JPH0471495A - Preparation of diacylglycerophospholipid - Google Patents

Abstract

PURPOSE:To prepare the subject lipid useful as a food emulsifier under low temperature and mild conditions in an impurity-free state by subjecting glycerophosphoric acid and a fatty acid to an esterification reaction using a specific lipase. CONSTITUTION:Glycerophosphoric acid or a salt or derivative thereof and a fatty acid are subjected to an esterification reaction employing both a lipase having a position specificity for triglycerides and a lipase free of the specificity to prepare the objective lipid. The ester of the fatty acid is preferably used instead of the fatty acid and the lipases immobilized on a carrier insoluble in water and organic solvents are also preferably employed as the lipases. Water or a lower alcohol is preferably removed from the reaction system.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing diacylglycerophospholipids,
Specifically, it relates to a method for producing diacylglycerophospholipids by acting on glycerophosphoric acid, a salt thereof, or a derivative thereof, and a fatty acid or a fatty acid ester with both a lipase that has positional specificity for triglycerides and a lipase that does not have specificity. be.

[Prior art and problems to be solved by the invention] Conventionally, methods for producing phospholipids include the acid anhydride method from glycerophosphoric acid and fatty acids such as glycerophosphorylcholine;
A method of producing phospholipids such as phosphatidylcholine by an acid chloride method or the like is known.

However, such a synthesis reaction based on a chemical reaction of phospholipids has the problem that fatty acids are degraded by a condensing agent and the like, and the reaction method is complicated and expensive.

In addition, as a method for obtaining phospholipids of natural animal and plant origin,
Extraction methods such as solvent fractionation and silicic acid column separation are commonly used, but in both cases it is difficult to separate pigments and mixed glycolipids, and a large amount of different types of solvents are required.

Furthermore, the fatty acids constituting the acyl groups of the phospholipids obtained in this way have a certain molecular weight distribution depending on their origin, and it is virtually impossible to obtain a phospholipid with a single fatty acid composition or with a fatty acid composition that meets the requirements. It was impossible.

On the other hand, lipase is used to hydrolyze ester bonds in triglycerides, diglycerides, monoglycerides, etc., and also to hydrolyze ester bonds at the sn-1 position of phospholipids.
3-position specific lipase hydrolyzes, and 5n
It has been reported that the ester bond at the 4-2 position is hydrolyzed by lipase, which has no position specificity. Therefore, studies have been made on the decomposition of phospholipids and their transesterification using this lipase. For example, a method for transesterification of phosphatidylcholine using polyalkylene glycol-modified lipase (Japanese Patent Application Laid-Open No. 63-1056
No. 86), or when the volume ratio of the organic solvent phase and the aqueous phase is 1:9 to
A method of carrying out a transesterification reaction using an enzyme (lipase) having the ability to transesterify microbial phospholipids in the range of 9:1 (Japanese Patent Application Laid-open No. 2-35093), and a report by Yagi et al. (Journal of Fermentation)
and Bi.

engineering, Vol. 69. Nα1
, 23-25.1990). As described above, the alkyl groups of phospholipids can be modified to some extent by transesterification. However, when natural phospholipids are used as raw materials, it is an equilibrium reaction with the composition of the alkyl groups, so it is necessary to make the composition of the alkyl groups uniform, or to
It was not possible to freely introduce a desired alkyl group into 1 and 5n-2). Furthermore, since decomposition reactions occur simultaneously, it is difficult to suppress them.

[Means to solve the problem]

As a result of intensive research by the present inventors to solve the above problems,
The present invention has now been completed.

That is, the present invention provides diacylglycerol, which is characterized by subjecting glycerophosphoric acid, salts thereof, or derivatives thereof and fatty acids to an ester synthesis reaction using both a lipase that has positional specificity for triglycerides and a lipase that does not have specificity. A method for producing a lipid, and a diacyl product characterized by transesterifying glycerophosphoric acid or its salts or derivatives thereof and a fatty acid ester using both a lipase that has positional specificity for triglycerides and a lipase that does not have specificity. A method for producing glycerophospholipids is provided.

Examples of the salt of glycerophosphoric acid used in the present invention include metal salts or ammonium salts of glycerophosphoric acid, such as disodium glycerophosphate and calcium glycerophosphate. In addition, examples of derivatives of glycerophosphoric acid include glycerophosphorylcholine and glycerophosphorylethanolamine, as well as derivatives represented by the following formula (1).

0-C1h H (wherein, X represents an alkyl group or alkenyl group having 1 to 24 carbon atoms which may have a substituent, a polyhydric alcohol residue, a sugar residue, or an alkylene oxide polymer residue. ) Specific examples of the derivative represented by formula (1) include derivatives represented by the following 2.

(1) Example HO-C)I in which x is an alkyl group or alkenyl group having 1 to 24 carbon atoms which may have a substituent.

H (wherein R represents an alkyl group or alkenyl group having 1 to 24 carbon atoms which may have a substituent.

(2) Example where X is a polyhydric alcohol residue HO-CH2 HO-CH.

HO-CHOH2COP 0CH2CHCH2 Kano OH0H (When X is a glycerin residue) HO-C1l□ )to-C1l O l H2C-0-P-OCH2CHCH3 0HOH (When x is a propylene glycol residue) (3) x is sugar Example 1 (0-Cut HO-CHO HzCOF OR' OH (wherein, P is a residue of glucose, fructose, galactose, sucrose, etc.) (4) where x is a residue of an alkylene oxide polymer Examples of groups include OH)10-CH2OH In the present invention, fatty acids include linear saturated fatty acids having about 6 to 24 carbon atoms, unsaturated fatty acids, highly unsaturated fatty acids,
Branched fatty acids are used. Further, as the fatty acid ester, a lower alcohol ester of a fatty acid is preferable, and an ester compound of the above-mentioned fatty acid and a straight-chain alcohol having 1 to 6 carbon atoms is particularly preferably used.

The lipase that can be used in the production of the glyc-heterophospholipid of the present invention is not limited to enzymes produced by microorganisms, but may be derived from animals or plants. For example, lipases with 1°3 position specificity include those of the genus Rhizopus, Mucor, Aspergillus, Chromobacterium, Penicillium, and porcine pancreatic lipase, while lipases without position specificity include those of the genus Candida. , Shutomonas genus, Streptomyces genus, Deotrichum genus, etc.

In the present invention, both a lipase that has position specificity for triglycerides and a lipase that does not have specificity are used for the reaction. In particular, by utilizing the fact that the monoacylation reaction activity of a lipase specific to the 1,3-position is higher than that of a lipase without position-specificity for glycerophosphoric acid, its salts, or its derivatives, After performing monoacylation to make the reaction system into a homogeneous phase, diacylglycerophospholipids can be produced more efficiently by performing diacylation under reduced pressure using a lipase without position specificity.

The ratio of lipase having positional specificity to triglyceride used in the present invention and lipase having no specificity is preferably in the range of former:latter=1:1 to 1:5, based on the lipase titer.

In the present invention, lipase immobilized on a carrier insoluble in water and organic solvents can also be used. The carrier used is preferably one that provides an immobilized enzyme that maintains high activity even under dehydration conditions, such as cation exchange resins, anion exchange resins, amphoteric ion exchange resins, chelate resins, and the like. In particular, resins having porous hydroxyl groups are preferred, and preferred resins include strongly basic anion exchange resins (■ type), glucamine type chelate resins, and the like.

As for the reaction system of the present invention, it is preferable to synthesize glycerineptinophospholipids by removing water or a lower alcohol, which is one of the products, from the reaction system during the entire reaction or during the reaction. Methods for removing water or lower alcohols from the system include conducting the reaction under reduced pressure or under a stream of inert gas such as nitrogen during the late stage of the reaction or throughout the entire reaction;
Examples include reactions by adding molecular sieves and dehydrating agents.

Further, glycerophosphoric acid, a salt thereof, or a derivative thereof may be reacted with a fatty acid or a fatty acid ester in powder form, or may be reacted as an aqueous solution of glycerophosphoric acid, a salt thereof, or a derivative thereof. Alternatively, the reaction may be carried out using a solvent that dissolves the fatty acid or its ester. In addition, when carrying out the reaction using a solvent, it is necessary to take a method such as removing the solvent under reduced pressure during the reaction. More specifically, the pH of the aqueous solution of glycerophosphoric acid, its salt, or its derivative is 2 to 10, preferably 5 to 8, and the concentration is preferably 10% or more, preferably close to a saturated solution. The reaction ratio of glycerophosphoric acid or its salt or its derivative and fatty acid or its ester should be twice or more in molar ratio, but when fatty acid or its ester is used as a dispersion medium or in order to speed up the reaction, it is sufficient. There is no problem in increasing the ratio. In addition, when the produced glycerophospholipid is recovered by solvent fractionation (acetone precipitation i#), it is preferable to suppress the amount of fatty acid or its ester used as a dispersion medium to about 10 times. In addition, when the fatty acid species used in the reaction has a low melting point as a dispersion medium, it is preferable to use triglycerides with the same fatty acid composition;
There are no particular restrictions as long as the solvent does not deactivate lipase. For example, nonpolar hexane, cyclohexane, benzene, toluene, etc., and halides such as chloroform, dichloroethane, etc. can also be used.

The reaction temperature is not particularly limited, but is 20 to 100°.
Any temperature that does not deactivate the enzyme is sufficient.

If a large amount of water is present in the initial stage of the enzyme reaction, it is preferable to suppress enzyme deactivation under mild conditions below 35°C; conversely, water and lower alcohols should be removed from the reaction system (in some cases,
It is desirable to carry out the reaction at as high a temperature as possible. In addition, when the fatty acid or its ester introduced into the glycerophospholipid is a highly unsaturated fatty acid, the reaction temperature is 70'C or less,
It is also preferable to add an antioxidant (for example, tocopherol) as much as possible. - For boat fishing, the temperature is 20-50'C when using free enzymes or bacterial powder, and 40-100'C when using immobilized enzymes or heat-resistant enzymes.
It is good to use it in

(Examples) Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Disodium glycerophosphate hexahydrate (manufactured by Kanto Kagaku ■)
After dissolving 10g in 5 parts of water, add oleic acid (manufactured by Tokyo Kasei)
After stirring and mixing 30 g under a nitrogen stream, add 100 g of enzyme derived from Rhizopus javonicus (Oliverase 4S, manufactured by Osaka Seiken).
After adding 0 U and reacting at 40°C for 12 hours, an enzyme derived from Cancida cylindrasse (Polysaccharide Sangyo, Lipase MY
) 2000 U was added and the reaction was incubated under reduced pressure for 24 h.
The reaction was carried out at 0°C.

After the reaction, 100 aZ of hexane was added, and the reaction product was filtered off to collect a hexane phase. After washing the hexane phase with a mixed solvent of 30 mZ of ethanol and 20 m7 of water, the hexane phase was removed under reduced pressure.

In order to remove unreacted fatty acids from this reaction product, it was stirred in cold acetone and then centrifuged to collect the precipitate. The product recovered was 2.7g.

A portion of this was taken and analyzed using high performance liquid chromatography (1lnisN Q N11z, manufactured by Gascro Industries, Ltd., elution conditions: acetonitrile:ethanol:10mM ammonium dihydrogen phosphate solution = 40:50:10). The result is phosphatidic acid (hereinafter referred to as PA) 11
%, lysophosphatidic acid (L-PA) 89%.

Example 2 The enzyme and reaction method were the same as in Example 1, except that 32 g of oleic acid ethyl ester was used instead of oleic acid.

The results were: recovered product 3.1 g, PA 23%, L-P 87
It was 7%.

Example 3.4 In order to examine the effect of immobilized enzymes, 1000 U of immobilized enzyme, which is an enzyme derived from Rhizopus javonicus (Osaka Saiken, Saiken 100) immobilized on porous anion resin, and lipase MY were immobilized on porous resin. Immobilized immobilized enzyme 2000
Using U, the substrates were glycerophosphate disodium salt and oleic acid (Example 3) or its ethyl ester (
Example 4) was used to carry out the reaction in the same manner as in Example 1.

The results are shown in Table 1 together with the results of Examples 1 and 2.

Comparative Example In Example 1, an enzyme derived from Cancida cylindrasose (Disaccharide Sangyo, Lipase MY) 200
The reaction was carried out under the same conditions as in Example 1 except that only 0 U was used, and the post-treatment was carried out in the same manner as in Example 1.

The results are shown in Table 1, 0.2 g of recovered product, PAO
%, L-PA 100%.

Table [Effects of the Invention] The method of the present invention has made it possible to produce impurity-free diacylglycerophospholipids at low temperatures and under mild conditions. Therefore, it has become easy to arbitrarily obtain diacylglycerophospholipids into which highly unsaturated alkyl groups have been introduced, and to obtain diacylglycerophospholipids having a certain alkyl composition.

As a result of the above, when used as an emulsifier in foods, cosmetics, etc., the concentration and range of use have been limited due to impurities such as coloring, odor, and glycolipids, but now we are no longer bound by these restrictions. Now available for use. In addition, phospholipids have been used as medicines and skin permeation agents through liposomes, etc., but with the present invention, it is possible to obtain phospholipids that are not naturally occurring or phospholipids with free fatty acid composition, and the stability can be adjusted. It has now become possible to freely obtain diacylglycerophospholipids with high skin permeation activity.

Claims (1)

[Claims] 1. Glycerophosphoric acid or its salts or its derivatives and fatty acids are subjected to an ester synthesis reaction using both a lipase that has positional specificity for triglycerides and a lipase that does not have specificity. Method 2 for producing diacylglycerophospholipids, characterized by transesterifying glycerophosphoric acid or its salts or its derivatives and a fatty acid ester using both a lipase that has positional specificity for triglycerides and a lipase that does not have specificity. A method for producing diacylglycerophospholipids. 3. The production method according to claim 1 or 2, characterized in that water or lower alcohol as a reaction product is removed from the reaction system. 4. The production method according to any one of claims 1 to 3, characterized in that the lipase is a lipase immobilized on a carrier insoluble in water and organic solvents.