JPH02200193A - Production of omega3 based highly unsaturated fatty acid-containing lipid - Google Patents

Abstract

PURPOSE:To make it possible to efficiently obtain the title lipid in large quantities by culturing a microorganism having omega3 based highly unsaturated fatty acid producing ability of the genus Conidiobolus in a culture medium containing alpha-linolenic acid (derivative). CONSTITUTION:0.1-10vol.% linseed oil, etc., is blended with a carbon source of glucose, nitrogen source of yeast extract, inorganic salts such as KH2PO4 to afford a culture medium. Then a microorganism having omega3 based highly unsaturated fatty acid producing ability, e.g. Conidiobolus nanodes is inoculated into the above-mentioned culture medium and subjected to aerobic spinner culture at 10-40 deg.C for 2-10 day to give a cultured product consisting of omega3 based highly unsaturated fatty acid-containing lipid containing 11,14,17-eicosatrienoic acid (20+ or -3C), etc., as a main ingredient and produced and accumulated in the culture medium. A bacterium cell is separated from the cultured product and the obtained bacterium cell is purified by solvent extraction, chromatography, etc., to collect the omega3 based highly unsaturated fatty acid-containing lipid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing a lipid containing omega-3 highly unsaturated fatty acids. Omega-3 highly unsaturated fatty acids such as eicosatrienoic acid, eicosatetraenoic acid, and eicosabencitric acid are useful as pharmaceuticals, biochemical reagents, and the like.

[Prior art and problems to be solved by the invention] As a fermentation production method for ω3-based highly unsaturated fatty acids, a method for producing eicosapentaenoic acid using a microorganism of the genus Mortinilla (Japanese Patent Application Laid-open No. 14697/1983) ) 2 Method for producing eicosaben citric acid using marine bacteria [Mude et al., Journal of the Japanese Society of Agricultural Chemistry, 62, p. 259 (1988)
) etc. have been reported. In addition, microorganisms of the genus Entomophsora and Conidiobolus of the order Hyperoptera produce eicosatetraenoic acid and eicosabencitric acid (J, P., Latge et al., J., Gen., Mi.
crobiol,, month u, p151~1.58(1
980)) is also known.

However, the yield of ω3 highly unsaturated fatty acids obtained by these methods is low, and there are problems in putting them into practical use.

[Means to solve the problem]

Therefore, the inventors of the present invention have developed a method for efficiently producing large quantities of lipids containing omega-3 polyunsaturated fatty acids using a fermentation method. The present invention was completed based on the discovery that α-linolenic acid, which serves as a protein, can be added to the medium and cultured in 'ζ.

That is, the present invention involves culturing a microorganism belonging to the genus Conidiobolas and having the ability to produce ω3-based highly unsaturated fatty acids in a medium containing α-linolenic acid or its derivatives, and producing ω from the culture.
The present invention provides a method for producing a lipid containing ω3 highly unsaturated fatty acids, which is characterized by collecting a lipid containing ω3 highly unsaturated fatty acids.

The ω3-based highly unsaturated fatty acids as used in the present invention are 11.14
, 17-eicosatrienoic acid (hereinafter referred to as C2063); 8.11.14.17-nicosatrienoic acid (hereinafter referred to as C10m4): 5.8.11.14
．． 17 eicosaben citric acid (hereinafter referred to as C!), etc.

The microorganism used in the present invention belongs to the genus Conidiobolus of the order Hyperoptera and has the ability to produce ω3 polyunsaturated fatty acids. Specifically, "Conidiobolus Nanodes vs.""Unlike a giant fish" CBS 183/62. Conidiobolus lamblausinis
Examples include TCC12585.

In addition to the above-mentioned microorganisms, mutant strains derived from these microorganisms having the ability to produce ω3-based highly unsaturated fatty acids as described above can also be used in the present invention.

The medium for culturing the above-mentioned microorganisms may be one that allows the microorganisms to grow well and produce the desired lipids (
A carbon source, a nitrogen source, inorganic salts, and if necessary, trace elements and nutrient sources suitable for the growth of microorganisms can also be added. As the carbon source, carbohydrates, fats and oils, etc. are used, and specific examples include glucose, soybean oil, safflower oil, and linseed oil. In addition, as a nitrogen source, organic nitrogen sources such as yeast extract, peptone, corn staple liquor, and soybean are preferable. Inorganic salts include KH2PO4,
Fe5Oa ・711zO, MgSO4・78zO,'
1 nsOa or the like is used.

In the present invention, it is necessary for the medium to contain α-linolenic acid or a derivative thereof. Sources of α-linolenic acid or its derivatives include linseed oil, ethyl ester of α-linolenic acid, sodium α-linolenic acid, and any other source containing α-linolenic acid can be used. . The amount of α-linolenic acid or its derivative added to the medium is, for example, 0.1 to 10% by volume when using linseed oil, preferably 3 to 5% by volume, and 0.1 to 10% by volume when using other materials. 1 to 10% by volume,
Preferably it is 2 to 5% by volume.

α-Linolenic acid or its derivatives may be added to the medium before or during the culture, but
It is preferable to add it within 113 days of culture.

The above-mentioned microorganisms are usually cultured in the above-mentioned liquid medium by shaking culture, aerated stirring culture, or the like.

Culture temperature is 10-40°C, preferably 20-30°C
The number of days of culture is 2 to 10 days, preferably 3 to 5 days.

In this way, 02°-3+czosn+c was added to the culture.
*. ,.

Since lipids containing such as
,.

Lipids containing Czoma, Cto, s, etc. may be collected.

When collecting, C2°S,,C,. “4+CZ.

5. However, since the culture contains unused α-linolenic acid and fats and oils added as a carbon source, bacterial cells are separated from the culture. C2゜aff+c2゜S6. C2゜,
.

It is preferable to collect lipids containing the following. 02°-31Cz from the lipid. -4, C2°, etc. can be collected by conventional methods such as solvent extraction and chromatography.

C Example] Next, the present invention will be explained by examples.

Example 1 A culture medium having the composition shown in Table 1 was added with a predetermined amount of linseed oil (valmitic acid 6%, stearic acid 3%, oleic acid 15%, linoleic acid IG%, α -linolenic acid (60%) in the medium! Five types of culture media were created. Pour 100 d of each medium into a 500 d Erlenmeyer flask, and add Conidiobolus lamblausinis ATC.
C12585 was inoculated and cultured with shaking at 30°C for 4 days.

Rabbit” - table K HIP 04 1.5 gM
gSOn 0.5g peptone
10g Yeast extract 5g F e SO47H2O0.01g Distilled water 1! After the culture was completed, the bacterial cells were collected by centrifugation, washed with phosphate buffer (pH 7,0), and collected by suction filtration. Put the bacteria into a stainless steel cup,
Glass beads, methanol, and chloroform were added, and the cells were disrupted using a homogenizer, and the lipids inside the cells were extracted.

Next, the extracted lipids were methyl esterified and subjected to capillary gas chromatography (column PE020M).
), the fatty acid composition was investigated. The results are shown in Table 2.

As is clear from the table, the bacterial cell yield reached almost the maximum when the amount of linseed oil added was 3% by volume, and when the amount of added flaxseed oil was 4% and 5% by volume.
There was almost no difference in capacity %.

C engineering, C2゜. 4 and C2°, were identified by the following method. 11-14-17-eicosatrienoic acid (CZ., ) and 5-8-11-1417
-Nicosapene citric acid (C2°5.) had the same retention time as these standards in capillary gas chromatography (column PE020M). Furthermore, when the sample and the methyl ester of lipid extracted from bacterial cells were mixed and analyzed by capillary gas chromatography, the peaks of the C2°, , and C2° fractions became large. On the other hand, a triene fraction, a tetraene fraction, and a pentaene fraction were separated by thin layer chromatography impregnated with silver nitrate. The triene fraction contains di-homo-T-linolenic acid, C2°, 3. γ-linolenic acid Contains γ-linolenic acid. The tetralaene fraction contained 6-9-12-15-octadecatetraenoic acid, arachidonic acid, and Czo-4. The pentaene fraction contains C2°8. was included.

Both of these methyl esterified products were separated into their respective fatty acids by capillary gas chromatography, then picolinyl derivatized, and identified by capillary gas mass spectra. As a result, C2°,, is 11
-14-17-eicosatrienoic acid, C2゜84 is 8-
11-14-17-nicosatetraenoic acid, C2°8.
was identified as 5-8-11-1417-nicosapencitric acid.

Comparative Example 1 A medium was prepared by adding 30 g/l of glucose as a carbon source to a medium having the composition shown in Table 1. This medium 10
0d was placed in a 500 ml Erlenmeyer flask, inoculated with Conidiobolus lamblausinis^TCC12585, and cultured with shaking at 30°C for 3 days.

After the culture was completed, the lipids in the bacterial cells were analyzed in the same manner as in Example 1, and the bacterial cell yield was 19 g/1. The oil content is 3.8g/l, and the temperature is C2°.

czo-4*c,. , were not detected.

Example 2 A medium was prepared by adding 30 g/j2 of glucose as a carbon source to a medium having the composition shown in Table 1. This medium 1.
00 ml was placed in a 500 ml regular Erlenmeyer flask, Conidiobolus lamblausinis ATCC 12585 was inoculated, and cultured with shaking at 30"C for 2 days. After 2 days of culture, linseed oil was added to medium II! to give a concentration of 1% by volume. The cells were added in the same manner as above, and cultured with shaking for an additional 2 days.

After the cultivation was completed, the lipids in the bacterial cells were analyzed in the same manner as in the example, and the C2゜53 was 0.85 g/l.
Czo-m is 0.09 g/i! , Czo-s is 0
．． 45g/I! , was obtained with a yield of .

Example 3 Six types of culture media were prepared by adding predetermined amounts of glucose and linseed oil shown in Table 3 to the culture medium having the composition shown in Table 1. Put 100 m of each medium into a 500 d Erlenmeyer flask,
Conidiobolus lamblausinis ATCC1258
5 was inoculated and cultured with shaking at 30°C for 4 days.

After the cultivation was completed, lipids within the bacterial cells were analyzed in the same manner as in Example 1. The results are shown in Table 3.

Example 4 A medium was prepared by adding linseed oil as a carbon source to a medium having the composition shown in Table 1 in an amount of 3% by volume per medium 11. Two 500-capacity Erlenmeyer flasks containing 100 volumes of this medium were prepared, and each was inoculated with the bacteria belonging to the genus Conidiobolus shown in Table 4, and cultured with shaking at 30"C for 4 days. Results. are shown in Table 4.

/ / / It is useful.

Claims (2)

[Claims] (1) Cultivate a microorganism belonging to the genus Conidiobolus and capable of producing ω3 highly unsaturated fatty acids in a medium containing α-linolenic acid or its derivatives, and collect lipids containing ω3 highly unsaturated fatty acids from the culture. A method for producing a lipid containing omega-3 highly unsaturated fatty acids. (2) The production method according to claim 1, wherein the ω3 highly unsaturated fatty acid contains eicosatrienoic acid as a main component.