JPS6368090A - Production of lipid containing arachidonic acid - Google Patents

Abstract

PURPOSE:To raise arachidonic acid content based on dried fungus cell weight and arachidonic acid content in lipid extracted from the fungus cell and to obtain high-purity arachidonic acid in high yield, by cultivating a fungus belonging to the genus Mortierella in a solid medium using the whole potato. CONSTITUTION:A fungus belonging to the genus Mortierella is cultivated in a solid medium using the whole potato to produce a fungus cell having lipid containing arachidonic acid. When a bivalent metal (e.g. Ca<++> or Mg<++>) is added to the solid medium and the fungus is cultivated, preferably the yield of arachidonic acid is improved. Potato, taro, sweet potato, etc., may be cited as the potatoes used as the medium and white potato is especially preferable.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing arachidonic acid-containing lipids, and more specifically, the present invention relates to a method for producing arachidonic acid-containing lipids, and more specifically, culturing filamentous fungi belonging to the genus Mortierella in a specific medium to determine the arachidonic acid content. This invention relates to a method for producing high fat content.

[Conventional technology]

Arachidonic acid is said to be a precursor of prostaglandins, thromboxanes, prostacyclin, leukotrienes, etc., which have strong and diverse physiological activities such as uterine muscle contraction/relaxation, vasodilation, and blood pressure lowering effects, and has attracted particular attention in recent years. There is. Arachidonic acid is widely distributed in the animal kingdom and has traditionally been isolated from lipids extracted from animal adrenal glands and liver.

However, the arachidonic acid content in these lipids is generally 5
%, the yield per dry cell weight is only 0.2% or less, and it is difficult to prepare large quantities of raw materials, so this extraction method cannot be called a useful method for producing arachidonic acid. .

On the other hand, methods for obtaining arachidonic acid by culturing various microorganisms capable of producing arachidonic acid have been proposed. For example, Japanese Patent Application Publication No. 52-64482, Japanese Patent Application Publication No. 51-64483
No. 52-64484, the genus Penicillium, the genus Talatosporium, the genus Mucor, the genus Fusarium,
A method is described for culturing microorganisms capable of producing arachidonic acid belonging to the genus Hormopuntrum, Aspergillus, or Rhodotorula in a medium containing hydrocarbons, carbohydrates, etc. as carbon sources, and collecting arachidonic acid from the culture. . However, the arachidonic acid content in the lipids obtained by this method is less than 7.5%, and the yield per dry bacterial cell is 1.
less than %.

Zygomycetes include fungi that produce arachidonic acid, including fungi belonging to the genera Entomophthora, Delacroixia, Conidiobolus, Phytium, and Phytophthora, which are filamentous fungi of the order Ekamida. 27.1% of the total fatty acids in lipids, B, 19.1% in Ibunobilis, C, 1 in Saxteriana.
It has been reported that arachidonic acid accounts for 8.8% (D. Tyrrell, Canadian Journal of Microbiology (2013).
Can, ,J., Microbiol,).

Vow, 13 (1967), 755-760)
. In addition, Nimoltierella and Renispora produce arachidonic acid, the lipid production amount of mycelia is 4.8%, and the content of arachidonic acid in lipids is 26.7% (R, H
, Haskins et al., Can.
JoMicrobiol, Vol. 10(1)
964), 187-195) and that the red alga Porphyrizoium cruentum produces arachidonic acid in a yield of less than 1% per dry cell weight [: T. J. hern) LA, Biotechnology and Bioengineering (Bio
technology and bioengineering
ring), Vol, XXV.

1057-1070 (1983)) has also been reported.

On the other hand, Suntory and Kyoto University cultured Maltii and Herrera elongata in liquid culture and produced 0.5-1.0 g/fl.
Arachidonic acid in culture solution (30.1% content in total fatty acids)
(Japan Society of Agricultural Chemistry, 1986 Conference, Abstracts P, 502) o Furthermore, Hiroshima University reported that Conidiobolus bacteria were treated with malt extract, yeast extract,
Cultured in polypeptone glucose culture solution, 0.8
Arachidonic acid content of g/Il in total fatty acids 24
．． 5%) (Japan Society of Agricultural Chemistry, Showa 6)
1st Annual Conference, Collection of Abstracts P, 502').

Furthermore, the present inventors have previously applied for a patent application (Japanese Patent Application No. 6O-218558) that there are strains of Mortierella filamentous fungi that produce lipids containing a high percentage of arachidonic acid.
), but no study was conducted regarding the production amount of arachidonic acid.

[Problem that the invention seeks to solve]

The purpose of the present invention is to have a high arachidonic acid content per dry bacterial weight, a high arachidonic acid content in lipids extracted from this bacterial body, and a high arachidonic acid content per medium, easy to separate and purify arachidonic acid, and high arachidonic acid content. The object of the present invention is to provide a method capable of obtaining pure arachidonic acid in high yield.

[Means for solving problems]

The object of the present invention is achieved by culturing a filamentous fungus belonging to the genus Mortierella that is capable of producing arachidonic acid in a specific medium.

That is, the present invention is characterized in that by culturing a filamentous fungus belonging to the genus Mortierella in a solid medium using whole potatoes, microbial cells having a lipid containing arachidonic acid are produced in the medium. This is a method for producing lipids.

Mortierella (Mortiella) advantageously used in the present invention
An example of an arachidonic acid-producing bacterium belonging to the genus Mortierella albina is Mortierella albina.
la alpina), Mortierella bainieri (
Mortierella bainieri), Mortierella x longata (Mortierella bainieri)
elongata), Mortierella exigua (
Mortierella exigua), Mortierella minutissima
Nutissima), Mortierella verticilla
ta), Mortiherella φhygrophila (Morti
erella hygrophila), Mortierella policephala (Mortierella polycephala)
ephala) and Mortierella rennisbora (
Examples include strains belonging to the species Mortierel La renispora). Specific examples of these bacteria include:
Mortierella albina
alpina) IPO8568゜8TCC16266,
ATCC32221, ATCC42430 Mortierella Bainier! ] (Mortierllabain
ieri) IPO8569 Mortierera・
Mortierella elongata
) IFO8570 Mortierella Exigua (Mort 1erel Iaexigua)
TFO8571 Mortierella minutissima (
Mortierella minutissima)
IFO8573 Mortierella varticillata (
Mortierella verticillata)
IFO8575 Mortierella. Hygrophila (
Mortierella hygrophila)
IFO5941 Mortierella policephala (Mor
tierella polycephala) IF
Examples include O6335. These bacteria were collected by the Institute of Fermentation (IFO) in Osaka City and the American Kuipfist Culture Collection (American Ty).
peCulture Co11ection, ATCC
) is a filamentous fungus listed in the bacterial strain list.

The above-mentioned filamentous fungi are cultured using a solid medium using whole potatoes. Potatoes used in the culture medium include potatoes, taro, sweet potatoes, mackerel, taro, Jerusalem artichoke, etc., and potatoes are most preferably used. Potatoes may or may not be peeled. To prepare a solid medium, add O-2 times, preferably 0-1 times water to potatoes cut into approximately 1 cm cubes, boil them, grind thoroughly with water, and add carbohydrates to 0-20%, preferably 0-20%. Add 2-10% and mix well. As carbohydrates,
Examples include glucose, fructose, saccharose, molasses, wood saccharification liquid, and starch liquefaction products. By adding a divalent metal as a minor additive component, the yield of arachidonic acid per medium can be further improved. Such divalent metals include, for example, Ca''+
Alternatively, Mg'' can be mentioned. [: The amount of a + '' added is 0.02-2 g/kg.

Preferably, 0.05-1 g/kg.
The amount added is 0.01-5g/kg, preferably 0.0
2-2 g/kg is good.

The initial pH of the culture is suitably 4.0 to 7.0, and the culture temperature is preferably 10 to 33°C, preferably 20 to 30°C.
Cultured for 20 days.

The filamentous fungi are cultivated by culturing under such aerobic conditions, and the produced lipids are largely contained within the fungal cells, so after separating the fungi from the culture and mechanically or physically grinding,
Extract with a solvent, supercritical carbon dioxide, etc. to obtain lipids with high arachidonic acid content.

The arachidonic acid content of the obtained lipid can be evaluated after hydrolysis, esterification, or transesterification using conventional methods. In addition, because the content of arachidonic acid in lipids is high, it is much easier and more economical than conventional methods to obtain the desired arachidonic acid or arachidonic acid ester using a solvent, chromatography fractionation, urea addition separation method, etc. Purification can be carried out. The yield of arachidonic acid or arachidonic acid ester reaches a maximum of 13.1 g/kg per solid medium, which means that productivity can be achieved about 13 times as much as when using a liquid medium.

〔Effect of the invention〕

According to the present invention, a lipid with a high content of arachidonic acid can be obtained, and arachidonic acid can be produced at a yield of about 13 times that of the conventional method per medium.

Since the content of arachidonic acid in lipids per medium is extremely high, purification of arachidonic acid can be carried out very easily and in a short time, and highly pure arachidonic acid can be produced in large quantities using a small-scale medium. Can be supplied at low cost. Therefore, using this as a raw material, prostaglandins, thromboxanes, prosfcyclines, leukotrienes, etc., which are utilized and expected to have various pharmacological activities, can be synthesized at a lower cost than before.

〔Example〕

The present invention will be explained in more detail with reference to Examples below.

Example 1 4 pieces of 600 g of peeled potatoes cut into 1 cm cubes
After soaking in 00 ml of water for 20 minutes, pass through a No. 32 mesh to form a gruel. 60 g of glucose is mixed with this and sterilized in an autoclave. Before cooling to room temperature, the gruel was poured into 70 80 ml Tl diameter sterile Petri dishes to prepare a solid medium. Thirty of the obtained media contained Mortierella albina (■F085
68), Mortierella albina (ATCC322)
21) and Mortierella elongata (' I F O
8570) were inoculated into platinum loops of 20 each, and incubated at 25℃ for 2 hours.
Cultured for 0 days. IFO8568 and ΔTCC32221
For each, 20 hyphae grown on the medium were collected. The remaining 10 sheets of IFO8568 and 2 of IFO8570
For 0 medium sheets, the medium was scraped off from the back of the fungal bed using a spatula, and the mycelium and fungal bed were harvested together. Hyphae (
Immediately after drying, the cells were ground with chloroform/methanol (2:1 v/v) in a mortar, followed by chloroform/methanol (2:1 v/v).
/v) total lipids were extracted. After the obtained lipid was methyl esterified using IJ methoxide, its fatty acid composition was analyzed by gas chromatography to determine the content of arachidonic acid, and the results shown in Table 1 were obtained.

In addition, CaCβ2・2H2 was added to the gruel prepared in the same manner as above.
0 at 735 mg/kg and MgCL.6 H2O.

IFO8568 was inoculated into 820 solid medium Petri dishes prepared by adding 400 mg/Kg individually, thoroughly mixing and sterilizing the cells, and cultured at 25° C. for 20 days.

Table 1 also shows the numerical values obtained by collecting the grown hyphae and fungal beds and treating them in the same manner.

On the other hand, malt agar medium 22. , 5 g and 32.5 g of the same Sabouraud agar medium were added to 500 mβ of distilled water, sterilized in an autoclave, and then dispensed into 25 Petri dishes each to prepare an agar medium. Mortierella albina F08568 was inoculated into a platinum loop, and after culturing at 25° C. for 20 days, mycelia were collected, dried, and treated in the same manner, and the results are also shown for comparison.

100g of potatoes cut into 1cm cubes and 500ml of water
After adding j2 and boiling for about 20 minutes, add 30 g of glucose and distilled water to the leachate obtained by filtering through a cloth to make a culture solution of 500 m j2. Pour 250 m J of the culture solution into a 5-shaped tube. After sterilization, Mortierella albina IF08568 was added. Inoculate and keep at 25℃
The cells were cultured with shaking for 20 days. The obtained bacterial cells were collected and washed by centrifugation, dried, and treated in the same manner as in Example 1. The results are shown in Table 1.

Although the lipids of mycelia growing on potato medium had a higher content of arachidonic acid than the lipids of the fungal bed, the bacterial yield was higher on the fungal bed. The yield of arachidonic acid is about 5 if only mycelium is used.
g/kg, (10 hyphae bed) is 10 g/kg or more, and Suntory/Kyoto University method (liquid culture/0.5-
The yield was 5-13 times higher than that of '1.0 g/1).

When calcium chloride and magnesium chloride were added to the culture medium, the yield of arachidonic acid increased by 27% and 18.4%, respectively, with the bacterial weight and methyl ester amount, and a remarkable effect of Ca'' and Mg++ was observed. Ta.

Claims (2)

[Claims] (1) Production of arachidonic acid-containing lipids, which is characterized by producing microbial cells containing arachidonic acid-containing lipids in the medium by culturing filamentous fungi belonging to the genus Mortierella in a solid medium using whole potatoes. Method. (2) The method for producing an arachidonic acid-containing lipid according to claim (1), which comprises adding a divalent metal to a solid medium and culturing it.