JPS63112694A - Separation and concentration of arachidonic acid and isomyristic acid - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to the use of Conidiobolus a (Conidiobolus a).
The present invention relates to a method for separating and concentrating arachidonic acid, isomyristic acid, or lower alcohol esters thereof from lipids obtained by culturing filamentous fungi.

(Prior Art) Arachidonic acid is currently well known as a precursor substance of prostaglandin, which is one of the most important physiologically active substances, and is accumulated in the body in the form of phospholipids. This phospholipid is an important component of biological membranes such as cell membranes, and arachidonic acid is excised from the phospholipid by the action of phospholipase and converted to prostaglandin in response to the needs of the living body or when it receives some stimulus. exert physiological functions. Therefore, arachidonic acid is a highly unsaturated fatty acid that is extremely important for living organisms.

In addition, isomyristic acid is a highly stable saturated fatty acid with a lower melting point than myristic acid, and is found in small amounts in the lipids of living organisms, and is a fatty acid that is expected to be used in cosmetics and the like.

Arachidonic acid and myristic acid are important lipids;
Various uses for it are being considered, such as medicines, reagents, health foods, etc., and its industrial development is expected.

(Problems to be solved by the invention) However, in any application, at present,
Because chemical synthesis is difficult, the raw material is obtained from Tenri (;'1).

``Arachidonic acid is made from very special raw materials, such as mammalian liver, kidney, heart, blood, or fish liver, and has not yet been applied industrially.Currently, it cannot be applied industrially. What is thought to be arachidonic acid is obtained from egg yolk lecithin, but since this also contains a large amount of other fatty acids 11, it requires considerable skill to separate.

Furthermore, isomyristic acid has conventionally been contained in only a small amount in fats such as butter, and it has been difficult to obtain it industrially.

The reason why it is difficult to apply it industrially is that it contains highly unsaturated acids, as shown by M. Kates, translated by Yamakawa et al., Lipid Research Methods, p. 65, 1975, Tokyo Kagaku Dojin. Lipid extraction from animal tissues, cell fractions, blood cells and blood cells is very? This is because, even when carried out on a scale of =kffi, complicated steps are required.

The simple gff process of arachidonic acid from the obtained lipid is complicated due to the presence of various highly unsaturated acids, and the movement of the double bonds and polymerization of arachidonic acid makes distillation difficult. be. In addition, there was the problem that it contained a large amount of arachidonic acid (2), and the other natural raw materials except egg yolk lecithin had the disadvantage that it required a lot of cost to store, transport, etc.

As mentioned above, isomyristic acid is naturally contained in only a small amount in butter, and because of its good physical properties, stability, and low melting point, it can be used in cosmetic base materials, soaps, surfactants, etc. Although it is possible, no attempt has been made to develop it industrially.

(Means for Solving the Problems) The present invention is intended to solve the above problems, and uses microorganisms for industrial application to produce arachidonic acid and isomyristic acid from inexpensive and easily available raw materials by simple operations. The purpose is to provide a method for separating and concentrating.

The present invention relates to the genus Conidiobolus
sSρ,) Lipids obtained by culturing filamentous fungi are hydrolyzed or alcoholysed with lower alcohols, resulting in lipid IJ
This method is characterized by separating and concentrating arachidonic acid, isomyristic acid, or lower alcohol esters thereof from j-acid or lower alcohol ester thereof by urea addition reaction and purification using a non-polar porous resin.

The filamentous fungi of the genus Conidiobolus used in the present invention are already known to accumulate arachidonic acid [Canadian Journal of Microbiology (C.
anadian journal of microb
iology 13. 755 (1967), same 1
7. 1115 (1971), 22. 105
8 (1976), Journal of Applied Batteriology
ied Bacteriology 54+ 85(
1983) ). However, all of the information presented in these papers is based on analysis and only shows the characteristics of seedlings.

The present inventors previously reported that Conidiobolus
When filamentous fungi (Obolus sp.) are cultured using organic nitrogen and carbohydrates as substrates, intracellular lipids accumulate in the dried bacterial cells by 30-50%, and arachidonic acid in the lipid fatty acids reaches 25-40%. I found out.

At that time, isomyristic acid accounted for 25-40%. The content value of arachidonic acid was higher than any value in the above paper.

The present invention uses this arachidonic acid- and isomyristic acid-containing bacterium. The bacterial cells removed from the culture solution by filtration or the like are extracted with a solvent, the obtained lipids are hydrolyzed or alcoholized with a lower alcohol, and then subjected to a urea addition reaction, resulting in urea addition of isomyristic acid or its lower alcohol ester. The product can be obtained efficiently (arachidonic acid and isomyristic acid can be separated).Next, a non-polar porous resin such as styrene-divinylbenzene resin is packed in a column, and an eluent such as an acetone-water system or a lower alcohol-water system is used. Arachidonic acid or its lower alcohol ester is purified to a high degree of purity by eluting the non-urea adduct using

In this case, purification can be achieved using acrylic or methacrylic porous resins, silica gel, silica gel derivatives, etc., but the throughput is a little low.

The lipid that is the raw material in the present invention is of the genus Conidiobolus (
It is obtained by solvent extraction of the cells obtained by culturing filamentous fungi of Conidiobolus sp.
This bacterial cell is Conidiobolus heterosporus (Conidiobolus 1leLeroso).
rus), Conidioborus lamprouges (C
onidiobolus (Lamkawa I), Conidiobolus eurymicus (Con1dfobolus)
Conidiobolus species can be mentioned.

The culture uses glucose, malt extract, sucrose, molasses, cornstap liquor, fructose, starch, etc. as a carbon source, and organic nitrogen such as peptone, meat extract, yeast extract, soy flour, etc. as a nitrogen source. It is carried out in a medium containing inorganic substances such as , magnesium, phosphoric acid, potassium, etc. and, if necessary, primary elements such as vitamins.

The pH should be slightly acidic, with a pH of 4 to 7. (Usually, culture is carried out using aerated liquid culture such as shaking culture. The number of culture days may be 2 to 7 days. After the culture is completed, the bacterial cells are removed by centrifugation^11 or vacuum filtration. Since this bacterial cell contains a large amount of arachidonic acid and isomyristic acid, it is necessary to extract it while avoiding exposure to oxygen as much as possible, but there is no need to dry the bacterial cell.

For extraction, lower alcohols such as methanol, ethanol, propatool, isopropanol, acetone, methyl ethyl ketone, chloroform, etc. are preferable, but hexane, petroleum ether, petroleum benzine, ether, benzene, etc. may be used in addition. These solvents are added alone or in combination to the bacterial cells, the lipids are extracted while stirring, and the bacterial cells are filtered. Repeat this several times to completely extract the lipids. All the extracts are collected and condensed to obtain the raw material lipid.

The raw material lipid is dissolved in a lower alcohol such as methanol, ethanol, propatool, etc., saponified by a conventional method with sodium hydroxide or potassium hydroxide, etc., and returned to acid with hydrochloric acid etc. to obtain a fatty acid, or the raw material lipid is dissolved in methanol, Alcoholysis is carried out at 20 to 60'C using potassium hydroxide, etc., as a catalyst, to form fatty acid lower alcoholysters.

The fatty acid or fatty acid lower alcohol ester is extracted with hexane or the like, washed with water, solvent removed, and purified.

In the urea addition reaction, fatty acids or fatty acid lower alcohol esters are added to a methanol-urea saturated 10 solution containing 1 to 8 times the amount of urea, and the mixture is cooled with stirring to precipitate crystals of the adduct. carried out by The crystals are filtered and separated into a filtrate and crystals.

The separated filtrate is concentrated, added with aqueous hydrochloric acid, extracted with hexane, and washed with water to obtain a fatty acid or fatty acid lower alcohol ester containing a large amount of arachidonic acid.

Alternatively, a fatty acid or fatty acid lower alcohol ester containing a large amount of isomyristic acid can be obtained by dissolving the separated adduct crystals in an aqueous hydrochloric acid solution, extracting with hexane, and washing with water. At that time, isomyristic acid can be concentrated to over 90% by extracting with hexane, then washing with water, and recrystallizing in hexane at 0 to 5°C in the case of fatty acids, and reduced pressure in the case of fatty acid lower alcohol esters. By distilling the isomyristic acid lower alcohol ester, 9
Can be concentrated to 0% or more. In the case of lower alcohol esters, recrystallization and precision may be combined.

The amount of urea used varies depending on the amount of lipid, treatment temperature, and amount of methanol, but is preferably 2 to 4 times the amount of lipid.

In the addition reaction, it is preferable to add the raw material lipid to a urea-saturated methanol solution at 50 to 60°C and slowly cool it to 5 to 10°C to react.The amount of methanol at this time is determined based on the amount of lipid The amount is preferably 10 to 20 times the amount.

The urea addition reaction can also be carried out by suspending urea in hexane, isooctane, etc. and using a small amount of methanol as a catalyst.

Next, a fatty acid containing a large amount of arachidonic acid or a fatty acid lower alcohol ester containing a large amount of arachidonic acid lower alcohol ester, crudely purified by urea addition reaction, is treated with acetone-water or lower alcohol-containing column in a column containing a non-polar porous resin. Purify using water etc. as an eluent. for example,
Fill a column with 500 ml of styrene-divinylbenzene resin (HP-20, manufactured by Mitsubishi Kasei), adsorb 10 g of crude arachidonic acid lower alcohol ester after addition of urea to the top of the column, and then add acetone/water = 80/20 (v
By flowing the eluate of /v), arachidonic acid or arachidonic acid lower alcohol ester can be purified while being checked by gas chromatography or the like.

As a result of the above operations, arachidonic acid or arachidonic acid lower alcohol ester can be concentrated to 70 to 80% by addition of urea, and can be concentrated to 95 to 99% by using a resin column.

(Effects of the Invention) According to the present invention, arachidonic acid, isomyristic acid, or their lower alcohol esters can be obtained by a simple method from microbial cells that can be mass-produced. It can be used effectively and inexpensively as a raw material.

(Example) The present invention will be specifically described below based on Examples.

Example 1 Glucose 30g 1 malt extract 30g, yeast extract 12g 1 ferrous sulfate 0.7g, magnesium sulfate 0
．． 5g, dipotassium hydrogen phosphate 1g, potassium chloride 0.
A culture medium was prepared by dissolving 5 g in 11 tap water. This medium was placed in a 500 m x wide-mouth Rubene 5 x IOM and sterilized in an autoclave at 120°C for 115 minutes. The pH of the medium was 5.6.

Here, Conidiobolus heterosporus (Conidiobolus) cultured on potato agar medium
Remove the spores of CC12941 with sterile water);
I was punished and inoculated. A strain cultured at 28°C with shaking at 130 rpm for 3 days was used as the bacterial strain, and 30I! Main culture was carried out in a jar fermenter.

The main culture was performed as follows.

In other words, the medium 201 has the same composition as that used in the seed culture.
The seeds were placed in a 306-volume jar fermenter, sterilized with Fuji air, and the above-mentioned 5 seeds were inoculated and allowed to 'E'ia for 5 days.

A turbine-type stirring blade was used, and the culture was carried out at a temperature of 28° C., an aeration amount of IVVM, and a rotation speed of 500 rpm.

The culture was completed after 5 days, and the bacterial cells were removed under wave pressure.

The amount of dry bacterial cells at this time was 35 g/(l).

After vacuum filtration, the bacterial cells remain as wet cells in a mixed solvent of chloroform/methanol = 2/1 57! The mixture was soaked in water and stirred with a stirrer for 2 hours (1 l). The extract was filtered, and the residue was extracted in the same manner as in step 31, and the procedure was repeated twice.

The extracts were combined, water-soluble substances were removed by Folch's method, and the chloroform layer was concentrated to obtain total lipids.

Most of these lipids were found to be neutral lipids by TLC (developing agent: chloroform/methanol/water = 65/25/4), most of which were triglycerides, and there were few polar lipids. The acid value was 3.6. The total amount of lipid obtained was 245 g.

Dissolve 245 g of total lipids in 21 methanol and 10
3 g of potassium hydroxide dissolved in 0 ml of methanol was added and metathurisis was carried out at 50° C. for 2 hours. After the reaction is complete,
Potassium hydroxide was neutralized with acetic acid and then evaporated to remove methanol. 50M hexane was added to the obtained methyl ester, and the mixture was washed with water. Hexane was evaporated to obtain 225 g of methyl ester. The composition of this methyl ester was as follows.

IsoC14,. ;29.6%, C1416; 5.1
%, isoC+S+. ; 10.1%, C13. ;3.
6%, C16. ;8.7%, C+s+o; 2
．． 0%, C18++; 3.3%, CI11+2;
2.2%-Cl11+3 i 1.2%% C
zo+ti o, ft%, arachidonic acid; 32.5%
, other 1.3% urea 675g at 50°C methanol 3.57! To? 8, and add 225g of methyl ester there.
added. The mixture was gradually cooled to 20° C. while stirring. The precipitated product 1111 was separated by filtration, and the filtrate was evaporated to remove methanol 11. At this time, most of the saturated acid, C, 8, acid, and some isoacids were added to the crystal side. The concentrated filtrate was slowly cooled to 10° C. while stirring, and the precipitated crystals were filtered off. Then '61. Hydrochloric acid acidic water was added to the liquid, and the mixture was extracted with hexane to obtain 83.6 g of methyl ester.

This composition is isoC44. (isomyristic acid); 5
．． 5%, Ia C15:o; 4.5%, Cr, z
y: 3.79% Czo++; 1.0%, Arachidon u; 74.9%, and 10.4%.

Further, the crystal side was decomposed with an acidic aqueous solution of hydrochloric acid and extracted with hexane to obtain 47.9 g of methyl ester. These three or
IsoC+aro (isomyristic acid) i64.2
%, isoC+s+o; 25.1%, Cl11+□;
2.4%, arachidonic acid: 3.5%, and others 4.7%.

Then, 83.6 g of methyl ester containing 74.9% arachidonic acid was adsorbed onto the upper part of a column containing 4J of )lr'-20 (manufactured by Mitsubishi Kasei) and having an inner diameter of I Q cm. A mixed solvent of acetone/water (8/2) was passed through the upper part of the column, and the arachidonic acid content was collected from the lower part of the column while being monitored by gas chromatography. Contains arachidonic acid 1
Collect over 98% of things? Agricultural shrinkage was performed to obtain 45.7 g of a product containing 99% arachidonic acid methyl ester.

Next, 99% arachidonic acid methyl ester was hydrolyzed with a potassium hydroxide-methanol solution to obtain 39.9 g of 99-6 arachidonic acid methyl ester.

Example 2 Satoshi obtained in the same manner as Example 1! 245 g was hydrolyzed in a conventional manner with aqueous methanol-potassium hydroxide solution, converted back to fatty acid with hydrochloric acid water, and extracted with hexane to obtain 1913 g of a fatty acid mixture. A urea addition reaction was carried out in the same manner as in Example 1, and from the filtrate side, isoC1410 (isomyristin i; 6.1%, isoC13,.; 4.3
%, C+a+s; 3.5%, C2゜+:+i
1.0%, arachidonic acid; 72.7%, other 12
．． 77.4 g of 4% Ill++ fat residue was obtained. From the crystal side, the same treatment as in Example 1 was carried out, and the isoCl410
(isomyristic acid); 66.2%, isoC+s+o
42.5 g of high-concentration isomyristic acid-containing fatty acid containing i 23.5%, C1n+z i 2.3%, arachidonic acid: 4.0%, and other 4.0% was obtained.

followed by fatty acids 77.4 containing 72.7% arachidonic acid;
t: was adsorbed onto the upper part of a column with an inner diameter of 10 can containing IIF'-20 (manufactured by Mitsubishi Kasei) 4β. Chee 1 from the top of the column. A mixed solvent of 7/3 ton/water is passed through, and arachidonic acid containing 99% or more is collected from the bottom of the column, and then 37.4. Arachidonic acid 99% fatty acid is 17.

Example 3 218 g of methyl ester was obtained in the same manner as in Example 1.

This methyl ester was subjected to a urea addition reaction in the same manner as in Example 1, and the filtrate was extracted with isoCI 4 +. :5.3%,・i
So C+s+o; 4.9%, C+n+*
i 3.0%, Czo+z; 1.0%, arachidonic acid; 76.1%, its +l! 19.79 koku t
81.3 g of methyl ester manufactured by lljn was obtained. IsoC14 was treated in the same manner as in Example 1 from the crystal side. ; 65
．． 6%, isoC+s+o i 22.2%, CI8+Z
i 2.4%, arachidonic acid; 3.3%, others 6.
5% high concentration imiristic acid containing methyl ester 48
．． 8g was obtained.

Next, 81.3 g of methyl ester containing 76.1% arachidonic acid was adsorbed onto the top of a 10 cm zero force column containing T (P-20 (manufactured by Mitsubishi Kasei) 41. From the top of the column, methanol/water = 9/ A mixed solvent of 1 was passed through the column, and arachidonic acid containing 99% or more was collected from the bottom of the column and concentrated to obtain 28.8 g of arachidonic acid 99% methyl ester.

Also, high concentration isomyristic acid-containing methyl ester 48.
By rectifying 8 g under reduced pressure, 91.2% of isomyristic acid, isoC12, was obtained. 22.8 g of 8.8% methyl ester were obtained.

Claims (6)

[Claims] (1) Genus Conidiobolus
s￥ sp. ) Arachidonic acid and isomyristic acid-containing lipids obtained by culturing filamentous fungi are hydrolyzed, the resulting fatty acids are subjected to a urea addition reaction, and on the other hand, crudely purified arachidonic acid is recovered from the non-urea adduct; A method for separating and concentrating arachidonic acid and isomyristic acid, which is characterized in that arachidonic acid is then purified using a non-polar porous resin, and on the other hand, isomyristic acid is obtained from a urea adduct. (2) The method for separating and concentrating arachidonic acid and isomyristic acid according to claim 1, wherein the non-polar porous resin is a styrene-divinylbenzene copolymer. (3) Separation and concentration of arachidonic acid and isomyristic acid according to claim 1 or 2 using an acetone-water solvent or a lower alcohol-water solvent as an eluent in purification using a non-polar porous resin. Method. (4) Genus Conidiobolus (￥Conidiobolu
s￥ sp. ) Lipids containing arachidonic acid and isomyristic acid obtained by culturing filamentous fungi are subjected to alcoholysis with lower alcohols, and the resulting fatty acid lower alcohol esters are subjected to urea addition reaction, while crudely purified from non-urea adducts. Arachidonic acid and isomyritic acid lower alcohol ester is recovered and then purified by a non-polar porous resin to obtain arachidonic acid lower alcohol ester, and on the other hand, isomyristic acid lower alcohol ester is obtained from the urea adduct. Method for separating and concentrating myristic acid. (5) The method for separating and concentrating arachidonic acid and isomyristic acid according to claim 4, wherein the non-polar porous resin is a styrene-divinylbenzene copolymer. (6) Separation and concentration of arachidonic acid and isomyristic acid according to claim 4 or 5, using an acetone-water solvent or a lower alcohol-water solvent as an eluent in purification using a non-polar porous resin. Method.