JP2848810B2 - Method for producing arachidonic acid-rich fat - Google Patents

Description

Description: TECHNICAL FIELD [0001] The present invention relates to a method for producing arachidonic acid by a fermentation method. [0002] Conventionally, as a method for producing arachidonic acid by a microorganism, a carbohydrate or a hydrocarbon is used as a carbon source, and penicillium ( Penicillum) is used as a microorganism.
ium) genus Aspergillus (Aspergillus) genus, Rhodotorula (Rhodotorula) genus, or Fuzaryumu (Fusarium) method using a microorganism belonging to the genus has been reported (JP-B 56-19231,56-19232,56-192
33). However, in either method, the yield is low, or the culture time is long, or
The process is complicated. SUMMARY OF THE INVENTION [0003] The present invention uses a Mortierella genus microorganism having the ability to produce arachidonic acid, using a low-cost conventional medium, and in a higher yield than the conventional method. Another object of the present invention is to provide a method capable of producing arachidonic acid by a simple process. [0004] The object of the present invention is to culture arachidonic acid or arachidonic acid by culturing a microorganism belonging to the genus Mortierella and having an arachidonic acid-producing ability in a medium containing a fatty acid, a fatty acid salt or an oil or fat. And a method for producing arachidonic acid or arachidonic acid-containing lipid, comprising collecting arachidonic acid or arachidonic acid-containing lipid. [0005] In the present invention, any microorganism that belongs to the genus Mortierella and has an arachidonic acid-producing ability can be used. Such microorganisms include, for example, Mortierella elongata
a elongata ) IFO 8570, Mortierella exigua IFO 8571, Mortierella hygrophila
IFO 5941 and the like. All of these strains can be obtained from the Fermentation Research Institute without any limitation. [0006] The strain Mortierella elongata SAM0219 isolated from the soil by the inventors of the present invention can also be used. Next, the bacteriological properties of the above-mentioned strain SAM 0219 (Microtechnical Laboratories No. 1239) will be described. Growth conditions in each medium Culture conditions: 25 ° C., in darkness The growth of the malt extract agar medium colony is good, and the colony on the second day of culture has a diameter of 2
8-31 mm, colonies on day 5 of culture are 65-72 m in diameter
m, colony is shallow-fissure, aerial mycelium is poorly developed, spore spore formation is good, spore stalk is odor similar to garlic, arising from aerial mycelium. [0007] 2. Potato / Glucose agar colonies grow well, colonies on day 2 of culture have a diameter of 2
7-31 mm, colonies on day 5 of culture are 75-80 m in diameter
m, colony has rose flower shape, aerial mycelium develops remarkably in the center of the colony, yellowish white or yellow on the reverse side of the colony, poor formation of spore spores, odor similar to garlic, slightly odor . 3. The growth of the Tzapek agar 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 5
0-53 mm, poor development of aerial hyphae, aerial mycelia may be tightly entangled. The formation of sporangia spores is very good and the spore stalks arise from aerial hyphae. Smell similar to garlic. [0008] 4. LCA agar medium (the method of preparing the medium is as follows:
Koichiro Miura, Mitsuyo Kudo, "Agar Medium for Imperfect Aquatic Bacteria," The Mycological Society of Japan, 11, 116-118, 19
Colony growth is good, colonies on day 2 of culture are 27-29 mm in diameter, and colonies on day 5 of culture are 64-66 in diameter.
mm, colonies show shallow fissures, aerial mycelium development is poor except in the center of the colonies, spore-spore formation is good. Spore stalks arise from aerial hyphae. Smell similar to garlic. Microscopic Observation By direct microscopic examination of microscopic specimens and colonies of each medium, the spore stalk, the manner of branching of the spore stalk, the spore sac, the spore spore, etc. were observed. Spore stalks are 87.5-320 in length
μm, the width is 3-7.5 μm at the base and tapers toward the tip, 1.0-2.5 μm. Spore stalks often branch off at the base. Spore sac is spherical, 15-30μ in diameter
m, leaving a slightly unclear color after withdrawal, containing a large number of spore-spores inside. Spore spores are oval, rarely kidney-shaped, surface smooth, 7.5-12.5 × 5-7.5 μm,
Chlamydospores are formed in relatively large numbers. May be linked alone or rarely. Occasionally, several hyphae are exposed.
Oval or subspherical, 12.5-30 x 7.5-15μ
m. Or 12.5-15 μm in diameter. No zygote is observed. [0010] 3. Physiological properties Optimal growth conditions pH: 6-9 Temperature: 20-30 ° C. Growth range pH: 4-10 Temperature: 5-40 ° C. According to the above mycological properties, the strain of the present invention (SAM-0219) ) Search for taxonomic position, JAvo
n Arx, “The Genera of Fungi sporulating in Pure Cu
lture, "3rd ed., J. Cramer, 1981 and KHDoms
ch, W.Gams, & T.-H.Anderson, “Compendium of Soil
According to Fungi, "Academic Press. 1980, the spore spore forms a spherical spore sac at the tip of the spore stalk, has no pillar axis, has no attached thread to the spore spore, and the cultured hypha becomes garlic. It is considered that this strain is a fungus belonging to the genus Mortierella because it produces a similar odor [0012] Thus, W. Gama, "A key to the species"
of Mortierella, "Persoonia 9 , 381-391, 1
Comparing the types of known Mortierella species with mycological properties according to 977, this strain has a colony that is not velvety, a cultured mycelium emits an odor similar to garlic, and a spore-scab having a length of 87. At 5-320 μm, branching occurs only in the lower part and does not branch in a tuft of grapes. The spore sac contains a large number of spore spores inside, thus Mortierella
Genus Mortierella Subgenus (Sugem. Morti-erella) Hygrophila
It is considered to be included in the knot (Sect. Hygrophila) . [0013] The Hygrophila section contains 22 species.
Comparing mycological properties with this strain and these 22 species, this strain is Mortierella zychae , M. elongatula , and M.
It is thought to be similar to the three species of elongata . So, KH
Domsch, W. Gams, & T.-H. Anderson, “Compendium of
Soll Fungi, "Academic Press, 1980, and W. Ga
ms, “Some new of noteworthy species of Mortierell
a, "Persoonia 9 , 111-140, 1976, and
G. Linnemann, " Mortierella Coemans 1863."
Zycha & R. Siepmann. “Mucorales Eine Beschreibung
Aller Gattungen und Arten dieser Pilzgruppe. ”Pp.
155-241, J. Cramer, 1969, the mycological properties of this strain and these three species were compared. This strain is clearly different from M. zychae in the length of the spore stalk, the width of the base, and the size of the spore sac. M.el
It differs from ongatula in the shape and size of the spore spores. With M. elongata , the spore scab is rather short, the form of chlamydospores may be rarely linked in an oval or subspherical shape, and sometimes chlamydospores sometimes emit several hyphae around,
However, the present inventors have determined that such a difference is not sufficient to make this strain different from Mortierella elongata . Therefore, the present inventors set this strain as Mo
rtierella elongata SAM 0219 was identified. S
AM 0219 strain was deposited on March 19, 1986 with the Research Institute of Microorganisms and Technology (FRI) of the Ministry of International Trade and Industry under the accession number FERM BP-1239. In order to culture the strain used in the present invention, a spore, hypha or a preculture obtained by pre-culturing the strain is inoculated into a liquid medium or solid medium and cultured. In the case of a liquid medium, any commonly used carbon source such as glucose, fructose, xylose, saccharose, maltose, soluble starch, molasses, glycerol, mannitol can be used as the carbon source, but is not limited thereto. is not. Peptone as a nitrogen source,
Yeast extract, malt extract, meat extract, casamino acids, in addition to natural nitrogen sources such as corn steep liquor, organic nitrogen sources such as urea, and sodium nitrate, ammonium nitrate,
An inorganic nitrogen source such as ammonium sulfate can be used. In addition, if necessary, inorganic salts such as phosphates, magnesium sulfate, iron sulfate, and copper sulfate, and vitamins can be used as trace nutrients. These medium components are not particularly limited as long as they do not impair the growth of microorganisms. In general, the carbon source is generally 0.1 to 30% by weight, preferably 1 to 1% by weight.
The concentration is 0% by weight and the concentration of the nitrogen source is 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
Is 4-10, preferably 6-9, aeration stirring culture,
Shake culture or static culture is performed. Culture is usually 2-10
Perform for days. When culturing in a solid medium, use bran, rice husk, rice bran or the like to which 50 to 100% by weight of water is added based on the weight of the solid, at 5 to 40 ° C., preferably 20 to 3 ° C.
Culture is performed at a temperature of 0 ° C. for 3 to 14 days. In this case, a nitrogen source, an inorganic salt, and a trace nutrient can be added to the medium as needed. In order to increase the production of arachidonic acid, a fatty acid such as oleic acid or linoleic acid or a salt thereof, for example, a sodium salt or a potassium salt: or an oil or fat such as olive oil, cottonseed oil or coconut oil alone or in the medium, or It is preferable that they be present in combination. These additives can be added to the medium before the start of the culture or the culture solution during the culture. These additives can be added all at once, or they can be added continuously or in multiple portions over time. During the culturing, it is preferable to add a fatty acid or a salt thereof, or an oil or fat. By culturing as described above, 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 cultivation, the cultured cells are obtained from the culture solution by conventional solid-liquid separation means such as centrifugation and filtration. The cells are thoroughly washed with water and preferably dried. Drying can be performed by freeze drying, air drying or the like. The dried cells are preferably extracted with an organic solvent under a nitrogen stream. As the organic solvent, ether, hexane, methanol, ethanol, chloroform, dichloromethane, petroleum ether, and the like can be used.Alternatively, methanol and petroleum ether are alternately extracted, and chloroform-methanol-water is extracted using a one-layer solvent. Can also give good results. By removing the organic solvent from the extract under reduced pressure, a lipid containing a high concentration of arachidonic acid can be obtained. In addition, extraction can be performed using wet cells instead of the above method. In this case, methanol,
A water-compatible solvent such as ethanol, or a water-compatible solvent mixture of these and water and / or another solvent is used. Other procedures are the same as above.
Arachidonic acid is contained in the lipid obtained as described above as a component of a lipid compound, for example, fat. These can be separated directly, but are preferably separated as esters with lower alcohols such as methyl arachidonic acid. By making such an ester, it can be easily separated from other lipid components, and other fatty acids produced during culture, such as palmitic acid, oleic acid, linoleic acid, etc. Arachidonic acid is esterified during the esterification of arachidonic acid). For example, in order to obtain the methyl ester of arachidonic acid, the extracted lipid is prepared by adding anhydrous methanol-hydrochloric acid 5%.
It is preferable to treat with BF ₃ -methanol 10% to 50% at room temperature for 1 to 24 hours. In order to recover arachidonic acid methyl ester from the above treated solution, it is preferable to extract with an organic solvent such as hexane, ether or ethyl acetate. Next, the 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 esters. This mixture contains, in addition to the intended methyl arachidonic acid ester, methyl palmitate, methyl stearate, methyl oleate, 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 the thus isolated methyl arachidonic acid, it may be hydrolyzed with an alkali and then extracted with an organic solvent such as ether or ethyl acetate. In order to collect arachidonic acid without passing through its methyl ester, the extracted lipid is alkali-decomposed (for example, with 5% sodium hydroxide at room temperature for 2 to 3 hours), and then, the fatty acid Extraction and purification can be performed by a method commonly used for extraction and purification. next,
The present invention will be described more specifically with reference to examples. Example 1 Glucose 5%, Peptone 0.5%, Yeast extract 0.3
Medium (pH 6.0) containing 50% and malt extract 0.3% 50
ml was placed in a 500 ml Sakaguchi flask and sterilized at 120 ° C. for 20 minutes. Mortierella Elongata SAM 02
One (1) loop of 19 (FERMBP-1239) was inoculated, and cultured with shaking at 28 ° C. for 5 days using a reciprocating shaker (110 rpm). After the culture, the cells were collected by filtration, washed sufficiently with water, and freeze-dried. As a result, 1.3 g of dried cells were obtained. From the cells, chloroform-methanol-
Extraction of total lipids by the Bligh & Dyer extraction method using a water-based solvent yielded 320 mg of lipids. The lipid was treated with anhydrous methanol-hydrochloric acid (95:
5) The mixture was treated at 20 ° C. for 3 hours to carry out methyl esterification of arachidonic acid. This was extracted with ether to obtain 200 mg of fatty acid methyl. The composition of this fatty acid methyl was analyzed by gas chromatography, and it was found that methyl palmitate 9%, methyl stearate 2%, methyl oleate 32%, methyl linoleate 9%, γ-linolenate 10%, methyl arachidonic acid 20% , Other 17
%. The mixed fatty acid methyl ester was separated by column chromatography, the methyl arachidonic acid fraction was separated, and the solvent was distilled off by a rotary evaporator to obtain 25 mg of purified methyl arachidonic acid. A comparison was made between this sample and a commercially available standard sample of methyl arachidonic acid by gas chromatography, high performance liquid chromatography, and mass spectrometry. The amounts of “methyl arachidonic acid” before and after purification were 0.84 mg / ml and 0.50 mg / ml per medium, respectively, and were 32 mg / g and 19 mg / g per dry cell, respectively. Example 2 5 l of a medium having the same composition as in Example 1 was charged into a 15 l jar fermenter, sterilized at 120 ° C. for 40 minutes, and then subjected to Mortierella elongata SAM 0219 (FERM BP).
-1239) was inoculated with 200 ml of the preculture solution. 30 ° C,
Aeration and agitation culture was performed for 3 days at an aeration rate of 0.5 v. Vm.
When extraction, hydrolysis, and methyl esterification were performed in the same manner as in Example 1, total fat 29 g, mixed fatty acid methyl 18
g was obtained. The composition of this product is methyl palmitate 8
%, Methyl stearate 1%, methyl oleate 29
%, Methyl linoleate 12%, methyl γ-linolenate 1
It was found that the content was 1%, methyl arachidonic acid 22%, and other 17%. The production amount of methyl arachidonic acid was 0.79 g / l per culture medium and 36 mg / g per dried cells. After completion of the cultivation, 4,350 ml of the culture filtrate obtained by filtration was dried and subjected to extraction, hydrolysis and methyl esterification in the same manner as in Example 1. As a result, a mixed fatty acid methyl ester containing 25% of methyl arachidonic acid was obtained. 156 mg were obtained. Example 3 Mortierella exigua, I
FO 8571) and Mortierella hygrophylla
Mortierella hygrophila , IFO 5941) were operated in the same manner as in Example 1 to obtain 72
mg and 95 mg of fatty acid methyl were obtained. When methyl arachidonic acid contained in these fatty acid methyls was isolated and purified, they were 12 mg and 20 mg, respectively. Example 4 Glucose 2%, yeast extract 1%, Tween 20
100 ml of a medium (pH 6.0) containing 0.2% and 0.5% of various hydrocarbons, sodium fatty acids, or fats and oils is added to 100 ml.
The mixture was placed in a ml-volume Meyer and sterilized at 120 ° C. for 20 minutes. Mortierella Elongata SAM 0219 (FERM
BP-1239) was inoculated with one loop of platinum loop and cultured at 28 ° C. for 5 days using a rotary shaker (200 rpm). The obtained cells were subjected to extraction, hydrolysis and methyl esterification 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 content, and the amount of methyl arachidonic acid produced per medium were as follows: Table 1 below. [Table 1] By adding hydrocarbons, fatty acids, fats and the like to the standard medium, the amount of arachidonic acid produced was increased by 10 to 80% as compared with the control non-added group. Example 5 A medium containing 2% glucose and 1% yeast extract (pH
6.0) Put 20 ml into a 100 ml Meyer, 120 ° C
For 20 minutes. Mortierella Elongata SA
M 0219 (FERM BP-1239) (1 platinum loop) was inoculated, and was inoculated on a rotary shaker (200 rpm).
After cultivation at 4 ° C. for 4 days, various sodium fatty acids or fats and oils 1
00 mg was sterilized at 120 ° C. for 15 minutes, added, and cultured in the same manner for 2 days. The obtained cells were subjected to extraction, hydrolysis and methyl esterification in the same manner as in Example 1. Various fatty acid sodium added to the medium,
Table 2 below shows the amount of methyl arachidonate produced per dry cell obtained and per medium for each of the fat and the oil. [Table 2] By adding fatty acids, fats and oils during the culture (after 4 days of culture), the amount of arachidonic acid produced increased by 10 to 60% as compared with the control-free group.

Claims (1)

(57) [Claims] A method for producing a fat containing arachidonic acid by culturing a microorganism belonging to the genus Mortierella (Mortierella) and having an arachidonic acid-producing ability in a medium containing an assimilable carbon source and an assimilable nitrogen source, comprising: In addition, by adding a fatty acid or a fatty acid salt or fat or oil as an additive, the amount of arachidonic acid constituting the fat per medium is increased by 10% or more as compared with the case where the additive is not added. For producing fat containing arachidonic acid.