JPH0734752B2 - Method for producing arachidonic acid and lipid containing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing arachidonic acid and a lipid containing the same by a fermentation method.

[Conventional technology]

Conventionally, as a method for producing arachidonic acid by a microorganism, a carbohydrate or a hydrocarbon is used as a carbon source, and as a microorganism, a genus Penicillium, a genus Aspergillus, and a rhodotora (Rhodoto).
A method of using a microorganism belonging to the genus rula or the genus Fusarium has been reported (Japanese Patent Publication No. 56-1923).
See 1,56-19232,56-19233).

However, in any method, the yield is low, the culture time is long, or the process is complicated.

Further, a method for producing arachidonic acid using a microorganism of the genus Mortierella is not known.

[Problems to be solved by the invention]

The present invention, using Mortierella microorganisms that were not previously known to have the ability to produce arachidonic acid, using an inexpensive conventional medium, in a shorter culture time than conventional methods, in high yield Moreover, it is intended to provide a method capable of producing arachidonic acid and a lipid containing the same by a simple process.

[Means for solving problems]

The above-mentioned purpose is to produce a arachidonic acid or a lipid containing arachidonic acid by culturing a microorganism belonging to the genus Mortierella and having arachidonic acid-producing ability, and collecting arachidonic acid. And a method for producing a lipid containing arachidonic acid, which comprises collecting a lipid containing arachidonic acid by culturing a microorganism belonging to the genus Mortierella and having an ability to produce arachidonic acid.

[Specific explanation]

In the present invention, any microorganism belonging to the genus Mortierella and capable of producing arachidonic acid can be used. Such microorganisms include, for example, Mortierella elongata IFO 857.
0, Mortierella exigu
a) IFO 8571, Mortierella hygrophyra (Mortier
ella hygrophila) IFO 5941 and the like.
Any of these strains can be obtained from the Fermentation Research Institute of Japan without any limitation.

In addition, the strain Mortiertilera elongata SAM 0219 (Miken Koken No. 1239) isolated by the present inventors from soil can also be used.

Next, the mycological properties of the above-mentioned strain SAM 0219 (Microtechnical Laboratory Article No. 1239) will be described.

Growth condition on each medium Culture conditions: 25 ℃, under dark 1. Malt extract agar medium Colonies grow well, colonies on the second day of culture are 28 in diameter.
-31 mm, colonies on day 5 of culture 65-72 mm in diameter, colonies show superficial cleavage, poor development of aerial hyphae, good formation of spores of ascospores, spores of spores derived from aerial hyphae, garlic Has a similar odor.

2. Potato-glucose agar medium Colonies grow well, colonies on the second day of culture are 27 in diameter.
-31 mm, diameter of colonies on the 5th day of culture is 75-80 mm, colonies have rose flower shape, aerial hyphae are remarkably developed in the center of the colony, the back side of the colony is yellowish white or yellow,
Poor sporangiospore formation, with a garlic-like odor, with a moderately strong odor.

3. Czapek agar medium The growth of colonies is relatively good, the diameter of the colonies on the second day of culture is 22-24 mm, and the diameter of the colonies on the fifth day of culture is 50-53.
mm, poor development of aerial hyphae, aerial hyphae may be tightly entangled. The formation of sporangiospores is very good and the sporangiophores arise from aerial hyphae. Has a odor similar to garlic.

4. LCA agar medium (medium preparation method was according to Koichiro Miura, Mitsuyo Kudo, “One Agar Medium for Incomplete Aquatic Bacteria”, Japan Society of Mycological Society, Vol. 11, pp. 116-118, 1970) Growth is good, the diameter of the colonies on the second day of culture is
27-29 mm, colonies on day 5 of culture 64-66 mm in diameter, colonies show superficial fissures, aerial hyphae are poorly developed except for the central part of the colonies, and spore formation is good. The sporangiophores arise from aerial hyphae. Has a odor similar to garlic.

Microscopic observation Using a microscopic sample of each medium and direct microscopic examination of colonies, spore-spore stalk, method of branching spore-spore stalk, sporangium, sporangio-spores, etc. were observed.

Sporum stalk length 87.5-320μm, width 3-7.5μ at the base
m, tapering toward the tip, becoming 1.0-2.5 μm. The sporangiophores often branch at the base. The sporangium is spherical and has a diameter
15-30 μm, containing many sporangial spores inside, leaving a slightly obscure color after withdrawal. Ascospores are elliptical, rarely kidney-shaped, smooth surface, 7.5-12.5 × 5-7.5 μm, and relatively many chlamydospores are formed. Alone, sometimes rarely chained. Occasionally, some hyphae are put around. Elliptical or subspherical, 12.5-30 × 7.5-15 μm. Or diameter 12.
5-15 μm. No zygospores are observed.

3. Physiological properties Optimal growth conditions pH: 6-9 Temperature: 20-30 ° C Growth range pH: 4-10 Temperature: 5-40 ° C The strain of the present invention (SAM-0219) according to the mycological properties above.
Search for taxonomic positions in JAvon Arx, “The Genera o
f Fungi sporulating in Pure Culture, "3rd ed., J. Cra
mer, 1981 and KHDomsch, W. Gams, & T.-H. Anderson,
According to “Compendium of Soil Fungi,” Academic Press.1980, a spherical sporangium is formed at the tip of the sporangial stalk, it has no pillar axis, there is no accessory thread in the sporangial spore, and it is cultured. This strain is considered to be a fungus belonging to the genus Mortierelia because the mycelium emits an odor similar to garlic.

So W. Gams, “A key to the species of Mortierel
la. "Morso known according to Persoonia 9 , 381-391, 1977
Comparing the species and mycological properties of the ierella genus, this strain shows that the colony is not velvety, the cultured hyphae give off an odor similar to garlic, and the sporangiophores are 87.5-320μ long.
In m, branching occurs only in the lower part, and it does not branch into a tuft of grapes,
Since the sporangia contains a large number of sporangial spores inside, the subgenus Mortierella (Sugem. Morti-e
rella) Hygrophila section (Sect. Hygrophila) is considered to be included. The Hygrophila section contains 22 species. Comparing the mycological properties with this strain and these 22 species, this strain shows Mortierella zychae, M.elongatula, and M.elongata.
It is considered to be similar to the three types. So KHDomsch,
W. Gams, & T, −H. Anderson, “Compendium of Soll Fung
i, “Academic Press, 1980, and W. Gams,“ Some new o
f noteworthy species of Mortierella, "Persoonia 9 ,
111-140, 1976, and G. Linnemann, "Mortierella Coe.
mans 1863. "H.Zycha &R.Siepmann." Mucorales Eine Bes
chreibung Aller Gattungen und Arten dieser Pilzgru
With reference to ppe. “pp.155-241, J. Cramer, 1969, this strain and these three species were compared with various mycological properties.
M. zychae is distinctly different in length and width of spore stalk and size of sporangium. It differs from M. elongaula in the shape and size of spores of ascospores. M. elongata is said to have a slightly short spore stalk, rarely chain chlamydospores with an elliptical or subspherical shape, and sometimes chlamydospores produce several hyphae around them. Although different in this respect, the present inventors have confirmed that such a difference makes this strain Mortierella elonga
It was judged to be insufficient to be a different species from ta. Therefore, the present inventors have confirmed that this strain is Mortierella elongata SAM.
It was identified as 0219. The SAM 0219 strain has been deposited on March 19, 1986 with the deposit number FERM BP-1239 at the Institute for Microbial Technology (FRI), Ministry of International Trade and Industry.

In order to culture the strain used in the present invention, spores, hyphae of the strain or a pre-cultured liquid obtained by culturing in advance is inoculated into a liquid medium or a solid medium and cultured. In the case of liquid medium, as the carbon source, any of commonly used ones such as glucose, fructose, xylose, sucrose, maltose, soluble starch, molasses, glycerol and mannitol can be used, but the carbon source is not limited to these. is not. As a nitrogen source, peptone, yeast extract,
In addition to natural nitrogen sources such as malt extract, meat extract, casamino acid and corn stapler, organic nitrogen sources such as urea and inorganic nitrogen sources such as sodium nitrate, ammonium nitrate and ammonium nitrate can be used. In addition, inorganic salts such as phosphates, magnesium sulfate, iron sulfate, copper sulfate and vitamins can be used as a trace nutrient source, if necessary.
These medium components are not particularly limited as long as the concentration does not impair the growth of microorganisms. In practice, it is generally preferable that the carbon source has a concentration of 0.1 to 30% by weight, preferably 1 to 10% by weight, and the nitrogen source has a concentration of 0.01 to 5% by weight, preferably 0.1 to 2% by weight.
The culture temperature is 5 to 40 ° C., preferably 20 to 30 ° C., and the pH of the medium is 4 to 10, preferably 6 to 9, and aeration stirring culture, shaking culture, or stationary culture is performed. Culture is usually 2
Do it for 10 days.

When culturing in a solid medium, 50 to 10 based on the solid weight
3% at a temperature of 5 to 40 ° C., preferably 20 to 30 ° C., using bran, chaff, kura-nuka etc. to which 0% by weight of water is added.
Incubate for ~ 14 days. In this case, if necessary, a nitrogen source, an inorganic salt and a micronutrient source can be added to the medium.

In order to increase the production of arachidonic acid, hydrocarbons such as hexadecane or octadecane; fatty acids such as oleic acid or linoleic acid or salts thereof such as sodium or potassium salts; or olive oil, cottonseed oil or coconut oil are added to the medium. It is preferable to allow such fats and oils to be present alone or in combination. These additives can be added to the medium before the start of the culture or the culture medium during the culture. These additives may be added all at once, or may be added continuously or in multiple portions over time. It is preferable to add a hydrocarbon, a fatty acid or a salt thereof, or an oil or fat before the start of culture, and it is preferable to add a fatty acid or a salt thereof, or an oil or fat during the culture.

By culturing in this manner, lipids containing arachidonic acid are produced and accumulated in the cells. When a liquid medium is used, arachidonic acid is collected from the cultured cells as follows.

After the completion of the culture, the cultured cells are obtained from the culture medium by a conventional solid-liquid separation means such as centrifugation and filtration. Wash the cells thoroughly with water,
It is preferably dried. Drying can be performed by freeze-drying, air-drying and the like. The dried cells are preferably subjected to extraction treatment with an organic solvent under a nitrogen stream. As the organic solvent, ether, hexane, methanol, ethanol, chloroform, dichloromethane, petroleum ether, etc. can be used, and by alternate extraction of methanol and petroleum ether, or extraction with a single-layer solvent of chloroform-methanol-water. Can also give good results. By distilling off the organic solvent from the extract under reduced pressure, a lipid containing a high concentration of arachidonic acid can be obtained.

In addition, the extraction can be performed using wet bacterial cells instead of the above method. In this case, a solvent compatible with water such as methanol or ethanol, or a mixed solvent compatible with water composed of these and water and / or another solvent is used. Other procedures are the same as above.

The lipid obtained as described above contains arachidonic acid as a constituent component of a lipid compound such as fat. These can be separated directly, but are preferably separated as an ester with a lower alcohol, for example, methyl arachidonate. By using such an ester, it can be easily separated from other lipid components,
Further, it can be easily separated from other fatty acids produced during the culture, such as palmitic acid, oleic acid, and linoleic acid (these are also esterified during the esterification of arachidonic acid). For example, in order to obtain the methyl ester of arachidonic acid, it is preferable to treat the extracted lipid with anhydrous methanol-hydrochloric acid 5% to 10%, BF ₃ -methanol 10% to 50% and the like at room temperature for 1 to 24 hours. .

In order to recover arachidonic acid methyl ester from the above-mentioned treatment liquid, it is preferable to extract it with an organic solvent such as hexane, ether or ethyl acetate. Next, this extract is dried with anhydrous sodium sulfate or the like, and the organic solvent is distilled off, preferably under reduced pressure, to obtain a mixture mainly composed of fatty acid ester. In addition to the target arachidonic acid methyl ester, this mixture contains palmitic acid methyl ester, stearic acid methyl ester, oleic acid methyl ester, and the like. In order to isolate arachidonic acid methyl ester from these fatty acid methyl ester mixtures, column chromatography, low temperature crystallization method, urea clathrate method and the like can be used alone or in combination.

In order to obtain arachidonic acid from methyl arachidonic acid thus isolated, hydrolysis with alkali is performed, and then ether,
It may be extracted with an organic solvent such as ethyl acetate.

In addition, in order to collect arachidonic acid without passing through its methyl ester, the extracted lipid is alkali-decomposed (for example, 5
% Sodium hydroxide at room temperature for 2 to 3 hours), and then the separated liquid can be extracted and purified by a method commonly used for the extraction and purification of fatty acids.

Next, the present invention will be described more specifically by way of examples.

Example 1. 50 ml of a medium (pH 6.0) containing 5% glucose, 0.5% peptone, 0.3% yeast extract and 0.3% epidermal extract was placed in a 500 ml Sakaguchi flask and sterilized at 120 ° C for 20 minutes. Mortierella elongata SAM 0219 (FERM BP-1239) 1 platinum loop inoculated and 28 ° C with reciprocal shaker (110 rpm)
The cells were cultivated with shaking for 5 days. After culturing, the cells were collected by filtration, washed thoroughly with water, and then freeze-dried. This gives 1.3g
To obtain dry cells. When the total lipids were extracted from these cells by the Bligh & Dyer extraction method using a single-layer solvent of chloroform-methanol-water, 320 mg of lipid was obtained. This lipid was treated with anhydrous methanol-hydrochloric acid (95: 5) at 20 ° C. for 3 hours to methylate arachidonic acid. 200 mg extracted with ether
Was obtained. The composition of this fatty acid methyl is 9% methyl palmitate, 2% methyl stearate, 32% oleate methyl by gas chromatography analysis.
%, Methyl linoleate 9%, γ-methyl linolenate 10
%, Methyl arachidonate 20%, and other 17%. This mixed fatty acid methyl was separated by column chromatography, the methyl arachidonic acid fraction was fractionated, and the solvent was distilled off by a rotary evaporator. As a result, 25 mg of purified methyl arachidonic acid was obtained.
When this standard product and a commercially available methyl arachidonic acid standard sample were compared by gas chromatography analysis, high performance liquid chromatography analysis and mass spectrometry, both were in agreement in all analyses. The amount of “methyl arachidonic acid” before and after purification was 0.84 mg / ml, 0.50 mg / ml, and 3 cells, respectively, per medium, respectively.
It was 2 mg / g and 19 mg / g.

Example 2 A medium 5 having the same composition as in Example 1 was placed in a 15 jar fermenter, sterilized at 120 ° C. for 40 minutes, and then precultured liquid 2 of Mortierella elongata SAM 0129 (FERM EP-1239).
00 ml was inoculated. 360 g of wet bacterial cells (dry weight 110
When g) was extracted, hydrolyzed and methyl esterified in the same manner as in Example 1, 29 g of total lipid and 18 g of mixed fatty acid methyl were obtained. The composition of this product is 8% methyl palmitate, 1% methyl stearate, 29% methyl oleate, 12% methyl linoleate, methyl γ-linolenate.
11%, methyl arachidonate 22%, and other 17%. The amount of methyl arachidonate produced was 0.79 g / g per medium and 36 mg / g per dry cell.

Also, after the completion of culture, the culture filtrate obtained by filtration was 4,350 m.
After drying l, extraction, hydrolysis and methyl esterification were carried out in the same manner as in Example 1 to obtain 156 mg of mixed fatty acid methyl containing 25% of methyl arachidonate.

Example 3 Mortierella exigua, IFO
8571) and Mortiere Hygrophila Mortiere
lla hygrophila, IFO 5941) was subjected to the same operations as in Example 1 to obtain 72 mg and 95 mg of fatty acid methyl, respectively.

Isolation and purification of methyl arachidonic acid contained in these fatty acid methyls gave 12 mg and 20 mg, respectively.

Example 4 Glucose 2%, yeast extract 1%, Tween 20 0.2% and various hydrocarbons, fatty acid sodium or fats and oils 0.5%
Add 20 ml of culture medium (pH 6.0) to a 100 ml volume Meyer, and
Sterilized at 0 ° C for 20 minutes. Mortierella Elongata S
AM 0129 (FERM BP-1239) 1 platinum loop was inoculated and cultured at 28 ° C. for 5 days on a rotary shaker (200 rpm). The obtained cells were extracted, hydrolyzed and methylesterified in the same manner as in Example 1. For each of the various hydrocarbons, fatty acid sodium, and fats and oils added to the medium, the obtained dry cell weight, total lipid amount, total fatty acid methyl amount, methyl arachidonic acid methyl content, and methyl arachidonic acid production amount per medium were as follows: It looks like the table.

By adding hydrocarbons, fatty acids, fats and oils to the frequency medium, the amount of arachidonic acid produced was higher than that in the control-free group.
Rose by 10-80%.

Example 5 A medium containing 2% glucose and 1% yeast extract (pH 6.
0) 20 ml was put in a 100 ml Mayer and sterilized at 120 ° C for 20 minutes. Mortierella Elongata SAM 0219 (FERM
BP-1239) 1 platinum loop was inoculated and cultivated on a rotary shaker (200 rpm) at 28 ° C for 4 days, then 100 mg of various fatty acid sodium or fats and oils were sterilized at 120 ° C for 15 minutes, then added, and similarly for 2 days. Cultured. The obtained cells were extracted, hydrolyzed and methylesterified in the same manner as in Example 1. The amounts of methyl arachidonic acid produced per the obtained dry cells and per medium for the various fatty acid sodium and fats and oils added to the medium are shown in the table below.

By adding fatty acids, fats and oils during the culture (after 4 days of culture), the production amount of arachidonic acid was increased by 10 to 60% as compared with the control-free group.

Claims (6)

[Claims] 1. A arachidonic acid or a lipid containing arachidonic acid is produced by culturing a microorganism belonging to the genus Mortierella and having arachidonic acid-producing ability.
Then, a method for producing arachidonic acid, which comprises collecting arachidonic acid. 2. The method according to claim 1, wherein a hydrocarbon, a fatty acid or a fatty acid salt, or an oil or fat is added to the medium before the start of culture. 3. The method according to claim 1, wherein a fatty acid, a fatty acid salt, or an oil is added to the culture medium during the culture. 4. A method for producing a lipid containing arachidonic acid, which comprises collecting a lipid containing arachidonic acid by culturing a microorganism belonging to the genus Mortierella and capable of producing arachidonic acid. 5. The method according to claim 4, wherein a hydrocarbon, a fatty acid or a fatty acid salt, or an oil or fat is added to the medium before the start of the culture. 6. The method according to claim 4, wherein a fatty acid, a fatty acid salt, or an oil or fat is added to the culture medium during the culture.