JPH04103558A - Method for obtaining eicosapentaenoic acid and its ester from marine microorganism - Google Patents

Abstract

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

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides eicosapentaenoic acid (hereinafter referred to as EPA).
The present invention relates to a method for selectively obtaining high concentrations of EPA and EPA esters from lipids of marine microorganisms rich in EPA and EPA esters.

[Prior Art] Polyunsaturated fatty acids represented by EPA are essential fatty acids for higher animals and play an important role in living organisms. In addition, various pharmacological effects of highly unsaturated fatty acids on the human body are known, and in particular, EPA is being considered as a drug for treating blood clots due to its serum lipid-improving effect and antithrombotic effect. There is a large demand for refined products.

Conventionally known methods for producing high-purity EPA include extraction and purification from fish oil or certain types of microorganisms. EPA-containing lipids can be purified using low-temperature fractional crystallization, urea addition, vacuum distillation, chromatography, etc., but EPA-containing lipids obtained from fish oil contain E.
Since it contains docosahexaenoic acid in addition to PA, the purification process requires complicated operations and expensive equipment. In addition, EPA-containing lipids produced by microorganisms do not have the characteristic odor of fish oil (and are relatively easy to separate due to their fatty acid composition, but the long cultivation time of microorganisms makes it difficult to improve productivity). Moreover, even with the purification methods using each of the above-mentioned methods, it is difficult to separate and purify highly pure EPA in large quantities at low cost.

[Problem that the invention seeks to solve]

The purpose of the present invention is to convert EPA produced by marine microorganisms into a fatty acid mixture and/or a fatty acid ester mixture containing EPA ester, and from there to produce EPA and/or esters in large quantities at low cost without deteriorating the target product under extremely mild conditions. Another object of the present invention is to provide a method for selectively separating and purifying only EPA ester.

[Means for Solving the Problems] Some of the present inventors have developed a method suitable for EPA production that is relatively easy to purify and can obtain highly pure EPA, and also has a short culture time and easy control. As a result of searching for bacteria in the ocean, we have discovered marine microorganisms that produce only EPA as a highly unsaturated fatty acid, as disclosed in Japanese Patent Application Laid-Open No. 2-23877. Such marine microorganisms have a total lipid content of 6 to 12% on a dry matter basis, which is higher than that produced by conventional microorganisms, and the fatty acid composition of the total lipid is EPA, which is the only polyunsaturated fatty acid, and the content is 25%.
The growth rate is as high as ~40%, and the growth rate is significantly faster than that of ordinary microorganisms or algae such as Chlorella, and it has the characteristic that a large amount of bacterial cells can be obtained in an extremely short period of time. EPA produced by these marine microorganisms can avoid the problems of stable supply when using fish oil as a source of EPA, and also has the conditions for easy purification as described above, making it suitable for industrial use. It is an advantageous source of EPA on a large scale.

On the other hand, some of the inventors also believe that highly unsaturated fatty acids, especially E
Focusing on PA, docosahexaenoic acid, arachidonic acid, etc., we have patented a technology to selectively obtain them by forming complexes with silver salts (patent application 106503/1999).
.

It is generally known that when an organic compound has an unsaturated bond, silver ions form a complex between π electrons and silver ions. Regarding this point, Japanese Patent Application Laid-Open No. 63-208549
Discloses a method for purifying highly unsaturated fatty acids such as eicosapentaene or derivatives such as their esters by fixing silver ions on an adsorbent and utilizing the difference in affinity between silver and unsaturated fatty acids. It's been done.

The present invention was completed based on these findings, and includes a fatty acid mixture containing EPA produced by marine microorganisms,
EPA is selectively extracted as a water-soluble silver-EPA complex into an aqueous layer by reacting an aqueous solution of a silver compound with a certain concentration or more that forms a complex with an unsaturated carbon bond, and the aqueous layer is separated and aqueous ammonia is added. It is based on selectively separating only EPA by dissociating it with a complex dissociating agent such as or with excess water. The present invention can be carried out not only with EPA but also with EPA ester, and alkyl esters such as methyl ester and ethyl ester are suitably applied, but are not particularly limited thereto.

To specifically explain the present invention, the marine microorganism to which the present invention is applied is not particularly limited to the genus, species, or strain, as long as it has a high EPA content, but it is usually of the genus Pseudomonas. , the genus Alteromonas, or the genus Shewanel la. EPA produced by marine microorganisms such as these
An aqueous solution of a silver compound that forms a complex with an unsaturated carbon bond is added to an EPA-containing fatty acid mixture obtained by saponifying the contained lipids or by acting with an enzyme, and an EPA ester-containing fatty acid ester mixture obtained by esterification. Add water-soluble silver-EPA and EP by stirring for 5 minutes to 4 hours.
Since a complex of A ester is formed, only this complex can be selectively separated from other fatty acids and other fatty acid esters and dissolved in the aqueous layer. Considering the stability of EPA and EPA ester, the solubility of the silver compound in water, and the rate of complex formation, the reaction temperature can be carried out at 0 to 100°C, but preferably around room temperature, and the reaction time is 10 minutes to 10 minutes. 2 hours is recommended.

Furthermore, in consideration of the oxidation stability of EPA and EPA ester and the stability of the silver compound, it is desirable to carry out the present invention under an inert gas atmosphere, such as nitrogen, while shielding from light. The silver compound capable of forming a complex with an unsaturated carbon bond is not particularly limited, but generally speaking, silver nitrate, silver perchlorate, etc. are preferably used. Considering cost and other aspects, silver nitrate is sufficient. The molar ratio of EPA and EPA ester to silver compound to form a complex is 1,30 to 50.
:1, and the concentration of the silver compound aqueous solution as a reagent ranges from about 0.1 mo to a saturated aqueous solution.

If the concentration is lower than that, the complex formation of silver-EPA and EPA ester is insufficient, and EPA and EPA ester do not become water-soluble. Considering the recovery rate of EPA and EPA ester, the molar ratio is preferably 1:15 to 1:1, and the concentration is preferably 1 to 10 m0/I2.

If a fat-soluble organic solvent is added to the reaction mixture containing the complex obtained above for extraction, other fatty acids and other fatty acid esters that do not form complexes with silver can be extracted and removed from the reaction system. can. The organic solvent used here is
It is preferable to use an organic solvent insoluble in water such as hexane or ether. The aqueous layer obtained by this operation contains silver-EP
It contains a complex of A and EPA ester, and by adding an excess of a complex dissociating agent such as ammonia water to this aqueous layer or diluting it with water, EPA and EPA ester can be liberated and recovered. Considering the recycling of silver compounds, it is desirable to dilute them with excess water. The amount of complex dissociating agent used and the amount of water to be diluted are 1 to 1 of the aqueous layer containing the complex.
00 times is used, but preferably 10 to 40 times. The liberated EPA and EPA ester are recovered by distillation with a water-insoluble organic solvent such as hexane or ether, and silver ions can be removed by washing the resulting organic layer with water and saturated saline.

Moreover, silver compounds can be recovered from a wastewater solution containing silver ions by distilling off water, and the wastewater solution can be reused.

Furthermore, in the present invention, the purity of EPA and EPA ester can be further increased by repeating the above separation and purification operations.

As explained in detail above, the present invention first obtains unsaturated fatty acids from an EPA-containing fatty acid mixture and/or an EPA ester-containing fatty acid ester mixture produced by marine microorganisms that produce EPA, which is attracting attention as a highly unsaturated fatty acid. A water-soluble silver compound capable of forming a complex with the bond and EPA and/or
Alternatively, a complex with EPA ester is formed, and secondly, using the water solubility of this complex, EPA and/or E
EPA from marine microorganisms is achieved by separating only the PA ester, and then adding an excess of a complex dissociating agent such as excess aqueous ammonia or excess water to dissociate the complex and liberate EPA and/or EPA ester. as well as/
Alternatively, the ester can be extracted, separated, and further purified.

[Example] The present invention will be explained in more detail with reference to Examples.

Example 1 Yeast extract 0.5%, peptone 1%, NaCβ 1.5%
Marine microorganisms (Alteromonas butrifaciae:/s 5CRC-28
71; Microtechnical Research Institute No. + GZ Ki No.) was cultured. After the culture was completed, the cells were collected by centrifugation, washed, and dried to obtain a powder of marine microorganism cells. After drying this powder, it was extracted with a mixed solvent of chloroform:methanol=2:1 to obtain a lipid.

The total lipid obtained by distilling the lipid under reduced pressure and removing the solvent is 0.
80°C in 95% ethanol containing 3N-NaOH
After saponifying the mixture for 1 hour, it was neutralized with 6N-HCl to produce a free fatty acid mixture. After methyl esterifying this fatty acid mixture with diazomethane, analysis using a gas chromatograph revealed that 18.5% of EPA was contained in the total fatty acids.

2.0602g of this fatty acid mixture was mixed with 1.5mj of hexane.
2 to make a hexane solution, and add 2.50 g of silver nitrate to this.
An aqueous solution prepared by dissolving 54 g of distilled water at 1.5 m° C. was added under nitrogen atmosphere and shielded from light, and the mixture was stirred for 2 hours. After the stirring was completed, the reaction solution was washed twice with 40 mj2 of hexane. Distilled water 4°mβ was added to the remaining aqueous layer and stirred for 1 hour to liberate the complexed fatty acids. The free fatty acids are converted into hexane 40
Extraction was carried out twice with rnβ, and the obtained hexane layers were combined, washed with distilled water and saturated saline, and dehydrated with magnesium sulfate. The hexane solution in which fatty acids were dissolved was concentrated under reduced pressure to obtain 131.1 mg of fatty acids. After methyl esterifying the fatty acid obtained with diazomethane, analysis using a gas chromatograph revealed that the purity of EPA was 94.9.
% (of total fatty acids).

Example 2 is an example of the method of the present invention in which a fatty acid methyl ester mixture is used to form a complex.

Methyl esterification was carried out by a conventional method using diazomethane, the extracted lipid used in Example 1, to obtain a fatty acid methyl ester mixture. When this fatty acid methyl varnish mixture was analyzed by gas chromatography, it was found that
It contained 18.7% (of all fatty acid methyl esters) of EPA methyl ester.

2.1063 g of the obtained fatty acid methyl ester mixture was dissolved in 1.5 m of hexane to obtain a hexane solution, and 2.5087 g of silver nitrate was added to 1.5 m of distilled water.
Exactly the same operation as described in Example 1 was performed using an aqueous solution dissolved in 5 mJ2, and after formation and dissociation of a complex, 140.3 mg of a fatty acid methyl ester mixture was obtained. When this was analyzed by gas chromatography, the purity of EPA methyl ester was 94.0%.

Example 3 Seawater medium containing 2% peptone and 0.5% yeast extract (p
H7,0) and marine microorganisms (Pseudomonas buterifaciens 5CRC-2878;
No. 3) was cultured. After the culture was completed, the cells were collected by centrifugation and extracted with a mixed solvent of chloroform and methanol (2=1). After extraction, the mixture was concentrated under reduced pressure to remove the solvent, and the resulting total lipids were ethyl esterified by a conventional method using sodium ethylate to obtain a fatty acid ethyl ester mixture.

When this fatty acid ethyl ester mixture was analyzed by gas chromatography, it was found that EPA ethyl ester was 20.6%.
(in total fatty acid ethyl esters).

4.2653 g of the obtained fatty acid ethyl ester mixture was dissolved in 3.0 mA of hexane to obtain a hexane solution, and 5.0865 g of silver nitrate was added to 3.0 mA of distilled water.
Exactly the same operation as described in Example 1 was performed using an aqueous solution dissolved in 0 mf2, and after formation and dissociation of a complex, 368.8 mg of a fatty acid ethyl ester mixture was obtained. When this was analyzed by gas chromatography, the purity of EPA ethyl ester was 96.3%.

Example 4 A seawater medium containing 3% peptone and 0.5% yeast extract (p
Marine microorganisms (Seawanella buterifaciens 5CRC-2874, Kaikoken Jokyo No. 1625) were cultured in the microorganisms (H7,0). After the culture was completed, the cells were collected by centrifugation and extracted with a mixed solvent of chloroform:methanol=2:l.

After extraction, the mixture was concentrated under reduced pressure to remove the solvent, and the resulting total lipids were ethyl esterified by a conventional method using sodium ethylate to obtain a fatty acid ethyl ester mixture. When this fatty acid ethyl ester mixture was analyzed by gas chromatography, it was found that it contained 18.3% (of all fatty acid ethyl esters) of EPA ethyl ester.

6.5148 g of the obtained fatty acid ethyl ester mixture was dissolved in hexane at 4.5 m°C to obtain a hexane solution, and 8.6513 g of silver nitrate was added to 4.5 m C of distilled water.
Exactly the same operation as described in Example 1 was carried out using an aqueous solution containing 5 mg of the fatty acid ethyl ester, and after formation and dissociation of a complex, 589.4 mg of a fatty acid ethyl ester mixture was obtained. When this was analyzed by gas chromatography, the purity of EPA ethyl ester was 95.6%.

[Effects of the Invention] The method of the present invention is significantly different from conventional methods in that it produces EPA and/or EPA without fishy odor, which is produced by marine microorganisms at almost room temperature, with simple operation and equipment, and at low cost.
Esters can be easily and selectively obtained.

Claims (2)

[Claims] (1) Extract lipids from marine microbial cells, bring the resulting fatty acid mixture and/or the fatty acid ester mixture obtained by transesterification into contact with a silver salt in an aqueous medium to form a complex, and then A method for obtaining eicosapentaenoic acid and/or its ester from marine microorganisms, which comprises adding a complex dissociating agent. (2) The method according to claim 1, wherein the concentration of the silver salt is 0.1 mol/l or more.