JP2012249597A - Method for producing phospholipid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a stable phospholipid of which hydrolysis is suppressed by deactivating remaining PLA2 (phospholipase A2) in a reaction solution in the production of the phospholipid by using the PLA2, and to provide a method for producing the phospholipid inexpensively.SOLUTION: The method for producing the phospholipid having a remaining phospholipase A2 activity of ≤10 units/g includes adding a glycerol solution of inorganic salts, a ≤4C alcohol, and an organic solvent immiscible with the glycerol and dissolving the phospholipid to the phospholipid obtained by an esterification reaction of a fatty acid with a lysophospholipid by the phospholipase A2, standing still the resultant product after being sufficiently stirred, and extracting the organic solvent layer.

Description

  The present invention relates to a phospholipid suitable for food and feed and a method for producing the same.

  Recent research on lipids revealed that highly unsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have various functions such as improved learning function, prevention of arteriosclerosis, and lipid metabolism improvement function. ing. In particular, when DHA is ingested in a form bound to a phospholipid such as phosphatidylcholine, it has a strong anti-oxidation property and stability as compared with the triglyceride type, and because it absorbs well, the physiological activity of DHA is easily expressed. It has become clear. EPA other than DHA and functional fatty acids such as conjugated linoleic acid and arachidonic acid can also be expected to increase physiological activity by binding to phospholipids.

  Therefore, a method for producing phospholipids having various functional fatty acids bound thereto has been disclosed. Above all, only raw materials that are inexpensive and stable in supply, and that can be used for food, are used, and phospholipids are produced with a high reaction rate. Patent Document 1 discloses a method capable of introducing a fatty acid at the 2-position, particularly a phospholipid in which a highly unsaturated fatty acid such as DHA is introduced at the 2-position, and a method for producing the same. Furthermore, after adding an alcohol having 4 or less carbon atoms, a solvent comprising a hydrocarbon solvent and / or a ketone solvent and / or an ester solvent is added to form a glycerin solution layer and a solvent layer, and phospholipids and fatty acids migrate. It is described that the target phospholipid can be obtained by separating (sorting) the solvent layer and purifying with a solvent or silica gel.

  However, compared to the phospholipid hydrolysis reaction, since a large amount of enzyme is required for the ester synthesis reaction of phospholipid, a large amount of phospholipase A2 (hereinafter also referred to as PLA2) remains, which causes problems such as allergies. May occur, and the phospholipid position 2 may hydrolyze over time.

  On the other hand, several techniques for inactivating residual PLA2 in a lysophospholipid solution obtained by a reaction using PLA2 due to allergy problems and the like have been disclosed. For example, in the example of patent document 2, it processed only with neutral protease, in patent document 3, it heat-processed at 70 degreeC or more of fresh water, and after processing with neutral protease only in the example of patent document 4 Through the protein removal process, if these treatments are performed in the phospholipid solution system obtained by the reaction using PLA2, the amount of PLA2 is reduced in any system, but the highly unsaturated fatty acid is deteriorated by heating, Alternatively, the fatty acid at the 2-position of the phospholipid is hydrolyzed by the residual PLA2 during the treatment.

  Patent Document 5 discloses that a lysophospholipid solution obtained by a reaction using PLA2 is treated with a mixed solution of a solvent and water, for example, ethanol or hexane, and an inorganic salt acetone solution. The PLA2 was not sufficiently removed due to the presence of moisture in the system.

JP 2010-68799 A JP-A 63-233750 JP 2002-165580 A JP 2003-93086 A International Publication No. 05/090587

  An object of the present invention is to produce a stable phospholipid in which hydrolysis of the phospholipid is suppressed by inexpensively producing the phospholipid by inactivating the residual PLA2 in the reaction solution in the production of the phospholipid using PLA2. Is to provide.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a phospholipid reaction solution obtained by a reaction between a fatty acid and lysophospholipid by phospholipase A2 contains an alcohol having 4 or less carbon atoms, an inorganic salt glycerin solution, and carbon. Add 5-5 hydrocarbon solvent, stir well and let stand, extract hydrocarbon solvent layer or treat with acidic protease, phospholipase A2 activity will be 10 units / g or less As a result, the present invention has been completed.

  That is, the first of the present invention relates to a phospholipid obtained by an esterification reaction of a fatty acid and lysophospholipid with phospholipase A2, and the remaining phospholipase A2 activity is 10 units / g or less. A preferred embodiment relates to a phospholipid as described above, wherein the remaining phospholipase A2 activity is 5 units / g or less. More preferably, the total amount of fatty acids having 4 or more double bonds and fatty acids having 2 or more conjugated double bonds in the total constituent fatty acids of the phospholipid is 15% by weight or more. About.

  The second aspect of the present invention is (1) a phospholipid obtained by esterification of a fatty acid and lysophospholipid with phospholipase A2, a glycerin solution of inorganic salts and an alcohol having 4 or less carbon atoms, and further phospholipid not miscible with glycerin. The present invention relates to a method for producing a phospholipid having a residual phospholipase A2 activity of 10 units / g or less, characterized in that an organic solvent that dissolves the phospholipase A2 is added and the mixture is sufficiently stirred and allowed to stand to extract the organic solvent layer. . A preferred embodiment relates to (2) a method for producing a phospholipid having a residual phospholipase A2 activity of 10 units / g or less, wherein the amount of water in the glycerol mixed solution of inorganic salts is 10% by weight or less. More preferably, (3) the remaining phospholipase A2 activity is 10 wherein the organic solvent that is immiscible with glycerin and dissolves the phospholipid is a hydrocarbon solvent and / or ether having 5 to 8 carbon atoms. A method for producing a phospholipid having a unit / g or less, (4) a phosphorus having a residual phospholipase A2 activity of 10 units / g or less, wherein the salt concentration in the glycerol solution is 0.2 to 40% by weight (5) The remaining phospholipase A2 activity as described above, wherein the inorganic salt is at least one selected from the group consisting of zinc sulfate, potassium chloride, magnesium chloride, magnesium sulfate, sodium chloride and calcium chloride. A method for producing a phospholipid of 10 units / g or less, (6) the remaining phospho, as described above, wherein the hydrocarbon solvent is hexane A method for producing a phospholipid having a ase A2 activity of 10 units / g or less, (7) a phospholipid having a residual phospholipase A2 activity of 10 units / g or less, wherein the alcohol having 4 or less carbon atoms is ethanol; (8) The method for producing a phospholipid according to the above, wherein the temperature during stirring is 40 to 60 ° C. and the remaining phospholipase A2 activity is 10 units / g or less.

  In the third aspect of the present invention, the remaining phospholipase A2 activity is 10 units / g or less, characterized in that a phospholipid reaction solution obtained by esterification of a fatty acid and lysophospholipid with phospholipase A2 is treated with an acidic protease. The present invention relates to a method for producing a phospholipid. A preferred embodiment relates to the above-mentioned method for producing a phospholipid having a residual phospholipase A2 activity of 10 units / g or less, characterized by treatment with an acidic protease followed by further treatment with a neutral protease.

  According to the present invention, in the production of phospholipids using PLA2, a stable phospholipid in which the remaining PLA2 in the reaction solution is deactivated and hydrolysis of the phospholipid is suppressed, and a method for producing the phospholipid at a low cost. Can be provided.

  Hereinafter, the present invention will be described in more detail. The phospholipid of the present invention is a phospholipid obtained by a reaction between a fatty acid and lysophospholipid by phospholipase A2, and is characterized in that the phospholipase A2 activity remaining in the phospholipid is 10 units / g or less. The smaller the amount of phospholipase A2 remaining in the phospholipid, the better. However, it is preferably 5 units / g or less, more preferably 1 unit / g or less.

  The total amount of fatty acids having 4 or more double bonds and conjugated fatty acids having 2 or more double bonds in the total constituent fatty acids of the phospholipid of the present invention is improved learning function, prevention of arteriosclerosis, lipid It is more preferable to increase the expression of various functions such as a function of improving metabolism, but it is more preferably 15% by weight or more. Examples of fatty acids having four or more double bonds include DHA, EPA, and arachidonic acid (ARA). Examples of fatty acids having two or more conjugated double bonds include conjugated linoleic acid and conjugated linolenic acid. Can be mentioned.

  The phospholipase A2 activity remaining in 1 gram of phospholipid can be measured using soybean lecithin as a substrate. Measurement is performed by adding a solution containing PLA2 to an aqueous solution in which soybean lecithin is dispersed, and measuring how much sodium hydroxide aqueous solution is required per minute in order to maintain a constant pH (for example, 8.0). be able to. At that time, the PLA2 activity remaining in the phospholipid is calculated using a calibration curve prepared in advance with an enzyme standard solution.

  The lysophospholipid in the present invention refers to a phospholipid obtained by removing the fatty acid at the 2-position, and means a lipid different from the phospholipid. As the lysophospholipid used in the present invention, a modified phospholipid can be used. From the viewpoint of availability, those derived from soybeans, rapeseed, and egg yolk are preferable, and those derived from soybeans from the price aspect. More preferably, other plant-derived lysophospholipids can also be used.

  The method of modifying the phospholipid to remove the fatty acid at the 2-position is not particularly limited as long as no harmful substance is used. For example, a method of hydrolyzing the fatty acid at the 2-position of phospholipid using phospholipase A2 or the like Etc. The phospholipid that can be used in this case is a molecule having a glycerin skeleton, a phosphate group, and two fatty acid esters, which can be a substrate for phospholipase A2, and does not include those having a sphingosine skeleton. Specific examples include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and the like.

  In the esterification reaction between the fatty acid and lysophospholipid of the present invention, the fatty acid to be introduced at the 2-position of the lysophospholipid is not particularly limited. However, fatty acids that can increase physiological activity are preferable from the viewpoint of health of consumers today. Examples include fatty acids having four or more bonds and fatty acids having two or more conjugated double bonds, and specifically, advanced conjugated linoleic acid, arachidonic acid, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), etc. An unsaturated fatty acid can be illustrated. As the DHA and EPA, those obtained by hydrolyzing fats and oils obtained mainly from marine animal oils and algae to form free fatty acids can be used.

  In the present invention, the highly unsaturated fatty acid means an unsaturated fatty acid having 3 or more carbon-carbon double bonds or conjugated linoleic acid.

  In addition, when it is difficult to obtain a naturally derived material alone, such as DHA, a mixture of fatty acids containing a desired fatty acid can be used. At that time, the content of the desired fatty acid in the fatty acid mixture is desirably approximately 20% by weight or more. In the case of DHA, the concentration of DHA in the DHA-containing fatty acid is preferably 20% by weight or more, and more preferably 45% by weight or more. In addition, solvent fractionation etc. may be performed after reaction by this invention, and the density | concentration of the phospholipid which is a reaction product may be raised.

The phospholipid of the present invention can be produced, for example, as follows.
<Phospholipid production method for reducing phospholipase A2 remaining in phospholipids using inorganic salts>
(Esterification reaction between lysophospholipid and fatty acid)
Any reaction system can be used as long as it can bind a fatty acid to lysophospholipid using phospholipase A2 as a catalyst. However, since it is highly reactive and can be used in foods, it is preferably carried out in a reaction system using glycerin as a solvent. . That is, the reaction can be carried out by adding appropriate amounts of lysophospholipid, fatty acid and phospholipase A2 to glycerin and stirring for 3 to 48 hours while maintaining the reaction solution at 30 to 60 ° C. It is preferable to add an additive for activating the reaction such as a calcium salt such as calcium chloride for activating the reaction, and other amino acids, to the reaction system.

(Removal of phospholipase A2 from the reaction solution in which phospholipids were generated)
The reaction solution containing the phospholipid obtained by the esterification reaction of the lysophospholipid and the fatty acid, the phospholipid isolated from the reaction solution, and the solution obtained by extracting the phospholipid from the reaction solution have 4 or less carbon atoms. Addition and mixing of glycerin mixed solution of alcohol and inorganic salt, and organic solvent that dissolves phospholipid without being mixed with glycerin. In addition, those addition amounts are appropriately adjusted when the reaction solution or the extraction solution contains the solvent.

  And after mixing, it heat-controls to 20-60 degreeC, and stirs strongly so that a nonpolar solvent and glycerol may not isolate | separate. Preferably, the temperature is adjusted to 40 to 60 ° C. Then, the mixture is allowed to stand for a while to form a glycerin solution layer and a solvent layer, and the solvent layer to which phospholipids and fatty acids have been transferred is separated (sorted). Such addition and preparative operation of the predetermined solvent may be repeated as appropriate in consideration of improvement in purity and work efficiency.

  When the solvent layer from which phospholipids or phospholipids and fatty acids have migrated is separated, the solvent can be distilled off to recover the phospholipids. After distilling off the solvent, it is preferable to recover the precipitated phospholipid by washing with a solvent that dissolves the fatty acid and water without dissolving the phospholipid. Here, acetone etc. are mentioned as a solvent which does not melt | dissolve a phospholipid but dissolves a fatty acid and water.

  Here, examples of the alcohol having 4 or less carbon atoms include methanol, ethanol, propanol and butanol, and at least one selected from the group can be used. Of these, ethanol is preferred because it has low toxicity and can be used as a food additive.

  The inorganic salt is not particularly limited as long as it is soluble in glycerin, but at least one selected from the group consisting of magnesium chloride, magnesium sulfate, zinc sulfate, potassium chloride, sodium chloride and calcium chloride is used. It is preferable. The inorganic salts are preferably mixed and used so as to be 0.2% by weight or more in the whole glycerin mixed solution. If the amount is less than 0.2% by weight, PLA2 may not be sufficiently removed. The higher the concentration of inorganic salts, the higher the PLA2 removal efficiency. If it exceeds 40% by weight, the viscosity of glycerin may increase, or inorganic salts may precipitate and become difficult to handle.

  Here, the preparation of the glycerin mixed solution of inorganic salts is preferably carried out after mixing the inorganic salts with an aqueous solution in consideration of the dispersibility of the salts in glycerin. Furthermore, the smaller the amount of water in the glycerin mixed solution mixed with inorganic salts, the better, but it is preferably 30% by weight or less, more preferably 10% by weight or less, and even more preferably 1% by weight or less. is there. If the amount of water in glycerin exceeds 30% by weight, PLA2 may not be sufficiently removed. Moreover, when adjusting the glycerol mixed solution after making inorganic salt into aqueous solution, it is preferable to use, after distilling off the water in a mixed solution.

  As an organic solvent that dissolves phospholipids without being mixed with glycerin, hydrocarbon solvents and ethers are preferable, and specific examples include heptane, hexane, pentane, diethyl ether, diisopropyl ether, and the like. Hexane is more preferable from the viewpoint.

<Method for Producing Phospholipid that Reduces Activity of Phospholipase A2 Remaining in Phospholipid Using Acid Protease>
(Esterification reaction between lysophospholipid and fatty acid)
This is the same as the phospholipid production method for reducing phospholipase A2 remaining in the phospholipid using the inorganic salts.

(Reduction of phospholipase A2 activity in the reaction solution in which phospholipids are produced)
The phospholipid obtained above is dissolved in an acidic aqueous solution preferably adjusted to pH 4.0 to 1.0, more preferably 3.0 to 1.5, and acidic protease is added. Examples of acidic proteases include animal-derived pepsin and Aspergillus niger-derived protease. The addition amount of the acidic protease is preferably 0.1 to 10 parts by weight with respect to 100 parts by weight of the acidic aqueous solution.

  After the addition of the acidic protease, the optimal temperature of the enzyme may be used, but it is preferable to carry out the enzyme reaction by stirring and maintaining at a temperature not exceeding 60 ° C. in order to prevent deterioration of the phospholipid. The reaction time depends on the amount of enzyme and reaction temperature, but is preferably 1 hour to 48 hours. If it is shorter than 1 hour, the phospholipase A2 activity reducing effect in the obtained phospholipid may be small. If it is longer than 48 hours, the phospholipid deteriorates, or the phospholipid aggregates or the protease is inactivated. The phospholipase A2 activity-reducing effect in the phospholipid obtained in this way may hit the head.

  In order to further reduce the phospholipase A2 activity, it is preferable to neutralize the pH to 4.0 to 8.0 after completion of the acidic protease reaction and add a neutral protease, more preferably 5. Neutralize to 0-7.0 and add neutral protease. Examples of neutral protease include those derived from Aspergillus Oryzae and Bacillus Subtilis.

  After adding the neutral protease, the optimal temperature of the enzyme may be used, but it is preferable to carry out the enzyme reaction by stirring and maintaining at a temperature not exceeding 60 ° C. in order to prevent phospholipid degradation. The reaction time depends on the amount of enzyme and reaction temperature, but is preferably 1 hour to 48 hours. If it is shorter than 1 hour, the phospholipase A2 activity reducing effect in the obtained phospholipid may be small. If it is longer than 48 hours, the phospholipid deteriorates, or the phospholipid aggregates or the protease is inactivated. The phospholipase A2 activity-reducing effect in the phospholipid obtained in this way may hit the head.

  After completion of the enzymatic reaction with acidic protease or acidic protease and neutral protease, phospholipid such as hexane is dissolved, 10 to 200 parts by weight of a solvent immiscible with water is added to 100 parts by weight of acidic aqueous solution and stirred, and the upper layer (Hexane layer) is collected, and the phospholipid can be extracted by removing the solvent in the upper layer. If it is difficult to extract phospholipids with hexane alone due to an increase in the viscosity of the acidic aqueous solution, the extraction efficiency may be improved by adding an alcohol solvent miscible with water such as ethanol. When adding ethanol, it is preferable to add 10-100 weight part with respect to 100 weight part of acidic aqueous solution. The phospholipase A2 activity in the phospholipid obtained by this treatment is greatly reduced.

  In the above, it is preferable to wash with acetone after removing the solvent of the upper layer. By washing with acetone, residual water, fatty acids released by hydrolysis, and the like can be removed.

  The method for producing phospholipids of the present invention can be suitably used for the production of phospholipids for food and feed. For this purpose, even raw materials that are not suitable for food and solvents such as toluene and formamide are used in the production process. If you don't. In addition, the phospholipid thus obtained, particularly one having a highly unsaturated fatty acid introduced at the 2-position thereof, can be suitably used as a highly functional edible or feed phospholipid.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples. In the examples, “parts” and “%” are based on weight.

<Method for measuring residual activity of PLA2>
400 ml of distilled water was added to 6 g of defatted soybean lecithin and stirred at room temperature for 30 minutes. A solution prepared by dissolving 0.7 g of sodium deoxycholate and 0.5 g of calcium chloride dihydrate in 11 ml of water was added to the defatted soybean lecithin solution, and homogenized at 8000 rpm for 15 minutes while cooling with ice. The obtained solution was used as an activity measurement solution. 20 ml of the activity measurement solution was weighed into a 50 ml sample bottle, and 1M sodium hydroxide aqueous solution was added to adjust the pH to 8.0. Disperse 20 mg of the phospholipid sample obtained in Examples and Comparative Examples in 1 ml of distilled water, add the solution to the activity measurement solution adjusted to pH 8.0, adjust the pH to 8.0 again, and adjust the pH to The titer per minute of 20 mM sodium hydroxide solution required to maintain 8.0 was measured. The PLA2 activity remaining in the phospholipid was calculated using a calibration curve prepared in advance with an enzyme standard solution.

<Analysis of fatty acid composition of phospholipid>
10 mg of the phospholipid obtained in Examples and Comparative Examples was dissolved in 2 ml of isooctane, 1 ml of a methanol solution of 0.2 M sodium methylate was added, and the mixture was heated at 60 ° C. with stirring for 10 minutes. The mixture was neutralized with acetic acid, water was added, and the upper isooctane layer was recovered and analyzed by gas chromatography. As the gas chromatograph, “5890 series II” manufactured by Agilent was used. The column was “DB-23” (length 30 m, inner diameter 0.25 mm, film thickness 0.25 μm) manufactured by Agilent, inlet temperature: 260 ° C., detection temperature: 260 ° C., oven temperature: constant 200 ° C. I went there.

(Production Example 1) Synthesis of Phospholipid In a 1 L glass reaction vessel, 200 g of glycerin (manufactured by Sakamoto Pharmaceutical Co., Ltd.), 15 g of lysophosphatidylcholine (“SLP-white lyso” manufactured by Sakai Oil Co., Ltd.), phospholipase A2 (manufactured by Sanyo Fine Co., Ltd. ”, Activity: 50,000 U / g), 6 g of DHA-containing triglyceride (Croda Japan“ Incromega DHA-J46 ”, DHA content: 49.7% by weight) by alkaline hydrolysis according to a conventional method 6 g of fatty acid, 6 g of glycine, 6 g of alanine 2 ml of 2M calcium chloride aqueous solution was added and reacted at 50 ° C. for 24 hours under reduced pressure of 300 Pa while stirring. After completion of the reaction, 100 ml of ethanol and 100 ml of hexane were added to form two layers. From this, the upper layer was recovered, and 15 g of a recovered product obtained by removing the solvent was obtained. To the recovered product, 50 ml of acetone was added and mixed well, and the precipitate obtained by cooling at 0 ° C. for 1 hour was recovered to obtain 10 g of phospholipid. The activity of PLA2 remaining in this phospholipid was 75 U / g. Of the fatty acid composition in the phospholipid, the DHA content was 16.5% by weight.

(Example 1) PLA2 reduction by treatment with inorganic salts, etc. 1 g of glycerin was added to 0.2 g of phospholipid, and 0.4 ml of 2M calcium chloride aqueous solution was added to prepare a glycerin solution. 1 ml of hexane and 1 ml of ethanol were added and stirred at room temperature for 1 hour. After completion of stirring, the mixture was allowed to stand and then the upper layer was collected. After the solvent was distilled off, 1 ml of acetone was added and the mixture was allowed to stand at 0 ° C. for 1 hour to collect a phospholipid precipitate. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

(Example 2) PLA2 reduction by treatment with inorganic salts and the like Phospholipids were treated in the same manner as in Example 1 except that the mixture was stirred at 50 ° C for 1 hour. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

(Example 3)
2 ml of 2M calcium chloride aqueous solution was added to 5 g of glycerin. This glycerin solution was dried at 80 ° C. and 500 Pa for 1 hour. A phospholipid sample (0.2 g) was dissolved in a mixed solvent of 1 ml of hexane and 1 ml of ethanol, and 1 g of the calcium chloride-glycerin solution prepared above was added, and the mixture was vigorously stirred so as not to separate the layers at room temperature for 1 hour. After completion of the stirring, the mixture was allowed to stand to recover the upper layer, the solvent was distilled off, 1 ml of acetone was added, and the mixture was allowed to stand at 0 ° C. for 1 hour to recover a phospholipid precipitate. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

Example 4
The phospholipid sample was treated in the same manner as in Example 3 except that the mixture was stirred at 50 ° C. for 1 hour, and the phospholipid was recovered. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

(Example 5)
After stirring for 1 hour in the same manner as in Example 4, the upper layer was recovered, the solvent was distilled off, and further hexane was added to remove the precipitated glycerin. The solvent was distilled off, 1 ml of acetone was added, and the mixture was allowed to stand at 0 ° C. for 1 hour to recover a phospholipid precipitate. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

(Example 6)
The phospholipid was treated in the same manner as in Example 5 except that 2M potassium chloride aqueous solution was used instead of 2M calcium chloride aqueous solution. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

(Example 7)
The treatment was performed in the same manner as in Example 5 except that a 2M sodium chloride aqueous solution was used instead of the 2M calcium chloride aqueous solution. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

(Example 8)
A phospholipid sample was treated in the same manner as in Example 3 except that 2M magnesium sulfate aqueous solution was used instead of 2M calcium chloride aqueous solution, isopropanol was used instead of ethanol and stirred at 50 ° C. for 1 hour. Was recovered. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

Example 9
Phospholipid samples were treated and phospholipids were recovered in the same manner as in Example 3 except that 2M magnesium chloride aqueous solution was used instead of 2M calcium chloride aqueous solution and diethyl ether was used instead of hexane. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

(Comparative Example 1)
1 g of glycerin was added to 0.2 g of a phospholipid sample, 1 ml of hexane and 1 ml of ethanol were added, and the mixture was stirred at 50 ° C. for 1 hour. After completion of the stirring, the mixture was allowed to stand to recover the upper layer, the solvent was distilled off, 1 ml of acetone was added, and the mixture was allowed to stand at 50 ° C. for 1 hour to recover a phospholipid precipitate. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

(Comparative Example 2)
In accordance with International Publication No. 05/090587, 0.2 g of phospholipid sample was dissolved in 1 ml of hexane and 1 ml of ethanol, and 1 ml of 2.6 M sodium chloride aqueous solution (water 1 ml, sodium chloride 150 mg) was added for 1 hour at 50 ° C. Stir. After completion of the stirring, the mixture was allowed to stand to recover the upper layer, the solvent was distilled off, 1 ml of acetone was added, and the mixture was allowed to stand at 0 ° C. for 1 hour to recover a phospholipid precipitate. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

(Comparative Example 3)
In accordance with International Publication No. 05/090587, 0.2 g of a phospholipid sample is dissolved in 1 ml of hexane and 1 ml of acetone, and 1 ml of 2.6 M sodium chloride aqueous solution (water 1 ml, sodium chloride 150 mg) is added at 50 ° C. for 1 hour. Stir. After completion of the stirring, the mixture was allowed to stand to recover the upper layer, the solvent was distilled off, 1 ml of acetone was added, and the mixture was allowed to stand at 0 ° C. for 1 hour to recover a phospholipid precipitate. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

(Comparative Example 4)
2 ml of 2M calcium chloride aqueous solution was added to 5 g of glycerin. This glycerin solution was dried at 80 ° C. and 500 Pa for 1 hour. A phospholipid sample (0.2 g) was dissolved in hexane (1 ml), the potassium chloride-glycerin solution (1 g) prepared above was added, and the mixture was vigorously stirred at 50 ° C. for 1 hour so as not to separate the layers. After completion of the stirring, the mixture was allowed to stand to recover the upper layer, and the solvent was distilled off. The solvent was distilled off, 1 ml of acetone was added, and the mixture was allowed to stand at 0 ° C. for 1 hour to recover a phospholipid precipitate. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

(Comparative Example 5)
2 ml of 2M calcium chloride aqueous solution was added to 5 g of glycerin. This glycerin solution was dried at 80 ° C. and 500 Pa for 1 hour. A phospholipid sample (0.2 g) was dissolved in 1 ml of diethyl ether, 1 g of the potassium chloride-glycerin solution prepared above was added, and the mixture was vigorously stirred so as not to separate the layers at room temperature for 1 hour. After completion of the stirring, the mixture was allowed to stand to recover the upper layer, and the solvent was distilled off. The solvent was distilled off, 1 ml of acetone was added, and the mixture was allowed to stand at 0 ° C. for 1 hour to recover a phospholipid precipitate. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

(Comparative Example 6)
2 ml of 2M aqueous magnesium sulfate solution was added to 5 g of glycerin. This glycerin solution was dried at 80 ° C. and 500 Pa for 1 hour. A 0.2 g phospholipid sample was dissolved in a mixed solvent of 1 ml of hexane and 1 ml of acetone, 1 g of the magnesium sulfate-glycerin solution prepared above was added, and the mixture was vigorously stirred so as not to separate the layers at 50 ° C. for 1 hour. After completion of the stirring, the mixture was allowed to stand to recover the upper layer, and the solvent was distilled off. The solvent was distilled off, 1 ml of acetone was added, and the mixture was allowed to stand at 0 ° C. for 1 hour to recover a phospholipid precipitate. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

(Comparative Example 7)
In accordance with Japanese Patent Publication No. 2002-165580, 2 ml of water was added to 0.2 g of a phospholipid sample and heated at 80 ° C. with stirring for 90 minutes. After completion of the stirring, 2 ml of hexane was added and stirred for 1 minute, and then allowed to stand to recover the upper layer. The solvent was distilled off, 1 ml of acetone was added, and the mixture was allowed to stand at 0 ° C. for 1 hour to recover a phospholipid precipitate. Table 1 shows the PLA2 residual activity in the collected phospholipid and the DHA content in the fatty acid composition of the phospholipid.

(Example 10) PLA2 reduction by enzyme treatment 1 g of the phospholipid obtained in Production Example 1 was dispersed in 10 ml of 0.2 M aqueous citric acid solution to a pH of 2.4. To this aqueous solution, 120 mg of acidic protease (“Orientase 20A” manufactured by HIBI) was added and stirred at 45 ° C. for 5 hours. After completion of the stirring, 5 ml of ethanol and 5 ml of hexane were added to the reaction solution, stirred and allowed to stand to recover the upper layer (hexane layer). The solvent in the upper layer was distilled off, 5 ml of acetone was added to the residue, and the mixture was allowed to stand at 0 ° C. for 1 hour to recover a phospholipid precipitate. The residual activity of PLA2 in the recovered phospholipid was 4 U / g, and the DHA content in the fatty acid composition of the phospholipid was 16.9% by weight.

(Example 11) PLA2 reduction by enzyme treatment 1 g of a phospholipid sample was dispersed in 10 ml of a 0.2 M aqueous citric acid solution to a pH of 2.4. To this aqueous solution, 120 mg of acidic protease (“Orientase 20A” manufactured by HIBI) was added and stirred at 45 ° C. for 5 hours. After completion of the stirring, 1M sodium hydroxide aqueous solution was added to the reaction solution to adjust the pH to 7.0, 120 mg of neutral protease (“Peptidase R” manufactured by Amano Enzyme) was added, and the mixture was stirred at 45 ° C. for 3 hours. After completion of the stirring, 5 ml of ethanol and 5 ml of hexane were added to the reaction solution, stirred and allowed to stand to recover the upper layer. The solvent in the upper layer was distilled off, 5 ml of acetone was added to the residue, and the mixture was allowed to stand at 0 ° C. for 1 hour to recover a phospholipid precipitate. The residual activity of PLA2 in the collected phospholipid was 1 U / g or less, and the DHA content in the fatty acid composition of the phospholipid was 17.1% by weight.

(Comparative Example 8) PLA2 reduction by enzyme treatment 1 g of a phospholipid sample was dispersed in 10 ml of water, 120 mg of protease (“Peptidase R” manufactured by Amano Enzyme) was added, and the mixture was stirred at 45 ° C. for 5 hours. After the completion of stirring, 5 ml of ethanol and 5 ml of hexane were added to the reaction solution, stirred for 1 minute, and allowed to stand to recover the upper layer. The solvent in the upper layer was distilled off, 5 ml of acetone was added to the residue, and the mixture was allowed to stand at 0 ° C. for 1 hour to recover a phospholipid precipitate. The residual activity of PLA2 in the collected phospholipid was 15 U / g, and the DHA content in the fatty acid composition of the phospholipid was 10.5% by weight.

Claims (13)

A phospholipid obtained by esterification of a fatty acid and lysophospholipid with phospholipase A2 and having a remaining phospholipase A2 activity of 10 units / g or less. The phospholipid according to claim 1, wherein the remaining phospholipase A2 activity is 5 units / g or less. The phosphorus according to claim 1 or 2, wherein the total amount of fatty acids having 4 or more double bonds and fatty acids having 2 or more conjugated double bonds in the total fatty acids of the phospholipid is 15% by weight or more. Lipids. To the phospholipid obtained by esterification of fatty acid and lysophospholipid with phospholipase A2, add a glycerin solution of inorganic salts and an alcohol having 4 or less carbon atoms, and an organic solvent that is immiscible with glycerin and dissolves the phospholipid. A method for producing a phospholipid having a residual phospholipase A2 activity of 10 units / g or less, wherein the organic solvent layer is extracted after stirring. The method for producing a phospholipid according to claim 4, wherein the amount of water in the glycerin mixed solution of inorganic salts is 10% by weight or less, and the remaining phospholipase A2 activity is 10 units / g or less. 6. The remaining phospholipase A2 activity of 10 units / percent of the organic solvent that dissolves phospholipids without being mixed with glycerin is a hydrocarbon solvent having 5 to 8 carbon atoms and / or ethers. The manufacturing method of the phospholipid which is below g. The method for producing a phospholipid having a residual phospholipase A2 activity of 10 units / g or less according to any one of claims 4 to 6, wherein the salt concentration in the glycerin solution is 0.2 to 40% by weight. The remaining phospholipase A2 activity according to any one of claims 4 to 7, wherein the inorganic salt is at least one selected from the group consisting of zinc sulfate, potassium chloride, magnesium chloride, magnesium sulfate, sodium chloride and calcium chloride. A method for producing a phospholipid of 10 units / g or less. The method for producing a phospholipid according to any one of claims 4 to 8, wherein the hydrocarbon solvent is hexane, and the remaining phospholipase A2 activity is 10 units / g or less. The method for producing a phospholipid according to any one of claims 4 to 9, wherein the alcohol having 4 or less carbon atoms is ethanol, wherein the remaining phospholipase A2 activity is 10 units / g or less. The manufacturing method of the phospholipid whose residual phospholipase A2 activity is 10 units / g or less in any one of Claims 4-10 whose temperature during stirring is 40-60 degreeC. A method for producing a phospholipid having a residual phospholipase A2 activity of 10 units / g or less, characterized by treating a phospholipid reaction solution obtained by esterification of a fatty acid and lysophospholipid with phospholipase A2 with an acidic protease. The method for producing a phospholipid according to claim 12, wherein the remaining phospholipase A2 activity is 10 units / g or less, wherein the phospholipase A2 activity is 10 units / g or less.