JPS61177990A - Production of highly unsaturated fatty acid - Google Patents

Abstract

PURPOSE:To produce a highly unsaturated fatty acid in high yield, by cultivating a protozoan of the genus Euglena aerobically in a specific culture medium. CONSTITUTION:A protozoan of the genus Euglena, e.g. Euglena gracilis Z strain, is inoculated into a culture medium containing a hydrocarbon or amino acid as a main carbon source and 0.5-5% fatty acid, e.g. linoleic acid, oleic acid or alpha-linolenic acid added thereto, and aerobically cultivated therein at 3.0-7.0pH and 20-33 deg.C for 3-6 days. Thus, the yield of bishomo-gamma-linolenic acid and arachidonic acid can be increased to 2-2.5 times based on the yield without adding the fatty acid and the yield of eicosapentaenoic acid can be increased to about 3 times.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for producing polyunsaturated fatty acids in high yields by culturing the protozoan Euglena in a culture medium at high yields. .

The highly unsaturated fatty acids produced in the present invention include bishomo-γ-linolenic acid (8,11,14-eicosatrienoic acid), arachidone@ (5,8,11,14-eicosatrienoic acid) and eicosatrienoic acid. Icosapentaenoic acid (5, 8,
11,14,17-eicosapentaenoic acid).

The bishomo-γ-linolenic acid, arachidonic acid, and eicosapencitric acid, together with linoleic acid, are essential fatty acids that cannot be synthesized in mammals. Bishomo-γ-linolenic acid is prostaglandin E1,
Arachidonic acid, such as F1a, is prostaglandin E2
, F21X% [2. Eicobentaenoic acids such as thromboxane A2 are prostaglandin Es N F
These highly unsaturated fatty acids are precursors of 3cXs Im, thromboxane A2, etc., and play extremely important physiological roles in living organisms.

Conventional technology It has been known for a long time that Euglena contains highly unsaturated fatty acids as its lipid constituent fatty acids.
al or 3 iological chemist
ry) Volume 239 (1964 > 2778 pages l-1ul
anicka.

It is described in the paper by E rain and 3 IOC. It is also known that arachidonic acid can be produced from cultured Euglena cells (Japanese Patent Publication No. 47-37030
Publication No.).

Problems to be Solved by the Invention However, in these techniques, the carbon source is derived from carbohydrates, amino acids, or the photosynthesis of Euglena itself, and in that case, the content of polyunsaturated fatty acids contained in the bacterial cells is few.

The present invention was made in view of the above circumstances, and it is an object of the present invention to provide a method for producing the above-mentioned polyunsaturated fatty acids in an extremely high yield from Euglena.

As a means to solve the problems, the present invention aerobically cultivates the protozoan Euglena in a medium containing carbohydrates or amino acids as the main carbon source and supplemented with fatty acids such as linoleic acid, oleic acid, or α-linolenic acid. It is characterized by:

That is, the present invention provides a method for culturing Euglena in a medium containing carbohydrates or amino acids as the main carbon source, in which fatty acids such as oleic acid, linoleic acid, and α-linolenic acid, which are considered to be precursors of highly unsaturated fatty acids, are added to the medium. It has been discovered that by adding these fatty acids to the bacterial cells, these fatty acids are efficiently taken up into the bacterial cells, undergo unsaturation and chain lengthening, and as a result, the content of the above-mentioned highly unsaturated fatty acids increases significantly. .

Euglena used in the present invention is a protozoan that belongs to the genus Euglena according to the zoological classification, and includes species, varieties, and mutant strains belonging thereto.

As a representative example, two strains of Euglena gracilis (
Euglena gracilis Z), Euglena gracilis bacillis variant strain (Euglena g
raciiis von bacillaris),
Euglena viridis
dis), Astasia Longa (AStaSial)
onga), etc.

Euglena also naturally inhabits natural water systems such as ponds and swamps, and these can also be collected and used. Various mutant strains obtained by treating these with known methods such as ultraviolet treatment, heat treatment, antibiotic treatment, and nitrosoguanidine treatment can also be used.

The basic medium used for culturing Euglena is Koren-Hallertner medium (Journal Opprotozoology).
Journal of ProtozooloOV)
Volume 14 (1967, enlarged page 17), Halatner medium (Journal Opbrotozoology Volume 6 <195)
9) (described on page 23) and the like can be used.

In addition, glucose, starch hydrolyzate, molasses hydrolyzate, glutamic acid, acetic acid, ethanol, etc. are used as carbon sources, and inorganic nitrogen sources such as ammonium nitrate, ammonium diphosphate, sodium nitrate, glutamic acid, aspartic acid, etc. are used as nitrogen sources. or organic nitrogen sources such as peptone, casamino acids, yeast extract, corn steep liquor, etc., in combination with inorganic salts such as calcium, magnesium, manganese, iron, etc., and vitamins B1 and 81.
A medium containing a small amount of 2 may also be used.

The appropriate culture temperature is 20 to 33°C, and the appropriate initial 1) H is 3.0 to 7.0. Cultivation may be carried out either under light irradiation or in darkness, but it is more preferable to culture in darkness.

Further, during culturing, it is preferable to perform shaking 50 to 250 times per minute or moderate aeration stirring.

By culturing under such aerobic conditions, Euglena grows to about 3
The stationary phase of growth is reached in 1 to 6 days, and the yield of cells is 10 to 20 g dry weight per 1 culture solution.

Examples of fatty acids to be added to the culture medium include oleic acid, linoleic acid, α-linolenic acid, and various metal salts thereof, which are considered to be precursors of highly unsaturated fatty acids. These fatty acids may be added at any time after the start of culture, or may be added to the medium from the beginning, but they may be added near the stationary phase of growth and the culture may be continued for roughly 3 to 6 days. It is preferable to do so.

The amount of fatty acids added is 0.5 to 5% of the medium, especially 0.5 to 5%.
2% is appropriate, but when added at the early stage of culture,
Addition of high concentration inhibits the growth of Euglena, so 1%
Hereinafter, it is preferably 0.2 to 0.5%. In addition, when adding near the stationary phase of growth, it is also effective to add a nutrient source at the same time as necessary.

Once highly unsaturated fatty acids have been accumulated in Euglena cell lipids in this way, the cells are separated from the culture medium by a method such as centrifugation, the lipids are extracted from the cells, and then the fatty acids are extracted from the lipids. Separate. To extract lipids from bacterial cells, for example, a mixture of chloroborum/methanol (volume ratio 1:1) is added to wet bacterial cells, and the mixture is homogenized using ultrasound to crush the bacterial cells and extract the lipids.

The process by which Euglena produces highly unsaturated fatty acids is thought to be due to the following process.

That is, oleic acid, linoleic acid, α-monosuronic acid, 11.14-eicosadienoic acid, 11.14
．． 17-eicosatrienoic acid bishomo-γ-
Linolenic acid 8, 11.14.17-Nicosatetraenoic acid Arachidonic acid Eicosapentaenoic acid When fatty acids such as oleic acid, linoleic acid, and α-linolenic acid are added to the culture medium of Euglena, these added fatty acids is taken up into the microbial cells and undergoes molecular chain elongation and desaturation according to the process described above, making it possible to increase the yield of polyunsaturated fatty acids.

Effects of the Invention According to the present invention, the yield of highly unsaturated fatty acids can be significantly increased by adding the fatty acids to the Euglena medium. That is, by adding oleic acid or linoleic acid to the medium, the yield of bishomo-γ-linolenic acid and arachidonic acid can be increased by about 2 to 2.5 times compared to that without adding fatty acids. Furthermore, by adding α-linolenic acid, the yield of eicosapencitric acid can be increased about three times.

Example Examples of the present invention will be shown below and compared with comparative examples.

Example-1 Two wild strains of Euglena gracilis were incubated in the dark for 5
inoculating Koren-Hallertner medium 100-mL with glucose as the main carbon source in a 0-mL long-neck shake flask,
At initial pH 3.5, at 28°C while shaking 120 times per minute.
Culture was carried out in After 3 days of culture, the cells were collected by centrifugation and transferred to 10011 Koren-Hallertner medium (p
1 g of oleic acid (oleic acid 93.5%, linoleic acid 1.7%, stearin 112.4%) manufactured by Nippon Oil & Fats Co., Ltd. was added, and the mixture was further suspended for 4 days at a temperature of 28%. The cells were cultured with shaking at ℃.

After culturing, the bacterial cells were collected by centrifugation, washed twice with 0.85% saline, measured for dry bacterial cell density, and then immersed in a mixture of OO form and methanol (1:1). Then, by homogenizing with ultrasound, the bacterial cells were disrupted and lipids were extracted at the same time.

Example 2 As the fatty acid added to the medium, linoleic acid manufactured by Wako Pure Chemical Industries, Ltd. (linoleic acid 88.7%, olein I!9.
8%, α-linolenic 110.5%, valmitic acid 0.6
Lipids were extracted in the same manner as in Example 1, except that 1 g of %) was used.

Example 3 As the fatty acid added to the medium, 1 g of α-linolenic acid (α-linolenic acid 72.7%, linoleic acid 19.7%, oleic acid 7°1%) manufactured by Hanka Kagaku Yakuhin Kogyo Co., Ltd. was used. Lipids were extracted in the same manner as in Example 1.

Comparative Example Lipids were extracted in the same manner as in Example 1, except that fatty acids were not added to the medium.

Analysis The lipids obtained in each Example and Comparative Example were saponified with an ethanolic 1N potassium hydroxide solution in a nitrogen stream, and unsaponifiables were extracted and removed with petroleum ether, and then acidified with hydrochloric acid (pH approximately 2). , fatty acids were extracted with ether. After washing the extracted fatty acids with water, they were methylated with diazomethane, and the composition of the fatty acids was analyzed by gas chromatography.

The dry cell mass and the yield of each fatty acid obtained from the analysis were as shown in the table.

BLA-bishomo-gamma-linolenic acid AA = arachidonic acid EPA-eicosapentaenoic acid The unit of dry bacterial mass is grams per 100 of the medium The unit of produced fat Mrlx is based on the result of milligrams per 100 of the medium. It can be seen that in the examples of the present invention, by adding fatty acids, the yield of highly unsaturated fatty acids is significantly increased compared to the comparative examples. In Examples 1 and 2, the yields of bishomo-γ-linolenic acid and arachidonic acid were increased by adding oleic acid or linoleic acid, especially in Example 2.
The effect is remarkable when linoleic acid is added.

It can also be seen that when α-linolenic acid was added in Example 3, the yield of eicosapentaenoic acid increased. In Example 3, the yield of arachidonic acid also increased, which is probably due to the fact that the added α-linolenic acid contained a considerable amount of linoleic acid and oleic acid as impurities.

Claims (1)

[Claims] 1. A highly sterile method characterized by culturing the protozoan Euglena aerobically in a medium containing carbohydrates or amino acids as the main carbon source and adding fatty acids such as linoleic acid, oleic acid or α-linolenic acid. Method for producing saturated fatty acids