-1 obtained by suspending Japanese red pine pollen in seawater in the Seto Inland Sea Nagashima area was used for seawater 1
A saccharide medium containing 60 g of glucose, 1 g of peptone, 1 g of potassium dihydrogen phosphate and 1 g of phosphorus per liter was inoculated, cultivated for 6 days under agitation under light irradiation at a temperature of 28 ° C. and 1,000 lux and stirring. Next, the culture solution was centrifuged to collect the microorganisms. FIG. 1 shows an electron micrograph of the surface of the microorganism thus obtained, and FIG. 2 shows a transmission electron micrograph of a section obtained by embedding the microorganism in a resin and sectioning it.

Next, the above-mentioned microorganisms were extracted with methyl alcohol, and this extract was passed through a column filled with silica gel (trade name "Wakogel", manufactured by Wako Pure Chemical Industries, Ltd.) and adsorbed. By developing with -methyl alcohol, 800 mg each of DHA and arachidonic acid and 580 mg each of astaxanthin and carotene were obtained. The contents of the microorganisms (DHA and arachidonic acid in the fatty acids, and astaxanthin and carotene in the microorganisms) were as follows. Product Concentration Astaxanthin 10% by mass Carotene 8% by mass DHA 45% by mass Arachidonic acid 30% by mass

Example 2 Next, 50 g of glucose and 1 peptone in 1 liter of seawater
g. and a medium containing 1 g of potassium dihydrogen phosphate, and a medium supplemented with 1 g of phosphorus, were used to culture Labyrinthura sp. CHN-1 under the same conditions as in Example 1.
Table 1 shows the results.

[0027]

[Table 1]

As can be seen from this table, the use of the medium without phosphorus compared to the case of using the medium without phosphorus
The concentration of carotenoids (astaxanthin + carotene) increases 1.7-fold, the concentration of arachidonic acid increases 20-fold, while the concentration of DHA decreases to 1/5.

Example 3 The experiment using the phosphorus-free medium of Example 2 was continued for 16 days, and biomass, total fatty acids, arachidonic acid and D
The production amount of HA over time was determined and is shown in FIG. 3 as a graph. As is clear from this figure, the DHA production hardly changes after 7 days, but the production of arachidonic acid sharply increases.

[0030]

Industrial Applicability According to the present invention, in particular, Labyrinthula itself containing a high content of arachidonic acid and carotenoid can be cultured in a large scale in a pure frame, and arachidonic acid and carotenoid can be easily extracted and separated therefrom with high yield. Arachidonic acid, carotenoids or DHA as desired
Can be obtained.

[Brief description of the drawings]

FIG. 1 Labyrinthura SP C obtained in Example 1
The electron microscope photograph figure of the surface of HN-1.

FIG. 2 Labyrinthura SP C obtained in Example 1
The electron microscope photograph figure of the section of HN-1.

FIG. 3 is a graph showing the time-dependent production of a product in a culture solution of the microorganism of the present invention.

[Procedure amendment]

[Submission date] July 16, 2002 (2002.7.1)
6)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0019

[Correction method] Change

[Correction contents]

That is, when an aqueous solution containing phosphorus is added to the medium, the phosphorus in the aqueous solution is taken up by the cells of Labyrinthula, which is an adsorbent, and the content of DHA, arachidonic acid, and carotenoid is determined by the microorganism ( dry mass ). 1g
300 μg of DHA, arachidonic acid and carotenoids in it
Increase to a high value. At that time, if the medium was changed to a culture solution containing no phosphorus, there was no phosphorus taken up by the cells of Labyrinthula, so that the DHA, arachidonic acid, and carotenoid contents were 380 μg / g of microorganism ( dry mass ).
To increase.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0025

[Correction method] Change

[Correction contents]

Next, the above-mentioned microorganisms were extracted with methyl alcohol, and this extract was passed through a column filled with silica gel (trade name "Wakogel", manufactured by Wako Pure Chemical Industries, Ltd.) and adsorbed. By developing with -methyl alcohol, 800 µg each of DHA and arachidonic acid and 580 µg each of astaxanthin and carotene were obtained. The contents based on the dry mass of the microorganism (DHA and arachidonic acid in the fatty acid, astaxanthin and carotene in the microorganism) were as follows. Product concentration Astaxanthin 1.0 % by mass Carotene 0.8 % by mass DHA 4.5 % by mass 3.0 % by mass of arachidonic acid

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0027

[Correction method] Change

[Correction contents]

[0027]

[Table 1]

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B064 AC38 AD88 CA05 CC04 CC12 CD02 CD09 CD20 CD21 CD30 CE08 CE09 CE10 4B065 AA58X AC14 BA22 BB02 BB15 BB24 BC03 BC05 BC08 BC48 BD14 BD16 CA09 CA13 CA41 CA43 CA44 CA46

Claims (3)

[Claims] 1. Labyrinthu (Labyrinthu)
la) a microorganism belonging to the genus Labyrinthura sp. CHN-1 (Labyrinth), which is a microorganism belonging to the genus and characterized by producing carotenoids and coloring cells red and producing arachidonic acid in phosphorus-free medium.
ula sp. CHN-1). 2. A method for preparing a Labyrinthula, comprising: suspending pine pollen in seawater collected from the Seto Inland Sea;
SP CHN-1 (Labyrinthulas)
p. A method for producing CHN-1). 3. A phosphorus-free medium containing saccharides,
Labyrinthura SP CHN-1 (Labyrin
thula sp. A method for producing arachidonic acid or carotenoid, comprising culturing CHN-1) and extracting arachidonic acid or carotenoid from the culture solution.