JPH07268382A - Production of fats and oils containing long-chain highly unsaturated fatty acid - Google Patents

Abstract

PURPOSE:To efficiently obtain the subject fats and oils without carrying out the deacidifying treatment by hydrolyzing the fats and oils with plural kinds of lipases and removing the liberated fatty acids other than long-chain highly unsaturated fatty acids and glycerol. CONSTITUTION:This method for producing fats and oils is to hydrolyze the fats and oils with two or more kinds of lipases such as lipases derived from the genera Rhizops, Chromobacterium, Mucor, Aspergillus, Achromobacter, Alcaligenes, Candida and Pseudomonas and remove the liberated fatty acids other than long-chain highly unsaturated fatty acids and glycerol.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a long-chain highly unsaturated fatty acid-containing oil or fat by hydrolyzing an oil or fat, particularly by hydrolyzing an oil or fat containing a small amount of a long-chain highly unsaturated fatty acid with an enzyme, The present invention relates to a method for producing fats and oils containing a large amount of long-chain highly unsaturated fatty acids.

[0002]

2. Description of the Related Art Recently, long-chain polyunsaturated fatty acids have attracted attention for their physiological activity and pharmacological effect on humans, and their utilization has been actively studied. Enzyme treatment with lipase has been mainly used for the preparation of this high-chain long-chain highly unsaturated fatty acid product. For example, it is prepared through treatments such as esterification of free fatty acid docosahexaenoic acid (DHA) and hydrolysis of fish oil triglyceride.

However, in esterification of the free fatty acid derivative DHA, the free fatty acid is liable to be decomposed, and many conditions need to be examined for practical use. Also,
Even when hydrolyzing fats and oils with lipase, when one type of enzyme is used, the hydrolysis of fats and oils containing a small amount of long-chain polyunsaturated fatty acid depends on the characteristics of the enzyme, and the reaction occurs when a predetermined concentration is reached. In order to reach the equilibrium, it is necessary to remove the produced fatty acid once and perform the enzyme treatment again, and there is a problem that the work is complicated.

[0004]

An object of the present invention is to provide a method for producing a fat or oil containing a large amount of long-chain polyunsaturated fatty acid, while reducing deoxidation treatment which is complicated in operation.

[0005]

As a result of earnest research in view of the above problems, the inventors of the present invention have found that lipases obtained from various biological species have different hydrolyzing abilities for fats and oils containing saturated or unsaturated fatty acids. For example, lipase derived from Rhizopus genus has a lower hydrolyzing power than lipase derived from Candida genus, but C18: 0, C
Regarding 20: 4 (or 22: 1) and C22: 5, the lipase derived from the genus Candida has a stronger action, and the lipase derived from the genus Chromobacterium contains C18: 4, C20: 4 (or
22: 1), C22: 4, and C22: 5 are found to have strong effects, and by utilizing their characteristics, if a plurality of types of lipases are combined to act on a substrate, deoxidation treatment is not performed. It was found that oils and fats containing a large amount of long-chain polyunsaturated fatty acids can be produced more than when one type of lipase is used, and the present invention has been completed.

That is, the present invention hydrolyzes fats and oils using two or more kinds of lipases to remove fatty acids other than the released long-chain highly unsaturated fatty acids and glycerin. It is a method for producing a saturated fatty acid-containing fat or oil. Hereinafter, the present invention will be described in detail. [1] Fats and Oils The fats and oils used in the present invention contain long-chain highly unsaturated fatty acids, and specifically, fish fat, whale fat, krill oil,
Marine chlorella oil and mixed fats and oils containing these fats and oils,
A conjugated isomerized oil or a partially hydrogenated oil can be used,
Particularly, it is preferable to use fish oil from the viewpoint of the content of long-chain highly unsaturated fatty acid. These fats and oils usually contain 15 to 55% by weight of long-chain highly unsaturated fatty acids.

In the present invention, the long-chain highly unsaturated fatty acid means a fatty acid having 18 or more carbon atoms per molecule and 3 or more double bonds. Less than
Abbreviated as PUFA. [2] Lipase Examples of the lipase used in the present invention include lipases derived from the genus Rhizopus, lipases derived from the genus Chromobacterium, lipases derived from the genus Mucor, lipases derived from the genus Aspergillus, lipases derived from the genus Achromobacter, and genus Alcaligenes. Lipases, lipases derived from the genus Candida, lipases derived from the genus Pseudomonas, and the like.

All of these lipases are commercially available. For example, lipases derived from the genus Rhizopus include lipase A-10 (manufactured by Nagase Biochemical), lipase B-4 (manufactured by Nagase Biochemical) and R lipase (manufactured by Boehringer Mannheim Yamauchi), lipase derived from the genus Candida. As lipase OF (manufactured by Meito Sangyo Co., Ltd.), lipase derived from genus Mucor (manufactured by Novo), lipase derived from Pseudomonas (manufactured by Toyobo), and lipase derived from Chromobacterium. Examples thereof include Chromobacterium lipase (manufactured by Bio Catalyst).

In the present invention, two or more of the above lipases are used in combination. The combination includes a lipase derived from the genus Candida (particularly lipase OF) and a lipase derived from the genus Rhizopus (particularly lipase A-10), a lipase derived from the genus Candida (particularly lipase OF) and a chromobacterium genus With lipase (especially chromobacterium lipase), and lipase derived from the genus Rhizopus (especially lipase A-1)
0) and a chromobacterium-derived lipase (among others, chromobacterium lipase) are preferred, and in particular, a lipase derived from the genus Candida (among others, lipase OF) and a lipase derived from the genus Rhizopus (among others, lipase A-10) Combinations are preferred. The mixing ratio of them depends on the combination of lipases, but is about 1: 2 to 1: 3 by weight. [3] Method for producing long-chain highly unsaturated fatty acid-containing glyceride An example of a method for producing a long-chain highly unsaturated fatty acid-containing oil and fat using the above-mentioned oil and lipase will be described.

The lipase is usually used by adding it to a solvent in which its activity can be sufficiently exhibited. As such a solvent, buffer solution, distilled water, well water or the like can be used. As the buffer solution, a phosphate buffer solution or an acetate buffer solution is preferable, and its concentration is preferably 1 to 50 mM and pH is 5.5 to 8.0. The amount of lipase used is determined by the weight ratio with respect to the amount of fats and oils, and 0.01 to 10% is preferable for each lipase, and especially 0.
1-2% is preferable.

Next, fats and oils are added to the above lipase-containing solvent. The amount of fat or oil added is preferably 10 to 400% by weight, and particularly preferably 50 to 100% by weight, based on the solvent. The reaction solution prepared as described above is stirred at preferably 20 to 60 ° C, particularly preferably 28 to 45 ° C for about 3 to 24 hours. It is not necessary to remove the fatty acids formed during the reaction.

After completion of the reaction, the produced fatty acid other than PUFA, glycerin, or the enzyme and salt after the reaction may be removed by a conventional method such as an alkaline deoxidation method. In this reaction, the ester bond between PUFA and glycerin in the constituent fatty acids of fats and oils is less likely to be hydrolyzed by lipase, and the decomposition rate is about 30-66% .However, fatty acids other than PUFA in the constituent fatty acids of fats and oils and glycerin Since the ester bond with and is hydrolyzed by lipase at a decomposition rate of about 59 to 90%, removing the decomposed fatty acids and glycerin results in a high concentration of PUFA in the oil and fat, resulting in a large amount of PUFA. It is possible to produce contained oils and fats (glycerides).

[0013]

The present invention will be described in more detail below with reference to examples, but these examples do not limit the scope of the present invention. (Example 1) Fish oil (docosahexaenoic acid (DHA) content: 2)
10% of well water containing 50 mg of lipase OF (manufactured by Meito Sangyo) and 150 mg of lipase A-10 (manufactured by Nagase Biochemical) in 5 g
Was added and reacted at 37 ° C. for 15 hours while stirring. After completion of the reaction, fatty acid, glycerin, enzyme and salts were removed by alkaline deoxidation method to obtain PUFA glyceride.

The yield of PUFA glyceride was 1.6 g, and the DHA content in the constituent fatty acids was 56%. (Comparative Example 1) A PUFA glyceride was produced in the same manner as in Example 1, except that Lipase A-10 was not used. The DHA content in the constituent fatty acids was 52%. (Comparative Example 2) A PUFA glyceride was produced in the same manner as in Example 1 except that lipase OF was not used. The DHA content in the constituent fatty acids was 43%.

Example 2 Fish oil refined oil (DHA content: 29)
%) 5 g, and 10 ml of well water containing 100 mg of lipase OF (manufactured by Meito Sangyo Co., Ltd.) and 150 mg of lipase A-10 (manufactured by Nagase Biochemical Co., Ltd.) was added to produce a PUFA glyceride in the same manner as in Example 1. The yield of PUFA glyceride is 1.4g, DHA in the constituent fatty acids
The content was 57%.

(Comparative Example 3) In Example 2, the lipase
A PUFA glyceride was produced in the same manner except that A-10 was not used. The DHA content in the constituent fatty acids was 51%. (Example 3) 10 mM phosphate buffer solution (pH) containing 50 mg of lipase OF (manufactured by Meito Sangyo Co., Ltd.) and 150 mg of lipase A-10 (manufactured by Nagase Biochemical Co., Ltd.) in 5 g of refined fish oil (DHA content: 29%) = 7.0) 10m
Then, PUFA glyceride was produced in the same manner as in Example 1.

The yield of PUFA glyceride was 1.5 g, and the DHA content in the constituent fatty acids was 58%. (Comparative Example 4) A PUFA glyceride was produced in the same manner as in Example 3, except that Lipase A-10 was not used. The DHA content in the constituent fatty acids was 53%. (Comparative Example 5) A PUFA glyceride was produced in the same manner as in Example 3, except that lipase OF was not used. The DHA content in the constituent fatty acids was 43%.

(Example 4) Fish oil refined oil (DHA content: 29)
%) 5 g, lipase OF (Meito Sangyo Co., Ltd.) 25 mg and chromobacterium lipase (Biocatalyst Co., Ltd.) 100 m
PUFA glyceride was produced in the same manner as in Example 1 by adding 10 ml of 10 mM phosphate buffer (pH = 7.0) containing g. The yield of PUFA glyceride was 1.5 g, and the DHA content in the constituent fatty acids was 51%.

Comparative Example 6 PUFA was prepared in the same manner as in Example 4, except that the chromobacterium lipase was not used.
A glyceride was produced. DHA content in constituent fatty acids is 48%
Met. (Comparative Example 7) A PUFA glyceride was produced in the same manner as in Example 4, except that lipase OF was not used. The content of DHA in the constituent fatty acids was 36%.

(Example 5) 5 g of fish oil (DHA content: 29%)
50 mg of Chromobacterium lipase (Biocatalyst) and lipase A-10 (Nagase Biochemical) 15
10 ml of well water containing 0 mg was added, and PUFA was added in the same manner as in Example 1.
A glyceride was produced. PUFA glyceride yield 1.7
g, the DHA content in the constituent fatty acids was 49%.

(Comparative Example 8) A PUFA glyceride was produced in the same manner as in Example 5, except that lipase A-10 was not used. The content of DHA in the constituent fatty acids was 36%. (Comparative Example 9) A PUFA glyceride was produced in the same manner as in Example 5, except that the chromobacterium lipase was not used. The DHA content in the constituent fatty acids was 43%.

As is clear from the above results, when two or more lipases are used in combination as in the present invention,
A synergistic effect is observed in the hydrolysis reaction, and fats and oils containing a large amount of PUFA can be obtained more efficiently than when lipase is used alone. (Example 6) Lipase O was added to 5 g of fish oil (DHA content: 27%) to obtain fats and oils having a DHA content of 50% or more in the constituent fatty acids.
10 ml of well water containing 50 mg of F (manufactured by Meito Sangyo Co., Ltd.) and 150 mg of lipase A-10 (manufactured by Nagase Biochemical Co., Ltd.) was added, and PUFA glyceride was manufactured in the same manner as in Example 1. DH in constituent fatty acids
The A content was 52%.

(Comparative Example 10) Using lipase alone,
The DHA content in the constituent fatty acids was 50 from the same fish oil as in Example 6.
In order to obtain the fats and oils of more than 100%, it carried out as follows. A PUFA glyceride was produced in the same manner as in Example 6 except that lipase A-10 was not used. The DHA content in the constituent fatty acids was 46%. Next, to the 2 g of fats and oils having a DHA content of 46% by the primary treatment, lipase O was added again.
5 ml of well water containing 20 mg of F (manufactured by Meito Sangyo Co., Ltd.) was added and reacted for 10 hours while stirring. After completion of the reaction, the DHA content in the constituent fatty acids of the oil / fat obtained by the same deoxidation treatment was 51%.

As is clear from Example 6 and Comparative Example 10, the use of a plurality of enzymes in combination is less complicated than the use of the enzymes alone, and the desired high PUFA level is achieved. Content fats and oils can be easily prepared. Reference Example 1 Various enzymes were tested by the method described below in order to investigate the decomposition characteristics of fatty acids constituting fats and oils.

Phosphate buffer solution (pH) containing 50 mg of lipase (lipase OF, lipase A-10, chromobacterium lipase, each alone) in 5 g of fish oil (DHA content: 29%)
7.0) 10 ml was added and reacted for 15 hours while stirring. After completion of the reaction, fatty acid, glycerin, enzyme and salts were removed by alkaline deoxidation method to obtain PUFA glyceride. In order to determine the absolute amount of fatty acids in oils and fats, first, the PU produced as a result of the hydrolysis reaction of the fish oil refined oil and lipase
Each FA glyceride was dissolved in ether and then esterified with methanol in an alkaline atmosphere to obtain a fatty acid ester. This was analyzed by gas chromatography to determine the fatty acid constituent ratio (F, F '). Generated
For the PUFA glyceride, the yield (Y) is obtained, the fatty acid constituent ratio (F) is multiplied by 90% of the yield (Y), and the absolute value is corrected by the molecular weight (M) of the constituent fatty acid (Formula 1). . On the other hand, since the raw material oil and fat is 5 g, 4.5 g, which is 90% thereof, was multiplied by the fatty acid constituent ratio (F ′) of the raw material and corrected by the molecular weight (M) of the constituent fatty acid to obtain an absolute amount (Equation 2). The reason for multiplying each by 90% is that the ratio of the constituent glycerin in the fats and oils is about 10%.

[F (%) × 0.9 Y (g)] / M × 1000 = D (mmol) ... Equation 1 [F ′ (%) × 4.5 (g)] / M × 1000 = C (mmol) Mathematical formula 2 The absolute amount (D) of the fatty acid remaining in the fat as a result of the reaction is divided by the absolute amount (C) of the fatty acid constituting the fat in the raw material, and multiplied by 100 to obtain the value from 100. The subtracted value was taken as the fatty acid decomposition rate (Formula 3).

100-[(D / C) × 100] = E (%) ... Equation 3 Table 1 shows the fatty acid decomposition rate in fats and oils for each enzyme. In this experiment, lipase was used as the lipase.
When OF is used, DH in the constituent fatty acids of the resulting fats and oils
The A content is 51.5% and the yield is 37.0%, and the lipase A-1
When using 0, the DHA content is 39.5% and the yield is 47.3%
And when using Chromobacterium lipase, the DHA content was 35.6% and the yield was 34.0%.

[0028]

[Table 1]

As is clear from Table 1, each lipase has different hydrolytic ability to various constituent fatty acids. Reference Example 2 In order to investigate the influence of various enzyme concentrations on the substrate, an experiment was conducted by the method described below.

Phosphate buffer solution (pH 7) containing 10, 20, 40, 100 and 200 mg of lipase (lipase OF, lipase A-10, chromobacterium lipase, each alone) in 5 g of fish oil (DHA content: 29%) .0) Add 10 ml and stir
Reacted for 15 hours. After completion of the reaction, fatty acid, glycerin, enzyme and salts were removed by alkaline deoxidation method to obtain PUFA glyceride. Table 2 shows the DHA content in the constituent fatty acids of the obtained fats and oils at each concentration of each lipase.

[0031]

[Table 2]

As is clear from Table 2, the effect of lipase was 0.2 to 2.0% by weight in lipase OF.
The concentration of A-10 was 0.4 to 2.0% by weight, and that of chromobacterium lipase was 0.1 to 0.4% by weight, regardless of the concentration, and it did not affect the constituent DHA content in the produced oil and fat.

[0033]

According to the present invention, fats and oils containing a large amount of long-chain highly unsaturated fatty acids can be efficiently produced without performing deoxidation treatment.

Claims (4)

[Claims] 1. A long-chain highly unsaturated fatty acid-containing oil or fat, characterized by hydrolyzing an oil or fat with two or more lipases to remove fatty acids other than the released long-chain highly unsaturated fatty acids and glycerin. Manufacturing method. 2. The above-mentioned two or more lipases are lipases derived from the genus Rhizopus, lipases derived from the genus Chromobacterium, lipases derived from the genus Mucor, lipases derived from the genus Aspergillus, lipases derived from the genus Achromobacter, and genus Alcaligenes. 2. The method according to claim 1, wherein the method is two or more selected from the group consisting of the lipase of No. 1, a lipase of the genus Candida and a lipase of the genus Pseudomonas. 3. The two or more lipases are a combination of a lipase derived from the genus Candida and a lipase derived from the genus Rhizopus, a combination of a lipase derived from the genus Candida and a lipase derived from the genus Chromobacterium, or the genus Rhizopus. The method according to claim 1, which is a combination of a lipase derived from a bacterium and a lipase derived from a genus Chromobacterium. 4. The oil and fat is at least one selected from the group consisting of fish oil, whale oil, krill oil and marine chlorella oil.
The method according to claim 1, which is a seed or a conjugated isomerized oil or a partially hydrogenated oil of these oils and fats.