JPH0216989A - Production of omega6-based unsaturated fatty acid-containing phospholipid - Google Patents

Abstract

PURPOSE:To obtain a phospholipid with a high content of an omega6-based unsaturated fatty acid by culturing a specific microorganism belonging to the genus Mucor or Conidiobolus in a culture medium containing a carbon source. CONSTITUTION:Mucor circinelloides, etc., of the genus Mucor or Conidiobolus heterosporus, etc., of the genus Conidiobolus is used as a microorganism having the ability to produce a phospholipid containing an omega6-based unsaturated fatty acid. The above-mentioned microorganism is cultured in a culture medium containing a carbon source. The production of the objective phospholipid is especially increased by adding >=18C unsaturated fatty acid-containing fats and oils, etc., as necessary, thereto. After completing the culture, the culture solution is separated by centrifugation, etc., to provide microbial cells containing the phospholipid. Furthermore, fractionation and purification can be carried out by column chromatography, etc., to readily afford the objective phospholipid with a high omega6-based unsaturated fatty acid content, such as phosphatidyl choline, having high biological activity.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing phospholipids containing ω6 unsaturated fatty acids. The present invention relates to a method for producing phospholipids containing unsaturated fatty acids.

[Prior Art and Problems to be Solved by the Invention] Phospholipids are contained in various living organisms including higher animals, and play an important role in living organisms mainly as constituents of cell membranes. Moreover, unsaturated fatty acids bound to phospholipids are liberated and converted into physiologically active substances such as prostaglandins due to some stimulus within various living organisms.

Currently, the phospholipids produced on an industrial scale are soybean phospholipids and egg yolk phospholipids. These are natural surfactants and are used in the fields of foods, medicines, etc. due to their emulsifying, dispersing, and wetting effects. However, these phospholipids have the disadvantage that they do not have sufficient physiological activity.

By the way, γ-linolenic acid and bis-homo-γ-linolenic acid are fatty acids that have physiologically active effects.

Unsaturated fatty acids such as arachidonic acid are known. As a method for producing phospholipids containing such unsaturated fatty acids, phospholipids containing γ-linolenic acid are extracted from lipids obtained by culturing microorganisms belonging to the genus Mortierella in a medium using carbohydrates as a carbon source. Minutes of phosphatidylcholine
A method of concentrating (Japanese Unexamined Patent Publication No. 62-254189) is known.

However, with this method, only a product with a low content of γ-linolenic acid can be obtained, and sufficient utilization cannot be expected.

[Means for Solving the Problems] Therefore, the present inventor searched for microorganisms that have the ability to produce phospholipids containing a high content of ω6 unsaturated fatty acids, and found that microorganisms belonging to the genus Mucor or Conidiobolus were found. He discovered that he possessed this ability and completed Zero Hatsutsui.

That is, the present invention involves culturing a microorganism that belongs to the genus Mucor rot or Conidiobolus and has the ability to produce spores containing ω6-based unsaturated fatty acids in a medium containing a carbon source, and extracts ω6 from the culture.
The present invention provides a method for producing phospholipids containing ω6-based unsaturated fatty acids, which is characterized by collecting phospholipids containing ω-6-based unsaturated fatty acids.

The microorganism used in the present invention belongs to the genus Mucor or Conidiobolus and has the ability to produce ω6 unsaturated fatty acids. Specifically, as a microorganism of the genus Mucor, Mucor circinelloides 1162 (FERM P-9
360), and Conidiobolus heterosporus (Coni+
Hobolus heterosporus) ATC
C12941, Conidiobolus glopuriferus (
Conidiobolus globuliferou
s) CB3218764. Conidiobolus nanodes C
Examples include B5183/62.

In addition to the above-mentioned microorganisms, mutant strains derived from these microorganisms that have the ability to produce lipids containing ω6 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 phospholipid, and contains a carbon source, a nitrogen source, inorganic salts, and, if necessary, amino acids suitable for the growth of the microorganisms. Those containing the following ingredients are used. As the carbon source, sugars such as glucose, starch, blackstrap molasses, and sodium acetate can be used, and sugars such as glucose are particularly preferred. Furthermore, in the case of the Mucor genus, safflower oil, sunflower oil, etc., which are high-linoleic acid-containing oils, are used, and in the case of the Conidiobolus genus, evening primrose oil, which is a γ-linolenic acid-containing fat, and Mucor and Mortierella genus are used. Microbial fats and oils extracted from filamentous fungi, especially those with a carbon number of 1
By adding fats and oils containing unsaturated fatty acids of 8 or more as necessary, the production amount of the desired phospholipid can be increased.

Further, nitrogen sources include ammonium salts, yeast extract, corn steep liquor, peptone, etc., and inorganic salts include magnesium salts, calcium salts, phosphates, iron salts, copper salts, etc.

During culture, the entire amount of carbon source may be added to the medium from the beginning, but it is possible to increase the production amount of phospholipids containing ω6 unsaturated fatty acids such as γ-linolenic acid by adding it at an appropriate time after the start of culture. I can do it. When adding a carbon source such as glucose to the medium from the beginning, if the initial addition amount is too large, it may have an adverse effect on the growth of microorganisms, so the amount is usually set at 10 to 250 gh. Furthermore, when adding a carbon source during culture, the timing and number of times may be determined as appropriate. For example, if the initial concentration of a carbon source such as glucose is
00~zoog#, and when most of the carbon source is consumed, increase the growth of bacterial cells by adding carbon source of about 50-IQOg/l (once or in several times). As a result, the production amount of phospholipids with a high content of ω6 unsaturated fatty acids can be increased.

Other culture conditions, such as temperature for 9 hours, etc., may be set so as to increase the production of the desired phospholipid, taking into consideration the properties of the microorganisms used. Usually 20-32
°C, preferably 25-30t, pH 3-7, preferably 3.5-6 nits, 60-120 hours, preferably 70-1
Just do it for 00 hours.

Phospholipids containing ω6 unsaturated fatty acids are usually accumulated in microbial cells, especially in cell membranes, so the culture solution is separated into solid and liquid by a conventional method such as centrifugation, and the microbial cells containing the lipids are separated. obtain. The content of ω6 unsaturated fatty acids in the phospholipids thus obtained is 38 to 56%. Furthermore, the bacterial cells can be washed and dried as necessary.

Furthermore, if necessary, the bacterial cells can be separated and purified by performing lipid extraction using a mixture of polar and non-polar organic solvents such as methanol, ethanol, dichloromethane, and chloroform in arbitrary proportions. The bacterial cells can be ground in advance in a ball mill and then the lipids extracted, or the cells can be ground in an organic solvent to extract the phospholipids. After removing the solvent from the extract, it is fractionated with acetone and acetone-insoluble substances are collected to obtain a crude phospholipid. Furthermore, all the obtained fractions are re-fractionated by silica gel column chromatography to obtain a phospholipid composition with a high content of ω6 unsaturated fatty acids. However, the separation method shown here is 1
This is an example, and the method of fractionating the phospholipid composition of the present invention is not limited to this. The content of ω6 unsaturated fatty acids in the phospholipids thus obtained is as high as 80 to 90%.

The phospholipid composition obtained by the above method contains phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, etc., and its constituent fatty acids include γ-linolenic acid,
Bispomo-γ-linolenic acid, arachidonic acid, etc. with 1 carbon number
It is an omega-6 highly unsaturated fatty acid with 8 or more double bonds and 3 or more double bonds.

Further, phosphatidylcholine, phosphatidylethanolamine, etc. can be made to have a roughly high content of ω6 unsaturated fatty acids by a conventional method such as column chromatography (liquid chromatography is particularly preferred). For example, phosphatidylcholine is fractionated into a fraction collector for each peak by preparative liquid column chromatography, and each of the obtained fractions is saponified and decomposed using conventional methods and converted into methyl ester. The composition is measured, and the one with a high concentration of γ-linolenic acid is selected as the target product. Similarly, phosphatidylethanolamine with a high concentration of γ-linolenic acid can be obtained. Furthermore, phosphatidylcholine and phosphatidylethanolamine with high concentrations of arachidonic acid and bis-homo-γ-linolenic acid can also be obtained in the same manner.

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

Example 1 Mucor culture medium shown in Table 1 (p) 14) 84! 1
I have been using a 0μ volume jar fermenter for a long time, and I have added Mucor circinelloides HUT 1121 (FEBMP-
9359) and cultured with aeration and agitation at 30°C for 3 days.

Male 1 table Mucor medium Conidiobolus medium Glucose 150 g # 30 g
/l Ammonium sulfate 19.8 g/ρ Yeast extract 0.6 g711 5 g
#! MgSO41, Og#1ghFeSO
440mg71 0.01g/j! peptone
10g/O KH2PO49, Og71
After the completion of the 3 g/l culture, the culture solution was T-filtered to collect the bacterial cells to obtain wet bacterial cells. 7 kg of this wet bacterial body was mixed with 20 Il of methanol.

It was crushed and extracted together. The obtained extract was concentrated to about 50011111 in an evaporator, and dichloromethane 30
0IIIJ was added to separate the two layers. The dichloromethane layer was then collected and concentrated to obtain a concentrate of approximately 501. Acetone was added at 400 mA to this, and the precipitate was treated with tp.

The precipitate was washed twice with acetone to obtain about 20 g of white crystals. The fatty acid composition of this product is shown in Table 3. Dissolve 2g of this in chloroform and perform // silica gel column chromatography (column: Fuco-
gel. (manufactured by Wako Pure Chemical Industries, Ltd.). The developing solvent was chloroform/methanol = () 4:1 (I+) 3:2.
(IIT) 1:4.

As a result, phosphatidylethanolamine is obtained from (I), phosphatidylcholine is obtained from (n), and phosphatidylcholine is obtained from (II+
), other phospholipids were fractionated.

Subsequently, 1 g of phosphatidylcholine was subjected to preparative liquid chromatography (column, oDs, m solvent; methanol/water niacetonitrile 90.5 nia/2.5 flow rate;
10 m1'/m1n), and a fraction of each Bi whole that could be detected with UV 205 mm was collected. After confirming that it was phosphatidylcholine using thin layer chromatography, we investigated the fatty acid composition using gas chromatography and found that 95% of the fractionated γ-linolenic acid was phosphatidylcholine.
400 mg of the contained phosphatidylcholine was obtained. Further, when phosphatidylethanolamine was separated by preparative liquid chromatography, about 2 oomg of phosphadecylethanolamine containing 90% or more of γ-linolenic acid was obtained. The results are shown in Table 2.

Table 2 Example 2 γ-linolenic acid-containing oil (43% nioleic acid, 11% linoleic acid, 8% γ-linolenic acid) was added to the Conidiobolus culture medium shown in Table 1. 3 voj for the medium! A medium was prepared in which the amount of 1% was added. This medium was inoculated with Conidiobolus heterosporus 8TCC12941, and cultured with shaking at 30°C for 2 days.

After the culture was completed, the bacterial cells were collected by centrifugation to obtain wet bacterial cells. When phospholipids were extracted from the wet bacterial cells in the same manner as in Example 1, 20 g of crude phospholipids were obtained. The fatty acid composition of this product is shown in Table 3.

Table 3 203 mg of lucorin was obtained. Furthermore, when 1 g of phosphatidylethanolamine was separated using preparative liquid chromatography, 50% of bis-homo-γ-linolenic acid was collected.
Phosphatidylethanolamine containing 12m or more
178 mg of phosphatidylethanolamine containing 80% or more of g and arachidonic acid was obtained. The results are shown in Table 4.

Table 4 When the obtained crude phospholipid was fractionated by silica gel column chromatography in the same manner as in Example 1, about 5 g of phosphatidylcholine and about 2 g of phosphatidylethanolamine were obtained.

When 1 g of the obtained phosphatidylcholine was separated by preparative liquid chromatography in the same manner as in Example 1, 24 mg of phosphatidylcholine containing 50% or more of bis-homo-γ-linolenic acid and 80% of arachidonic acid were separated.
[Effects of the Invention] According to the present invention, it is possible to obtain phospholipids containing a high content of ω6-based unsaturated fatty acids, and by further fractionating and purifying them by column chromatography etc. Phospholipids such as bosphadicylcholine and phosphatidylethanolamine, which have a high content of unsaturated fatty acids, can be easily obtained.

The obtained phospholipids have high physiological activity and are used as pharmaceuticals.

Raw material for liposomes 1 It is expected to be used in the fields of food, cosmetics, etc.

Claims (2)

[Claims] (1) Belongs to the genus Mucor or Conidiobolus, ω
An ω6 unsaturated fatty acid characterized by culturing a microorganism capable of producing spores containing a 6 unsaturated fatty acid in a medium containing a carbon source, and collecting phospholipids containing an ω6 unsaturated fatty acid from the culture. Method for producing phospholipid content. (2) The production method according to claim 1, wherein the ω6 unsaturated fatty acid is any one of γ-linolenic acid, bis-homo-γ-linolenic acid, and arachidonic acid, and the phospholipid is phosphatidylcholine or phosphatidylethanolamine.