JP2801024B2 - Method for producing phospholipid hydrolyzate - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for hydrolyzing phospholipids by the action of phospholipase, and particularly to a method for producing a hydrolyzed phospholipid hydrolyzate by a simple operation. It relates to a good manufacturing method. More specifically, the present invention relates to a method for producing phosphatidic acid (hereinafter abbreviated as PA) hydrolyzed by a simple operation in a high yield.

[Problems to be solved by conventional technology and invention]

Phospholipids are basic substances of the components of biological membranes, and are one of the lipids having functions that control the basics of life activities such as protection of cell tissues, transmission of information, and control of mass transfer.

In recent years, the phenomenon that vesicles (or liposomes) formed by phospholipids having a bilayer membrane-forming ability have a function of enclosing various functional substances has begun to attract attention in academic and industrial fields. Its application is expected as a drug delivery system).

The present inventors have been continuously studying the use of such high-functional lipids in the food field, but recently, by using PA, which is one of the phospholipids, is excellent in releasability without oil splash. Cooking oil has been successfully completed (Japanese
1-27431).

Further, examples of the use of PA in the industrial field include, for example, improvement of dough physical properties in a baking process (JP-A-58-51853), and production of an emulsifier comprising a complex of PA and Tween (JP-A-62-20483).
No. 8), etc. for the food industry and for pharmaceuticals
No. 105222, No. 5-1582, No. 56-127308, No. 60-255728
No.), use in cosmetics (JP-A-59-27809). Application to chemical products (JP-A-53-108503, JP-A-60-243171) and the like are being studied for use in various industrial fields.

However, since PA itself is contained only in a small amount in lecithin, which is a by-product of oil production, it is extremely difficult to extract it with high purity, and an industrial production method has not yet been established. Therefore, the use of PA is still limited compared to the use of lecithin.

Studies on phospholipase D (hereinafter abbreviated as PL-D), which catalyzes a reaction for producing PA by hydrolyzing phospholipids, have been made for a long time. Further, as a factor for activating PL-D, an organic solvent, a metal ion, a surfactant, and the like have been found.For example, ethyl ether is often used as an organic solvent, and esters such as ethyl acetate, dibutyl ketone, and the like. Ketones have also been found to activate PL-D [M. Kates, Can. J. Biochem. Physiol., Vol. 35, No. 1,
27 (1975)].

However, such a conventional reaction system has many problems for industrial production of PA. For example, ethyl ether has a low flash point and is regulated by the Fire Service Act as a dangerous special flammable substance.In addition, there is a risk that explosion may occur when ethyl ether is distilled off due to accumulation of peroxide. Safety measures are required.

Further, as another method, phospholipase and other water-soluble reaction components are dissolved in water, and a phospholipid that is hardly soluble in water is dissolved in an organic solvent (eg, ether, hexane, etc.),
There was a method in which the desired reaction was caused by mixing the two reaction component solutions obtained with vigorous stirring, but this method requires continuous strong stirring to sufficiently contact the enzyme and the substrate component. (If the stirring is stopped, the reaction will hardly proceed due to separation into two layers of an aqueous solution layer and an organic solvent layer), and even if the stirring is performed, the reaction will be at the liquid-liquid contact interface, resulting in poor reaction efficiency. There was a problem.

In order to solve these problems, use of a surfactant such as triton and sodium dodecyl sulfate has been proposed.
However, this method is also not preferred because these active agents remain in the product. On the other hand, esters and ketones can be used relatively safely compared to ethyl ether, but since the hydrolysis reaction of phospholipids by PL-D proceeds in an emulsified state, the viscosity of the reaction system increases as the reaction progresses. After the end of the reaction
There is a problem that layer separation in the PA recovery process becomes difficult, yield is reduced, and industrial use is largely hindered.

[Means for solving the problem]

Under such circumstances, the present inventors have proposed a method for producing a phospholipid hydrolyzate such as PA by modifying a phospholipid,
By using an organic mixed solvent and water as the reaction solvent, a phospholipase is allowed to act in a two-phase system of water and the organic mixed solvent to carry out the hydrolysis reaction of the phospholipid, thereby obtaining a sufficient reaction rate and after the reaction is completed. The inventors have found that almost all of the modified phospholipid hydrolyzate can be recovered from the organic solvent layer, and have completed the present invention.

That is, the present invention provides, in the hydrolysis of phospholipids by phospholipase, water, a phospholipid, a lower fatty acid ester of a lower alcohol, an organic mixed solvent comprising a mixture of a phospholipid and a water-immiscible organic solvent. An object of the present invention is to provide a method for producing a phospholipid hydrolyzate, which comprises reacting a phospholipase with a reaction mixture system containing a phospholipase to cause a reaction, and then recovering the phospholipid hydrolyzate from an organic solvent layer.

 Hereinafter, the present invention will be described in detail.

Examples of the phospholipid used in the present invention include phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and the like, and a mixture thereof. You may.

The constituent fatty acids of these phospholipids are the same or different, and are saturated or unsaturated fatty acids having 8 to 24 carbon atoms, such as caproic acid, caprylic acid, lauric acid, myristic acid, palmitic acid, stearic acid, and behenic acid. Acid, arachidic acid, palmito oleic acid, oleic acid, linoleic acid, α- and γ-linoleic acid, erucic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid,
And tetracosatetraenoic acid. These phospholipids may be extracts or concentrates from nature or synthetic products, and are not particularly limited.

PL-D is preferable as the phospholipase used in the present invention. PL-D includes enzymes of microbial origin. The enzyme may be separated or extracted from the microbial cells and used as it is or immobilized, or the microbial cells may be used as it is. The amount of the enzyme used is 0.01 to 1000 units per gram of phospholipid, preferably 0.1 to 1 unit.
0 units can be exemplified. One unit of the enzyme activity unit referred to herein indicates an amount of an enzyme that hydrolyzes 1 μmole of phosphatidylcholine per minute. Examples of PL-D derived from microorganisms include PL-D derived from Streptomyces.

Further, in order to maintain the activity of the phospholipase for a long period of time, it can be used in the form of an immobilized enzyme. As a carrier for immobilization, cellulose, dextran,
Polystyrene, polyacrylamide, polyvinyl alcohol, ion exchange resins, magnetic substances, alumina, photocrosslinkable resins, alginates, various gelling agents, and the like are used. In the present invention, the enzyme or the enzyme extract can be adsorbed, ion-bonded, covalently bonded or entrapped on these immobilization carriers and used as a granular, membrane- or sheet-shaped immobilized enzyme for the reaction. .

The organic mixed solvent used in the present invention is a mixture of a lower fatty acid ester of a lower alcohol and an organic solvent that dissolves phospholipids and is immiscible with water.

As the lower fatty acid ester of the lower alcohol, an ester of a primary alcohol having 1 to 6 carbon atoms and a fatty acid having 1 to 6 carbon atoms is preferable, and as constituent fatty acids of the ester, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, In addition to hexanoic acid and the like, branched fatty acids such as isobutyric acid may be used, and unsaturated fatty acids such as acrylic acid may be used. Examples of the lower alcohol constituting the ester include methanol, ethanol, n-propanol, n-butanol, n-pentanol, n-hexanol and the like, and branched alcohols such as isopropanol and the like. Particularly preferred lower fatty acid esters of these lower alcohols include, for example, ethyl acetate, butyl acetate, ethyl propionate, ethyl butyrate and the like.

As the organic solvent which dissolves the phospholipid and is immiscible with water used in the present invention, hydrocarbons having a melting point of 40 ° C. or less, a liquid at the reaction temperature and a solubility in water of 5% or less are preferable, and pentane And alicyclic hydrocarbons such as cycloheptane and aromatic hydrocarbons such as benzene, in addition to aliphatic saturated hydrocarbons such as hexane and octane, and aliphatic unsaturated hydrocarbons such as hexane and decene.
Halogenated hydrocarbons can also be used and have 1 carbon atom.
Examples include chlorination, bromination, iodide and the like of straight-chain or branched alkanes of No. 8 to 8, but chloroform, carbon tetrachloride and methylene chloride are particularly preferred. One or more of these water-immiscible organic solvents can be used.

Regarding the mixing ratio of the lower fatty acid ester of the lower alcohol and the water-immiscible organic solvent that dissolves the phospholipid, when the water-immiscible organic solvent that dissolves the phospholipid is in a large excess, the enzyme activity decreases. On the other hand, if the amount is small, the solvent layer and the aqueous layer are emulsified, and it becomes difficult to separate the product. Therefore, based on 1 part by volume of lower fatty acid esters of lower alcohol, 0.05 to 100 parts by volume, preferably 0.1 to 20 parts by volume, more preferably 1 to 10 parts by volume of a water-immiscible organic solvent for dissolving phospholipids.
The capacitance part is appropriate. In addition, in the reaction system, the ratio of the organic mixed solvent to water is not preferable if the amount of the organic mixed solvent is too small because the reaction system becomes a strong emulsified state, and if the amount is too large, it is economically disadvantageous. On the other hand, 1 to 20 parts by volume, preferably 1.1 to 5 parts by volume of an organic mixed solvent is suitable.

In the method of the present invention, it is preferable to add salts activating phospholipase to the reaction system. Suitable salts for activating phospholipase include highly water-soluble salts such as alkaline earth metal chlorides such as calcium chloride and magnesium chloride, and alkali metal chlorides such as sodium chloride and potassium chloride. It is not limited. These salts have a weight ratio to phospholipid 1
It is appropriate to add 0.01-20, preferably 0.1-10.

In the method of the present invention, the reaction temperature may be selected so as not to deactivate the enzyme. For example, the reaction temperature can be in the range of 5 to 90 ° C, but it is usually preferably 20 to 60 ° C.

Although the reaction time varies depending on the amount of the enzyme used, it is usually preferable to set the reaction to be completed in 2 to 72 hours.

After completion of the reaction, the organic solvent layer and the aqueous layer are separated, and a phospholipid hydrolyzate such as PA is recovered from the organic solvent layer. In the method of the present invention, the phase separation between the organic solvent layer and the aqueous layer is extremely short (5.
Within minutes). As a method for recovering the phospholipid hydrolyzate, a known method can be applied. For example, a method of removing the solvent from the organic solvent layer under reduced pressure, or a method of adding acetone to the organic solvent layer and collecting the precipitate as a precipitate can be used.

Thus, a phospholipid hydrolyzate such as PA modified by phospholipase can be easily obtained in high yield.

〔Example〕

Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples.

Example 1 4.0 g of acetone precipitate of soybean lecithin (trade name: SLP-W-SP manufactured by True Lecithin Industry Co., Ltd.) as a raw material phospholipid was dissolved in a mixed solvent of 20 ml of ethyl acetate and 40 ml of hexane.
As the enzyme, 10 units of Streptomyces genus PL-D (manufactured by Toyo Brewery Co., Ltd.) were used per 1 g of the starting phospholipid. 97.6 mg of the enzyme and calcium chloride were dissolved in 40 ml of a 0.1 M Tris-HCL buffer (pH 8.0), and the mixture was added to a mixed solvent in which the raw material phospholipid was dissolved to start the reaction. The mixture was stirred in a closed vessel at a reaction temperature of 37 ° C. for 30 hours. The reaction solution after completion of the reaction was easily separated by standing still (the time required for the separation was 3 minutes).

The organic solvent layer was taken out and the organic solvent was distilled off by an evaporator to obtain a modified phospholipid. Its composition was determined by liquid chromatography (Lich-rosorb NH2 column, UV detector). Table 1 shows the yield, the PA content in the modified phospholipid, and the time required for phase separation.

Examples 2 to 4 60 ml of an organic mixed solvent was mixed in a volume ratio of ethyl acetate and hexane.
1: 1 (Example 2), 1: 5 (Example 3), 1: 9 (Example 4)
The reaction was carried out in the same manner as in Example 1 except that
The yield, the PA content in the modified phospholipid, and the time required for phase separation were determined. The results are shown in Table 1.

Comparative Example 1 The reaction was carried out in the same manner as in Example 1 except that 60 ml of hexane was used instead of 60 ml of the organic mixed solvent, and the yield, the PA content in the modified phospholipid, and the time required for phase separation were determined. The results are shown in Table 1.

Example 5 20 ml of butyl acetate and heptane 40 as an organic mixed solvent
The reaction was carried out in the same manner as in Example 1 except that ml was used. After completion of the reaction, 60 ml of acetone was added, and PA was recovered by precipitation. After drying, the yield, the PA content in the modified phospholipid, and the time required for layer separation were determined in the same manner as in Example 1. The results are shown in Table 2.

Example 6 100 g of the raw material phospholipid used in Example 1 was extracted at 40 ° C. with 2000 ml of ethanol to obtain a phospholipid extract. The reaction was carried out in the same manner as in Example 1 except that this phospholipid extract was used in place of the starting phospholipid, and the yield, the PA content in the modified phospholipid, and the time required for phase separation were determined. Table 3 shows the results.

Example 7 PL-D used in Example 1 in 1 g of a weakly basic anion exchange resin
2000 units were immobilized by a covalent bond method using glutaraldehyde to obtain an immobilized enzyme of PL-D. The reaction was carried out under the same conditions as in Example 1 except that this immobilized enzyme was used in place of PL-D in Example 1, and the yield, the PA content in the modified phospholipid, and the time required for phase separation were determined. The results are shown in Table 1.

Comparative Example 2 Example 1 except that only 60 ml of ethyl acetate was used as a solvent.
The reaction was carried out in the same manner as described above, however, the reaction system was in a strong emulsified state, and no separation was observed even after 24 hours. When a part of this emulsion was collected and its composition was determined, a slight progress of the reaction was observed.

Table 1 shows the results obtained for the yield, the PA content in the modified phospholipid, and the time required for phase separation.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References Enzyme Handbook (First Edition, Second Printing), Asakura Shoten, pp. 451-452, “Phospho Lipase D,” published March 1, 1983 (58) Survey Field (Int.Cl. ⁶ , DB name) C12P 9/00 CA (STN) REGISTRY (STN)

Claims (5)

(57) [Claims] 1. A method for hydrolyzing phospholipids using phospholipase, comprising an organic mixed solvent comprising a mixture of water, a phospholipid, a lower fatty acid ester of a lower alcohol, and a water-immiscible organic solvent in which the phospholipid is dissolved. A method for producing a phospholipid hydrolyzate, comprising reacting phospholipase on a reaction mixture system to be reacted to cause the reaction, and then recovering the phospholipid hydrolyzate from the organic solvent layer. 2. The method according to claim 1, wherein the phospholipid hydrolyzate is phosphatidic acid. 3. The method according to claim 1, wherein the lower fatty acid ester of the lower alcohol is an ester of a primary alcohol having 1 to 6 carbon atoms and a carboxylic acid having 1 to 6 carbon atoms. 4. The method according to claim 1, wherein the amount of the water-immiscible organic solvent is 0.005 to 100 parts by volume per 1 part by volume of the lower fatty acid ester of the lower alcohol. 5. The method according to claim 1, wherein the water-immiscible solvent is a hydrocarbon or a halogenated hydrocarbon having a melting point of 40 ° C. or lower.