JP2730081B2 - Method for producing arachidonic acid-containing fats and oils by microorganism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention uses arachidonic acid, which is an important regulator in vivo as a precursor of prostaglandins and leukotrienes, using a microorganism, The present invention relates to a method for mass-producing quickly and inexpensively.

Although (prior art) usually arachidonic acid are incorporated in β-position of phospholipids of the cell membrane in vivo, physical, chemical, or immunological stimulation are cut by phospholipase A _2. Then, they are sequentially converted into prostaglandins or leukotrienes by lipoxygenase, cyclooxygenase or prostaglandin synthetase, and exert their respective physiological activities.

These prostaglandins and leukotrienes are greatly expected as remedies for allergies, viral diseases, senile dementia, etc., which have become social problems as modern diseases, and research is rapidly progressing. Therefore, arachidonic acid, which is a precursor of prostaglandins and leukotrienes, is an essential messenger in living organisms, and its importance is incalculable.

As described above, arachidonic acid, which is a precursor of prostaglandins that plays an important role in a living body, is contained in a living body, for example, in the lung, brain, heart, blood, or yolk. From there, extraction and separation and purification by column chromatography or distillation were attempted. Also, some total chemical synthesis methods have been tried.

(Problem to be Solved by the Invention) The substance containing arachidonic acid exists only in a specific site such as brain, lung or blood, and is also contained in egg yolk in food, All are contained only in trace amounts.

Moreover, with the above substances, the collection of samples is extremely restricted, and the substances themselves are very susceptible to spoilage and mass production is difficult. In addition, the synthesis of arachidonic acid is extremely difficult in the synthesis, and none of them has been applied industrially at present.

An object of the present invention is to provide a method for inexpensively producing an oil or fat containing a high concentration of arachidonic acid using a microorganism.

(Means for Solving the Problems) The present invention relates to a genus Entomophthora sp.
By culturing filamentous fungi in a medium containing an organic polybasic acid, arachidonic acid is accumulated at a high concentration in the cells,
This is a method for producing arachidonic acid-containing fats and oils, which is collected.

The genus Entomoftra, a kind of fungal microorganism,
It is known that arachidonic acid accumulates in cells,
At this time, a small amount of arachidonic acid is accumulated.

On the other hand, the present invention is to accumulate fats and oils containing a large amount of arachidonic acid in cells of the genus Entomoftra.

The strain to be used in the present invention is Eentalis of the genus Entomoftra "ATCC14290, 32890", and all are filamentous fungi described in the ATCC catalog (strain list).

The filamentous fungi include glucose, sucrose, fructose, maltose, glycerin, molasses, starch, corn steep liquor, olive oil, coconut oil and the like as a carbon source, and yeast extract, peptone, meat extract, soybean as a nitrogen source. It grows in organic nitrogen such as flour, defatted soy flour, gluten, and gelatin. At this time, the carbon source is
It is used at 3 to 20%, but preferably 5 to 15%.

In the present invention, the organic polybasic acid added to the medium is
For example, citric acid, isocitric acid, succinic acid, fumaric acid,
Malic acid and the like, and the addition amount is 0.01-1%, preferably 0.1%.
It is 3 to 1%. Other non-base salts, for example, iron sulfate,
Magnesium sulfate, zinc sulfate, potassium chloride, monopotassium phosphate, dipotassium phosphate and the like are used. In addition, a trace element may be added as needed.

In the culture of the filamentous fungus, liquid culture is usually performed with aeration and stirring. Stirring speed is 300 ~ 900rpm, aeration is 0.5 ~ 2vvm
Therefore, it is necessary to perform sufficient ventilation (kla = 600 to 1000 hr ⁻¹ ), and air or pure oxygen may be used. The culturing temperature is 20 to 33 ° C., and the culturing period is 3 to 7 days. At this time, pH
Is weakly acidic, and is preferably adjusted to 5 to 6. After completion of the culture, the cells are separated by filtration or centrifugation, and the cells are sufficiently washed. Then, fats and oils are extracted from the cells with chloroform, methanol, ethanol, propanol, butanol, hexane, acetone, and a mixed solution thereof. further,
The crude lipid was separated by a distribution method such as Folch or the like, then methylesterified, and analyzed by a mass spectrometer with gas chromatography (GC-MS).

According to the present invention, for example, cells 40g /, lipids 16g /
The amount of arachidonic acid reached 4.8 g / (30% arachidonic acid / total lipid). The fatty acids contained no branched fatty acids at all, and mainly C _{16: 1} and C _{18: 1} as unsaturated fatty acids other than arachidonic acid.

(Effects of the Invention) According to the present invention, since microorganisms are used, oils and fats containing arachidonic acid at a high concentration can be easily obtained. It is possible to supply.

(Examples) Hereinafter, the present invention will be specifically described based on Examples.
The present invention is not limited to this.

Example 1 A liquid medium was prepared by dissolving 5 g of glucose, 2.4 g of an acid extract, 0.05 g of iron sulfate, 0.01 g of magnesium sulfate, 0.1 g of potassium chloride, 0.1 g of monopotassium phosphate and 0.5 g of succinic acid in 100 ml of deionized water. Prepare this in 500ml Sakaguchi Corben, 120
The autoclave was autoclaved at 20 ° C for 20 minutes, and the pH was adjusted to 5.4. Only spores of Entomophthora exitalis ATCC14290 cultured in a potato agar medium in advance were suspended in sterilized water, and this was inoculated into Sakaguchi Kolben with 100 ml of the above liquid medium in which glucose was changed to 5 g.
What was shake-cultured at 8 ° C. for 3 days was used as an inoculum. This inoculum was newly inoculated into Sakaguchi Kolben containing 100 ml of the above liquid medium, and cultured with shaking at 28 ° C. for 4 days.

After completion of the culture, the cells were collected, and lipids were extracted twice while stirring with a chloroform-methanol (1: 2) solvent.
The extracts were combined, water-soluble substances were removed by the method of Forti, and the chloroform layer was concentrated to obtain total lipids. At this time, the amount of the dried cells was 30 g /, and the total lipid amount was 9 g /, which is about 30% of the dried cells. A part of this total lipid was taken, methanol-BF ₃ was added, methylated, and analyzed by gas chromatography. As a result, the target arachidonic acid was contained in about 20% of the total lipid amount.

Example 2 6 g of olive oil, 3.6 g of defatted soy flour, 0.05 g of iron sulfide, 0.01 g of magnesium sulfate, 0.1 g of potassium chloride, 0.1 g of monopotassium phosphate and 0.2 g of isocitric acid were dissolved in 50 ml of demineralized water,
A liquid medium was used. Put this in 500ml Sakaguchi Corben,
Hereinafter, the cells were cultured and analyzed based on Example 1. At this time, the dry cells were 35 g /, and the total lipid was 10.5 g /, which is about 30% of the dry cells. A part of this total lipid was taken, methanol-BF ₃ was added, methylated while boiling, and analyzed by gas chromatography. As a result, the target arachidonic acid was contained in about 30% of the total lipid amount.

Example 3 300 g glucose, 216 g defatted soy flour, 3 g iron sulfate, 0.6 g magnesium sulfate, 3 g potassium chloride, 6 g monopotassium phosphate,
20 g of citric acid was dissolved in 6 demineralized water to obtain a liquid medium. Put this into a 10 jar fermenter, 120
The autoclave was autoclaved at 20 ° C for 20 minutes, and the pH was adjusted to 5.4. In advance, only spores of Entomophthora exitalis ATCC32890 cultured on a potato agar medium were suspended in sterilized water, and this was suspended in the liquid medium 10 of Example 1.
The inoculum was inoculated into Sakaguchi Koruben containing 0 ml and cultured with shaking at 28 ° C. for 3 days to obtain a seed. The inoculum was inoculated into the above-mentioned liquid medium 10 jar fermenter at 28 ° C and 600 rp.
m, 1 vvm for 4 days. During this time, the glucose concentration was kept constant, and eventually reached a concentration of 120 g /.

After completion of the culture, the cells were collected and lipids were extracted twice with hexane and chloroform. After the combined extracts were concentrated and washed with water, the chloroform layer was concentrated to obtain total lipids. At this time, the amount of the dried cells was 40 g /, and the total lipid amount was about 35% of the dried cells.
%, Which was 14 g /%. A part of this total lipid was taken, methanol-BF ₃ was added, methylated while boiling, and analyzed by gas chromatography. As a result, the target arachidonic acid was contained in about 25% of the total lipid amount.

Example 4 In Example 3, culture was carried out at a rotation speed of 600 rpm, air was changed to pure oxygen, and aeration was 1 vvm for 4 days.

After completion of the culture, lipids were extracted by the above method. At this time, the amount of dried cells was 50 g /, and the total lipid amount was about 40% of that of the dried cells.
%, Which was 20 g /%. As a result of lipid analysis, the target arachidonic acid contained about 30% of the total lipid amount, and the dry 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 completion of the culture, lipids were extracted by the above method.
At this time, the amount of the dried cells was 50 g /, and the total lipid amount was 20 g /, which is about 40% of the dried cells. As a result of lipid analysis, the target arachidonic acid contained about 35% of the total lipid amount, the growth of the cells was remarkably good, and the arachidonic acid production increased.

Comparative Example The same operation was performed as in Example 1 except that citric acid was not added. The amount of dried cells obtained at this time was 25 g /, and the total lipid amount was 5 g /, which is about 20% of the dried cells. The lipid analysis showed that about 15% of the target total arachidonic acid lipid was
%, And the overall arachidonic acid yield was significantly reduced.

Claims (1)

(57) [Claims] 1. The genus Entomophthora sp.
By culturing filamentous fungi in a medium containing an organic polybasic acid, arachidonic acid is accumulated at a high concentration in the cells,
A method for producing arachidonic acid-containing fats and oils, which comprises collecting the oil.