KR20030084200A - Method for producing phosphatidylethanolamine and lysophosphatidylethanolamine using non-organic solvent system and a water-soluble composition comprising the lysophosphatidylethanolamine produced by the method - Google Patents

Abstract

PURPOSE: Methods for producing phosphatidylethanolamine(PE) and lysophosphatidylethanolamine(LPE) using a non-organic solvent system and a water-soluble composition comprising the lysophosphatidylethanolamine produced by the same method are provided, thereby producing high purity PE and LPE without using an organic solvent. CONSTITUTION: A method for producing phosphatidylethanolamine(PE) and lysophosphatidylethanolamine(LPE) using a non-organic solvent system is characterized by reacting lecithin with ethanolamine in the presence of phospholipase D, wherein the lecithin contains at least one selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylic acid and phosphatidylinositol; and the reaction is carried out under the non-organic solvent system containing potassium chloride and buffer solution. A method for producing phosphatidylethanolamine(PE) and lysophosphatidylethanolamine(LPE) using a non-organic solvent system is characterized by the steps of: reacting lecithin with ethanolamine in the presence of phospholipase D; and adding phospholipase A into the reaction resulting product and reacting them, wherein the lecithin contains at least one selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylic acid and phosphatidylinositol; and the reaction is carried out under the non-organic solvent system containing potassium chloride and buffer solution.

Description

Method for producing phosphatidylethanolamine and lysophosphatidylethanolamine using non-organic solvent system and a water-soluble in a method of preparing phosphatidylethanolamine and lysophosphatidylethanolamine in a non-organic solvent system composition comprising the lysophosphatidylethanolamine produced by the method}

The present invention relates to a process for preparing phosphatidylethanolamine, a process for preparing lysophosphatidylethanolamine, and a water-soluble composition comprising lysophosphatidylethanolamine prepared by the method, and more particularly, to phosphatidylcholine, phosphatidyl in a non-organic solvent system. Phosphatidylethanolamine or lysophosphatidyl by treating phospholipid D alone or in combination with phospholipase A (A1 or A2) to a natural or synthetic lecithin comprising at least one ethanolamine, phosphatidyl acid, phosphatidylinositol, etc. It relates to a method for producing ethanolamine. In more detail, lecithin including at least one of phosphatidylcholine, phosphatidylethanolamine, phosphatidyl acid, phosphatidylinositol, and the like is added to a buffer containing calcium chloride at a concentration of 1 to 50%, and phospholipase D alone or phospho Treatment with lipase A (A1 or A2) relates to a process for producing phosphatidylethanolamine or lysophosphatidylethanolamine.

Lysophosphatidylethanolamine is known to play a very important role in ripening and senescence of fruits. It is known that lysophosphatidylethanolamine can inhibit the aging of leaves and fruits of tomatoes. Therefore, treatment of lysophosphatidyl ethanolamine after tomato harvesting can extend the shelf life of fruits. Known (US Pat. No. 5,110,341, US Pat. No. 5,126,155). In addition, it is known that the treatment of lysophosphatidylethanolamine on apples promotes anthocyanin formation on the skin and inhibits the loss of firmness during storage of harvested apples. This action is known to be associated with a role in lowering the respiration rate of fruits such as apples, cranberries, tomatoes, and the like and promoting or inhibiting ethylene gas formation (Farag). , KM and JP Palta, "Stimulation of Ethylene Production by Erea, Thidiazoron, and Lysophosphatidylethanolamine and Possible sites of this stimulation" Annual meeting of the American Society of Plant Physiologists, April 1989).

Lysophosphatidylethanolamine solutions adjusted to appropriate concentrations are also used as a means of extending the life of cut flowers (HortScience 32 (5): 888-890, 1997). Generally, silver thiosulfate solution containing sugar has been used for this purpose because it suppresses aging of flowers by treating the harvested flowers for about 20 hours or more. However, the use of silver ions in this solution has caused environmental pollution and has recently been reluctant to be used in the United States. Naturally purified lysophosphatidylethanolamine is also known to play a role in increasing the shelf life of cut flowers in vases similar to silver thiosulfate solutions. In this field, the use of lysophosphatidylethanolamine is active. It is being promoted.

In order to extend the life of harvested crops, lysophosphatidylethanolamine is actually used as a method of immersing the crop in a solution in which lysophosphatidylethanolamine is dissolved or spraying the solution on the crop. When lysophosphatidylethanolamine is dissolved in water to make a uniform formulation and good efficacy. Therefore, the greater the effect of lysophosphatidylethanolamine in water, the greater the effect.

To date, no method of producing high-purity lysophosphatidylethanolamine has been developed industrially, and a small amount of separation and purification using silica gel column chromatography has been performed in a laboratory. It is sold in small quantities only for reagents by Avanti Polar Lipids, Inc or Sigma, and is very expensive. The production of lysophosphatidylethanolamine using column chromatography is very difficult because of the similar movement of lysophosphatidylcholine in the column. In addition, when performing column chromatography using a low toxicity organic solvent such as hexane or ethanol as a single solvent, purification is very difficult because lysophosphatidylethanolamine has a very low solubility in an organic solvent. Good results can be obtained only by purification by column chromatography using solvent systems known to be very toxic, such as chloroform, benzene and methanol, but in this case yields are poor and expensive to purify. Therefore, there is an urgent need for a method with good yield and easy purification.

In general, soybean lecithin and egg yolk lecithin are the starting materials of the invention for preparing lysophospholipids, but are not limited to these raw materials. Crude soybean lecithin (commonly referred to as crude lecithin), produced as a by-product of the soybean oil manufacturing process, is 60-70% polar lipids (phospholipids / glycolipids), 27-39% soybean oil, 1-3% water, It is composed of 0.5 ~ 3% of other ingredients. The polar lipids are purified by removing soybean oil, which is a neutral lipid contained in crude lecithin, and the purified composition is 22-30% phosphatidylcholine (PC), 2-5% lysophosphatidylcholine (LPC). , 16-22% phosphatidylethanolamine (PE), 0.5-2% lysophosphatidylethanolamine (LPE), 0.5-8% phosphatitidic acid (PA), 0.1-3% Phosphatidylserine, 6-15% phosphatidylinositol, and the like. Egg yolk lecithin also contains 73-83% phosphatidylcholine (PC), 2-5% lysophosphatidylcholine (LPC), 13-17% phosphatidylethanolamine (PE), 1-3% lysophosphatidylethanolamine (LPE), etc. Consists of. Thus, since lecithin contains a very small amount of lysophosphatidylethanolamine, it is almost impossible to separate commercially from lysophosphatidylethanolamine directly from these lecithins. Therefore, lysophosphatidylethanolamine was prepared by reacting lecithin with ethanolamine in the presence of phospholipase D and phospholipase A, but there was a high demand for a preparation method with high yield and easy purification.

In general, in the synthesis of phospholipid using phospholipase D, since phospholipid is insoluble in water, it is generally synthesized in a two-phase system using an organic solvent such as ethyl acetate, diethyl ether, and a buffer solution. However, lecithin with 20-40% phosphatidylcholine and 20-30% phosphatidylethanolamine (e.g. Xindong SOYA LECITHIN POWDER 95, CENTRAL SOYA's FP 40, D, RP40, etc.) is insoluble in ethyl acetate. There was a problem. In particular, in an ideal system composed of ethyl acetate-water, when lecithin is used at a certain concentration, stirring is not smooth, lecithin aggregation occurs, reactivity is low, and enzyme inactivation is large. In the case of diethyl ether, high volatility and low boiling point make mass production impossible. In the case of other toluene, benzene, etc., since the enzyme inactivation is severe and the solvent is toxic, there is a problem that the remaining organic solvent after the reaction affects the separation and purification. Hexane is a very efficient solvent that can dissolve lecithin, but has a disadvantage in that the reactivity is very poor in the synthesis reaction.

The present invention is to solve the above problems, it is an object of the present invention to provide a method for the efficient synthesis and separation purification in the production of phosphatidylethanolamine and lysophosphatidylethanolamine. Without the use of toxic organic solvents, the inactivation of the enzyme is not severe and the aim is to provide a safe and efficient method. In addition, since the organic solvent is not used during the reaction, the separation and purification process after the reaction is simplified, and a method of recovering the organic solvent used in the separation and purification is provided. It is another object of the present invention to prepare phosphatidylethanolamine and lysophosphatidylethanolamine using a non-organic solvent system that is very safe and highly reactive from not only medium and low purity lecithin but also high purity lecithin. The present invention also provides a method for removing the enzyme and ethanolamine used in the reaction, the choline generated after the reaction and the remaining ethanolamine remaining after the reaction by washing with water the reactant, which forms a salt after phosphatidylethanolamine synthesis.

In order to achieve the above object, the method for preparing phosphatidylethanolamine according to the present invention comprises the step of reacting lecithin and ethanolamine in the presence of phospholipase D, wherein the lecithin Silver comprises at least one of phosphatidylcholine, phosphatidylethanolamine, phosphatidyl acid and phosphatidylinositol, and the reaction is characterized in that it is performed in a non-organic solvent system comprising calcium chloride and a buffer solution.

Further, the method for producing lysophosphatidylethanolamine according to the present invention is a method for producing lysophosphatidylethanolamine from lecithin and ethanolamine by treating phospholipase D and phospholipase A, wherein the lecithin is phosphatidylcholine, phosphatidylethanol At least one of amine, phosphatidylic acid and phosphatidylinositol, wherein the reaction is carried out in a non-organic solvent system comprising calcium chloride and a buffer solution.

One embodiment of the above-described method for preparing lysophosphatidylethanolamine according to the present invention comprises the steps of first reacting lecithin and ethanolamine in the presence of phospholipase D; And phospholipase A in the reactant.

In one embodiment of the above methods, the non-organic solvent system is characterized by consisting only of calcium chloride and buffer solution.

In one embodiment of the above methods, the lecithin is characterized in that the reaction is added in an amount of 1 to 50% relative to the buffer solution.

In one embodiment of the above methods, the pH of the buffer is characterized in that in the range of 4 to 8.

In one embodiment of the above methods, the temperature of the reaction is characterized in that the range of 20 ℃ to 60 ℃.

In one embodiment of the above methods, the lecithin is one selected from the group consisting of soybean lecithin, rape lecithin, fish-derived lecithin, mollusk-derived lecithin and egg yolk lecithin, wherein the fatty acid ring has 6 to 30 carbon atoms, Mono-, polyunsaturated fatty acids, or sodium salts, potassium salts, magnesium salts, ammonium salts, phosphates, hydrochlorides and sulfates.

In one embodiment of the above methods, the phospholipase D is characterized in that it is of microbial origin or of plant origin.

In the method for preparing the phosphatidylethanolamine, after the step of reacting with the phospholipase D, the reaction solution in which the calcium salt is formed by washing with water or a buffer solution is washed with water or a buffer solution and choline, which is an enzyme and a reaction byproduct in the reaction solution. , Inositol and ethanolamine, which are unreacted substrates, are further included.

The method for producing lysophosphatidylethanolamine is characterized in that it further comprises the step of extracting, separating and purifying with ethanol after the step of reacting with the phospholipase A.

The method for preparing lysophosphatidylethanolamine is characterized in that it further comprises the step of extracting, separating and purifying with hexane, ethanol and water after the reaction with the phospholipase A.

The method for preparing lysophosphatidylethanolamine may further include the step of pulverizing the purified lysophosphatidylethanolamine by acetone after the extraction, separation and purification.

The above-mentioned methods further comprise the step of changing the composition of the fatty acid by adding hydrogen to the raw material lecithin, phosphatidylethanolamine prepared by the method or lysophosphatidylethanolamine prepared by the method.

In addition, the composition according to the invention is characterized in that it comprises phosphatidylethanolamine or lysophosphatidylethanolamine prepared by the method according to the above methods.

Hereinafter, the preparation method according to the present invention and a composition comprising lysophosphatidylethanolamine prepared by the method will be described in more detail.

In the present invention, a buffer solution in which calcium chloride was dissolved was used as the reaction medium system. According to the present invention, a phosphatidylethanolamine is prepared by substituting a commercially available polar portion of lecithin with ethanolamine using phospholipase D derived from microorganisms or plants, and washing the reactant produced as a calcium salt with water. By removing the by-products such as choline, the by-product generated during the reaction, the enzyme used in the transfer reaction, and ethanolamine remaining after the reaction, impurities were not mixed in the product, thereby simplifying the separation and purification process.

In order to obtain lysophosphatidylethanolamine, a lecithin containing washed phosphatidylethanolamine is added and reacted with a buffer solution containing phospholipase A and calcium chloride.

In the above production method, a buffer solution such as an acetic acid buffer solution or a phosphate buffer solution, such as pH 4-8, more preferably pH 5-7, is used as the buffer solution.

The lecithin may be a vegetable or animal lecithin having a fatty acid ring having the same or different saturated, mono- and polyunsaturated fatty acids having 6 to 30 carbon atoms, and may be a form of a salt by changing the composition of the fatty acid by adding hydrogen to the lecithin. Can be. The salt may be used without any problem as long as it is in the form of a physiologically acceptable salt, and specific salts include sodium salt, potassium salt, magnesium salt, ammonium salt, phosphate salt, hydrochloride salt and sulfate salt. Among these salts, sodium salts and potassium salts are preferred.

In addition, the pH of the buffer solution is in the range of 4 to 8, the reaction temperature in the method is preferably in the range of 20 ℃ to 60 ℃.

The phospholipase D according to the present invention may be derived from microorganisms or from plants.

In more detail, the ethanolamine is added to a reaction tank equipped with a stirring device and a temperature control device, and an acetic acid buffer solution or a phosphate buffer solution (pH 4-8, more preferably pH 5-7, about 0.2 M ) Is added about 3 to 6 times the lecithin as the reaction substrate. The reaction can be carried out in a range of 20 to 50 ° C. as necessary. To the buffer in which ethanolamine is dissolved is added an enzyme having the activity of phospholipase D as necessary. Thereafter, the lecithin is dissolved in an appropriate concentration and added, followed by stirring for 5 hours to 24 hours. After completion of the reaction, the reaction solution was filtered under reduced pressure, washed with addition of clean water, and washed with water under reduced pressure filtration two to three times. The phosphatidylethanolamine-containing lecithin thus obtained is dried under reduced pressure, or further powdered using acetone treatment or the like to obtain a phospholipid containing a high concentration of phosphatidylethanolamine.

To obtain lysophosphatidylethanolamine, 2 to 4 times the buffer solution containing calcium chloride is added to the washed phospholipid after the reaction, and the required amount of phospholipase A is added and stirred for 5 to 12 hours. Thereafter, the fatty acid is removed from the reaction solution and a phospholipid containing a high concentration of lysophosphatidylethanolamine is obtained. Here, there may be various methods for separating and purifying lysophosphatidylethanolamine.

First, the extraction method using ethanol, after completion of the reaction is added to the reactant so that the concentration of ethanol is 70-90%, and then stirred and heated to about 60 to 75 ℃, the extract is filtered or left to remove the upper portion Concentrate on evaporation. Thereafter, acetone treatment is used to remove and powder the fatty acids to obtain a high concentration of lysophosphatidylethanolamine. As a method of removing fatty acids, extraction methods using supercritical fluid extraction or high pressure propane gas may be used instead of acetone.

Another method is to extract using nucleic acid and ethanol. After completion of the enzyme reaction, the reaction solution is added with nucleic acid, ethanol and water, stirred and left to stand for phase separation and the upper layer is concentrated. Thereafter, acetone treatment is used to remove and powder the fatty acids to obtain a high concentration of lysophosphatidylethanolamine.

In addition, the fatty acid composition may be changed by hydrogenation of the prepared phosphatidylethanolamine or lysophosphatidylethanolamine. In the case of the hydrogenation reaction, phosphatidylethanolamine or lysophosphatidylethanolamine synthesized by raw material lecithin or a transfer reaction is dissolved in ethanol at a concentration of 10 to 20%, and 4% palladium carbon is added to 3.3 to 5% of the total. Then, the mixture was blown three times at a hydrogen pressure of 40 psi or more, and finally reacted for about 6 to 15 hours at a temperature of 300 to 900 rpm at 50 ° C. at a hydrogen pressure of 40 to 60 psi, depending on the desired degree of hydrogenation. Raw material lecithin, phosphatidylethanolamine or lysophosphatidylethanolamine can be obtained.

The present invention will be described in more detail with reference to the following examples.

<Measurement conditions>

HPLC analysis was used to determine the contents of phosphatidylethanolamine and lysophosphatidylethanolamine, and the conditions were as follows. LiChrosphere 100 diol (Merck, 4m × 125 mm) for HPLC analysis was used, phase A was hexane / isopropanol / acetic acid / triethylamine = 82: 17: 1: 0.08, phase B isopropanol / water / acetic acid / tri Use a concentration gradient of two solvent systems with ethylamine = 85: 14: 1: 0.08. The time program is shown in Table 1 below.

Minutes Flow (mL / min) % A % B 0 1.5 95 5 23 1.5 60 40 28 1.5 0 100 29 1.5 0 100 39 1.5 95 5

The flow rate was 1.5 ml per minute, the ELSD detector was used as a detector, the detector temperature was 65 ℃, the gas pressure was 1.9 L / min.

<Example 1>

60 g of ethanolamine and 8.8 g of calcium chloride were added to 1 l of sodium acetate buffer solution (pH 5.6), and actinomycetes-derived phospholipase D 2000unit was added thereto, and stirred at 40 ° C. to completely dissolve the ethanolamine, followed by lecithin (FP 40, 200 g of CENTRAL SOYA, 40% of phosphatidylcholine) were added and reacted for 12 hours while stirring at 200 rpm. After the reaction was completed, 3 liters of clean water was added, stirred, and filtered. 3 L of water was added thereto, stirred, and filtered. The resultant was dried under reduced pressure. The yield was 90%, and the phosphatidylethanolamine content was 70%.

<Example 2>

Subsequent to the transfer reaction in Example 1, phospholipase A was further treated to prepare lysophosphatidylethanolamine.

In other words, 60 g of ethanolamine and 8.8 g of calcium chloride were added to 1 L of sodium acetate buffer solution (pH 5.6), and actinomycetes-derived phospholipase D 2000unit was added thereto, and stirred at 40 ° C. to completely dissolve the ethanolamine, followed by lecithin (FP). 40 g of CENTRAL SOYA, phosphatidylcholine content 40%) was added thereto, and the reaction was carried out for 12 hours while stirring at 200 rpm. After the reaction was completed, 3 liters of clean water was added, stirred, and filtered. After adding 2 liters of water, stirring and filtration twice, 400 ml of sodium acetate buffer (pH 5.6) was added, and 3.5 g of calcium chloride and pancreatic phospholipase A2 (8,000 IU / ml, trade name LECITASE 10L) were added. , 6 ml of NOVO NORDISK Co.) was added and reacted at 37 ° C. for 6 hours. After completion of the reaction, 1000, 600 and 400 ml of hexane, ethanol and water were added, stirred for 30 minutes, and left to stand. After phase separation, the upper layer was removed, concentrated by evaporation, and then powdered using 1 L of acetone. The yield was 35% and the content of lysophosphatidylethanolamine was 70%.

<Example 3>

Ethanol and acetone were used for separation and purification of lysophosphatidylethanolamine after completion of the reaction in Example 2.

60 g of ethanolamine and 8.8 g of calcium chloride were added to 1 L of sodium acetate buffer solution (pH 5.6), and actinomycetes-derived phospholipase D 2000unit was added thereto, and stirred at 40 ° C. to completely dissolve the ethanolamine, followed by lecithin (FP 40, 200 g of CENTRAL SOYA company, phosphatidylcholine content 40%) was added and reacted for 12 hours while stirring at 200 rpm. After the reaction was completed, 3 liters of clean water was added, stirred, and filtered. After adding 2 liters of water, stirring and filtration twice, 400 ml of sodium acetate buffer (pH 5.6) was added, 3.5 g of calcium chloride and pancreatic phospholipase A2 (8,000 IU / ml, trade name LECITASE 10L) , 6 ml of NOVO NORDISK Co.) was added and reacted at 37 ° C. for 6 hours. After the reaction was completed, 1.6 liters of ethanol was added thereto, stirred and extracted with heating at 70 ° C., followed by filtration. The filtrate was concentrated by evaporation and stirred twice with 1 L of acetone to remove fatty acids and powdered lysophosphatidylethanolamine. The yield was 40% and the content of lysophosphatidylethanolamine was 64%.

<Example 4>

After the completion of the reaction in Example 2 for the separation and purification of lysophosphatidylethanolamine was dissolved in ethanol without using acetone and then cooled.

60 g of ethanolamine and 8.8 g of calcium chloride were added to 1 l of sodium acetate buffer solution (pH 5.6), and actinomycetes-derived phospholipase D 2000unit was added thereto, and stirred at 40 ° C. to completely dissolve the ethanolamine, followed by lecithin (FP 40, 200 g of CENTRAL SOYA, 40% of phosphatidylcholine) were added and reacted for 12 hours while stirring at 200 rpm. After the reaction was completed, 3 liters of clean water was added, stirred, and filtered. After adding 2 liters of water, stirring and filtration twice, 400 ml of sodium acetate buffer (pH 5.6) was added, and 3.5 g of calcium chloride and pancreatic phospholipase A2 (8,000 IU / ml, trade name LECITASE 10L) were added. , 6 ml of NOVO NORDISK Co.) was added and reacted at 37 ° C. for 6 hours. After the completion of the reaction, 1.6 liters of ethanol was added thereto, stirred and extracted with heating at 70 ° C., and then cooled and precipitated at −10 ° C. and filtered. At this time, the yield was 30%, and the content of lysophosphatidylethanolamine was 90.2%.

Example 5

In the reaction in Example 1, lecithin derived from egg yolk was used as a raw material. 100 g of ethanolamine and 8.8 g of calcium chloride were added to 1 L of sodium acetate buffer solution (pH 5.6), 2000 units of actinomycetes-derived phospholipase D were added, and stirred at 40 ° C. to completely dissolve the ethanolamine, followed by lecithin (PL95, Doosan). 200 g of Biotech Co., Ltd., phosphatidylcholine content 75%) was added and reacted for 12 hours while stirring at 200 rpm. After the reaction was completed, 3 liters of clean water was added, stirred, and filtered. 3 L of water was added thereto, stirred, and filtered. The resultant was dried twice under reduced pressure. The yield was 90%, and the phosphatidylethanolamine content was 90%.

<Example 6>

In the reaction in Example 3, lecithin derived from egg yolk was used as a raw material. That is, 100 g of ethanolamine and 8.8 g of calcium chloride were added to 1 L of sodium acetate buffer solution (pH 5.6), and Actinomycetes-derived phospholipase D 2000 unit was added thereto, and stirred at 40 ° C. to completely dissolve the ethanolamine, followed by lecithin ( 200 g of PL95, Doosan Biotech Co., Ltd., phosphatidylcholine content 75%) was added thereto and reacted for 12 hours while stirring at 200 rpm. After the reaction was completed, 3 liters of clean water was added, stirred, and filtered. After adding 3 liters of water, stirring and filtration twice, 400 ml of sodium acetate buffer (pH 5.6) was added, and 3.5 g of calcium chloride and phospholipase A2 from pig pancreas (8,000 IU / ml) 10 ml of LECITASE 10L, NOVO NORDISK Co.) was added and reacted at 37 ° C. for 6 hours. After the completion of the reaction, 1.6 liters of ethanol was added, followed by extraction with stirring while heating to 80 ° C, followed by filtration. The filtrate was concentrated by evaporation and stirred twice with 1 L of acetone to remove fatty acids and powdered lysophosphatidylethanolamine. The yield was 60% and the content of lysophosphatidylethanolamine was 94%.

<Example 7>

In addition to phospholipase D derived from actinomycetes as an enzyme source, phospholipase D derived from plants can also be used. Plant-derived phospholipase D includes cabbage, soybean, and the like. In this example, an example of synthesizing phosphatidylethanolamine using a cabbage extract showing the activity of phospholipase D is shown. Add 60 g of ethanolamine and 8.8 g of calcium chloride to 1 L of sodium acetate buffer (pH 5.6), add 2000 units of cabbage extract with phospholipase D activity prepared above, and add 200 g of lecithin (CENTROLEX D, CENTRAL SOYA) to 200 RPM. The reaction was stirred for 12 hours. After the reaction was completed, 3 liters of clean water was added thereto, stirred, and filtered. After adding and stirring 3 L of water, the mixture was filtered twice, and 1000, 600, and 400 ml of hexane, ethanol, and water were added, stirred for 30 minutes, and left to stand. After phase separation, the upper layer was removed, concentrated by evaporation, and then powdered using 1 L of acetone. The yield was 83% and the phosphatidylethanolamine content was 50%.

<Example 8>

1 kg of lysophosphatidylethanolamine obtained in Examples 2 and 3 was dissolved in ethanol at a concentration of 10%, 5% palladium carbon was added so as to be 2.5% by weight of the whole, and then about 15 hours at 50 ° C at a hydrogen pressure of 14.7 psi. By the reaction, almost all fatty acids were hydrogenated to obtain 952 g of lysophosphatidylethanolamine composed of saturated fatty acids.

Example 9

10 g of lysophosphatidylethanolamine obtained in Examples 2 and 3 was added to 100 ml of water and stirred to obtain a 10% clear aqueous solution of lysophosphatidylethanolamine.

When using the production method according to the present invention, since only the buffer solution without the use of an organic solvent, there is no risk of the use of toxic organic solvents, the difficulty of purification, enzyme inactivation is not severe and the reaction efficiency is high. In particular, since the organic solvent is not used during the reaction, the separation and purification process after the reaction is simplified, and the recovery of the solvent is easier even when the organic solvent is used in the separation and purification. In addition, both high and low purity lecithin (50% or less of the lecithin content) as well as high purity lecithin can be smoothly dispersed. Also, by synthesizing phosphatidylethanolamine with water, the reaction product that forms calcium salt can be washed with water to remove enzymes and ethanolamine used in the reaction and choline produced after the reaction, so that impurities such as enzymes, ethanolamine and choline are not mixed in the product. can do.

Claims (15)

In the method for producing a phosphatidyl ethanolamine comprising the step of reacting lecithin and ethanolamine in the presence of phospholipase D, The lecithin comprises at least one of phosphatidylcholine, phosphatidylethanolamine, phosphatidyl acid and phosphatidylinositol, The reaction is a method for producing phosphatidylethanolamine, characterized in that in a non-organic solvent system containing calcium chloride and a buffer solution. A process for treating phospholipase D and phospholipase A to produce lysophosphatidylethanolamine from lecithin and ethanolamine, The lecithin comprises at least one of phosphatidylcholine, phosphatidylethanolamine, phosphatidyl acid and phosphatidylinositol, The reaction is a method for producing lysophosphatidylethanol amine, characterized in that in a non-organic solvent system containing calcium chloride and a buffer solution. The method of claim 2, The method, First reacting lecithin with ethanolamine in the presence of phospholipase D; And adding phospholipase A to the reactant, followed by secondary reaction. The method of claim 1, wherein the non-organic solvent system comprises only calcium chloride and a buffer solution. The method according to any one of claims 1 to 3, wherein the lecithin is added and reacted with the buffer solution in an amount of 1 to 50%. The method according to any one of claims 1 to 3, wherein the pH of the buffer solution is in the range of 4 to 8. The process according to any one of claims 1 to 3, wherein the temperature of the reaction is in the range of 20 ° C to 60 ° C. The lecithin according to any one of claims 1 to 3, wherein the lecithin is selected from the group consisting of soybean lecithin, rapeseed lecithin, fish-derived lecithin, mollusk-derived lecithin and egg yolk lecithin, wherein the fatty acid ring has 6 to 6 carbon atoms. 30 saturated, mono-, polyunsaturated fatty acids, or a sodium salt, potassium salt, magnesium salt, ammonium salt, phosphate salt, hydrochloride salt and sulfate process. The method according to any one of claims 1 to 3, wherein the phospholipase D is derived from a microorganism or a plant. According to claim 1 or 3, After the step of reacting with the phospholipase D, the reaction solution formed calcium salt by the reaction washed with water or buffer solution choline, which is an enzyme and reaction by-product in the reaction solution, And removing inositol and ethanolamine which are unreacted substrates. The method of claim 3, further comprising extracting, separating, and purifying with ethanol after the reaction with phospholipase A. The method of claim 3, further comprising extracting, separating, and purifying with hexane, ethanol, and water after the reaction with phospholipase A. 5. 13. The method of claim 11 or 12, further comprising the step of pulverizing the purified lysophosphatidylethanolamine by acetone after the extracting, separating and purifying the lysophosphatidyl ethanol. Process for the preparation of amines. The method of claim 1, further comprising the step of changing the composition of the fatty acid by adding hydrogen to the raw material lecithin, phosphatidylethanolamine prepared by the method or lysophosphatidylethanolamine prepared by the method. The manufacturing method to make. A water-soluble composition comprising phosphatidylethanolamine or lysophosphatidylethanolamine prepared by the method according to any one of claims 1 to 14.