JPS62278991A - Production of highly unsaturated fatty acid - Google Patents

Abstract

PURPOSE:To produce the titled substance under mild condition, by decomposing a natural oil or fat containing a highly unsaturated fatty acid with a lipase obtained from a lipase-producing microbial strain and separating the highly unsaturated fatty acid in the form of free fatty acid. CONSTITUTION:A natural oil or fat containing a 18-24C highly unsaturated fatty acid having 3-6 double bonds, e.g. fish oil, whale oil, etc., is treated with a lipase produced by a lipase-producing microbial strain such as Chromobacterium viscosum, Pseudomonas fluorescens, Aspergillus niger, etc. The reaction is carried out by mixing a natural oil or fat with water, a raw oil or fat and a lipase and reacting the components under temperature and pressure condition suitable for enzymatic decomposition. The highly unsaturated fatty acid is separated from the reaction system by extracting with a solvent such as methanol/chloroform.

Description

Detailed Description of the Invention 3. Detailed Description of the Invention [Field of Industrial Application] The present invention is directed to the use of highly inorganic compounds such as eicosapentaenoic acid (hereinafter abbreviated as EPA) and docosahexaenoic acid (hereinafter abbreviated as DHA). Natural fats and oils containing saturated fatty acids (hereinafter abbreviated as PUFA) as a fatty acid component (for example.

The present invention relates to a method for producing PUFA by causing lipase to act on (fish oil, whale oil, fats and oils derived from microorganisms).

[Conventional technology]

EPA and DHA have an inhibitory effect on platelet aggregation, and research by Dr. Dyerberg et al. suggests that they may be useful as preventive agents for cardiovascular diseases such as cerebral thrombosis and myocardial infarction. EPA also has the ability to lower cholesterol in the blood, and its activity is said to be about four times that of linoleic acid, which is currently used as a cholesterol-reducing agent.

As described above, PUFA has attracted attention for its pharmacological effects.

Conventional methods for producing PUFA include decomposing fats and oils containing PUFA by hydrolysis or transesterification to produce PUFA as free fatty acids (hereinafter abbreviated as FFΔ).
There is a way to get it. Among these, hydrolysis methods include high-temperature and high-pressure methods (250° C., 250 atm), saponification and decomposition methods, and transesterification methods include metatrilysis and ethanolysis. On the other hand, a method has been proposed for concentrating PUFA by hydrolyzing fish oil with lipase obtained from Candida cylindrasse (Japanese Patent Laid-Open No. 16579/1989).
No. 6).

[Problem that the invention seeks to solve]

However, since PUFA has a structure with a large number of carbon atoms and many double bonds, it is extremely unstable and easily oxidized to light, heat, oxygen, etc., and easily generates lipid peroxide. When lipid peroxide occurs, it has a negative effect on the human body,
It can cause diarrhea, vomiting, fever, and in the worst cases, death.

When decomposed by conventional hydrolysis or transesterification methods, it is very easily oxidized by heat, catalysts, etc., and polymerization and peroxides are likely to occur.

Furthermore, saponification decomposition methods and transesterification methods that use alkali are highly likely to cause denaturation such as movement of double bonds.

Therefore, when decomposing fats and oils containing PUFA, it is necessary to carry out the reaction at normal temperature and normal pressure under neutral conditions.

In addition, a method using lipase as an application of conventional enzymatic hydrolysis to fats and oils containing PUFA (Japanese Patent Laid-Open No. 58-1
No. 65796) utilizes the property that glycerides containing PUFA are difficult to be hydrolyzed by lipase.
Ordinary fatty acids with 18 carbon atoms or less are decomposed by lipase to form fatty acids, and PUFA is separated undecomposed as glycerides.

The glyceride containing PUFA is then decomposed using a conventional method to obtain PUFA, and the PUF is extracted using lipase.
No study has been made to decompose A into fatty acids.

The present invention allows PUFA, which is unstable to heat, light, air, etc., to be obtained as FFA by a gentle method that does not cause double bond transition, etc. 1. It is possible to produce PUFA that has original physiological activity. The purpose of this study is to propose a method for producing highly unsaturated fatty acids.

[Means for solving problems]

The present invention decomposes natural oils and fats containing highly unsaturated fatty acids as fatty acid components with lipase obtained from lipase-producing bacteria belonging to the genus Chromobacterium, Pseudomonas, or Aspergillus, and then converts the highly unsaturated fatty acids into free fatty acids. This is a method for producing highly unsaturated fatty acids, which is characterized by separating.

In the present invention, PUFA includes carbon atoms of 18 to 24,
There are long-chain highly unsaturated fatty acids having 3 to 6 double bonds, examples of which include EPA and DHA, as well as γ-lylunic acid and arachidonic acid. The raw material oil and fat in the present invention is a natural oil and fat containing these PUFAs as fatty acid components.

Examples include fish oil, whale oil, evening primrose seed oil, pine nut oil, and oils and fats derived from microorganisms.

The lipase used in the present invention is of the genus Chromobacterium,
These lipases are obtained from lipase-producing bacteria belonging to the genus Pseudomonas or Aspergillus.7 These lipases do not have specificity for the position of glycerin. Examples of the above-mentioned lipase-producing bacteria include Chromobacterium viscosum, Pseudomonas fluorescens, and Aspergillus niger. The previously used lipases obtained from Candida cylindranose or Mucor minihai cannot be used because they have poor decomposition properties for fats and oils containing PUFA.5 The above lipase used in the present invention is much more effective than conventional ones. Shows high PUFA degradability.

To decompose raw oils and fats such as fish oil using lipase.

Water is necessary to exhibit its activity, and the appropriate amount is 30 to 70% by weight, preferably about 50% by weight, based on the natural oil or fat. The appropriate amount of lipase to be used is usually about 10 to 10,000 units, preferably about 100 to 500 units per gram of natural oil or fat.

The decomposition method is to mix the above raw materials lipase, lipase and water.
The reaction can be carried out for 24 to 72 hours by stirring under neutral conditions at a mild temperature and pressure suitable for enzymatic decomposition.

In this reaction, the raw fat or oil is hydrolyzed by lipase and PUFA is liberated as FFA, so PUFA is separated by solvent extraction or the like. For solvent extraction, a solvent that dissolves PUFA, such as a methanol/chloroform solvent, can be used.

〔Effect of the invention〕

In the production method of the present invention, the raw material fats and oils are decomposed using lipase obtained from a specific bacterium, so extremely unstable PUFA can be processed under mild conditions to obtain FFA. It is possible to produce active PUFA without denaturing it.

〔Example〕

The present invention will be explained below using examples. In each example, % indicates weight %.

Example 1 Fish oil log and purified water (sterilized distilled water, hereinafter referred to as "distilled water") from 4 companies were placed in a screw tube with a capacity of 50 mu, and 1000 units of enzyme obtained from Taromobacterium viscosum were dissolved in 1 mg of purified water. was added, and the reaction was carried out for about 48 hours by rotating and stirring at a speed of 500 revolutions per minute in a constant temperature bath at 45°C. After solvent extraction of the decomposition product with methanol/chloroform-based solvent, FFA is separated from the solvent,
Composition analysis was performed by gas chromatography under the following measurement conditions. The results are calculated based on the amount of EPA and DH from the raw fats and oils.
The recovery rate of A is shown in Table 1. The peroxide value (hereinafter abbreviated as PCM) according to the basic oil and fat analysis test method established by the Japan Oil Chemists' Association was 3.2, with almost no change observed.

Gas chromatography measurement conditions Column: Capillary chromatography column Salmon 3000A (manufactured by Shimadzu Corporation).

(φ0.24mo+X50m) Inlet, detector temperature = 250℃ Column temperature: 180℃ Constant temperature Carrier gas: He; 25kPa, Air;
30kPa.

It, ; 30 kPa Detection path: FID Example 2 A reaction was carried out under the same conditions as in Example 1 using lipase obtained from Pseudomonas fluorescens. The results are shown in Table 1.

Comparative Example 1 A reaction was carried out under the same conditions as in Example 1 using lipase obtained from Candida cylindracea. The results are shown in Table 1.

Example 3 A reaction was carried out under the same conditions as in Example 1 using lipase obtained from Aspergillus niger.

The results are shown in Table 1.

Comparative Example 2 A reaction was carried out under the same conditions as in Example 1 using lipase obtained from Mucor Minihigh. The results are shown in Table 1.

Table 1 From the above results, it can be seen that the recovery rate of PUFA in the examples is much higher than that in the comparative examples.

Claims (3)

[Claims] (1) After decomposing natural oils and fats containing highly unsaturated fatty acids as fatty acid components with lipase obtained from lipase-producing bacteria belonging to the genus Chromobacterium, Pseudomonas, or Aspergillus, highly unsaturated fatty acids are converted into free fatty acids. A method for producing highly unsaturated fatty acids, characterized by separating them. (2) The manufacturing method according to claim 1, wherein the highly unsaturated fatty acid has 18 to 24 carbon atoms and 3 to 6 double bonds. (3) Claim 1 or 2, wherein the lipase-producing bacterium is Chromobacterium viscosum, Pseudomonas fluorescens, or Aspergillus niger.
Manufacturing method described in section.