JP6763521B2 - 2-DHA-lysophosphatidylcholine-containing lipid composition and method for producing the same - Google Patents

Description

The present invention relates to a lipid composition containing lysophosphatidylcholine in which docosahexaenoic acid (DHA) is bound to the 2-position of the glycerin skeleton, and a method for producing the same.

Phospholipids are a general term for lipids having a phosphate ester moiety in their structure, and because they have amphipathic properties, they are used as major constituents of cell membranes together with glycolipids and cholesterol in the living body. Phospholipids in which one of the constituent fatty acids is hydrolyzed are particularly called lysophospholipids and exhibit various physiological activities in the living body. Phospholipids are also industrially used as emulsifiers.

On the other hand, DHA is a kind of polyunsaturated fatty acid having 22 carbon atoms having 6 unsaturated bonds, and is known to have excellent physiological activities such as improvement of memory learning ability, anti-allergy and anti-tumor. ing. In the human body, it exists in the form of DHA-bound phospholipid as a constituent fatty acid of phospholipid, and has various physiological functions. For example, it has been reported that lysophospholipids (2-DHA-lysophospholipids) in which DHA is bound to the 2-position of the glycerin skeleton, especially 2-DHA-lysophosphatidylcholine, are excellent in DHA transport ability to the brain and erythrocyte deformability. There is.

From the above, it is said that 2-DHA-lysophospholipid, particularly 2-DHA-lysophosphatidylcholine or a lipid composition containing a large amount thereof, has high added value in the fields of medicine and food as an excellent emulsifier with physiological activity of DHA. Be expected.

As a method for preparing a lipid composition containing 2-DHA-lysophospholipid, DHA can be prepared by using a suitable organic solvent such as isopropanol from fish such as sardines, mackerel and salmon or aquatic resources containing a large amount of DHA such as squid. A lipid mixture containing a phospholipid (2-DHA-phospholipid) bound to the 2-position of the glycerin skeleton is extracted, and lipase is allowed to act on the lipid mixture to convert it into lysophospholipid, and then solvent partitioning is repeated to perform 2-DHA. -A method for recovering a lipid composition containing a lysophospholipid is known (for example, Patent Document 1). However, in such a method, the content of lysophospholipid and the content of DHA in the lipid composition were not high, and neither the physiological activity nor the emulsifying ability was satisfactory.

Japanese Unexamined Patent Publication No. 9-206088

An object of the present invention is to provide a method for preparing a lipid composition containing a large amount of 2-DHA-lysophospholipid, particularly 2-DHA-lysophosphatidylcholine.

As a result of various studies on a new method for preparing a lipid composition instead of solvent partitioning, the present inventor has excellent emulsifying ability containing a large amount of 2-DHA-lysophosphatidylcholine by using an ion exchange resin and silka gel in combination. It was found that the lipid composition could be prepared, and the following inventions were completed.

1) The following steps (1) to (4)
(1) Step of lipase-treating a lipid mixture containing 2-DHA-phosphatidylcholine (2) A solution prepared by dissolving the lipid mixture after the lipase treatment in a lower alcohol having a concentration of 95 v / v% or more and a strong base ion exchange resin. The process of contacting and adsorbing lipids on a strong base ion exchange resin,
(3) A step of eluting the lipid adsorbed on the strong base ion exchange resin with a lower alcohol having a concentration of 90 v / v% or less, and (4) subjecting the eluted lipid to silica gel chromatography to 2-DHA-lysophosphatidylcholine. The lysophosphatidylcholine content is 70% by weight or more based on the weight of the composition, and the DHA content is 35 based on the weight of the fatty acid of the lipid composition, which comprises the step of recovering the passage liquid containing the above to obtain a lipid composition. A method for producing a 2-DHA-lysophosphatidylcholine-containing lipid composition in an amount of% by weight or more.
2) The production method according to 1), wherein the step (2) further comprises an activated carbon treatment of a lipid mixture dissolved in a lower alcohol having a concentration of 95 v / v% or more.
3) The production method according to 1) or 2), wherein the lipid mixture containing 2-DHA-phosphatidylcholine in step (1) is a lipid mixture derived from squid or salmon.
4) 2-DHA-lysophosphatidylcholine-containing lipid composition in which the lysophosphatidylcholine content is 70% by weight or more based on the weight of the composition and the DHA content is 35% by weight or more based on the fatty acid weight of the lipid composition. object.
5) The lipid composition according to 4), wherein the content of phosphatidylethanolamine and lysophosphatidylethanolamine is 2% by weight or less based on the weight of the composition.
6) The lipid composition according to 4) or 5), wherein the content of cholesterol and free fatty acid is 10% by weight or less based on the weight of the composition.
7) An emulsifier containing the lipid composition according to any one of 4) to 6).

According to the production method of the present invention, a high-value-added lipid composition having beneficial physiological activity based on DHA and excellent emulsifying ability can be produced in large quantities from a lipid mixture containing 2-DHA-phosphatidylcholine at low cost. it can.

The first aspect of the present invention is the following steps (1) to (4).
(1) Step of lipase-treating a lipid mixture containing 2-DHA-phosphatidylcholine (2) A solution prepared by dissolving the lipid mixture after the lipase treatment in a lower alcohol having a concentration of 95 v / v% or more and a strong base ion exchange resin. The process of contacting and adsorbing lipids on a strong base ion exchange resin,
(3) A step of eluting the lipid adsorbed on the strong base ion exchange resin with a lower alcohol having a concentration of 90 v / v% or less, and (4) subjecting the eluted lipid to silica gel chromatography to 2-DHA-lysophosphatidylcholine. The lysophosphatidylcholine content is 70% by weight or more based on the weight of the composition, and the DHA content is 35 based on the weight of the fatty acid of the lipid composition, which comprises the step of recovering the passage liquid containing the above to obtain a lipid composition. The present invention relates to a method for producing a 2-DHA-lysophosphatidylcholine-containing lipid composition which is by weight% or more.

A first aspect of the present invention comprises the step (1) of lipase treating a lipid mixture containing 2-DHA-phosphatidylcholine.

In step (1), 2-DHA-phosphatidylcholine, a lipid mixture recovered from a natural resource containing phosphatidylcholine in which DHA is bound to the 2-position of the glycerin skeleton, is utilized as a target for lipase treatment. Examples include fish tissues such as bonito, tuna, sardines, mackerel and salmon, and fishery resources containing DHA such as swordtip squid, squid, mica, Japanese flying squid, firefly squid and squid, and suitable organic solvents. A lipid mixture that can be recovered by means such as extraction using squid can be mentioned. Also available in the present invention is a lipid mixture that can be recovered from eggs laid by poultry fed a feed containing DHA-containing oil by extraction with a suitable organic solvent.

The content of phosphatidylcholine in the lipid mixture used as a raw material in the step (1) is 10% by weight or more, preferably 20% by weight or more, and more preferably 40% by weight or more with respect to the entire mixture. The DHA content in the lipid mixture is 5% by weight or more, preferably 10% by weight or more, more preferably 20% by weight or more, still more preferably 30% by weight or more, based on the total fatty acid weight in the mixture. .. In addition, the lipid mixture may contain phospholipids other than 2-DHA-phosphatidylcholine, free fatty acids, neutral lipids and other lipids, proteins, amino acids, nucleic acids, etc., and may be in the form of a solution or suspension. May be good.

In the present invention, extraction using an organic solvent from fish skins, ara, squid skins, etc., which are marine wastes, preferably from squid skins or salmon heads, and most preferably from squid skins. The lipid mixture recovered by is utilized. Examples of the organic solvent include ethanol, hexane, isopropyl alcohol, ethyl acetate, acetone, ether, chloroform, methanol, butanol, or a mixture thereof.

2-DHA-phospholipids in such lipid mixtures recovered from natural resources are converted to 2-DHA-lysophospholipids by lipase treatment. The lipase used in the present invention is a 1,3-position specific lipase that hydrolyzes the ester bond at the sn-1 or sn-3 position of the glyceride skeleton, a phospholipase A1 that specifically hydrolyzes the sn-1 position, or A lipase having similar specificity or a lipase having a low activity of hydrolyzing the ester bond of a long-chain unsaturated fatty acid is selected. Such lipases are well known to those skilled in the art, and examples of 1- and 3-position specific lipases include lipase A-10D (Nagasechemtex Co., Ltd.), lipozyme RMIM (Novozymes), lipozyme TLIM (Novozymes), and paratase 20000L. (Novozymes), Lipase A "Amano" 6 (Amano Enzyme), Lipase R "Amano" (Amano Enzyme), Lipase DF "Amano" 15 (Amano Enzyme), etc., and hydrolyzed ester bonds of long-chain unsaturated fatty acids Examples of lipases having low activity include lipase AY "Amano" 30SD (Amano Enzyme), lipase MER "Amano" (Amano Enzyme), and Lipase OF (Meishu Sangyo).

The recovery and lipase treatment of a lipid mixture containing 2-DHA-phosphatidylcholine from marine resources are described in, for example, Patent Document 1 (Japanese Patent Laid-Open No. 9-206088), Patent Document 2 (Japanese Patent Laid-Open No. 2003-93086) and the like. It can be done under certain conditions or conditions modified within the normal practicability of those skilled in the art.

For example, to recover the lipid mixture, an alcohol solution having a concentration of 95 v / v% or more, which is about 10 times as much, is added to the marine resources powdered by freeze-drying or the like, and the mixture is stirred at room temperature to about 60 ° C. for several hours and then the alcohol solution. It can be done by collecting. Further, the same reaction as described above can be carried out in an aqueous system containing no alcohol. The lipase treatment may be carried out under any conditions as long as the fatty acids are hydrolyzed, but preferably the reaction temperature is 0 to 50 ° C., the reaction time is 10 minutes to 48 hours, and the pH of the reaction solution is in the range of 3 to 8. It may be adjusted as appropriate. The lipase treatment can be terminated by inactivating the enzyme by heating or adding an organic solvent, or by removing the enzyme by filtration.

The reaction solution after the lipase treatment can be used as it is, or after performing solvent extraction, concentration treatment using an evaporator or other equipment, or the like, in the next step (2).

In the first aspect of the present invention, a solution in which a lipid mixture after lipase treatment is dissolved in a lower alcohol having a concentration of 95 v / v% or more is brought into contact with a strong base ion exchange resin to convert the lipid into a strong base ion exchange resin. Including the step (2) of adsorbing to.

Step (2) is a batch-type step in which a solution prepared by dissolving the lipid mixture treated with lipase in step (1) in a lower alcohol of 95 v / v% or more and a strong base ion exchange resin are brought into contact with each other by stirring in a tank. However, a suitable column packed with a strong base ion exchange resin using a lower alcohol having a concentration of 95 v / v% or more as a column solvent is put into a lower alcohol having a concentration of 95 v / v% or more in the lipase in step (1). It is preferable to perform column chromatography in which a solution in which the treated lipid mixture is dissolved is circulated and a lipid containing 2-DHA-lysophospholipid is adsorbed on a strongly basic ion exchange resin.

Examples of the lower alcohol having a concentration of 95 v / v% or more include solutions of methanol, ethanol and isopropanol, and ethanol, particularly ethanol having a concentration of 97 v / v% called industrial ethanol or pure ethanol is preferable.

As the strongly basic ion exchange resin that can be used in the step (2), a type I strongly basic ion exchange resin having a quaternary ammonium group introduced as a functional group, for example, a trimethylammonium group, or a type II strong having a dimethylethanolammonium group introduced therein. Basic ion exchange resins and the like can be used. Specific examples of such strongly basic ion exchange resins include PA306S and other strongly basic ion exchange resins "Diaion (trademark) series" (Mitsubishi Chemical), IRA400J, IRA402BL and other type I strongly basic anions. Exchange resin (organo), IRA410J, IRA411 and other type II strongly basic anion exchange resins (organo), DOWNEX ™ 1x2 and other type I strongly basic anion exchange resins (Dow Chemical), etc. Can be mentioned. The amount of the resin used may be appropriately determined according to the amount of the lipid mixture after the lipase treatment and the amount of the solution. For example, 1 to 20 times, preferably 2 to 10 times the amount of the lipid mixture is used. ..

When the adsorption of the lipid to the strong base ion exchange resin is completed, the resin is separated from the solution, and preferably 95 v / v% or more of a lower alcohol is appropriately distributed to wash away the undesired components, and then the next step (3). ).

In step (2), the solution is further treated with activated carbon before contacting the solution obtained by dissolving the lipase-treated lipid mixture with a lower alcohol having a concentration of 95 v / v% or more with the strongly basic ion exchange resin. It is preferable to include it. The type of activated carbon used is not particularly limited, and the amount of activated carbon may be 5 to 20 parts by weight, preferably 5 to 10 parts by weight, per 1 part by weight of the lipid mixture.

The first aspect of the present invention includes a step (3) of eluting the lipid adsorbed on the strong base ion exchange resin with a lower alcohol having a concentration of 90 v / v% or less. The step (3) may be a batch-type step in which the strong base ion exchange resin on which the lipid is adsorbed is stirred in a lower alcohol having a concentration of 90 v / v% or less, and the column is filled with the ion exchange resin of the step (2). If so, a column chromatography step may be performed in which a lower alcohol having a concentration of 90 v / v% or less is circulated in the column to elute the lipid from the strong base ion exchange resin. Preferably, step (3) is a column chromatography step.

The lower alcohol for elution is a lower alcohol aqueous solution having a concentration of 75 to 90 v / v%, preferably 80 to 90 v / v%, and more preferably 85 to 90 v / v%. At the time of elution, the lower alcohol concentration may be switched from 95 v / v% or more in step (2) to the above elution concentration at once, or from 95 v / v% or more to the above elution concentration continuously or stepwise. May be good.

When the step (3) is a batch type step, it is preferable that the elution is performed a plurality of times and the eluates collected in each are collectively subjected to the next step. When the step (3) is column chromatography, it is preferable to circulate a lower alcohol solution corresponding to 2 to 5 times the column volume and collect the eluate. The recovered eluate can be used as it is or in the next step after being concentrated by using an evaporator or other equipment.

The first aspect of the present invention comprises a step (4) of subjecting the lipid eluted in the step (3) to silica gel chromatography and recovering a passing solution containing 2-DHA-lysophosphatidylcholine to obtain a lipid composition. ..

In step (4), silica gel filled with the eluate containing the lipid eluted in step (3) as it is, or a solution obtained by re-dissolving the lipid preferably concentrated or dried from the eluate with lower alcohol in an appropriate column. Silica gel column chromatography is preferred, in which the passing liquid fraction of the column is recovered as a lipid composition containing 2-DHA-lysophosphatidylcholine. Silica gel does not require any special material and may be any silica gel normally used in biochemical applications. The amount of silica gel may be appropriately determined according to the amount of lipid to be distributed in the column and the amount of solution. For example, 1 to 20 times, preferably 2 to 10 times, amount of silica gel is used.

The concentration of the lower alcohol to be distributed on the silica gel in the step (4) may be the same as that used when eluting the lipid in the step (3). Further, it is preferable that the eluate of the step (3) containing the lipid or the lower alcohol solution in which the lipid is dissolved is applied to the silica gel column, and then the lower alcohol solution is further circulated to collect the passing liquid.

The passing liquid from silica gel contains a lipid having a characteristic that the content of lysophosphatidylcholine is 70% by weight or more and 35% by weight or more of the fatty acid in the lipid is DHA. This solution can be concentrated or dried by a suitable method, and such a concentrate or a dry matter can also be utilized as a lipid composition having the above characteristics.

The lipid composition produced by the method according to the first aspect of the present invention has a lysophosphatidylcholine content of 70% by weight or more based on the weight of the composition, and a DHA content based on the weight of the fatty acid of the lipid composition. On the other hand, it has a feature of 35% by weight or more. In addition, in this specification, a fatty acid in a composition is used as a term referring to both a free fatty acid contained in a lipid composition and a fatty acid in an ester-bonded state with a lipid.

The lipid composition produced by the method of the first aspect contains 70% by weight or more, preferably 75% by weight or more, more preferably 80% by weight or more of lysophosphatidylcholine, while the total content of cholesterol and free fatty acids. Is 10% by weight or less, preferably 5% by weight or less, and the total content of phosphatidylethanolamine and lysophosphatidylethanolamine is 2% by weight or less, preferably 1% by weight or less, more preferably general phosphorus described later. It is below the detection limit of the lipid analysis method. Further, the lipid composition is characterized in that DHA is 35% by weight or more, preferably 50% by weight or more, more preferably 60% by weight or more, and even more preferably 70% by weight or more of the fatty acids contained therein. ..

The contents of phospholipids and fatty acids can be measured by analytical methods known to those skilled in the art. For example, a liquid chromatography method using a silica gel-based column, a TLC method and a TLC-FID (thin layer chromatography-flame ionization detection) method are used as methods for analyzing phospholipids, and a gas chromatography method is used as a method for quantifying fatty acids. Can be mentioned.

Further, when the lipid mixture used as a raw material in the step (1) contains EPA, such as a lipid mixture recovered from the skin of squid or the head of a salmon by extraction with an organic solvent, the first embodiment. The lipid composition produced by the above method may also contain EPA. For example, when the total fatty acid when the fatty acid composition of the lipid composition is analyzed by gas chromatography is 100% by weight, DHA accounts for 35% by weight or more and EPA accounts for about 10 to 20% by weight, and palmitic acid or Only small amounts of saturated fatty acids such as oleic acid are detected.

EPA is a kind of polyunsaturated fatty acid, and is known to have a preventive or ameliorating effect on myocardial infarction, ischemic heart disease, arteriosclerotic, cerebral infarction, stroke, thrombotic hyperlipidemia and the like. The lipid composition produced by the method of the first aspect of the present invention contains DHA and EPA, and in particular, by containing DHA as 2-DHA-lysophosphatidylcholine, various physiological activities possessed by DHA and EPA. It is expected that it will demonstrate.

In addition, the lipid composition produced by the method according to the first aspect of the present invention has a high emulsifying ability that the critical micelle concentration at 25 ° C. is on the order of ^10-4 wt%.

The lipid composition produced by the method according to the first aspect of the present invention directly corresponds to the lipid composition according to the second aspect of the present invention. That is, the lipid composition according to the second aspect of the present invention is typically a lipid composition that can be produced by the production method according to the first aspect, particularly a lipid composition derived from aquatic resources.

The lipid composition according to the second aspect of the present invention is suitable for use in fields where phospholipids, particularly lysophospholipids, are required, and is typically used as an active ingredient or emulsifier in the pharmaceutical field or the food field including health foods. be able to.

In the pharmaceutical field, high-purity lysophospholipids, particularly 2-DHA-lysophosphatidylcholine, can be used as active ingredients for therapeutic agents or therapeutic compositions for effective diseases, as well as suspensions and suspensions for injection. It can be used in combination with other drugs and / or pharmaceutically acceptable excipients as a lipid or emulsifier in the preparation of agents, liposome preparations, ointments and other preparations.

In the food field, it can be edible as it is as a lipid, can also be used as an emulsifier for food production, and has functionality expected to have physiological activity of high-purity lysophospholipids, especially 2-DHA-lysophosphatidylcholine. It can also be used as an active ingredient in foods. There are no particular restrictions on the type and form of the food, and it can be used by adding it to beverages, general processed foods containing seasonings, nutritional supplements, supplements, and the like. In addition, the content of the lipid composition of the present invention in the food may be appropriately adjusted as long as the form of the food and the purpose of use of the lipid composition are not impaired.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

<Example 1 Purification of squid-derived lysophospholipid by the column method of the present invention>
1) Extraction of phospholipids from squid and lipase treatment 191 kg of crushed and dried squid from Hokkaido squid was placed in a 900 L extraction tank. About 1000 L of 95% isopropanol was placed in a 5 m ³ tank with a stirring blade kept at 55 ° C., and extraction was performed for 4 hours while circulating the solvent in the tank in the extraction tank. After collecting the extract, the residual squid meal was washed with 200 L of isopropanol and combined with the above extract. The extract was desolvated under reduced pressure to give a concentrated solution containing about 50 L of phospholipid (estimated crude phospholipid content 15.4 kg).

Of this extract, 1/3 was dried with an evaporator to prepare a crude phospholipid of about 4.6 kg for use as a sample without an enzymatic reaction for a later test. In the remaining 2/3 concentrate, 3.0 kg of lipase A-10D derived from Rhizopus japonicus (Nagasechemtex Co., Ltd., a lipase that acts on the sn-1, 3 position of triacylglycerol, but of phospholipids Lipase treatment was performed by adding (retaining the phosphoripase A1 activity acting at the 1st position) and 100 L of water and stirring at 30 ° C. overnight. The liquid after the enzyme treatment was extracted with 40 kg of butanol, desolvated under reduced pressure, and dried to dryness to obtain about 10.8 kg of crude lysophospholipid derived from squid.

2) Purification of lysophospholipid 120 ml of strong base ion exchange resin (Diaion PA306S) is placed on a glass column (inner diameter 30 mm x length 400 mm), and silica gel (AGC SI-) is placed on another glass column (inner diameter 30 mm x length 400 mm). 100 ml of Tech.KK.D-50-120A) was filled, and 200 ml of 97% ethanol was flowed through each column.

50.70 g of the squid-derived crude lysophospholipid obtained in 1) was dissolved in 260 ml of 97% ethanol, and this solution was first flowed on a column of a strongly basic ion exchange resin to adsorb the lysophospholipid on the resin. After washing with 97% ethanol in an amount twice the volume of the resin, the lysophospholipid was eluted with 85% ethanol in an amount twice the volume of the resin. The eluate was further poured on silica gel (AGC SI-Tech.KK.D-50-120A), and the passing solution was collected and concentrated to obtain 22.13 g of a purified lysophospholipid, which is the lipid composition of the present invention. ..

<Example 2 Purification of squid-derived lysophospholipid by the column method of the present invention>
100 ml of strong base ion exchange resin (Diaion PA306S) is placed on a glass column (inner diameter 30 mm x length 400 mm), and silica gel (AGC SI-Tech.KK. D-) is placed on another glass column (inner diameter 30 mm x length 400 mm). 100-60A) 100 ml was filled and 200 ml of 97% ethanol was flowed through each column.

30.68 g of squid-derived crude lysophospholipid obtained in 1) of Example 1 was dissolved in 200 ml of 97% ethanol, and this solution was first flowed on a strong base ion exchange resin column to adsorb the lysophospholipid on the resin. .. After washing with 97% ethanol in an amount twice the volume of the resin, the lysophospholipid was eluted with 85% ethanol in an amount twice the volume of the resin. The eluate was further poured on a silica gel (AGC SI-Tech.KK. D-100-60A) column, and the passing solution was collected and concentrated to obtain 13.81 g of a purified lysoline lipid, which is the lipid composition of the present invention. It was.

<Example 3 Purification of squid-derived lysophospholipid by the column method of the present invention>
3000 ml of strong base ion exchange resin (Diaion PA306S) was placed on a glass column (inner diameter 95 mm x length 600 mm), and silica gel (Kanto Chemical KK silica gel 60-40) was placed on another glass column (inner diameter 95 mm x length 600 mm). -50 μm) 2500 ml was filled and 3000 ml of 97% ethanol was flowed through each column.

1002 g of squid-derived crude lysophospholipid obtained in 1) of Example 1 was dissolved in 4000 ml of 97% ethanol, and this solution was first flowed on a strong base ion exchange resin column to adsorb the lysophospholipid on the resin. After washing with 97% ethanol in an amount twice the volume of the resin, the lysophospholipid was eluted with 85% ethanol in an amount twice the volume of the resin. The eluate was further poured on a silica gel (Kanto Chemical KK silica gel 60-40-50 μm) column, and the passing solution was collected and concentrated to obtain 352.0 g of a purified lysophospholipid, which is the lipid composition of the present invention. ..

<Example 4 Purification of squid-derived lysophospholipid by the column method of the present invention>
125 kg of squid-derived crude lysophospholipid obtained in the same manner as in 1) of Example 1 was dissolved in 400 L of 97% ethanol, 5 to 10% of activated carbon was added, and the mixture was stirred at room temperature for 30 minutes. The mixed solution was filtered using a diatomaceous earth precoated filter press to remove activated carbon, and the filtrate was recovered.

The collected filtrate was passed through a column packed with 500 L of a strong base ion exchange resin (Diaion PA306S), and the lysophospholipid was adsorbed on the resin. After washing with 97% ethanol in an amount twice the volume of the resin, the lysophospholipid was eluted with 85% ethanol in an amount twice the volume of the resin. The eluate was further poured on a silica gel (AGC SI-Tech.KK. D-100-60A) column, and the passing solution was collected and concentrated to obtain about 50 kg of a purified lysoline lipid, which is the lipid composition of the present invention. ..

<Comparative example Purification of squid-derived lysophospholipids by solvent partitioning method>
8 ml of n-hexane: ethanol: water (10: 1: 1 by volume) was added to 335 mg of the crude lysophospholipid derived from squid obtained in 1) of Example 1 to separate layers, and the n-hexane layer was removed. It was distributed once. Next, 3.5 ml of n-hexane was added to separate the layers, and the n-hexane layer was removed. The same process was repeated with 3 ml, 2.5 ml, and 2 ml of n-hexane (add a small amount of ethanol if the viscosity of the liquid phase became high and the layer did not separate), and the ethanol / aqueous layer obtained after 5 times of partitioning was recovered. By concentrating, a purified lysophospholipid by the solvent partitioning method was obtained.
Here, for comparison, a purified lysophospholipid fractioned once or three times with a solvent was also prepared.

<Test Example 1 Fatty acid composition and lipid class composition of squid-derived phospholipids>
The purified squid-derived lysophospholipids obtained in Examples and Comparative Examples, and each sample in the preparation step thereof were methyl esterified with 2N Methanolic Sodium Hydroxide and 2nd Methanolic Hydrogen Chloride, and then gas chromatographed. Graffiti (Column: G column G-300, PEG-20M, 40 m x 1.2 mm, film thickness 0.5 μm, Chemical Substance Evaluation and Research Organization; Equipment name: GC-353, GL Science Co., Ltd .; Column temperature: 190 The fatty acid composition was analyzed by ° C.; injection temperature: 250 ° C.; detector temperature: 250 ° C.; carrier gas: helium 10 ml / min; detector: FID).

Further, the crude phospholipid (sample before enzyme treatment of Example 1) and the crude lysophospholipid (sample after enzyme treatment of Example 1) were developed by TLC, and phosphatidylcholine (PC) or lysophosphatidylcholine (LPC) was developed. The fatty acid composition of the PC sample and the LPC sample obtained by scraping the spots of)) was also analyzed in the same manner.

The results are shown in Table 1 in% by weight. Purification by the column method increased the content of polyunsaturated fatty acids. Moreover, when the PC sample and the LPC sample were compared, the content of polyunsaturated fatty acids, particularly DHA, was nearly doubled by the enzyme treatment.

＊１ リン脂質に結合している脂肪酸全ての合計値
＊２ 遊離脂肪酸とリン脂質に結合している脂肪酸全てとの合計値

* 1 Total value of all fatty acids bound to phospholipids * 2 Total value of all fatty acids bound to phospholipids and free fatty acids

In addition, the lipid class composition of these samples was analyzed by the TLC-FID method using Iatroscan (LSI Medience Co., Ltd.). After dissolving the sample in chloroform: methanol (2: 1, v / v) to a concentration of 1%, 5 micron was spotted on the chromatorod SIII. Chloroform: methanol: water = 45:20: 2 (v / v) was developed twice (45 minutes × 2 times), and then analyzed by iatroscan (detector: FID). Since the obtained data is proportional to the number of carbon atoms having a CH bond, it was corrected by the number of carbon atoms and the average molecular weight, and converted to% by weight.

The results are shown in Table 2 in% by weight. In the conventional solvent fractionation method, phosphatidylethanolamine (PE) and lysophosphatidylethanolamine (LPE) remained even after the fractionation was repeated 5 times, whereas in the column method of the present invention, PE and LPE remained. Below the detection limit, the amount of cholesterol + fatty acid was also reduced to less than half that of the conventional method, and the ratio of LPC was increased to more than 80%. The analysis values of the purified lysophospholipids of Example 4 are shown in the table, but the purified lysophospholipids of Examples 1 to 3 had the same composition.

<Test Example 2 Evaluation of emulsion stability of purified squid-derived lysophospholipid>
Using the squid-derived lysophospholipid purified products obtained in Example 4 and Comparative Example as an emulsifier, an emulsified liquid dressing-like salad seasoning was prepared, and its emulsification stability was evaluated. After mixing each emulsifier with water so that the amount of the emulsifier was 0.25 g and the total amount was 1.3 g, the mixture was left to dissolve overnight. Table 3 shows the types of emulsifiers used and the amount of water blended. Total PL (phospholipid) content was measured by iatroscan.

After adding 1.5 g of rice vinegar and 0.2 g of salt to this mixture, 7 g of Chilean cultured coho salmon meal oil (commercially available coho salmon is freeze-dried, crushed, and then extracted with hexane / isopropanol (2: 1). The oil was recovered and deoxidized) was added in 3 portions to homogenize (homogenizer VH-10, shaft generator S10N-10G (all from AS ONE Corporation)). After emulsification by homogenization at 11,400 rpm, additional emulsification was performed by homogenization at 20,450 rpm for 30 seconds. After nitrogen substitution with a nitrogen gas generator, the mixture was left at room temperature for 2 hours and then left at 80 ° C. for 14 days to examine the time at which the emulsification began to break and the time at which the emulsification completely broke. Heating at 80 ° C. is an accelerated test to break the emulsification quickly.

The results are shown in Table 4. The lysophosphatid purified by the column method of the present invention was far superior in emulsion stability to existing emulsifiers containing commercially available lysolecithin derived from soybean and purified products by the solvent fractionation method.

<Test Example 3 Evaluation of emulsifying properties of purified lysophospholipids>
An oil containing phospholipids was prepared from the head of salmon in the same manner as in 1) of Example 1 and subjected to enzymatic treatment, and crude phospholipids derived from salmon containing LPC and LPE to the same extent (of which, as constituent fatty acids of LPC). DHA 54.3% by weight and EPA 15.3% by weight) were obtained. This crude lysophospholipid was subjected to purification by the column method in the same manner as in Examples 1 and 2) and Examples 2 to 4, to obtain a salmon-derived lysophospholipid purified product mainly composed of LPC.

In order to evaluate the emulsification characteristics of this salmon-derived crude lysophospholipid and lysophospholipid purified product, and the squid-derived lysophospholipid purified product obtained in Example 4, the critical micelle concentration (CMC) was determined by Kyowa Surface Chemistry's surface tension meter CAVP-. It was measured at 25 ° C. by the suspension plate method using A3. CMC is the minimum surfactant concentration required for micelle formation, and the smaller the value, the higher the ability as a surfactant.

The bending point of the graph with the emulsifier concentration (% by weight) on the horizontal axis and the surface tension (mN / m) on the vertical axis was calculated as CMC. When there were multiple bending points for one emulsifier, the smallest bending point was used for evaluation.

The results are shown in Table 5. The CMC of the crude salmon-derived crude lysophospholipid was 100 times higher than that of the existing emulsifier, sucrose fatty acid ester. On the other hand, the CMC of the salmon-derived lysophospholipid purified product and the squid-derived lysophospholipid purified product were both at the same level as the CMC of the sucrose fatty acid ester. Was also confirmed to exhibit excellent emulsifying properties.

Claims (3)

The following steps (1) to (4)
(1) Step of lipase-treating a lipid mixture containing 2-DHA-phosphatidylcholine (2) A solution prepared by dissolving the lipid mixture after the lipase treatment in a lower alcohol having a concentration of 95 v / v% or more and a strong base ion exchange resin. The process of contacting and adsorbing lipids on a strong base ion exchange resin,
(3) A step of eluting the lipid adsorbed on the strong base ion exchange resin with a lower alcohol having a concentration of 90 v / v% or less, and (4) subjecting the eluted lipid to silica gel chromatography to 2-DHA-lysophosphatidylcholine. The lysophosphatidylcholine content is 70% by weight or more based on the weight of the composition, and the DHA content is 35 based on the weight of the fatty acid of the lipid composition, which comprises the step of recovering the passage liquid containing the above to obtain a lipid composition. A method for producing a 2-DHA-lysophosphatidylcholine-containing lipid composition in an amount of% by weight or more. The production method according to claim 1, wherein the step (2) further comprises an activated carbon treatment of a lipid mixture dissolved in a lower alcohol having a concentration of 95 v / v% or more. The production method according to claim 1 or 2, wherein the lipid mixture containing 2-DHA-phosphatidylcholine in the step (1) is a lipid mixture derived from squid or salmon.