JPH02186946A - Method for modifying phospholipid - Google Patents

Abstract

PURPOSE:To obtain a modified phospholipid useful as a raw material for phosphatidic acid by treating a phospholipid (mixture) with crushed oil seeds. CONSTITUTION:The objective phospholipid obtained by treating a phospholipid or/and a mixture thereof (e.g. phosphatidyl ethanolamine or phosphatidyl choline) with crushed oil seeds (preferably soybean, rapeseed, sunflower, peanut, cottonseed, safflower, mustard, sesame, olive or corn).

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to modifying phospholipids or/and phospholipid mixtures by treating the phospholipids or/and phospholipid mixtures with crushed oilseeds. It is about the method.

[Conventional technology]

Phospholipids are basic substances of biological membrane components, and are one of the lipids that have functions that govern the basics of life activities, such as protecting cell tissues, transmitting information, and controlling mass transfer.

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. Its application as a drug delivery IJ 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), they succeeded in creating a cooking oil with excellent mold release properties without oil splash (Patent application No. 62-243494).
issue).

Furthermore, examples of the use of PA in industrial fields include, for example, improving the physical properties of dough in the bread-making process (Japanese Patent Application Laid-Open No. 58-51853), and production of emulsifiers made of PA and zein complexes (Japanese Patent Application Laid-Open No. 62-204838). For use in the food industry, such as No.
Use in pharmaceuticals (JP-A-54-105222, JP-A No. 55)
-11582, 56-127308, 60-2
No. 55728), use in cosmetics (JP-A-59-278)
No. 09), Application to chemical products (JP-A-53-108503)
No. 60-243171), and its use in various industrial fields is being considered.

[Problem to be solved by the invention]

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 such circumstances, the present invention aims to provide (1) a method for modifying phospholipids, which comprises treating phospholipids or/and phospholipid mixtures with crushed oilseeds.

More specifically, when producing PA from a phospholipid or/and a phospholipid mixture, a destroyed oilseed product is used and a specific organic solvent or an alkali metal salt or alkaline earth metal salt of a carboxylic acid or/and a carboxylic acid is used. This article relates to a method for producing high-purity FA on an industrial scale with extremely few by-products such as unhydrolyzed products by carrying out a hydrolysis reaction in an aqueous solution of a mixture of alkali metal salts or alkaline earth metal salts. be.

Phospholipids used in the present invention include, for example, phosphatidylethanolamine (PE), phosphatidylcholine (PC)
, phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (P
G), etc., and a mixture thereof may be used.

The constituent fatty acids of these phospholipids are the same or different types, and are saturated or unsaturated fatty acid acyl residues having 8 to 24 carbon atoms, such as caproic acid, caprylic acid, lauric acid, myristic acid, and palmitic acid. , stearic acid, behenic acid, alaxic acid, palmitooleic acid, oleic acid, linoleic acid, α- and r-lyluic acid, erucic acid, arachidonic acid, eicosapencitric acid, docosahexaenoic acid, tetracosatetraenoic acid, etc. can be given.

These phospholipids may be natural extracts, concentrates, or synthetic products, and are not particularly limited.

The organic solvent used in the present invention is one or two selected from alkyl esters of carboxylic acids, alkanes, aliphatic hydrocarbons, alicyclic hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, etc. having a melting point of 40°C or less. The above can be used in combination. For example, the alkyl ester of carboxylic acid has 2 to 2 carbon atoms.
Alkyl (straight chain or branched alkyl having 1 to 8 carbon atoms) esters of 6 straight chain or branched fatty acids, such as methyl acetate, ethyl acetate, methyl propionate, methyl butyrate, methyl valerate, methyl caproate, etc. Methyl acetate is particularly preferred. Examples of aliphatic hydrocarbons include straight chain or branched aliphatic hydrocarbons having 6 to 12 carbon atoms, with hexane, heptane, and petroleum ether being particularly preferred. As an alicyclic hydrocarbon, carbon number is 6
-12 unsubstituted or substituted or alicyclic hydrocarbons, 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 carbon atoms of 1 to 8.
chloroform, carbon tetrachloride,
Methylene chloride is preferred. Furthermore, linear or branched lower alcohols having 1 to 5 carbon atoms such as methanol and ethanol can also be used. Ethyl acetate is most preferred in achieving the present invention.

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 straight 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, acetic acid, propionic acid, and/or aromatic carboxylic acids such as benzoic acid. , acetic acid is more preferred. In addition, examples of alkali metals include sodium, charcoal, etc., examples of alkaline earth metals include barium, magnesium, calcium, etc., and halides, carbonates, phosphates, etc. of these metals. In the reaction in such a system, the pH of the reaction solution is important, and it is preferable to carry out the reaction within the range of pH = 4.0 to 7.5, but if the pH is 6.0±0.5
The range is more preferable.

Suitable oilseeds for use in the present invention include soybean, rapeseed, sunflower, peanut, cottonseed, safflower, mustard, sesame, olive, and corn, and more preferably soybean.

The crushed oilseed product can be obtained by pulverizing the above-mentioned oil seeds by physical treatment, and the resulting pulverized product can be directly subjected to the reaction. There are no particular restrictions on the particle size of the pulverized material, but any pulverized material preferably falls within the range of 5 mm or less.

As for the pulverization method, any apparatus, method, etc. known in the art can be used.

Furthermore, for the purpose of increasing the enzyme activity of the crushed oilseeds, plant growth promoters (e.g., growth hormones such as gibberellins and auxins such as indoleacetic acid, divalent ions such as calcium ions and barium ions) may be added as necessary. halides, carbonates, phosphates of typical metal ions and/or transition metal ions such as manganese, lanthanum, cerium, etc.
sulfate, etc.) at a concentration of 0.1 to 10% based on the raw material phospholipid,
Preferably, it may be added in an amount of 0.1 to 5%.

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

A slurry is prepared by stirring and mixing 10 to 100% by weight of crushed oilseeds with respect to phospholipids (commercially available plant-based or animal-based defatted lecithin, commercially available crude lecithin, etc.), and alkyl esters of carboxylic acids are prepared. , preferably lower alkyl (C, -C,) ester of acetic acid or propionic acid,
Weight ratio of 0.5 to 20, preferably 1 to 1 of phospholipid
．． The reaction is carried out by adding 0-10.

Or 0.05 to 1. OM, preferably 0.1-0.
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 reaction mixture has a reaction temperature of 10 to 60°C, especially 20 to 50°C.
The reaction is completed by continuing stirring at ℃ for several hours or more.

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 crushed products, and the composition of the solvent and reaction solution used. This can be arbitrarily adjusted within the range where the effect of the station departure method is achieved.

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 this method is as follows:
For example, by using an analysis method such as thin layer chromatography (TLC) or high performance liquid chromatography (HPLC), the progress of the reaction can be grasped, and the reaction time can thereby be controlled.

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, and in addition, it is a PI that is difficult to decompose using an enzymatic reaction method. is almost completely hydrolyzed.

Therefore, according to the present invention, there is provided a manufacturing method that allows high-purity PA to be easily separated on an industrial scale.

〔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.

Reference Example (Preparation of crushed oilseed) Soybean was used as the oilseed. That is, U.S. soybeans (Ohio, Glycine max (L, )) 200
g was crushed using a 2-1 Fung blender. The crushed material was passed through a sieve (Mesh No. 20) to remove impurities. The obtained crushed soybean product was used in the following Examples.

Example 1 Into a 500d40 flask equipped with a stirring device, 25 g of commercially available defatted lecithin (trade name 5LP-WSP, manufactured by True Lecithin Industry ■) was placed, and 25 g of the crushed soybean product prepared in Reference Example was added. While stirring the mixture, add 250ml of ethyl acetate, and add 32.5ml of ion-exchanged water.
g and continued stirring. The reaction mixture was heated at 30°C for 20
The mixture was stirred for an hour.

Remove the ethyl acetate layer (A) from the reaction product and leave the residue (B)
was extracted twice with chloroform/methanol (2/1 v/v). The extraction solution containing B is subjected to Folch distribution,
Phospholipid crude product C was obtained by removing chloroform/methanol together with the residue obtained by removing ethyl acetate from the previously obtained A.

By adding 5 times the volume of acetone to C, stirring, and leaving it to stand still, a precipitate (D>21 g) was obtained.D was further washed with water-cooled acetone and then dried under reduced pressure.The obtained D The composition of phospholipids was identified by HPLC (UV detection type).

The results are shown in Table 1.

Comparative Example 1 20 g of commercially available defatted lecithin (manufactured by True Lecithin Industries) was placed in a 500mj' 4-day flask equipped with a stirring device, and IM Tris/HCl buffer (pH 8,0
Add m1 and more ether 340rd! Add and stir. Furthermore, 150 d of calcium chloride aqueous solution (IMa degree) was added, and then phospholipase D of microbial origin (manufactured by Toyo Jozo Co., Ltd., Streptomyces Chrom-mof
uscus origin) aqueous solution 150mj! (phospholipid 1
15 units per g) and the temperature of the reaction mixture was increased to 3.
Stirring was continued for 14 hours while maintaining the temperature at 0°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 composition of the obtained phospholipid (17 g) was identified by HPLC (UV detection type). The results are shown in Table 1.

Example 2 25 g of commercially available defatted lecithin (trade name 5LP-WSP: manufactured by True Lecithin Kogyo@) was placed in a 500 d 4-day flask using a stirrer, and 25 g of the crushed soybean prepared in Reference Example was added. While stirring, add 0.1M sodium acetate/acetic acid buffer (pH 6,
0) 100rnl and 32.5 g of a 50mM calcium chloride aqueous solution were added and stirring was continued. Reaction mixture at 30℃
The mixture was stirred for 20 hours.

Residue 1 (B) was taken out from the reaction product and extracted twice with chloroform/methanol (2/1 Shiba). The extracted solution containing B was subjected to Folch partitioning to remove chloroform/methanol to obtain a crude phospholipid product G. 21 g of precipitate (H) was obtained by adding 5 times the volume of acetone to G, stirring, and then allowing it to stand still. H was further washed with water-cooled acetone and then dried under reduced pressure. The composition of the phospholipid for the obtained H was determined by HPLC (tJV detection type)
It was identified. The results are shown in Table 1.

Table (Unit: 2%) Measured by HPLC Kimoto LPC: IJ Sophosphatidylcholine PC, P
E, PI, PA: Example 3 as described above In Example 1, rapeseed, sunflower, peanut, olive, and corn were used as oil seeds instead of soybean seeds (the same weight of each as in the case of soybeans was used), and others. All hydrolysis was performed under the same conditions. The composition of the obtained modified phospholipid is shown in Table 2.

Example 4 In Example 2, rapeseed, sunflower, peanut, olive, and corn were used as oil seeds instead of soybean seeds (the same weight of each as in the case of soybeans was used), and all other hydrolysis was performed under the same conditions. Ta. The composition of the obtained modified phospholipid is shown in Table 2.

Comparative Example 2 In Example 3, instead of oilseeds, plant juice obtained by filtration from crushed plants (cabbage) was used.The amount of plants used was the same weight as the oilseeds) All other conditions were the same. Hydrolysis was carried out below. The composition of the phospholipids is shown in Table 2.

Comparative Example 3 In Example 4, instead of oilseeds, plant juice obtained by filtration from crushed plants (cabbage) was used (the amount of plants used was the same weight as the oilseeds). Hydrolysis was carried out under the same conditions. The composition of the phospholipids is shown in Table 2.

Table 2 Comparison procedure for crushed fried oil seeds Shufushosho (self-proposal) ■, Indication of the case Patent application No. 1-3509 No. 2, Name of the invention Method for modifying phospholipids 3, Person making the amendment Relationship with the case Patent applicant (091) Kao Co., Ltd. Company 4, agent (unit: %) Kimoto is the same as in Table 1

Claims (1)

[Scope of Claims] 1. A method for modifying phospholipids, which comprises treating phospholipids or/and a phospholipid mixture with crushed oilseeds. 2. A method for producing phosphatidic acid, which comprises reacting a phospholipid or/and a phospholipid mixture with a crushed oilseed product in the presence of an organic solvent. 3. Phospholipids or/and phospholipid mixtures and crushed oilseeds in the presence of alkali metal salts or alkaline earth metal salts of carboxylic acids or/and mixtures of carboxylic acids and alkali metal salts or alkaline earth metal salts. A method for producing phosphatidic acid, which comprises reacting it. 4. The method for producing phosphatidic acid according to claim 2, wherein the organic solvent is an alkyl ester of a carboxylic acid having a melting point of 40°C or less. 5. The method for producing phosphatidic acid according to claim 3, wherein the carboxylic acid is an aliphatic or/and aromatic carboxylic acid having a total number of carbon atoms of 2 to 8. 6. The production of phosphatidic acid according to any one of claims 1 to 5, wherein the oilseed is selected from the group consisting of soybean, rapeseed, sunflower, peanut, cottonseed, safflower, mustard, sesame, olive, and corn. Law.