JPH0471496A - Preparation of phospholipid - Google Patents

Abstract

PURPOSE:To prepare the subject phospholipid not containing impurities and useful as an emulsifier for foods, etc., at a low temperature under a mild condition by performing an ester exchange reaction while the reaction system is maintained at a reaction system containing a fine amount of water. CONSTITUTION:When a phospholipid having a single fatty acid composition and an arbitrary fatty acid or a lower alcohol ester thereof are subjected to an ester exchange reaction in the presence of a 1,3-position specific lipase, the reaction system is maintained at a reaction system containing a fine amount of water to provide the objective fatty acid composition. The phospholipid having the single fatty acid composition is preferably a diacylglycerophospholipid.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for producing phospholipids, and more specifically, the present invention relates to a method for producing phospholipids. The present invention relates to a method for producing phospholipids having a free fatty acid composition that can be widely used in pharmaceuticals and magnetic recording media that utilize a unique dispersion effect in magnetic powder.

[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 hydrolyzes ester bonds in triglycerides, diglycerides, monoglycerides, etc., and also hydrolyzes ester bonds at the 5n-1 position of phospholipids.
3-position specific lipase hydrolyzes, and 5n
It has been reported that the ester bond at the -1-2 positions is hydrolyzed by a lipase with no position specificity.

Therefore, studies have been made on the decomposition of phospholipids and their transesterification using this lipase. for example,
Method for transesterification of phosphatidylcholine using polyalkylene gelylcol-modified lipase (JP-A-63-105
686), or when the volume ratio of the organic solvent phase to the aqueous phase is 1=9.
A method of carrying out a transesterification reaction using an enzyme (lipase) capable of transesterifying microbial phospholipids in the range of ~9:l (Japanese Unexamined Patent Publication No. 15093/1993), and a report by Yagi et al. (Journal of Fermentatio
n andBioengineering, Vol.
69. Nal, 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 and to distinguish between specific parts (sn-1 and 5n-2). It was not possible to freely introduce a desired alkyl group into Furthermore, since decomposition reactions occur simultaneously, it is difficult to suppress them.

In addition, by using lipase, it was possible to transesterify polyunsaturated fatty acids at low temperatures without degrading them; Because of this, the reaction equilibrium tilted toward hydrolysis or remained at transesterification.

Moreover, in the transesterification reaction, the decomposition reaction occurs simultaneously, so the recovery rate of phospholipids inevitably decreases.

[Means for Solving the Problems] As a result of intensive research to solve the above problems, the present inventors have found that
The present invention has now been completed.

That is, the present invention combines a phospholipid having a single fatty acid composition and any fatty acid or lower alcohol ester thereof,
1. Provides a method for producing a phospholipid having a desired fatty acid composition, which is characterized in that the transesterification reaction is carried out while maintaining the reaction system in a slightly aqueous reaction system in the presence of a 3-position specific lipase. It is something to do.

The phospholipid having a single fatty acid composition used in the present invention is not particularly limited, and may be obtained by any method. For example, in the presence of lipase or esterase, a diacylglycerophospholipid having a single fatty acid composition is produced by ester synthesis or transesterification of a monofatty acid or its lower alcohol ester and glycerophosphoric acid or its salt or its derivative. Examples include those obtained by a method of producing a phospholipid, or a method of obtaining a phospholipid having a single fatty acid composition by a chemical synthesis method.

Examples of the above-mentioned salts of glycerophosphate include metal salts or ammonium salts of glycerophosphate, 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).

Specific examples of the derivative represented by +1O-CI+□ 2N) include derivatives represented by the following formula.

(1) Examples where X is an alkyl group or alkenyl group having 1 to 24 carbon atoms which may have a substituent HOCHl HO-C)I O I HtCOP OR OH (wherein, P has a substituent and It represents an alkyl group or alkenyl group having 1 to 24 carbon atoms.

(2) Example 0-CH2 in which x is a polyhydric alcohol residue (wherein, group or alkylene oxide polymer residue.) OHOH01l (When X is a glycerin residue) 1 (OCHz +1O-C8O OH011 (When X is a propylene glycol residue) (3) x is a sugar residue Example +1O-C11□ +1O-CII O 11□C-0-P-0-R" OH (In the formula, ρ is a residue such as glucose, fructose, galactose, sucrose, etc.) (4) Examples of alkylene oxide polymer residues +1O-CFI2 +1O-C110 11□COP O(CIIzCHzO), 11H 1(0-CI+□ HOCHOCH3 HzCOP O(CHzCtlO)1.HON In the present invention, fatty acids with a carbon number of 6 ~24 straight chain saturated fatty acids, unsaturated fatty acids, highly unsaturated fatty acids,
Branched fatty acids are used. In addition, as lower alcohol esters of fatty acids, the above fatty acids and straight chain carbon atoms of 1 to 6 carbon atoms can be used.
Ester compounds of alcohols are preferably used.

The lipases having 1- and 3-position specificity used in the present invention include lipases of the genus Rhizopus, genus Mucor, genus Aspergillus, genus Chromobacterium, genus Penicillium, and porcine pancreatic lipase. Furthermore, lipases and esterases that can be used in the production of diacylglycerophospholipids are not limited to enzymes produced by microorganisms, but may be those of animal or plant origin. For example, a lipase with positional specificity at position 1.3 as described above, or a lipase with low positional specificity such as those of the genus Candida, Shutomonas, Streptomyces, and Deotrichum can be used.

Particularly in the present invention, during the production of diacylglycerophospholipids, it is preferable to use both a lipase that has positional specificity for triglycerides and a lipase that does not have specificity for the reaction. In particular, taking advantage of the fact that the monoacylation reaction activity of the 1.3-position specific lipase for glycerophosphoric acid, its salts, or its derivatives is higher than that of non-position-specific lipase, first, the 1.3-position position-specific lipase is 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.

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 anion exchange resins, cation exchange resins, amphoteric ion exchange resins, chelate resins, etc. Resins having porous hydroxyl groups are preferred, and preferred resins include strongly basic anion exchange resins (■ type), glucamine type chelate resins, and the like.

In the present invention, when a phospholipid having a single fatty acid composition and any fatty acid or its lower alcohol ester are transesterified in the presence of a 1,3-position specific lipase, the produced water is Alternatively, it is necessary to remove the lower alcohol from the reaction system and conduct the reaction while maintaining the reaction system as a slightly aqueous reaction system. In the present invention, the slightly aqueous system refers to a system in which the water content in the entire reaction system is more than 0% and 10% or less, preferably more than 0% and 5% or less.

Furthermore, even when producing a diacylglycerophospholipid having a single fatty acid composition, it is preferable to carry out the reaction while removing water or lower alcohol produced from the reaction system.

Methods for removing water or lower alcohols from the system include conducting the reaction under reduced pressure or a stream of inert gas such as nitrogen during the late stage of the reaction or throughout the entire reaction, or by adding molecular sieves or dehydrating agents. For example, how to do this.

In addition, when producing diacylglycerophospholipids having a single fatty acid composition, glycerophosphoric acid or its salts or its derivatives may be reacted with fatty acids or fatty acid esters in powder form, but glycerophosphoric acid or its salts or its derivatives may be reacted with fatty acids or fatty acid esters. The reaction may be carried out as an aqueous solution of the derivative. 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 10% or more, preferably closer 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), 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 as a dispersion medium has a low melting point,
A method using triglycerides having the same fatty acid composition is preferred, but there are no particular restrictions as long as the solvent dissolves and disperses fatty acids and their esters and does not deactivate lipase or esterase.

For example, nonpolar hexane, cyclohexane, benzene,
Toluene etc. and halides such as chloroform and dichloroethane 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 at the beginning of the enzyme reaction, it is preferable to suppress the enzyme deactivation under mild conditions below 35°C.On the contrary, when water and lower alcohols are removed from the reaction system,
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 best to use it with C.

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

Reference example Disodium glycerophosphate hexahydrate (manufactured by Kanto Kagaku ■)
After dissolving 10 g in 5 d of water, 32 g of ethyl oleate (manufactured by Tokyo Kasei Q-1) was stirred and mixed under a nitrogen stream, and then an enzyme derived from Rhizopus javonicus (manufactured by Osaka Seiken, Oliverase 4) was dissolved in 5 d of water.
After adding 100 OIJ of S) immobilized on a porous resin and reacting at 40°C for 12 hours, 50 U of immobilized enzyme derived from Cancida antarctica (manufactured by lJovo) was added, and the reaction was continued under reduced pressure for 24 hours. The reaction was carried out at 50°C for an hour.

After the reaction, 100 d of hexane was added, and the reaction product was filtered off to collect a hexane phase. 50 m of the hexane phase
After washing with water from step 1, 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 4.2 g.

A portion of this was analyzed using high performance liquid chromatography (Llnisil Q Nil□, manufactured by Gascro Industries, Ltd., elution conditions: acetonitrile: ethanol: 10 mM ammonium dihydrogen phosphate solution - 40: 50: 10). The result is phosphatidic acid (PA) 88%
, 12% lysophosphadecic acid (L-PA).

This phospholipid was dissolved in chloroform and eluted with chloroform and methanol = 2:3 using a silica gel column.The phospholipids were collected and 3 g of 98% phosphatidic acid was collected.
I got it.

Example 1 10 g of ethyl laurate was added to 2 g of phosphatidic acid whose constituent fatty acid was only oleic acid obtained in Reference Example,
Enzyme derived from Rhizopus javonicus (Osaka Seiken, Olivase 4S, moisture 7.5%) was added while stirring under a nitrogen stream.
100OU was added and reacted at 50°C for 24 hours.

After the reaction, 100 d of hexane was added, and the reaction product was filtered off to collect a hexane phase. 50 m of the hexane phase
After washing with water from step 1, 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.

A portion of this was analyzed using high performance liquid chromatography (Unisil Q NH2, manufactured by Gascro Industries, Ltd., elution conditions: acetonitrile: ethanol: 10 mM ammonium dihydrogen phosphate solution - 40: 50: 10). The results showed that α-lauroyl-β-oleoyl-glycerol phosphate was 34%, and unreacted dioleoylglycerol phosphate was 66%.

Example 2 In Example 1, in order to examine the effect of the immobilized enzyme, an enzyme derived from Rhizopus japonicus (manufactured by Osaka Seiken, Saiken 1) was used.
An immobilized enzyme (00) immobilized on a porous anionic resin (
The reaction was carried out under the same conditions except that 1000 U (10% water content) was used, and the post-treatment and analysis were carried out in the same manner as in Example 1.

The results showed that α-lauroyl-β-oleoylglycerol phosphate was 63%, and unreacted dioleoylglycerol phosphate was 37%.

Example 3 In Example 2, 13g of ethyl stearate was used instead of ethyl laurate, and 100ml of hexane was used as the solvent.
The reaction was carried out under the same conditions except that f was used, and the post-treatment and analysis were carried out in the same manner as in Example 2.

The results showed that α-stearoyl-β-oleoylglycerol phosphate was 47%, and unreacted dioleoylglycerol phosphate was 53%.

Comparative Example In Example 1, Rhizopus javonicus-derived enzyme (Oliverase 4S, manufactured by Osaka Seiken) was replaced with Cansida.
Enzyme derived from cylindranthe (polysaccharide industry, lipase?'l
Y) The reaction was carried out under the same conditions except that 2000 U was used. After the reaction, post-treatment and analysis were performed in the same manner as in Example 1.

The results showed that α-lauroyl-β-oleoylglycerol phosphate was 3.9%, α-oleoyl β-lauroyl-glycerol phosphate was 1.9%, and α,β-dilauroylglycerol phosphate M was 0.9%. 6%, unreacted dioleoylglycerol phosphate 44.4%, β-oleoylglycerol phosphate 47.7%, other lysophospholipids 1.5
%Met.

〔Effect of the invention〕

By the method of the present invention, it has become possible to produce impurity-free phospholipids having the desired fatty acid composition at low temperatures and under mild conditions. Therefore, it has become easy to arbitrarily obtain phospholipids into which highly unsaturated alkyl groups have been introduced, and to obtain phospholipids 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 dispersants in magnetic recording media, etc., but by manually producing phospholipids that are not naturally occurring or have a free fatty acid composition, it is possible to improve purity, high temperature, and wide range pH. It has now become possible to freely obtain phospholipids with high emulsion stability.

Claims (1)

[Claims] 1. When carrying out a transesterification reaction between a phospholipid having a single fatty acid composition and any fatty acid or its lower alcohol ester in the presence of a 1,3-position specific lipase, the reaction system A method for producing a phospholipid having a desired fatty acid composition, the method comprising performing the reaction while maintaining a slightly aqueous reaction system. 2. In the presence of lipase or esterase, a diacylglycerophospholipid having a single fatty acid composition is produced by ester synthesis or transesterification of a single fatty acid or its lower alcohol ester and glycerophosphoric acid or its salt or its derivative. After production, this diacylglycerophospholipid with a single fatty acid composition and any fatty acid or its lower alcohol ester are combined in the presence of a 1,3-position specific lipase to keep the water content of the reaction system at a slight level. A method for producing a glycerophospholipid having a desired fatty acid composition, the method comprising carrying out a transesterification reaction. 3. The production method according to claim 2, wherein both a lipase with triglyceride position specificity and a lipase without specificity are used as lipases when producing diacylglycerophospholipids having a single fatty acid composition. 4. The production method according to claim 2 or 3, characterized in that during the production of diacylglycerophospholipids having a single fatty acid composition, the reaction is carried out while removing generated water or lower alcohol from the reaction system. 5. The production method according to any one of claims 1 to 4, characterized in that the lipase used is a lipase immobilized on a carrier insoluble in water and organic solvents.