JPH0286789A - Production of fats and oils containing arachidonic acid by microorganism - Google Patents

Abstract

PURPOSE:To readily mass produce fats and oils containing a high concentration of arachidonic acid useful as the raw material for medicines, health foods, etc., at an industrially low cost by using mold fungi belonging to Entomophthora. CONSTITUTION:Mold fungi (e.g., ATCC 32890) belonging to Entomophthora genus are cultured in a culture medium containing a organic polybasic acid for accumulation of arachidonic acid in the fungal cells in a high concentration and the cultured fungi are collected, thus obtaining the objective fats and oils.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention is directed to the production of arachidonic acid, which is attracting attention as a precursor of prostaglandins and leukotrienes, which are important regulatory factors in living organisms, using microorganisms. It relates to a method for mass production quickly and inexpensively.

(Prior Art) Arachidonic acid is normally incorporated into the β-position of phospholipids in cell membranes in vivo, but is excised by phospholipase A2 upon physical, chemical, or immunological stimulation. Next, they are sequentially converted into prostaglandins or leukotrienes by lipoxygenase, cyclooxygenase, or prostaglandin synthetase, and exhibit their respective physiological activities.

These prostaglandins and leukotrienes are
It is highly anticipated as a therapeutic agent for modern social problems such as allergies, viral diseases, and senile dementia, and research is progressing rapidly. Therefore, arachidonic acid, which is a precursor of prostaglandins and leukotrienes, is an essential messenger for living organisms, and its importance is immeasurable.

Arachidonic acid, which is a precursor of prostaglandins that play an important role in the body as described above, is contained in the body, for example, in the lungs, brain, heart, blood, or egg yolk. From this, attempts were made to extract and separate and purify it by column chromatography or distillation. In addition, several total chemical synthesis methods have also been attempted.

(Problems to be Solved by the Invention) Substances containing arachidonic acid exist only in specific areas such as the brain, lungs, or blood, and are also contained in egg yolks in 11 foods. All of them are contained in only trace amounts.

Moreover, with the above-mentioned materials, sample collection is extremely limited, and the materials themselves are highly perishable, making mass production difficult. Furthermore, the structure of arachidonic acid makes it extremely difficult to synthesize, so at present none of them have been applied industrially.

An object of the present invention is to provide a method for inexpensively producing fats and oils containing a high concentration of arachidonic acid using microorganisms.

(Means for Solving the Problems) The present invention relates to Entomo htho
A method for producing an arachidonic acid-containing oil or fat, which comprises culturing a filamentous fungus in a medium supplemented with an organic polybasic acid, thereby accumulating arachidonic acid at a high concentration within the bacterial body, and collecting the arachidonic acid. It is.

Entomophthora, which is a type of microorganism of the genus Fungi, is known to accumulate arachidonic acid in its bacterial cells, and at this time, a small amount of arachidonic acid is accumulated.

In contrast, the present invention allows fats and oils containing a large amount of arachidonic acid to accumulate in the cells of Entomophthora.

The strain used in the present invention is Entomophthora extalis rATcc14290.32890J, and both are filamentous fungi listed in the ATCC catalog (list of bacterial strains).

The above filamentous fungi use glucose, sucrose, fructose, maltose, glycerin, blackstrap molasses,
Starch, corn steep liquor, olive oil, coconut oil, etc. as a nitrogen source, yeast extract, peptone,
Organic substances such as meat extract, soybean flour, defatted soybean flour, gluten, and gelatin grow in nitrogen. At this time, the carbon source is 3~
It is used at 20%, but preferably from 5 to 15%.

In the present invention, the organic polybasic acid added to the medium includes, for example, citric acid, isocitric acid, succinic acid, fumaric acid, malic acid, etc., and the amount added is 0.01 to 1%, preferably 0.63 to 1%. be. Other non-base salts that can be used include, for example, iron sulfate, magnesium sulfate, zinc sulfate, potassium chloride, potassium phosphate, dipotassium phosphate, and the like. In addition, trace elements may be added as necessary.

The above-mentioned filamentous fungi are usually cultured in liquid with aeration and agitation. The stirring speed was 300 to 900 rpm, and the aeration rate was 0.5 to 2 νvm to ensure sufficient ventilation (kla = 6
00 to 1000 hr-'), and air or pure oxygen may be used. Culture temperature is 20-33
The culture period is 3 to 7 days at °C. At this time, the pH is preferably slightly acidic, preferably adjusted to 5-6. After culturing, isolate the bacterial cells by filtration or centrifugation, wash the bacterial cells thoroughly, and then extract fats and oils from the bacterial cells using chloroform, methanol, ethanol, propatool, butanol, hexane, acetone, or a mixture thereof. . Furthermore, after separating the crude lipids using a partition method such as Folch, methyl esterification is performed, and a mass spectrometer equipped with a gas chromatograph (G
C-MS), etc.

According to the present invention, for example, 40 g of bacterial cells/1, 16 lipids
g1ll? :! , 7-7-F-)'nic acid is 4.8 g/
l! (30% arachidonic acid/total lipid).

Containing fatty acids do not contain any branched fatty acids, and as unsaturated fatty acids, in addition to arachidonic acid, Cl611
% Cl1ll+ was mainly contained.

(Effects of the Invention) According to the present invention, since microorganisms are used, fats and oils containing arachidonic acid in high concentrations can be easily obtained, and can be used industrially at low cost and in large quantities as raw materials for pharmaceuticals, health foods, cosmetics, etc. It is possible to supply.

(Examples) The present invention will be specifically described below based on Examples, but the present invention is not limited thereto.

Example 1 Glucose 5g, yeast extract 2.4g, iron sulfate 0.05
g, magnesium sulfate 0.01g, potassium chloride 0.1
g, 0.1 g of potassium phosphate, 0.5 g of succinic acid to 10
It was dissolved in 0- demineralized water to prepare a liquid medium. This is 500
- The mixture was placed in a Sakalokolben, sterilized with steam at 120°C for 20 minutes, and the pH was adjusted to 5.4. Entomophthora extalis (E
ntomo hthora exitalis) A
Only the spores of TCC14290 were suspended in sterilized water, and this was inoculated into the above-mentioned liquid medium 100-containing Sakalokolben containing 5 g of glucose, and cultured with shaking at 28°C for 3 days, which was used as an inoculum. Newly mentioned liquid medium 1001
This inoculum was inoculated into Sakalocolben containing n1, and cultured with shaking at 28°C for 4 days.

After culturing, collect the bacteria and add chloroform-methanol (1
: 2) Lipids were extracted twice with the solvent of the system while stirring.

The extracts were combined and water-soluble substances were removed using Forte's method, and the chloroform layer was concentrated to obtain total lipids. The amount of dry bacterial cells at this time was 30 g//l, and the total lipid amount was 9 g/β, which is about 30% of the dry bacterial cells. A portion of this total lipid was added with methanol-BF3, methylated, and analyzed by gas chromatography. As a result, the desired arachidonic acid content was about 20% of the total lipid amount.

Example 2 Olive oil 6g, defatted soybean flour 3.6g, iron sulfide 0.05
g, magnesium sulfate 0.01g, potassium chloride O, I
g, potassium phosphate O, Ig, and 0.2 g of isocitric acid were dissolved in 5 Qml of demineralized water to prepare a liquid medium. This was placed in a 500 ml Sakalokolben, cultured and analyzed based on Example 1 below. The dry bacterial mass at this time was 35g/
l, and the total lipid is 10.5, which is about 30% of the dry bacterial cell.
g/l. Take a part of this total lipid and methanol-
BF was added, methylated while boiling, and analyzed by gas chromatography. As a result, the desired arachidonic acid content was about 30% of the total lipid amount.

Example 3 Glucose 300g, defatted soybean flour 216g, iron sulfate 3g
, 0.6 g of magnesium sulfate, 3 g of potassium chloride, 6 g of potassium phosphate, and 20 g of citric acid were dissolved in 6β demineralized water to prepare a liquid medium. This was placed in a 101 jar fermenter, sterilized with autoclaved steam at 120°C for 20 minutes, and pn
was adjusted to 5.4. Entomophthora extalis (Entomo
hthora exitalis) AT CC32
Only 890 spores were suspended in sterilized water, and this was inoculated into the liquid medium 100-containing Sakalocolben prepared in Example 1, and the spores were incubated at 28°C.
The inoculum was cultured with shaking for 3 days. This inoculum was newly inoculated into the aforementioned liquid medium 10j2 jar fermentor and cultured at 28° C., 600 rpm, and 1 vvm for 4 days. During this period, the glucose concentration was kept constant and finally reached 120 g/l.

After the culture was completed, the bacteria were collected and the lipids were extracted twice with hexane and chloroform. After concentrating the extracts together and washing with water,
The chloroform layer was concentrated to obtain total lipids. The amount of dry bacterial cells at this time was 40 g/l, and the total lipid amount was 14 g/l, which is about 35% of the dry bacterial cells.

Take a part of this total lipid, add methanol-BFff,
The mixture was methylated while boiling and analyzed using a gas chromatograph. As a result, the desired arachidonic acid content was about 25% of the total lipid amount.

Example 4 In Example 3, culture was carried out for 4 days at a rotational speed of 60 rpm, air replaced with pure oxygen, and aeration rate of 1 m.

After the culture was completed, lipids were extracted using the method described above. The amount of dry bacterial cells at this time was 50 g/l, and the total lipid amount was 20 g/l, which is about 40% of the dry bacterial cells. A lipid analysis revealed that the target arachidonic acid accounts for approximately 30% of the total lipid content.
%, and dry bacterial cells, lipid content, and arachidonic acid production increased compared to air alone.

Example 5 In Example 4, citric acid was increased to 0.5% and cultured for 3 days. After the culture was completed, lipids were extracted using the method described above.

The amount of dry bacterial cells at this time was 50 g/l, and the total lipid amount was 20 g/β, which was about 40% of the dry bacterial cells. A lipid analysis revealed that the desired arachidonic acid content was approximately 35% of the total lipid content, and the growth of the bacterial cells was extremely good.
Arachidonic acid production increased.

Comparative example The same procedure as in Example 1 was carried out without adding citric acid.

The amount of dry bacterial cells obtained at this time was 25 g/l, and the total lipid amount was 5 g/l, which is about 20% of the dry bacterial cells.

A lipid analysis revealed that the amount of arachidonic acid was about 15% of the total target arachidonic acid lipid, and the overall arachidonic acid yield was significantly reduced.

Claims (1)

[Claims] Entomophthora spp.
p. ) A method for producing arachidonic acid-containing fats and oils, which comprises culturing filamentous fungi in a medium supplemented with an organic polybasic acid, thereby accumulating arachidonic acid at a high concentration within the microbial cells, and collecting the arachidonic acid.