JPH02312551A - Modification of phospholipid - Google Patents

Abstract

PURPOSE:To obtain a modified phospholipid in high purity and yield under mild reaction condition by treating a phospholipid with two specific kinds of enzymes. CONSTITUTION:The objective phospholipid can be produced by treating a phospholipid with (A) an enzyme capable of hydrolyzing a phospholipid into phosphatidic acid and a nitrogen containing base (preferably phospholipase D) and (B) an enzyme capable of hydrolyzing a phospholipid into a diglyceride and a phosphoryl base (preferably phospholipase C, phosphodiesterase or acidic phosphatase). The treatment is preferably hydrolysis.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention is directed to the use of phospholipids (in the present invention, phospholipids refer to phospholipids or/and phospholipid mixtures unless otherwise specified), This invention relates to a method for modifying phospholipids by treating them with an enzyme that hydrolyzes them into acids and nitrogen-containing bases, and an enzyme that hydrolyzes phospholipids into diglycerides and phosphoryl bases.

[Prior art and problems to be solved by the invention] Phospholipids are basic substances of biological membrane components, and have functions that govern the basics of life activities, such as protecting cell tissues, transmitting information, and controlling mass transfer. It is a type of lipid.

In recent years, the phenomenon that vesicles (or liposomes) formed by phospholipids that have the ability to form bilayer membranes have the ability to encapsulate various functional substances has begun to attract academic and industrial attention, and for example, in the pharmaceutical and medical fields, DDS ( Its application as a drug delivery system is expected.

The present inventors have been studying the use of such highly functional lipids in the food field, and recently found that
By using the seed phosphatidic acid (hereinafter abbreviated as PA), we succeeded in creating a cooking oil with excellent mold release properties without oil splatter (Japanese Patent Application Laid-Open No. 1-27431).
.

Furthermore, examples of the use of PA in the industrial field include improving the physical properties of dough in the bread making process (Japanese Patent Application Laid-Open No. 58-51853), and manufacturing an emulsifier consisting of a PA and Zein complex (Japanese Patent Application Laid-Open No. 62-204838). ), etc., in the food industry, and in pharmaceuticals (JP-A No. 54-105222, No. 55-11)
No. 582, No. 56-127308, No. 60-2557
28), cosmetics (Japanese Patent Application Laid-open No. 59-27809), and chemical products (Japanese Patent Application Laid-open Nos. 53-108503 and 60-243171). It is being considered.

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

[Means to solve the problem]

Under the present circumstances, the present inventors have completed the present invention as a result of intensive research to solve the above problems.

That is, the present invention provides a method for treating phospholipids with an enzyme that hydrolyzes phospholipids into phosphatidic acid and nitrogen-containing bases, and an enzyme that hydrolyzes phospholipids into diglycerides and phosphoryl bases. The present invention provides a modification method.

More specifically, when producing PA from phospholipids, an enzyme that hydrolyzes phospholipids into PA and nitrogen-containing bases and an enzyme that hydrolyzes phospholipids into diglycerides and phosphoryl bases are used separately or simultaneously. The present invention provides a method for producing highly pure and highly soluble PA by carrying out a hydrolysis reaction, with extremely few by-products such as unhydrolyzed products.

Examples of enzymes that hydrolyze phospholipids into PA and nitrogenous bases include phospholipase D (derived from microorganisms and/or plants).
Phospholipase C (hereinafter abbreviated as PL-C), which is also derived from microorganisms and/or animals and/or plants, is suitable as an enzyme that hydrolyzes phospholipids into diglyceride and phosphoryl base. , phosphogesterase, acid phosphatase, and alkaline phosphatase, or a combination of two or more thereof can be used.

In particular, in the present invention, an enzyme that specifically acts on phosphatidylinositol and hydrolyzes phospholipids into diglyceride and phosphorylinositol is suitable as phospholipase C.

Furthermore, in order to maintain the activity of the enzyme described above over a long period of time, it can also be used in the form of an immobilized enzyme. As the immobilization carrier, cellulose, dextran, polystyrene, polyacrylamide, polyvinyl alcohol, ion exchange resin, magnetic material, alumina, photocrosslinkable resin, alginate, various gelling agents, etc. are used. In the present invention, the enzyme acid or the enzyme extract is adsorbed on these immobilization carriers,
It can be used in reactions with immobilized enzymes in the form of particles, membranes, or sheets by ionic bonding, covalent bonding, or entrapment.

Examples of the phospholipids used in the present invention include phosphatidylethanolamine (hereinafter abbreviated as PE), phosphatidylserine (hereinafter abbreviated as PS), phosphatidylglycerol (hereinafter abbreviated as PC), and phosphatidylinositol (hereinafter abbreviated as PI). It is essentially a mixture of these.

The constituent fatty acids of these phospholipids are the same or different, saturated or unsaturated fatty acids having 8 to 24 carbon atoms, such as caproic acid, caprylic acid, lauric acid, myristic acid, valmitic acid, stearic acid, Examples include behenic acid, arachidonic acid, palmitoleic acid, oleic acid, linoleic acid, α- and T-liluic acid, erucic acid, arachidonic acid, eicosabencitric acid, docosahexaenoic acid, tetracosatetraenoic acid, and the like. These phospholipids may be natural extracts, concentrates, or synthetic products, and are not particularly limited.

Next, in order to carry out the reaction efficiently, hydrolysis is carried out in a specific organic solvent or an aqueous solution of an alkali metal salt or alkaline earth metal salt of carboxylic acid or/and a mixture of carboxylic acid and alkali metal salt or alkaline earth metal salt. It is better to react. Furthermore, a more efficient reaction rate can be obtained by carrying out the reaction in the presence of an alkali metal salt or/and an alkaline earth metal salt.

The organic solvent used in the present invention is an alkyl ester of a carboxylic acid having a melting point of 40°C or more, an alkane, an aliphatic hydrocarbon,
It can be used singly or in combination of two or more of alicyclic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, and the like. For example, as an alkyl ester of carboxylic acid, carbon number 2
Alkyl (straight chain or branched alkyl having 1 to 8 carbon atoms) esters of ~6 straight chain or branched fatty acids, including methyl acetate, methyl acetate, methyl propionate, methyl butyrate, methyl valerate, methyl caproate. etc. can be used. Examples of aliphatic hydrocarbons include straight chain or branched aliphatic hydrocarbons having 6 to 12 carbon atoms, particularly hexane,
Heptane, petroleum ether are preferred. Examples of alicyclic hydrocarbons include unsubstituted or substituted alicyclic hydrocarbons having 6 to 12 carbon atoms, with cyclohexane, methylcyclohexane, and cyclooctane being particularly preferred. Examples of aromatic hydrocarbons include unsubstituted or substituted aromatic hydrocarbons having 6 to 12 carbon atoms, with benzene, toluene, and xylene being particularly preferred. Furthermore, halogenated hydrocarbons include chlorination of straight chain or branched alkanes having 1 to 8 carbon atoms;
Examples include brominated and iodized compounds, with chloroform, carbon tetrachloride, and methylene chloride being particularly preferred. Furthermore, linear or branched lower alcohols having 1 to 5 carbon atoms such as methanol and ethanol, and ethers such as diethyl ether and dimethyl ether can also be used.

In the alkali metal salt or alkaline earth metal salt of carboxylic acid and/or the alkali metal salt or alkaline earth metal salt of carboxylic acid used in the present invention, the carboxylic acid is a linear or branched type having 2 to 8 carbon atoms. aliphatic carboxylic acids and/or aromatic carboxylic acids having 7 to 12 carbon atoms, including aliphatic carboxylic acids such as acetic acid, butyric acid, propionic acid, and/or aromatic carboxylic acids such as benzoic acid. , aliphatic carboxylic acids are more preferred. In addition, examples of alkali metals include sodium and potassium J1, and examples of alkaline earth metals include barium, magnesium,
Examples include calcium, and halides, carbonates, phosphates, etc. of these metals. In reactions in such systems, the pl+ of the reaction solution is important, and pH = 4.0.
It is preferable to carry out the reaction within the range of 9.5 to 9.5.

The production of PA by the hydrolysis reaction of phospholipids according to the method of the present invention is carried out, for example, as follows.

Phospholipids (commercially available plant-based or animal-based defatted lecithin, commercially available crude lecithin, etc.), an enzyme that hydrolyzes phospholipids into PA and nitrogenous bases, and an enzyme that hydrolyzes phospholipids into diglycerides and phosphoryl bases. are added and mixed simultaneously or separately, and an alkyl ester of carboxylic acid, preferably a lower alkyl (1 to 3 carbon atoms) ester of acetic acid or propionic acid, is added at a weight ratio of 0.00 to 1 phospholipid.
The reaction is carried out by adding 5 to 20, preferably 1.0 to 10.

Alternatively, 0.05 to 1.0M, preferably 0.1 to 0.0M.
An aqueous solution (hereinafter referred to as reaction solution) of an alkali metal salt or alkaline earth metal salt of carboxylic acid (or a mixture of a carboxylic acid and an alkali metal salt or alkaline earth metal salt) at a concentration of 5M was added to phospholipid 1. 1.0 to 3 in weight ratio
The reaction is carried out by adding 0, preferably 2.0 to 10.

The amounts of the two types of enzymes mentioned above need to be at concentrations that allow the hydrolysis reaction to proceed sufficiently. Specifically, for 1 g of phospholipid used in the reaction, 0.01 to 100 of an enzyme that hydrolyzes phospholipid into PA and nitrogen-containing base is added.
0 units (more preferably 0.1 to 500 units)
and 0.01 to 1000 units (more preferably 0.1 to 500 units) of an enzyme that hydrolyzes phospholipids into diglycerides and phosphoryl bases may be added simultaneously or separately. When the two types of enzymes described above are added separately, the order does not matter.

One enzyme unit here refers to phospholipid PA
and enzymes that hydrolyze nitrogenous bases at a rate of 1 per minute.
It represents the amount of enzyme that hydrolyzes μ5 ole of phosphatidylcholine, and for enzymes that hydrolyze phospholipids into diglyceride and phosphoryl base, it represents the amount of enzyme that hydrolyzes 1 μ5 ole of phosphatidylcholine per minute.

Regarding the specific method of the reaction of the present invention, there are no restrictions on the order of addition of substances involved in the reaction such as raw material phospholipids, solvents, and enzymes, and the composition of the solvent and reaction solution used. Training can be carried out as desired within the scope of the effectiveness of the method.

The PA produced by the production method of the present invention can be easily separated and obtained from the reaction slurry by a conventional method, for example, by subjecting it to purification treatments such as solvent extraction and solvent fractionation.

The reaction process of the phospholipid hydrolysis reaction in the method of the present invention can be monitored by using an analysis method such as thin layer chromatography (TLC) or high performance liquid chromatography (HPLC). You can also control time.

〔Effect of the invention〕

According to the present invention, the target PA is produced with high purity and high yield under mild reaction conditions such as room temperature, normal pressure, and neutrality.

Furthermore, a particularly noteworthy effect of the present invention is that diglyceride, a by-product of phospholipids, serves as an extremely excellent solubilizer for PA obtained in the present invention. The solubilizing effect of diglyceride on PA enables PA to be widely used in various oil-based products, and the functions of oil and fat products can be significantly improved.

Therefore, according to the present invention, there is provided a manufacturing method that allows PA to be easily separated on an industrial scale with high purity and good solubility in fats and oils.

〔Example〕

The method of the present invention will be explained in detail below using Examples, Comparative Examples, etc., but the present invention is not limited thereto.

Example 1 20 g of commercially available defatted lecithin (manufactured by True Lecithin Industries) was placed in a 500 nt Z 4-day flask equipped with a stirring device, and
0.1M) Lis-hydrochloric acid buffer (pH 6.0-8.0)
1250- and then hexane/ethyl acetate (2/
1v/v) 340- is added and stirred. Furthermore, a calcium chloride aqueous solution (LM concentration) 150- was added, and then PL-D of microbial origin (manufactured by Toyo Jozo ■, Strepto-my.
ces Chromofuscus) and PI-specific PL-C (produced by Sarapolo Beer ■, Bacillus
Turingogenesis) aqueous solution 150 af
(15.3 units per gram of phospholipid each) were added and stirring continued for 14 hours while maintaining the temperature of the reaction mixture at 30'C. After the reaction, the reaction product was allowed to stand and the solvent layer was isolated.

The solvent in the solvent layer was distilled off under reduced pressure. The PA content in the obtained phospholipid (17 g) was determined by HPLC (UV detection).
It was measured with The results are shown in Table 1.

Example 2 After reacting only PL-D under the same conditions as in Example 1, the solvent layer was isolated. 20g of the product after solvent distillation is 0.1M
The mixture was dispersed and dissolved in borate buffer (pH 17,5) 90mZ, PI-specific PL-C was added, and the mixture was reacted at 37°C for 220 hours. The reaction product is chloroform/methanol (
2/IV/V), the extract was subjected to Folch partitioning, and chloroform/methanol was removed to obtain 12 g of phospholipid product.

The PA content in the obtained phospholipids was measured by HPLC (UV detection). The results are shown in Table 1.

Examples 3 to 5 Phosphogesterase (Wako Pure Chemicals Reagent, derived from pit viper venom) instead of PL-C used in Example 1 [Example 3]
, acid phosphatase (manufactured by Fluka AG, derived from wheat embryo) 1 Example 41, and alkaline phosphatase (Wako Pure Chemicals reagent, derived from calf intestine) [Example 51] were used to carry out the reaction in the same manner as in Example 1. 0.1M each) Lis-hydrochloric acid buffer (pH 8,8), 0.1M acetate buffer (pH 5)
,0), 0゜5M Tris-HCl buffer (pH 9,0)
The amount of each enzyme is 0.5.5.5 per gram of phospholipid.
0 units were used. The PA content in the obtained phospholipids was measured by HPLC (UV detection). The results are shown in Table 1.

Example 6 PL used in Example 1 for 1 g of weakly basic anion exchange resin
-D and PiC, 200 units each, were immobilized by a covalent bonding method using glutaraldehyde to obtain immobilized enzymes of PL-D, PI, and -C.

This immobilized enzyme was mixed with PL-[1 and Pi, -C of Example 1.
The reaction was carried out under the same conditions as in Example 1 except that . The results are shown in Table 1.

Comparative Example 1 5ooIn! equipped with a stirring device! Put 201 g of commercially available defatted lecithin (manufactured by True Lecithin Industries) into a 40 flask, add 0.1 M 1-Lys-hydrochloric acid buffer (pH 8,0
) I250m/ is added, and further ether 340m/ is added and stirred. Furthermore, calcium chloride aqueous solution (IM concentration) 15
0- was added, and then phospholipase D of microbial origin (manufactured by Toyo Jozo Co., Ltd., Streptomyces Chromo
150 m/g (15 units/g of phospholipid) of an aqueous solution of A. fuscus) was added and stirring continued for 14 hours while maintaining the temperature of the reaction mixture at 30'C. After the reaction, the reaction product was allowed to stand and the ether layer was isolated.

Ether was distilled off from the ether layer under reduced pressure. The PA content in the obtained phospholipid (17 g) was determined by HPLC (
UV detection type). The results are shown in Table 1.

Table 1 * Measured by HPLC (unit: 2%) When the solubility of these phospholipid products in fats and oils was examined after being left at 25°C for 24 hours, the solubility of Comparative Example 1 was extremely poor. (Precipitates were noticeably observed)
In contrast, Examples 1 to 6 of the present invention all showed good solubility (no precipitates were formed at all).

Applicant's agent Kaoru Furuya B Procedural amendment (spontaneous) March 1990 7I] 1. Indication of the case Japanese Patent Application No. 1-133654 2. Name of the invention Method for modifying phospholipids 3. Person making the amendment Relationship to the case Patent applicant (091) Kao Corporation 4, Agent 5, Name of the invention in the specification to be amended, Scope of Claims and Detailed Description of the Invention column 6, Contents of the amendment ( 1. The name of the invention has been corrected to ``Method for producing modified phospholipids J.''

(1) The scope of claims has been amended as shown in the attached sheet.

(1) Specification page 2 lines 8-9 “Method for modifying phospholipids”
was corrected to "method for producing modified phospholipids."

(1) On page 4, line 11, “method for modifying phospholipids” was corrected to “method for producing modified phospholipids.”

2. Claim 1: A modification characterized in that phospholipids are treated with an enzyme that hydrolyzes phospholipids into phosphatidic acid and nitrogen-containing bases, and an enzyme that hydrolyzes phospholipids into diglycerides and phosphoryl bases. 1-1-2 -' Notification Law.

2. The method according to claim 1, wherein the cytalline 2 U method is a method for producing phosphatidic acid by hydrolysis of phospholipids.

3. The method according to claim 1 or 2, wherein the enzyme that hydrolyzes phospholipids into phosphatidic acid and nitrogen-containing bases is phospholipase D.

4. The enzyme that hydrolyzes phospholipids into diglyceride and phosphoryl base is one or two selected from phospholipase C, phosphogesterase, and acid phosphatase.
The method according to claim 1 or 2, wherein the enzyme is more than one species.

Claims (1)

[Claims] 1. A phospholipid characterized by treating a phospholipid with an enzyme that hydrolyzes the phospholipid into phosphatidic acid and a nitrogen-containing base, and an enzyme that hydrolyzes the phospholipid into a diglyceride and a phosphoryl base. Lipid modification method. 2. The method according to claim 1, wherein the method for modifying phospholipids is a method for producing phosphatidic acid by hydrolyzing phospholipids. 3. Claim 1 or 2, wherein the enzyme that hydrolyzes phospholipids into phosphatidic acid and nitrogen-containing bases is phospholipase D.
Method described. 4. The method according to claim 1 or 2, wherein the enzyme that hydrolyzes phospholipids into diglycerides and phosphoryl bases is one or more enzymes selected from phospholipase C, phosphodiesterase, and acid phosphatase.