JP2582622B2 - Production of polyunsaturated fatty acids by filamentous fungi - Google Patents

Description

The present invention relates to a method for producing polyunsaturated fatty acids by culturing filamentous fungi at a nutrient doubling value and obtaining a lipid having a higher content of polyunsaturated fatty acids from the culture. .

The polyunsaturated fatty acid produced in the present invention has a carbon number
18-22, fatty acids having 3 or more unsaturated double bonds, specifically, for example, γ-linolenic acid (9,12,15-octadecatrienoic acid), dihomo-γ-linolenic acid (8,11 , 14-eicosatrienoic acid), arachidonic acid (5,8,11,14-eicosatetraenoic acid), EPA (5,8,11,14,17-eicosapentaenoic acid), DTA (7,10, 13,16-docosatetraenoic acid), DPA (4,7,10,13,16- or 7,10,13,16,19-docosapentaenoic acid) and DHA (4,7,10,13,16 , 19-docosahexaenoic acid) and the like.

These highly unsaturated fatty acids are essential fatty acids that cannot be synthesized in the body in mammals. Dihomo
etc. γ- linolenic acid prostaglandin E _1, F _1, arachidonic acid is prostaglandin _{E 2, F 2, I 2} , such as thromboxane A _2, EPA is prostaglandin _{E 3, F 3, I 3} , known as the respective precursor such as thromboxane a _3, at the same time is important, polyunsaturated fatty acids themselves, are known to exhibit a number of physiological activity in vivo.
For example, EPA, DHA, and the like have been noted for their effects on antithrombotic effects, heart disease, serum total cholesterol lowering effects, and the like.

[Conventional technology and its problems]

Among these polyunsaturated fatty acids, microorganisms containing arachidonic acid include, in addition to protozoa Euglena, fungi such as Conidiobolus, Tricnothecium, and E.
Genus ntomophthora, Phytophthora, Saprolegnia, Pyt
The genus hium, the genus Mortierella and the genus Blastocladiella are known. These microorganisms simultaneously contain dihomo-γ-linolenic acid. Microorganisms containing EPA include filamentous fungi such as Phytophthora, Saprolegnia, and Pythium.
The genus Mortierella has been reported (HBWHITE and
SSPWELL Biochim. Biophys. Acta116: 391 (1966), DT
yrell Can. J. Microbiol. 13: 755 (1967) and the like]. Others
Arachidonic acid can be obtained by adding hydrocarbons during culturing, as filamentous fungi Aspergillus, Fusar
The genera genus Penicillium, Porphyridium, Mucor and Rhizopus are known (Japanese Patent Application Laid-Open No. 56-19231).

In addition, polyunsaturated fatty acids such as DHA are extracted from fish oil. However, the content of the fatty acids in fish oil (whale oil, sardine oil) is low, and polyunsaturated fatty acids themselves are easily oxidatively decomposed. It is difficult to obtain the desired highly unsaturated fatty acid with high purity.

[Means for solving the problem]

Under such circumstances, the present inventors considered that it is most advantageous to produce polyunsaturated fatty acids using microorganisms, and as a result of intensive studies on filamentous fungi having the ability to produce lipids having a high content of polyunsaturated fatty acids. American Type Culture Co
thraustotheca clavata ATCC 34112, Ac, stored in the LLection (ATCC) and listed in the catalog
hlya bisexualis ATCC 14524, Haliphthorous philippi
nensis ATCC 58303, Rizophydium littoreum ATCC 3610
0 and Japonochytrium sp. ATCC 28207 produce polyunsaturated fatty acids, and the present invention has been made based on this finding.

That is, the present invention relates to the genera Thraustotheca, Achlya, Hal
A filamentous fungus belonging to the genus iphthorous, Rizophydium or Japonochytrium, culturing a filamentous fungus having a high unsaturated fatty acid content and having a high lipid-producing ability, and collecting a lipid having a high unsaturated fatty acid content from the culture. Is a method for producing polyunsaturated fatty acids.

The composition of the medium for culturing the filamentous fungus is not particularly limited. However, as the carbon source, glucose, ethanol, glycerol and the like are preferable, and the amount used is usually in the range of 0.5 to 20% by weight. As the nitrogen source, it is preferable to use an organic nitrogen source such as yeast extract, malt extract, peptone, etc. In addition, inorganic nitrogen such as nitrate can also be used, and the amount used is usually in the range of 0.1 to 10% by weight. is there. Further, in addition to these components, the addition of trace elements such as amino acids and vitamins and inorganic salts are effective in promoting the growth of bacteria.

The pH of the medium is 4-10, preferably 5-8, and the culture temperature is 10
The temperature is preferably in the range of ~ 35 ° C, and the culturing time is required about 2 to 15 days.

The cultivation is usually performed in a liquid culture medium by a method such as stationary culture, shaking culture, or aeration and stirring culture.

Cells are collected from the thus obtained culture by filtration, centrifugation, or the like, and lipids are extracted from the cells. Extraction of lipids is performed by a conventional method, for example, by solvent extraction.

〔Example〕

Example 1. Thraustotheca clavata AT in the following organic nutrient medium 1
After inoculating CC 34112 and culturing with shaking at 25 ° C. for 7 days, the cells were collected by filtration and freeze-dried. As a result, 5.8 g of dried cells were obtained. After the dried cells were pulverized and extracted and purified by the Folch method, the solvent was distilled off under reduced pressure, and the total lipid was measured by a gravimetric method. As a result, 0.5 g of crude lipid was obtained.

Medium Glucose 10g / Yeast extract 3g / Malt's extract 3g / Tryptone 5g / Distilled water 1 pH 7.0 Lipids extracted and purified from cells were partially methylesterified, and fatty acid composition was analyzed by gas chromatography. . The results are shown in Table 1.

In addition, each peak obtained by gas chromatography was identified by mass spectrometry. Gas chromatographic analysis conditions Column packing Diasolid ZF Column temperature 200 ° C Carrier gas N ₂ Detector temperature 250 ° C Detector FID injection temperature 250 ° C Column 1.5m × 3φ The arachidonic acid content per dry cell is 21.6 mg / g.

 The EPA content per dry cell is 4.6 mg / g.

Example 2. Achlya bisexualis ATCC 1 in the following organic nutrient medium 1
After inoculating 4524, the same operation as in Example 1 was performed, and the fatty acid composition was analyzed.

 The results are shown in Table-2.

Medium Glucose 10g / Yeast extract 3g / Malt's extract 3g / Polypeptone 5g / Distilled water 1 pH 6.5 Example 3. Haliphthorous philippine was added to the following organic nutrient medium 1.
nsis ATCC 58303 was inoculated, and thereafter the same operation as in Example 1 was performed to analyze the fatty acid composition. The results are shown in Table-3.

Medium Glucose 5g / Yeast extract 1g / Polypeptone 1g / Artificial seawater 1 pH 6.5 Example 4. Rizophydium littoreum AT in the following organic nutrient medium 1
After inoculating CC 36100, the same operation as in Example 1 was performed, and the fatty acid composition was analyzed. The results are shown in Table-4.

Medium Glucose 5g / Yeast extract 1g / Polypeptone 1g / Artificial seawater 1 pH 6.5 Example 5. Japonochytrium sp. ATCC 2 was added to the following organic nutrient medium 1.
8207, and the same operation as in Example 1 was carried out.
7 g of dried cells were obtained, followed by 0.14 g of crude lipid. Further, the fatty acid composition of this lipid was analyzed in the same manner as in Example 1.

 The results are shown in Table-5.

Medium Glucose 10g / Yeast extract 3g / Malt's extract 3g / Peptone 1g / Artificial seawater 1 pH 7.0

Claims (1)

(57) [Claims] 1. The genus Thraustotheca, the genus Achlya, the Haliphthoro
By culturing a filamentous fungus belonging to the genus Rizophydium or Japonochytrium and having a lipid-producing ability with a high content of highly unsaturated fatty acids, and collecting a lipid with a high content of a highly unsaturated fatty acid from the culture, a filamentous fungus characterized by A method for producing polyunsaturated fatty acids.