JPS6318475B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention involves culturing filamentous fungi of the genus Morteierella in a medium containing hydrocarbons as a carbon source, and culturing lipids [neutral lipids (oils, etc.),
The present invention relates to a method for producing polar lipids (phospholipids, glycolipids). Muyur is a microorganism containing γ-linolenic acid that has been reported to date. Globosus (linolenic acid content relative to total fatty acids: neutral lipids 12.3%,
Polar lipids 25.7% (hereinafter simply the content of neutral lipids and polar lipids), Muyur. pusillus (neutral lipid
1.5%, polar lipids 1.4%) [ROMumma etal,
Lipids, 6 , 584 (1971)], Euanephora cucurbitarum (total lipids 10.8%) [HBWhite, Jr.SS
Rowell, Biochim. Biophys. Acta, 116 , 388.
(1966)], Pythium tevarianum (total lipid 4.5
%), Saprolegnia. litoralis (total fat 2.8
%), Rhizopus. Stronifa (15.6% total fat),
Rhizopus. Alhizas (9.8% total fat), Piymyces. Brakeethreanus (total fat 5.4%), Muyur. J. jabanica (total fat 13.7%), Heliyusteilum. Piriforme (total fat 8.5%) [R.Shaw,
Biochim, Biophys. Acta, 98 , 230 (1965)], Entomophthora. Coronata (2.2% total fat) [RO
Mumma, T., E. BruszewsKi, Lipids, 5 , 915
(1970)], but in all of these, the carbon source is carbohydrate, and the content is high in some polar lipids, but the γ-linolenic acid content in all lipids is high. 10. Only a few percent. As a result of research on filamentous fungi that produce lipids with a high content of γ-linolenic acid, the present invention found that certain filamentous fungi belonging to the genus Morteierella can be cultured in a medium using carbohydrates as a carbon source by changing the nitrogen source concentration, culture temperature, etc. Even when using a medium containing hydrocarbons as a carbon source, the content of γ-linolenic acid in the total fatty acids within the bacterial cells was generally less than 5%, and was around 10% at most. In cultured bacterial cells, the γ-linolenic acid content is generally 23% of the fatty acid composition of total lipids.
The present invention has been completed based on the discovery that lipids can be produced at a high value of 40% for polar lipids. That is, the present invention cultivates a specific filamentous fungus belonging to the genus Morteierella in a medium containing hydrocarbons as a carbon source, and the culture produces lipids with a high content of γ-linolenic acid [neutral lipids (fats and oils, etc.), polar lipids (phosphorus, etc.)]. lipids,
glycolipids)]. The fungus used in the present invention is ramaniana var. anglispora [IFO.8187] belonging to the genus Mortierella. The above-mentioned bacteria are filamentous bacteria stored at the Fermentation Research Institute and listed in the IFO catalog (strain inventory). Hydrocarbons that are carbon sources for the medium for culturing the filamentous fungi include, for example, kerosene (n-decane 80%
), n-alkane (C ₁₀ - C ₁₅ ), n-decane,
Undecane, hexadecane, pentadecane, etc. are used. It is preferable to use 10 to 30 ml of hydrocarbon in medium 1. In addition, as a nitrogen source, for example,
Inorganic nitrogen sources such _{as NH4NO3} , ( _NH4 ) _{2SO4 ,} etc.
Alternatively, organic nitrogen sources such as urea, peptone, yeast extract, corn stave liquor, etc. are used.
Examples of inorganic salts include KH ₂ PO ₄ , K ₂ HPO ₄ ,
NaCl, _FeSO4・_7H2O , _MgSO4・_7H2O ,
ZnSO ₄ .7H ₂ O etc. are used. Add trace elements and other nutritional sources as necessary. The above filamentous fungi are usually cultured statically in a liquid medium.
This is done by shaking culture, aerated agitation culture, etc.
The pH of the medium is preferably 4.0 to 6.0, and the culture is usually carried out at 10 to 33°C for 5 to 30 days. Thus, γ in the culture
-Produces lipids with high linolenic acid content,
Collecting lipids from a culture When collecting lipids, since lipids are contained in the cells of filamentous fungi, it is preferable to separate the cells from the culture and collect the lipids from the cells. be. The lipids are collected according to conventional methods, such as by solvent extraction. Thus, according to the present invention, it is possible to produce lipids with a high content of γ-linolenic acid using hydrocarbons as carbon sources. γ-linolenic acid [18:3(6,
[9, 12)] is a fatty acid (essential fatty acid) that cannot be synthesized in the mammalian body, and is required in the diet, along with linoleic acid. This is because γ-linolenic acid is a precursor that becomes bishomo-γ-linolenic acid and then arachidonic acid in the body, and bishomo-γ-linolenic acid and arachidonic acid are respectively prostaglandins, E ₁ , E _1d and _E2 ,
This is because it becomes E _2d and plays an extremely important physiological role in living organisms. Linolenic acid produced from plant seeds is α-linolenic acid [18:3 (9,
12, 15)], and there are few that produce γ-linolenic acid, and in this sense, the production of lipids with a high γ-linolenic acid content is also important in terms of the production of γ-linolenic acid. It has meaning. Next, examples of the present invention will be shown, but the present invention is not limited thereto. Example 1 12.5 ml of n-decane, 2 g of KH ₂ PO ₄ , MgSO ₄ .
_7H2O 0.3g, NaCl 0.1g, _FeSO4・_7H2O 10mg,
_CaCl2・_2H2O 10mg, _CuSO4・_5H2O 0.2mg,
_ZnSO4・_7H2O 1.0mg, _MnCl2・_4H2O 1.0mg,
Deionized 2 mg of Thiamine-HCl, 0.02 mg of D-Biotin, and 0.91 g of NH ₄ NO ₃ as a nitrogen source, [C/N ratio (weight of carbon atoms in n-decane/weight of nitrogen atoms in nitrogen source) was 24.1]. Mix in 1000ml of water and adjust the pH
Adjusted to 4.6. 400 ml of this synthetic medium was placed in Erlenmeyer flask No. 1, each strain was inoculated, and cultured with shaking at 150 rpm at a predetermined temperature for a predetermined period of time.
After culturing, bacterial cells were collected by filtration or centrifugation. A portion of the cells was accurately weighed to determine the water content, dried overnight at 120°C in a constant temperature bath to determine the water content, and lipids were extracted from the remaining bacterial cells. To extract lipids from the bacterial cells, a mixture of chloroform and methanol (2:1 v/v) was added to the remaining wet bacterial cells and homogenized in the presence of glass beads, thereby crushing the bacterial cells and extracting the lipids at the same time. In order to perform the extraction completely, this was repeated five times and all the extracts were collected. After the above extract was purified by Floch's partition washing method, the solvent was distilled off under reduced pressure, and the total lipid amount was measured gravimetrically. A portion of the produced lipids was extracted from the bacterial cells and accurately weighed, and after methyl esterification, the fatty acid composition was analyzed by gas chromatography. The remaining lipids were separated into neutral lipids and polar lipids by column chromatography using UNISIL as a packing material and chloroform and methanol as developing solvents, and the abundance of each was determined, and each lipid was also analyzed by gas chromatography. The fatty acid composition was determined. The following table shows the lipid production amount and γ-linolenic acid content of various Morteierella filamentous fungi determined by this method. From the results in Table 1, Lamaniana angrispora of the genus Morteiera has a higher total lipid production amount for the bacterial body than other bacteria of the genus Morteiera.
Moreover, since the production rate of γ-linolenic acid is high, γ-linolenic acid can be efficiently produced. 【table】

Claims (1)

[Claims] 1. A method for producing a lipid, which comprises culturing a Lamaniana anglispora strain belonging to the genus Morteierella in a medium using hydrocarbon as a carbon source, and collecting a lipid containing γ-linolenic acid from the culture.