WO2007063886A1 - Method for producing phospholipid containing polyvalent unsaturated fatty acid, and phospholipid containing polyvalent unsaturated fatty acid obtained by such method - Google Patents

Method for producing phospholipid containing polyvalent unsaturated fatty acid, and phospholipid containing polyvalent unsaturated fatty acid obtained by such method Download PDF

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Publication number
WO2007063886A1
WO2007063886A1 PCT/JP2006/323790 JP2006323790W WO2007063886A1 WO 2007063886 A1 WO2007063886 A1 WO 2007063886A1 JP 2006323790 W JP2006323790 W JP 2006323790W WO 2007063886 A1 WO2007063886 A1 WO 2007063886A1
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Prior art keywords
fatty acid
phospholipid
polyunsaturated fatty
unsaturated fatty
antioxidant
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PCT/JP2006/323790
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French (fr)
Japanese (ja)
Inventor
Naruyuki Taniwaki
Xiaoli Liu
Tetsuyuki Sasaki
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Nagase Chemtex Corporation
Wakunaga Pharmaceutical Co., Ltd.
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Priority to JP2007547965A priority Critical patent/JPWO2007063886A1/en
Publication of WO2007063886A1 publication Critical patent/WO2007063886A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
    • A23D9/013—Other fatty acid esters, e.g. phosphatides
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B5/00—Preserving by using additives, e.g. anti-oxidants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6481—Phosphoglycerides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P9/00—Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen

Definitions

  • the present invention relates to a method for producing a polyunsaturated fatty acid-containing phospholipid using phospholipase D. More specifically, in the method for producing a polyunsaturated fatty acid-containing phospholipid using phospholipase D, an antioxidant is added, and the blending ratio of the nonpolar solvent and the polar solvent, and the phospholipid and the hydroxyl group-containing compound are added.
  • the present invention relates to a production method in which the reaction is carried out with a limited mixing ratio. Furthermore, the present invention relates to a polyunsaturated fatty acid-containing phospholipid obtained by the production method and an oil containing the polyunsaturated fatty acid-containing phospholipid.
  • Phospholipids are known to have various medicinal effects depending on their types, and many studies have been conducted so far.
  • docosahexaenoic acid-containing phosphatidylserine which is one of phospholipids having polyunsaturated fatty acid residues, has the effect of improving learning ability and improving intellectual ability to adapt to environmental changes. It has been reported (see Patent Document 1).
  • Phospholipids are known to be obtained by a base exchange reaction using phospholipase D (hereinafter abbreviated as PLD) as a catalyst.
  • PLD catalyzes a base exchange reaction between a phospholipid such as phosphatidylcholine (hereinafter abbreviated as PC), phosphatidylethanolamine (hereinafter abbreviated as PE), and an alcohol as a receptor.
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • an alcohol as a receptor.
  • PG phosphatidylglycerol
  • PS phosphatidylserine
  • PA phosphatidic acid
  • the base exchange reaction of phospholipids using PLD is carried out in a two-phase system that also has an aqueous phase and an organic solvent compatibility, but it is a component of human-dose components such as docosahexaenoic acid-containing phosphatidylserine. It is not desirable to use organic solvents that are harmful to the human body for production. A method of performing a base exchange reaction without using an organic solvent can be used.
  • Such a method includes a method in which an enzyme is dissolved in an aqueous medium containing a phospholipid suspension of a phospholipid and a reagent having a hydroxyl group, and then the reaction is performed by adding silica gel and stirring.
  • Patent Document 2 See Patent Document 2, and a method has been proposed in which hexane, acetone, which is safe for use in food applications as an organic solvent, is used, and the reaction is performed in a reaction solvent composed of hexane, acetone, and water (see Patent Document 3). ).
  • a method has been devised that suppresses the hydrolysis reaction by minimizing the amount of water (10% or less).
  • a force mainly containing a receptor such as PLD or alcohol in the aqueous phase for example, a method of adding an aqueous phase containing these components in a reverse micelle and adding it to the reaction system (see Patent Document 4)
  • Patent Document 5 There is a method in which a phospholipid and a receptor are reacted with each other in a state where the water content in the reaction system is 1% by weight or less by contacting with a PLD in which the phospholipid and the receptor are adsorbed on a carrier.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2005-104893
  • Patent Document 2 Japanese Translation of Special Publication 2003-505035
  • Patent Document 3 Japanese Patent Laid-Open No. 2001-186898
  • Patent Document 4 Japanese Patent Publication No. 7-16426
  • Patent Document 5 Japanese Patent Publication No. 3-67676
  • Patent Document 6 Japanese Patent Application Laid-Open No. 2003-304814
  • the present invention provides a method for producing polyunsaturated fatty acid-containing phospholipids in a two-phase system.
  • a production method for suppressing oxidation of saturated fatty acid a polyunsaturated fatty acid-containing phospholipid obtained by the production method, and an oil / fat containing the polyunsaturated fatty acid-containing phospholipid.
  • the present invention comprises a step of reacting a polyunsaturated fatty acid-containing phospholipid and a hydroxyl group-containing compound in a two-phase system comprising a nonpolar solvent, a polar solvent and water in the presence of an antioxidant and phospholipase D.
  • the content of the polar solvent is more than 20% by volume and 80% or less based on the total amount of the nonpolar solvent and the polar solvent, and the hydroxyl group-containing compound for phospholipids
  • the present invention relates to a method for producing a polyunsaturated fatty acid-containing phospholipid, characterized in that the molar ratio is from 4 to 30.
  • the antioxidant is a fat-soluble antioxidant.
  • the fat-soluble antioxidant is vitamin E or a derivative thereof.
  • the water content is 30 to 200 on a volume basis with respect to the total amount of the nonpolar solvent and the polar solvent.
  • the polyunsaturated fatty acid-containing phospholipid is preferably squid-derived lecithin.
  • the present invention also relates to a polyunsaturated fatty acid-containing phospholipid obtained by the production method.
  • composition ratio (n-3 / n-6) of the n-3 polyunsaturated fatty acid and the n-6 polyunsaturated fatty acid constituting the polyunsaturated fatty acid-containing phospholipid is 25 or more. I prefer to be there.
  • the present invention also relates to an oil or fat comprising the polyunsaturated fatty acid-containing phospholipid.
  • the method for producing a polyunsaturated fatty acid-containing phospholipid of the present invention comprises a polyunsaturated fatty acid-containing compound in a two-phase system comprising a nonpolar solvent, a polar solvent and hydraulic power in the presence of an antioxidant and phospholipase D. It comprises reacting a phospholipid with a hydroxyl group-containing compound.
  • the polyunsaturated fatty acid-containing phospholipid used in the present invention is a phospholipid to which a residue of a polyunsaturated fatty acid is bound.
  • a natural product containing phospholipids of any origin an extract from the natural product, a purified product of the extract, a synthetic phospholipid, etc. are preferably used.
  • Examples of such phospholipids are phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PE PI) and mixtures thereof.
  • polyunsaturated fatty acids include known ones such as linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, and arachidonic acid.
  • the above phospholipids may be used alone or in combination of two or more.
  • lecithin derived from plants such as soybeans, rapeseed, rice, sunflower, palm, etc .; egg yolk, tuna, squid, sardine, bonito, salmon roe, krill
  • lecithin derived from animals such as scallops; lecithin derived from microorganisms such as yeast;
  • squid-derived lecithin is particularly preferred because of the high proportion of n-3 polyunsaturated fatty acids in the phospholipid.
  • the purity of the phospholipid is preferably 20 to 99.5% by mass based on the total mass of the compound.
  • the phospholipid is preferably used in an amount of 0.1 to 60% by mass in this reaction system.
  • the antioxidant used in the present invention is not particularly limited, and examples thereof include a fat-soluble antioxidant and a water-soluble antioxidant.
  • Examples of the fat-soluble antioxidant include coenzyme Q10, vitamin A, vitamin E, and derivatives thereof.
  • examples of the water-soluble antioxidant include superoxide dismutase (SOD), glutathione, vitamin C, grape seed extract, pycnogenol, strength techin, austaxanthin and amino acids.
  • SOD superoxide dismutase
  • glutathione glutathione
  • vitamin C grape seed extract
  • pycnogenol strength techin
  • austaxanthin amino acids.
  • Vitamin E or a derivative thereof, for which an antioxidant is preferred is more preferred.
  • examples of the vitamin E derivative include tocopherol nicotinate and tocopherol acetate.
  • the antioxidant may be any extract from a natural product, a product obtained by purifying the extract, or a synthetic product.
  • vitamin E or a derivative thereof is extracted from vegetable oil or the like. Can also be used.
  • the antioxidant may be liquid regardless of purity, or may be solid as long as it becomes liquid during the base exchange reaction.
  • the addition amount of the antioxidant is preferably 1 to 15 parts by weight with respect to 100 parts by weight of the polyunsaturated fatty acid-containing phospholipid.
  • the addition amount is less than 1 part by weight and exceeds 15 parts by weight, the aqueous phase and the organic solvent phase are not sufficiently separated, and separation and purification of phospholipids becomes difficult. Anti-wrinkle effect may not be sufficient. More preferably, it is 2.5 to 15 parts by weight.
  • the antioxidant is preferably added before starting the reaction. If it is added before the start of the base exchange reaction, it is possible to prevent acidity of the phospholipid. Furthermore, the aqueous phase and the organic solvent phase are well separated, and separation and purification of the phospholipid after the reaction is easy.
  • the nonpolar solvent used in the present invention is not particularly limited, and examples thereof include aliphatic hydrocarbons such as heptane, hexane, and petroleum ether; cyclic aliphatic hydrocarbons such as cyclopentane and cyclohexane; , Ethers such as tetrahydrofuran; methyl acetate, vinegar Examples thereof include esters such as oxyethyl; halogenated hydrocarbons such as tetrasalt carbon and carbon form.
  • heptane and hexane are preferable.
  • the polar solvent is not particularly limited, and examples thereof include ketones such as acetone, methyl ethyl ketone, and ethyl ketone.
  • acetone is preferable.
  • the content of the polar solvent in the two-phase system consisting of the nonpolar solvent, the polar solvent and the hydraulic power is 20 on a volume basis with respect to the total amount of the nonpolar solvent and the polar solvent.
  • the polyunsaturated fatty acid-containing phospholipid is reacted with the hydroxyl group-containing compound in an amount of more than% and not more than 80%.
  • the content of the polar solvent is 20% or less or exceeds 80%, the yield of the target phospholipid is lowered.
  • the content of the polar solvent is preferably 25 to 50%, more preferably 25 to 40%.
  • the phospholipid is dissolved in an organic solvent phase consisting of a nonpolar solvent and a polar solvent during the base exchange reaction, and the nonpolar solvent and the polar solvent.
  • the organic solvent is usually 1 to: L OOml with respect to phospholipid lg.
  • Examples of water used in the production method of the present invention include ion-exchanged water, purified water, distilled water, and tap water. Furthermore, acetic acid or the like may be contained in these to form a buffer solution for pH adjustment. Preferably, ion exchange water, purified water or distilled water is used.
  • the content of water is preferably 30 to 250% on a volume basis with respect to the total of the nonpolar solvent and the polar solvent, and more preferably 30 to 200%. More preferably, it is 50 to 150%.
  • Examples of the hydroxyl group-containing compound used in the present invention include alcohols, nitrogen-containing alcohols, saccharides, polyols, and hydroxy cyclic compounds.
  • alcohols examples include methanol, ethanol, propanol, ascorbic acid and the like.
  • nitrogen-containing alcohols include amino acids such as serine; 1-amino-2-propanol.
  • saccharide examples include adenosine, guanosine, inosine, xanthosine, and deoxyade.
  • nucleosides such as nocine and deoxyguanosine; glucose, trehalose, N-acetyl-D darcosamine and the like.
  • polyols examples include glycerol, ethylene glycol, propylene glycol and the like.
  • hydroxy cyclic compound examples include oxalic acid, arbutin and the like.
  • the reaction is preferably performed in a state where the hydroxyl group-containing compound is dissolved in water.
  • the ratio between the hydroxyl group-containing compound and water is not particularly limited. For example, when serine is used as the hydroxyl group-containing compound, 3 to 20 ml of water is added to serine lg. I like it!
  • the production method of the present invention is carried out under conditions where the molar ratio of the hydroxyl group-containing compound to the polyunsaturated fatty acid-containing phospholipid is 4 or more and 30 or less. If the molar ratio of the hydroxyl group-containing compound is less than 4, the yield of the phospholipid produced will be low.
  • the molar ratio is preferably 6 or more, more preferably 8 or more. On the other hand, if the molar ratio exceeds 30, the cost increases, which is unrealistic.
  • the production method of the present invention is carried out in the presence of phospholipase D (PLD).
  • PLD phospholipase D
  • the PLD is not particularly limited as long as it can hydrolyze and Z or base-exchange the phospholipid base moiety, and examples thereof include plant-derived PLD and microorganism-derived PLD.
  • Plant-derived PLDs include cabbage and soybean-derived PLDs, and microorganism-derived PLDs include actinomycetes and filamentous fungal PLDs.
  • the concentration of PLD in this reaction system is preferably 5 to 200 U, more preferably 20 to 100 U, relative to phospholipid lg. 1U uses 95% soybean phosphatidylcholine as a substrate, and 0.2M acetate buffer (pH 4.0, 10 mM CaCl, 1.3% Tri ton X-100) containing 0.16% substrate concentration.
  • the base exchange reaction is preferably performed under the conditions of pH 3.5 to 10, and more preferably pH 4 to 9.
  • the reaction temperature is preferably 10 to 40 ° C, more preferably 20 to 30 ° C. Further, since the reaction system is a two-phase system, it is preferable to stir the reaction system during the base exchange reaction.
  • PC, PE, etc. in polyunsaturated fatty acid-containing phospholipids PS and the like are obtained by reaction with a hydroxyl group-containing compound such as serine.
  • the PLD is deactivated by a treatment such as heating, and the organic solvent layer and the aqueous layer are separated by a centrifugal method or the like to obtain the organic solvent layer.
  • the organic solvent is removed under reduced pressure. Concentrate by removing.
  • crystallization is performed with acetone or ethanol, a solid is obtained by solid-liquid separation, and dried, whereby the polyunsaturated fatty acid-containing phospholipid produced by the production method of the present invention can be isolated.
  • the polyunsaturated fatty acid-containing phospholipid of the present invention is obtained by the above production method.
  • Kisaen acid to docosapentaenoic constituting the phospholipids eicosapentaenoic acid, such as n- 3 polyunsaturated fatty acids and Reno one Le acids
  • Ya Arakidon acids such as a-linolenic acid n-
  • the composition ratio of 6-series polyunsaturated fatty acids (n-3Zn-6) force 25 is preferably 25 or more, more preferably 30 or more.
  • composition ratio (n-3Zn-6) By setting the composition ratio (n-3Zn-6) to less than 25, excessive intake of n-6 polyunsaturated fatty acids in the current diet can be avoided, and the risk of heart disease can be suppressed.
  • the upper limit the higher the value, the better, considering the current dietary fatty acid intake rate, which is not particularly limited.
  • the fat and oil of the present invention is not particularly limited as a lipid other than the phospholipid and may contain a lipid other than the phospholipid as long as it contains the polyunsaturated fatty acid-containing phospholipid. It is not a thing.
  • the ratio of the polyunsaturated fatty acid-containing phospholipid in the fat is 10 to: LOO mass%, but the higher the ratio of phospholipid, the more effective, so preferably 20 to: LOO mass%, more preferably Is 30-100%.
  • the polyunsaturated fatty acid-containing phospholipids and fats and oils of the present invention are tablets, granules, powders, turnips.
  • Various forms of preparations such as cell agents and soft capsules can be prepared. Since the phospholipids and fats and oils of the present invention have high purity and can be easily powdered, they are suitable for production of tablets, granules, powders, etc., and are provided only in powder form such as powdered milk. It is easy to add to the product.
  • known excipients, disintegrants, lubricants and the like can be used, and they may be blended with water-soluble drugs (herbal extracts, vitamin C, etc.).
  • the blending ratio of the nonpolar solvent and the polar solvent and the blending ratio of the polyunsaturated fatty acid-containing phospholipid and the hydroxyl group-containing compound are limited.
  • the base exchange reaction proceeds efficiently, so that a large amount of the hydroxyl group-containing compound can be added, and the yield of the target phospholipid can be increased. it can.
  • an antioxidant added to the reaction system, oxidation of polyunsaturated fatty acids is suppressed without reducing the yield of phospholipids, and the aqueous phase and organic solvent phase are well separated after the reaction. Therefore, separation and purification of the phospholipid to be produced becomes simple and high-purity phospholipid can be obtained.
  • Riken E Oil 600 (Vitamin E manufactured by Riken Vitamin Co., Ltd.) was added at 0 to 10 parts by weight at a concentration shown in Table 1 with respect to 100 parts by weight of lecithin to obtain a lecithin solution.
  • this solution is referred to as a lecithin solution.
  • L-serine manufactured by Kyowa Hakko was dissolved in 0.6 M sodium acetate buffer (PH 4.0) to a concentration of 25% (weight Z volume).
  • this solution is referred to as a serine solution.
  • the lecithin solution and the serine solution prepared as described above were used as a molar ratio of lecithin and serine.
  • phospholipase D manufactured by Nagase ChemteX
  • Mobile phase: Acetonitrile: Methanol: 1 OmM Ammonium dihydrogen phosphate 619: 291: 90
  • the PS production rate represents the amount of PS produced relative to the amount of PC and PE before the reaction.
  • PS generation rate (Amount of generated PS Z amount of PC + PE before reaction) X 100
  • the sample was mixed with 1 ml of a 5 mg / ml black mouth form solution and mixed with 1 ml of methanol containing 10% hydrogen chloride, and treated at 80 ° C. for 3 hours. Further, 1 ml of hexane and 6 ml of distilled water were added and mixed well, and then the hexane'form layer separated by centrifugation was collected and used for GC.
  • Carrier gas helium
  • Gas pressure Helium (carrier gas) 150 kPa, nitrogen (make-up gas) 75 kPa, hydrogen 60 kPa, air 50 kPa
  • a serine solution was prepared in the same manner as in Example 1.
  • the prepared lecithin solution and serine solution were mixed with lecithin and serine in molar ratio.
  • phospholipase D manufactured by Nagase ChemteX
  • the following formulation containing PS obtained in Example 2 was mixed with a V-type mixer and tableted with a tableting machine to obtain tablets with a diameter of 7.5 mm and 200 mg.
  • the following formulation containing PS obtained in Example 2 was mixed with a stirring granulator, and purified water of 5% of the powder weight was added to perform stirring granulation.
  • the granulated product was dried with a fluid bed dryer to obtain good fine granules.
  • the blending ratio of the nonpolar solvent and the polar solvent and the blending ratio of the polyunsaturated fatty acid-containing phospholipid and the hydroxyl group-containing compound are limited.
  • the base exchange reaction proceeds efficiently, so that a large amount of the hydroxyl group-containing compound can be added, and the yield of the target phospholipid can be increased. it can.
  • an antioxidant added to the reaction system, oxidation of polyunsaturated fatty acids is suppressed, and the aqueous phase and organic solvent phase are well separated after the reaction. Becomes simple and high-purity phospholipid is obtained.

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Abstract

Disclosed is a method for producing a phospholipid containing a polyvalent unsaturated fatty acid in a two-phase system, wherein the yield of the produced phospholipid is high, isolation of the product is easy, and oxidation of the polyvalent unsaturated fatty acid is suppressed. Specifically disclosed is a method for producing a phospholipid containing a polyvalent unsaturated fatty acid, which is characterized by comprising a step wherein a phospholipid containing a polyvalent unsaturated fatty acid and a hydroxyl group-containing compound are reacted in a two-phase system composed of a nonpolar solvent, a polar solvent and water in the presence of an antioxidant and phospholipase D. This method for producing a phospholipid containing a polyvalent unsaturated fatty acid is further characterized in that the polar solvent content relative to the total of the nonpolar solvent and the polar solvent is more than 20% but not more than 80% on a volume basis, and the molar ratio of the hydroxyl group-containing compound to the phospholipid is not less than 4 but not more than 30.

Description

明 細 書  Specification
多価不飽和脂肪酸含有リン脂質の製造方法及び該製造方法により得ら れる多価不飽和脂肪酸含有リン脂質  Method for producing polyunsaturated fatty acid-containing phospholipid and polyunsaturated fatty acid-containing phospholipid obtained by the production method
技術分野  Technical field
[0001] 本発明は、ホスホリパーゼ Dを用いた多価不飽和脂肪酸含有リン脂質の製造方法に 関する。更に詳しくは、ホスホリパーゼ Dを用いた多価不飽和脂肪酸含有リン脂質の 製造方法において、抗酸化剤を添加し、更に非極性溶媒と極性溶媒との配合比及 びリン脂質とヒドロキシル基含有化合物との配合比を限定して反応を行う製造方法に 関する。更に本製造方法によって得られた多価不飽和脂肪酸含有リン脂質および該 多価不飽和脂肪酸含有リン脂質を含む油脂に関する。  The present invention relates to a method for producing a polyunsaturated fatty acid-containing phospholipid using phospholipase D. More specifically, in the method for producing a polyunsaturated fatty acid-containing phospholipid using phospholipase D, an antioxidant is added, and the blending ratio of the nonpolar solvent and the polar solvent, and the phospholipid and the hydroxyl group-containing compound are added. The present invention relates to a production method in which the reaction is carried out with a limited mixing ratio. Furthermore, the present invention relates to a polyunsaturated fatty acid-containing phospholipid obtained by the production method and an oil containing the polyunsaturated fatty acid-containing phospholipid.
背景技術  Background art
[0002] リン脂質には、その種類に応じて様々な薬効があることが知られており、これまでに多 くの研究がなされている。例えば、多価不飽和脂肪酸残基を有するリン脂質の 1種で あるドコサへキサェン酸含有ホスファチジルセリンには、学習能の向上効果や環境の 変化に順応する知能力を向上させる効果があることが報告されている (特許文献 1参 照)。  [0002] Phospholipids are known to have various medicinal effects depending on their types, and many studies have been conducted so far. For example, docosahexaenoic acid-containing phosphatidylserine, which is one of phospholipids having polyunsaturated fatty acid residues, has the effect of improving learning ability and improving intellectual ability to adapt to environmental changes. It has been reported (see Patent Document 1).
[0003] リン脂質は、ホスホリパーゼ D (以下、 PLDと略す)を触媒とする塩基交換反応により 得られることが知られている。 PLDは、例えば、ホスファチジルコリン（以下、 PCと略 す）、ホスファチジルエタノールァミン (以下、 PEと略す)等のようなリン脂質と、受容体 であるアルコールとの間の塩基交換反応を触媒する。例えば、アルコールとしてダリ セロールを用いた場合には、ホスファチジルグリセロール（以下、 PGと略す）が生産さ れる。また、アルコールとしてセリンを用いた場合には、ホスファチジルセリン（以下、 P Sと略す）が生産される。一方、受容体としてアルコールの代わりに水を用いた場合に は、加水分解反応が進行し、ホスファチジン酸 (以下、 PAと略す）が生産される。  [0003] Phospholipids are known to be obtained by a base exchange reaction using phospholipase D (hereinafter abbreviated as PLD) as a catalyst. PLD catalyzes a base exchange reaction between a phospholipid such as phosphatidylcholine (hereinafter abbreviated as PC), phosphatidylethanolamine (hereinafter abbreviated as PE), and an alcohol as a receptor. For example, when daricerol is used as alcohol, phosphatidylglycerol (hereinafter abbreviated as PG) is produced. When serine is used as the alcohol, phosphatidylserine (hereinafter abbreviated as PS) is produced. On the other hand, when water is used as an acceptor instead of alcohol, hydrolysis proceeds and phosphatidic acid (hereinafter abbreviated as PA) is produced.
[0004] 一般的に、 PLDを用いたリン脂質の塩基交換反応は、水相及び有機溶媒相力もなる 二相系で行われるが、ドコサへキサェン酸含有ホスファチジルセリン等の人間が服用 する成分の製造に、人体に有害な有機溶媒を用いるのは好ましくないため、このよう な有機溶媒を用いずに塩基交換反応を行う方法が利用されて ヽる。 [0004] Generally, the base exchange reaction of phospholipids using PLD is carried out in a two-phase system that also has an aqueous phase and an organic solvent compatibility, but it is a component of human-dose components such as docosahexaenoic acid-containing phosphatidylserine. It is not desirable to use organic solvents that are harmful to the human body for production. A method of performing a base exchange reaction without using an organic solvent can be used.
[0005] このような方法としては、リン脂質のリボソーム懸濁物、ヒドロキシル基を有する試薬を 含有する水性媒体に酵素を溶解し、次いでシリカゲルを添加して攪拌することにより 反応を行う方法や (特許文献 2参照)、有機溶媒として食品用途に用いても安全なへ キサン、アセトンを用い、へキサン、アセトン及び水からなる反応溶媒中で反応を行う 方法が提案されている (特許文献 3参照)。しかしながら、これらの方法では、反応系 に水を多く含むと副反応である加水分解反応が進行し、目的とするリン脂質の収率 が充分でなぐまた生成するリン脂質の分離が困難になる、多価不飽和脂肪酸残基 が酸ィ匕する等の問題もあった。  [0005] Such a method includes a method in which an enzyme is dissolved in an aqueous medium containing a phospholipid suspension of a phospholipid and a reagent having a hydroxyl group, and then the reaction is performed by adding silica gel and stirring. (See Patent Document 2), and a method has been proposed in which hexane, acetone, which is safe for use in food applications as an organic solvent, is used, and the reaction is performed in a reaction solvent composed of hexane, acetone, and water (see Patent Document 3). ). However, in these methods, if the reaction system contains a large amount of water, the hydrolysis reaction, which is a side reaction, proceeds, the target phospholipid yield is not sufficient, and it is difficult to separate the produced phospholipid. There were also problems such as polyunsaturated fatty acid residues acidifying.
[0006] 加水分解反応を抑制する方法や塩基交換反応を促進する方法としては様々な報告 力 されており、水系の場合、塩類 (カルシウム塩等)や界面活性剤を添加する方法 が知られている。  [0006] Various methods have been reported as a method for suppressing a hydrolysis reaction and a method for promoting a base exchange reaction. In the case of an aqueous system, a method of adding a salt (calcium salt or the like) or a surfactant is known. Yes.
二相系においては、水の量を最小限（10%以下）にして加水分解反応を抑制する方 法が考案されている。二相系の場合、主として水相に PLD、アルコール等の受容体 等が含まれる力 例えばこれらの成分を含む水相を逆ミセル中に封入して反応系に 添加する方法 (特許文献 4参照）、リン脂質と受容体とを担体に吸着させた PLDに接 触させて、反応系中の水分含量が 1重量％以下の状態でリン脂質と受容体とを反応 させる方法がある（特許文献 5参照)。  In a two-phase system, a method has been devised that suppresses the hydrolysis reaction by minimizing the amount of water (10% or less). In the case of a two-phase system, a force mainly containing a receptor such as PLD or alcohol in the aqueous phase, for example, a method of adding an aqueous phase containing these components in a reverse micelle and adding it to the reaction system (see Patent Document 4) There is a method in which a phospholipid and a receptor are reacted with each other in a state where the water content in the reaction system is 1% by weight or less by contacting with a PLD in which the phospholipid and the receptor are adsorbed on a carrier (Patent Document 5). reference).
しかしながら、これらの方法で加水分解反応は抑制されたが、目的とするリン脂質の 収率が充分ではなぐ更に収率を上げることが望まれている。また、リン脂質の分離 や多価不飽和脂肪酸が酸化する問題にっ 、ては、何ら改善されて 、な 、。  However, although the hydrolysis reaction was suppressed by these methods, it is desired to further increase the yield of the target phospholipid, which is not sufficient. In addition, the problem of phospholipid separation and oxidation of polyunsaturated fatty acids has been improved.
[0007] 塩基交換反応での収率を更に上げるためには、アルコール等の受容体の添加量を 増やす必要があるが、受容体の水への溶解度に限界があることから、受容体の添カロ 量を増やすと必然的に水の量も増加せざるを得ないという問題がある。  [0007] In order to further increase the yield in the base exchange reaction, it is necessary to increase the amount of the acceptor such as alcohol. However, since the solubility of the acceptor in water is limited, it is necessary to add the acceptor. Increasing the amount of calories inevitably increases the amount of water.
[0008] 一方、塩基交換反応後の生成物の分離については、塩基交換反応による PSの製造 方法において、濃縮工程又は溶媒除去工程の前に、塩基交換反応で得られた PS含 有溶液に希釈油を添加して、流動性のよ！ヽ PS組成物を効率よく得る方法が報告さ れている (特許文献 6参照)。 しかしながら、塩基交換反応での収率がよぐ生成物の分離が簡便で、かつ多価不 飽和脂肪酸の酸ィ匕を抑制する方法はまだ知られて 、な 、。 [0008] On the other hand, for the separation of the product after the base exchange reaction, in the PS production method by the base exchange reaction, it is diluted with the PS-containing solution obtained by the base exchange reaction before the concentration step or the solvent removal step. Add oil and it's fluid! (2) A method for efficiently obtaining a PS composition has been reported (see Patent Document 6). However, there is still no known method for easily separating the product having a good yield in the base exchange reaction and suppressing the acidity of the polyunsaturated fatty acid.
[0009] 特許文献 1 :特開 2005— 104893号公報  Patent Document 1: Japanese Patent Application Laid-Open No. 2005-104893
特許文献 2：特表 2003 - 502035号公報  Patent Document 2: Japanese Translation of Special Publication 2003-505035
特許文献 3：特開 2001— 186898号公報  Patent Document 3: Japanese Patent Laid-Open No. 2001-186898
特許文献 4：特公平 7 - 16426号公報  Patent Document 4: Japanese Patent Publication No. 7-16426
特許文献 5：特公平 3 - 67676号公報  Patent Document 5: Japanese Patent Publication No. 3-67676
特許文献 6：特開 2003 - 304814号公報  Patent Document 6: Japanese Patent Application Laid-Open No. 2003-304814
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0010] 本発明は、上記現状に鑑み、二相系での多価不飽和脂肪酸含有リン脂質の製造方 法において、生成するリン脂質の収率がよぐ分離が簡便で、更に多価不飽和脂肪 酸の酸化を抑制する製造方法、該製造方法によって得られた多価不飽和脂肪酸含 有リン脂質、及び、該多価不飽和脂肪酸含有リン脂質を含む油脂を提供する。 [0010] In view of the above situation, the present invention provides a method for producing polyunsaturated fatty acid-containing phospholipids in a two-phase system. Provided are a production method for suppressing oxidation of saturated fatty acid, a polyunsaturated fatty acid-containing phospholipid obtained by the production method, and an oil / fat containing the polyunsaturated fatty acid-containing phospholipid.
課題を解決するための手段  Means for solving the problem
[0011] 本発明者らは、前述の課題を解決すべく鋭意検討を重ねた結果、反応系中に抗酸 ィ匕剤を添加し、更に反応系中の非極性溶媒と極性溶媒との配合比、及び、多価不飽 和脂肪酸含有リン脂質とヒドロキシル基含有化合物との配合比を限定することにより、 収率を下げることなく生成するリン脂質の分離が容易になり、更に多価不飽和脂肪酸 の酸ィ匕が抑制されることを見出した。 [0011] As a result of intensive studies to solve the above-mentioned problems, the present inventors have added an antioxidant and added a nonpolar solvent and a polar solvent in the reaction system. Ratio and the compounding ratio of the polyunsaturated fatty acid-containing phospholipid and the hydroxyl group-containing compound makes it easy to separate the phospholipids produced without reducing the yield, and further, polyunsaturated It was found that the fatty acid acidity was suppressed.
すなわち本発明は、多価不飽和脂肪酸含有リン脂質とヒドロキシル基含有化合物と を、抗酸化剤とホスホリパーゼ Dの存在下、非極性溶媒、極性溶媒及び水からなる二 相系中で反応させる工程を含み、極性溶媒の含有量が、非極性溶媒及び極性溶媒 の合計量に対して、容積基準で 20%を超え、かつ 80%以下の量であり、リン脂質に 対するヒドロキシル基含有ィ匕合物のモル比が 4以上 30以下であることを特徴とする多 価不飽和脂肪酸含有リン脂質の製造方法に関する。  That is, the present invention comprises a step of reacting a polyunsaturated fatty acid-containing phospholipid and a hydroxyl group-containing compound in a two-phase system comprising a nonpolar solvent, a polar solvent and water in the presence of an antioxidant and phospholipase D. The content of the polar solvent is more than 20% by volume and 80% or less based on the total amount of the nonpolar solvent and the polar solvent, and the hydroxyl group-containing compound for phospholipids The present invention relates to a method for producing a polyunsaturated fatty acid-containing phospholipid, characterized in that the molar ratio is from 4 to 30.
[0012] 前記抗酸化剤は脂溶性抗酸化剤であることが好ま ヽ。 [0012] Preferably, the antioxidant is a fat-soluble antioxidant.
[0013] 前記脂溶性抗酸化剤がビタミン E又はその誘導体であることが好ま 、。 [0014] 水の含有量が、非極性溶媒及び極性溶媒の合計量に対して、容積基準で 30〜200[0013] Preferably, the fat-soluble antioxidant is vitamin E or a derivative thereof. [0014] The water content is 30 to 200 on a volume basis with respect to the total amount of the nonpolar solvent and the polar solvent.
%であることが好ましい。 % Is preferred.
[0015] 塩基交換反応を開始する前に抗酸化剤を添加することが好ましい。 [0015] It is preferable to add an antioxidant before starting the base exchange reaction.
[0016] 前記多価不飽和脂肪酸含有リン脂質がイカ由来のレシチンであることが好ましい。 [0016] The polyunsaturated fatty acid-containing phospholipid is preferably squid-derived lecithin.
[0017] また、本発明は、前記製造方法により得られた多価不飽和脂肪酸含有リン脂質に関 する。 [0017] The present invention also relates to a polyunsaturated fatty acid-containing phospholipid obtained by the production method.
[0018] 前記多価不飽和脂肪酸含有リン脂質を構成する n— 3系多価不飽和脂肪酸と n— 6 系多価不飽和脂肪酸の構成比 (n - 3/n-6)が 25以上であることが好まし 、。  [0018] The composition ratio (n-3 / n-6) of the n-3 polyunsaturated fatty acid and the n-6 polyunsaturated fatty acid constituting the polyunsaturated fatty acid-containing phospholipid is 25 or more. I prefer to be there.
[0019] また、本発明は、前記多価不飽和脂肪酸含有リン脂質を含む油脂に関する。  [0019] The present invention also relates to an oil or fat comprising the polyunsaturated fatty acid-containing phospholipid.
[0020] 以下に本発明を詳細に説明する。  [0020] The present invention is described in detail below.
本発明の多価不飽和脂肪酸含有リン脂質の製造方法は、抗酸化剤とホスホリパーゼ Dの存在下、非極性溶媒、極性溶媒及び水力ゝらなる二相系中で、多価不飽和脂肪 酸含有リン脂質とヒドロキシル基含有ィ匕合物とを反応させることからなる。  The method for producing a polyunsaturated fatty acid-containing phospholipid of the present invention comprises a polyunsaturated fatty acid-containing compound in a two-phase system comprising a nonpolar solvent, a polar solvent and hydraulic power in the presence of an antioxidant and phospholipase D. It comprises reacting a phospholipid with a hydroxyl group-containing compound.
本発明で用いられる多価不飽和脂肪酸含有リン脂質とは、多価不飽和脂肪酸の残 基が結合したリン脂質である。上記多価不飽和脂肪酸含有リン脂質としてはいかなる 起源のものでもよぐリン脂質を含む天然物、天然物からの抽出物、当該抽出物を精 製したもの、合成リン脂質等が好適に用いられる。このようなリン脂質の例としては、 多価不飽和脂肪酸の残基が結合したホスファチジルエタノールァミン (PE)、ホスファ チジルコリン（PC)、ホスファチジルセリン（PS)、ホスファチジルグリセロール（PG)、 ホスファチジルイノシトール (PI)、これらの混合物等が挙げられる。多価不飽和脂肪 酸としては公知のものが挙げられ、例えばリノレン酸、エイコサペンタエン酸、ドコサへ キサェン酸、リノール酸、ァラキドン酸等が挙げられる。上記リン脂質は単独で用いて もよいし、 2種以上を同時に用いてもよい。一般的には、大豆、菜種、米、ひまわり、 パーム等の植物由来のレシチン;卵黄、マグロ、イカ、イワシ、カツォ、イクラ、ォキアミ The polyunsaturated fatty acid-containing phospholipid used in the present invention is a phospholipid to which a residue of a polyunsaturated fatty acid is bound. As the polyunsaturated fatty acid-containing phospholipid, a natural product containing phospholipids of any origin, an extract from the natural product, a purified product of the extract, a synthetic phospholipid, etc. are preferably used. . Examples of such phospholipids are phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PE PI) and mixtures thereof. Examples of polyunsaturated fatty acids include known ones such as linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, and arachidonic acid. The above phospholipids may be used alone or in combination of two or more. Generally, lecithin derived from plants such as soybeans, rapeseed, rice, sunflower, palm, etc .; egg yolk, tuna, squid, sardine, bonito, salmon roe, krill
、ホタテ等の動物由来のレシチン；酵母等の微生物由来のレシチン；水添レシチンの ようなカ卩ェレシチン等が用いられる。 And lecithin derived from animals such as scallops; lecithin derived from microorganisms such as yeast;
[0021] これらの多価不飽和脂肪酸含有リン脂質のなかでも、リン脂質中の n— 3系多価不飽 和脂肪酸の割合が高 ヽと 、う理由から、イカ由来のレシチンが特に好ま 、。 [0022] リン脂質の純度は、好ましくは、化合物の全質量を基準として 20〜99. 5質量％であ る。また、上記リン脂質は、本反応系中で 0. 1〜60質量％用いるのが好ましい。 [0021] Among these polyunsaturated fatty acid-containing phospholipids, squid-derived lecithin is particularly preferred because of the high proportion of n-3 polyunsaturated fatty acids in the phospholipid. . [0022] The purity of the phospholipid is preferably 20 to 99.5% by mass based on the total mass of the compound. The phospholipid is preferably used in an amount of 0.1 to 60% by mass in this reaction system.
[0023] 本発明で用いる抗酸化剤としては特に限定されないが、脂溶性抗酸化剤または水溶 性抗酸化剤が挙げられる。  [0023] The antioxidant used in the present invention is not particularly limited, and examples thereof include a fat-soluble antioxidant and a water-soluble antioxidant.
上記脂溶性抗酸化剤としては、例えば、コェンザィム Q10、ビタミン A、ビタミン E及び その誘導体等が挙げられる。上記水溶性抗酸化剤としては、スーパーォキシドデイス ムターゼ（SOD)、グルタチオン、ビタミン C、ぶどう種子エキス、ピクノジェノール、力 テキン、ァスタキサンチン、アミノ酸等が挙げられる。上記抗酸化剤としては、脂溶性 であるリン脂質を構成する多価不飽和脂肪酸の酸ィ匕を防止できるという理由から、ま た本反応後に水相の分離がし易いという理由から、脂溶性抗酸化剤が好ましぐビタ ミン E又はその誘導体がより好ましい。上記ビタミン Eの誘導体としては例えば、ニコ チン酸トコフエロール、酢酸トコフエロール等が挙げられる。  Examples of the fat-soluble antioxidant include coenzyme Q10, vitamin A, vitamin E, and derivatives thereof. Examples of the water-soluble antioxidant include superoxide dismutase (SOD), glutathione, vitamin C, grape seed extract, pycnogenol, strength techin, austaxanthin and amino acids. As the above antioxidant, it is possible to prevent the oxidation of the polyunsaturated fatty acid constituting the lipid-soluble phospholipid, and because the aqueous phase can be easily separated after the reaction. Vitamin E or a derivative thereof, for which an antioxidant is preferred, is more preferred. Examples of the vitamin E derivative include tocopherol nicotinate and tocopherol acetate.
上記抗酸化剤は、天然物からの抽出物、当該抽出物を精製したもの、合成製品等の 何れを用いてもよぐ例えば、ビタミン E又はその誘導体としては、植物性油脂等から 抽出したものを用いることもできる。  The antioxidant may be any extract from a natural product, a product obtained by purifying the extract, or a synthetic product. For example, vitamin E or a derivative thereof is extracted from vegetable oil or the like. Can also be used.
また、上記抗酸化剤は、純度に関わらず、液状であってもよいし、塩基交換反応時に 液状となるものであれば固体であってもよ 、。  The antioxidant may be liquid regardless of purity, or may be solid as long as it becomes liquid during the base exchange reaction.
[0024] 抗酸化剤の添加量は、多価不飽和脂肪酸含有リン脂質 100重量部に対して、 1〜1 5重量部が好ましい。添加量が 1重量部未満である力 15重量部を超えると、水相と 有機溶媒相が充分に分離せず、リン脂質の分離精製が困難となる場合や、多価不 飽和脂肪酸の酸ィ匕防止効果が充分でない場合がある。より好ましくは、 2. 5〜15重 量部である。 [0024] The addition amount of the antioxidant is preferably 1 to 15 parts by weight with respect to 100 parts by weight of the polyunsaturated fatty acid-containing phospholipid. When the addition amount is less than 1 part by weight and exceeds 15 parts by weight, the aqueous phase and the organic solvent phase are not sufficiently separated, and separation and purification of phospholipids becomes difficult. Anti-wrinkle effect may not be sufficient. More preferably, it is 2.5 to 15 parts by weight.
抗酸化剤は、本発明においては反応を開始する前に添加することが好ましい。塩基 交換反応の開始前に添加すると、リン脂質の酸ィ匕を防ぐことができ、さらに水相と有 機溶媒相が良好に分離し、反応後のリン脂質の分離精製が容易である。  In the present invention, the antioxidant is preferably added before starting the reaction. If it is added before the start of the base exchange reaction, it is possible to prevent acidity of the phospholipid. Furthermore, the aqueous phase and the organic solvent phase are well separated, and separation and purification of the phospholipid after the reaction is easy.
[0025] 本発明で用いる非極性溶媒としては特に限定されず、例えば、ヘプタン、へキサン、 石油エーテル等の脂肪族炭化水素；シクロペンタン、シクロへキサン等の環状脂肪 族炭化水素；ジェチルエーテル、テトラヒドロフラン等のエーテル類;酢酸メチル、酢 酸ェチル等のエステル類；四塩ィ匕炭素、クロ口ホルム等のハロゲンィ匕炭化水素類等 が挙げられる。上記非極性溶媒としては、ヘプタン、へキサンが好ましい。 [0025] The nonpolar solvent used in the present invention is not particularly limited, and examples thereof include aliphatic hydrocarbons such as heptane, hexane, and petroleum ether; cyclic aliphatic hydrocarbons such as cyclopentane and cyclohexane; , Ethers such as tetrahydrofuran; methyl acetate, vinegar Examples thereof include esters such as oxyethyl; halogenated hydrocarbons such as tetrasalt carbon and carbon form. As the nonpolar solvent, heptane and hexane are preferable.
上記極性溶媒としては特に限定されず、例えば、アセトン、メチルェチルケトン、ジェ チルケトン等のケトン類等が挙げられる。上記極性溶媒としては、アセトンが好ましい  The polar solvent is not particularly limited, and examples thereof include ketones such as acetone, methyl ethyl ketone, and ethyl ketone. As the polar solvent, acetone is preferable.
[0026] 本発明の製造方法では、非極性溶媒、極性溶媒及び水力ゝらなる二相系中で、極性 溶媒の含有量が、非極性溶媒及び極性溶媒の合計量に対して容積基準で 20%を 超え、かつ 80%以下の量で、多価不飽和脂肪酸含有リン脂質とヒドロキシル基含有 化合物とを反応させる。極性溶媒の含有量が 20%以下であるか、 80%を超えると、 目的リン脂質の収率が低くなる。上記極性溶媒の含有量は、好ましくは 25〜50%で あり、より好ましくは 25〜40%である。 [0026] In the production method of the present invention, the content of the polar solvent in the two-phase system consisting of the nonpolar solvent, the polar solvent and the hydraulic power is 20 on a volume basis with respect to the total amount of the nonpolar solvent and the polar solvent. The polyunsaturated fatty acid-containing phospholipid is reacted with the hydroxyl group-containing compound in an amount of more than% and not more than 80%. When the content of the polar solvent is 20% or less or exceeds 80%, the yield of the target phospholipid is lowered. The content of the polar solvent is preferably 25 to 50%, more preferably 25 to 40%.
なお、本発明の製造方法では、塩基交換反応時にリン脂質が非極性溶媒及び極性 溶媒カゝらなる有機溶媒相に溶解していることが好ましぐリン脂質と非極性溶媒及び 極性溶媒との割合は特に限定されないが、通常、リン脂質 lgに対して有機溶媒が 1 〜： L OOmlである。  In the production method of the present invention, it is preferable that the phospholipid is dissolved in an organic solvent phase consisting of a nonpolar solvent and a polar solvent during the base exchange reaction, and the nonpolar solvent and the polar solvent. Although the ratio is not particularly limited, the organic solvent is usually 1 to: L OOml with respect to phospholipid lg.
[0027] 本発明の製造方法で用いる水としては、イオン交換水、精製水、蒸留水、水道水等 が挙げられる。さらに、これらに酢酸等を含有させて pH調整のための緩衝液としても よい。好ましくは、イオン交換水、精製水又は蒸留水が用いられる。本発明の製造方 法では、水の含有量が、非極性溶媒及び極性溶媒の合計に対して容積基準で 30〜 250%であること力 S好ましく、 30〜200%であることがより好ましい。更に好ましくは、 50〜150%である。  [0027] Examples of water used in the production method of the present invention include ion-exchanged water, purified water, distilled water, and tap water. Furthermore, acetic acid or the like may be contained in these to form a buffer solution for pH adjustment. Preferably, ion exchange water, purified water or distilled water is used. In the production method of the present invention, the content of water is preferably 30 to 250% on a volume basis with respect to the total of the nonpolar solvent and the polar solvent, and more preferably 30 to 200%. More preferably, it is 50 to 150%.
[0028] 本発明で用いるヒドロキシル基含有ィ匕合物としては、例えば、アルコール類、含窒素 アルコール類、糖類、ポリオール類、ヒドロキシ環状ィ匕合物等が挙げられる。  [0028] Examples of the hydroxyl group-containing compound used in the present invention include alcohols, nitrogen-containing alcohols, saccharides, polyols, and hydroxy cyclic compounds.
アルコール類としては、例えば、メタノール、エタノール、プロパノール、ァスコルビン 酸等が挙げられる。  Examples of alcohols include methanol, ethanol, propanol, ascorbic acid and the like.
含窒素アルコール類としては、例えば、セリンなどのアミノ酸； 1—アミノー 2—プロパノ ールなどが挙げられる。  Examples of nitrogen-containing alcohols include amino acids such as serine; 1-amino-2-propanol.
糖類としては、例えば、アデノシン、グアノシン、イノシン、キサントシン、デォキシアデ ノシン、デォキシグアノシン等のヌクレオシド；グルコース、トレハロース、 N ァセチル D ダルコサミン等が挙げられる。 Examples of the saccharide include adenosine, guanosine, inosine, xanthosine, and deoxyade. And nucleosides such as nocine and deoxyguanosine; glucose, trehalose, N-acetyl-D darcosamine and the like.
ポリオール類としては、例えば、グリセロール、エチレングリコール、プロピレングリコー ル等が挙げられる。  Examples of polyols include glycerol, ethylene glycol, propylene glycol and the like.
ヒドロキシ環状ィ匕合物としては、例えば、麹酸、アルブチン等が挙げられる。  Examples of the hydroxy cyclic compound include oxalic acid, arbutin and the like.
本発明の製造方法では、ヒドロキシル基含有ィ匕合物が水に溶解した状態で反応を行 うことが好ましい。ヒドロキシル基含有ィ匕合物と水との割合は特に限定されないが、例 えばヒドロキシル基含有ィ匕合物としてセリンを用いる場合、セリン lgに対して、 3〜20 mlの水を配合するのが好まし!/、。  In the production method of the present invention, the reaction is preferably performed in a state where the hydroxyl group-containing compound is dissolved in water. The ratio between the hydroxyl group-containing compound and water is not particularly limited. For example, when serine is used as the hydroxyl group-containing compound, 3 to 20 ml of water is added to serine lg. I like it!
[0029] 本発明の製造方法では、多価不飽和脂肪酸含有リン脂質に対するヒドロキシル基含 有ィ匕合物のモル比が 4以上、 30以下の条件下で行う。ヒドロキシル基含有化合物の モル比が 4未満であると、生成するリン脂質の収率が低くなる。上記モル比は、 6以上 が好ましぐ 8以上がより好ましい。また、上記モル比が 30を超えると、コストが高くなり 、非現実である。 [0029] The production method of the present invention is carried out under conditions where the molar ratio of the hydroxyl group-containing compound to the polyunsaturated fatty acid-containing phospholipid is 4 or more and 30 or less. If the molar ratio of the hydroxyl group-containing compound is less than 4, the yield of the phospholipid produced will be low. The molar ratio is preferably 6 or more, more preferably 8 or more. On the other hand, if the molar ratio exceeds 30, the cost increases, which is unrealistic.
[0030] 本発明の製造方法では、ホスホリパーゼ D (PLD)の存在下で行う。上記 PLDとして は、リン脂質の塩基部分を加水分解、及び Zまたは塩基交換できるものであれば特 に限定されず、例えば植物由来の PLD、微生物由来の PLD等が挙げられる。植物 由来の PLDとしてはキャベツ、大豆由来の PLD等が挙げられ、微生物由来の PLDと しては放線菌、糸状菌由来の PLD等が挙げられる。  [0030] The production method of the present invention is carried out in the presence of phospholipase D (PLD). The PLD is not particularly limited as long as it can hydrolyze and Z or base-exchange the phospholipid base moiety, and examples thereof include plant-derived PLD and microorganism-derived PLD. Plant-derived PLDs include cabbage and soybean-derived PLDs, and microorganism-derived PLDs include actinomycetes and filamentous fungal PLDs.
本反応系中における PLDの濃度は、リン脂質 lgに対し、好ましくは 5〜200U、より 好ましくは 20〜100Uである。なお、 1Uは、 95%大豆ホスファチジルコリンを基質と し、基質濃度 0. 16%の 0. 2M酢酸緩衝液（pH4. 0、 10mMの CaCl、 1. 3%の Tri ton X— 100を含む）を 37°Cにて反応させた時、 1分間に 1 molのコリンを遊離す る酵素量である。  The concentration of PLD in this reaction system is preferably 5 to 200 U, more preferably 20 to 100 U, relative to phospholipid lg. 1U uses 95% soybean phosphatidylcholine as a substrate, and 0.2M acetate buffer (pH 4.0, 10 mM CaCl, 1.3% Tri ton X-100) containing 0.16% substrate concentration. The amount of enzyme that liberates 1 mol of choline per minute when reacted at 37 ° C.
[0031] 上記塩基交換反応は、 pH3. 5〜10の条件で行うことが好ましぐ pH4〜9がより好ま しい。反応温度は、 10〜40°Cが好ましぐ 20〜30°Cがより好ましい。また、反応系が 二相系であることから、塩基交換反応を行う間は反応系を攪拌することが好ましい。  [0031] The base exchange reaction is preferably performed under the conditions of pH 3.5 to 10, and more preferably pH 4 to 9. The reaction temperature is preferably 10 to 40 ° C, more preferably 20 to 30 ° C. Further, since the reaction system is a two-phase system, it is preferable to stir the reaction system during the base exchange reaction.
[0032] 本発明の製造方法により、例えば多価不飽和脂肪酸含有リン脂質中の PC、 PE等と 、セリン等のヒドロキシル基含有ィ匕合物とが反応して PS等が得られる。 塩基交換反応を行った後は、例えば加熱等の処理で PLDを失活させ、遠心分離法 等により有機溶媒層と水層を分離して有機溶媒層を得たあと、有機溶媒を減圧下で 除去することによって濃縮する。次いで、アセトン又はエタノールで晶析を行い、固液 分離によって固形物を得、乾燥することにより、本発明の製造方法で生成した多価不 飽和脂肪酸含有リン脂質を単離することができる。 [0032] By the production method of the present invention, for example, PC, PE, etc. in polyunsaturated fatty acid-containing phospholipids PS and the like are obtained by reaction with a hydroxyl group-containing compound such as serine. After performing the base exchange reaction, for example, the PLD is deactivated by a treatment such as heating, and the organic solvent layer and the aqueous layer are separated by a centrifugal method or the like to obtain the organic solvent layer. Then, the organic solvent is removed under reduced pressure. Concentrate by removing. Next, crystallization is performed with acetone or ethanol, a solid is obtained by solid-liquid separation, and dried, whereby the polyunsaturated fatty acid-containing phospholipid produced by the production method of the present invention can be isolated.
[0033] これまで二相系中での塩基交換反応では、反応系中の水によって加水分解反応も 進むことから、目的とするリン脂質の収率が充分でなぐまた水によって多価不飽和 脂肪酸が酸化する、水相と有機溶媒相が乳化するため反応後にリン脂質の分離精 製が困難であるといった問題があった。本発明の製造方法では、反応系中に抗酸化 剤を添加することにより、反応系中に水が多く含まれていても、リン脂質の収率を下げ ることなく反応後に水相と有機溶媒相が良好に分離し、また多価不飽和脂肪酸の酸 ィ匕も抑制される。 [0033] So far, in the base exchange reaction in the two-phase system, the hydrolysis reaction proceeds with water in the reaction system, so that the yield of the target phospholipid is not sufficient, and the polyunsaturated fatty acid is also lost with water. Oxidizes, and the aqueous phase and the organic solvent phase are emulsified, making it difficult to separate and purify phospholipids after the reaction. In the production method of the present invention, by adding an antioxidant to the reaction system, even if the reaction system contains a large amount of water, the aqueous phase and the organic solvent are reacted after the reaction without reducing the yield of phospholipid. The phases are well separated and the acidity of polyunsaturated fatty acids is also suppressed.
[0034] 本発明の多価不飽和脂肪酸含有リン脂質は、前記製造方法により得られたものであ る。多価不飽和脂肪酸含有リン脂質において、リン脂質を構成するドコサへキサェン 酸、エイコサペンタエン酸、 a リノレン酸などの n— 3系多価不飽和脂肪酸とリノ一 ル酸ゃァラキドン酸などの n— 6系多価不飽和脂肪酸の構成比 (n— 3Zn— 6)力 2 5以上であることが好ましぐ 30以上であることがより好ましい。該構成比（n—3Zn— 6)を 25未満とすることにより、現在の食生活における n— 6系多価不飽和脂肪酸の 過剰摂取を避け、心臓病の危険性を抑えることができる。上限については、とくに限 定されるものではなぐ現在の食事中の脂肪酸摂取割合から考えれば、値は高けれ ば高いほどよい。 [0034] The polyunsaturated fatty acid-containing phospholipid of the present invention is obtained by the above production method. In polyunsaturated fatty acid-containing phospholipids, Kisaen acid to docosapentaenoic constituting the phospholipids, eicosapentaenoic acid, such as n- 3 polyunsaturated fatty acids and Reno one Le acids Ya Arakidon acids such as a-linolenic acid n- The composition ratio of 6-series polyunsaturated fatty acids (n-3Zn-6) force 25 is preferably 25 or more, more preferably 30 or more. By setting the composition ratio (n-3Zn-6) to less than 25, excessive intake of n-6 polyunsaturated fatty acids in the current diet can be avoided, and the risk of heart disease can be suppressed. Regarding the upper limit, the higher the value, the better, considering the current dietary fatty acid intake rate, which is not particularly limited.
[0035] 本発明の油脂は、前記多価不飽和脂肪酸含有リン脂質を含んでいればよぐ該リン 脂質以外の脂質を含んでいても良ぐ該リン脂質以外の脂質としてはとくに限定され るものではない。該油脂中の多価不飽和脂肪酸含有リン脂質の割合は、 10〜： LOO 質量％であるが、リン脂質の割合が高いほど有効であるので、好ましくは 20〜： LOO質 量％、さらに好ましくは、 30〜100%である。  [0035] The fat and oil of the present invention is not particularly limited as a lipid other than the phospholipid and may contain a lipid other than the phospholipid as long as it contains the polyunsaturated fatty acid-containing phospholipid. It is not a thing. The ratio of the polyunsaturated fatty acid-containing phospholipid in the fat is 10 to: LOO mass%, but the higher the ratio of phospholipid, the more effective, so preferably 20 to: LOO mass%, more preferably Is 30-100%.
[0036] 本発明の多価不飽和脂肪酸含有リン脂質および油脂は、錠剤、顆粒剤、散剤、カブ セル剤、ソフトカプセル剤等の各種の形態の製剤とすることができる。本発明のリン脂 質および油脂は高純度であり、容易に粉状にすることができるので、錠剤、顆粒剤、 散剤などの製造に好適であり、粉ミルク等のように粉体のみで提供される製品への加 ェが容易になる。前記製剤において、公知の賦形剤、崩壊剤、滑沢剤等を用いるこ とができ、水溶性薬物（生薬エキス、ビタミン Cなど）と配合してもよい。 [0036] The polyunsaturated fatty acid-containing phospholipids and fats and oils of the present invention are tablets, granules, powders, turnips. Various forms of preparations such as cell agents and soft capsules can be prepared. Since the phospholipids and fats and oils of the present invention have high purity and can be easily powdered, they are suitable for production of tablets, granules, powders, etc., and are provided only in powder form such as powdered milk. It is easy to add to the product. In the preparation, known excipients, disintegrants, lubricants and the like can be used, and they may be blended with water-soluble drugs (herbal extracts, vitamin C, etc.).
発明の効果  The invention's effect
[0037] 本発明の多価不飽和脂肪酸含有リン脂質の製造方法では、非極性溶媒と極性溶媒 との配合比、多価不飽和脂肪酸含有リン脂質とヒドロキシル基含有化合物との配合 比を限定することにより、反応系に水が多く含まれていても効率よく塩基交換反応が 進むので、ヒドロキシル基含有ィ匕合物を多く配合することができ、目的とするリン脂質 の収率を上げることができる。更に、反応系に抗酸化剤を添加することにより、リン脂 質の収率を下げることなく多価不飽和脂肪酸の酸化が抑制され、また、反応後も水 相と有機溶媒相が良好に分離するので、生成するリン脂質の分離精製が簡便となり 、高純度のリン脂質が得られる。  [0037] In the method for producing a polyunsaturated fatty acid-containing phospholipid of the present invention, the blending ratio of the nonpolar solvent and the polar solvent and the blending ratio of the polyunsaturated fatty acid-containing phospholipid and the hydroxyl group-containing compound are limited. As a result, even if the reaction system contains a large amount of water, the base exchange reaction proceeds efficiently, so that a large amount of the hydroxyl group-containing compound can be added, and the yield of the target phospholipid can be increased. it can. Furthermore, by adding an antioxidant to the reaction system, oxidation of polyunsaturated fatty acids is suppressed without reducing the yield of phospholipids, and the aqueous phase and organic solvent phase are well separated after the reaction. Therefore, separation and purification of the phospholipid to be produced becomes simple and high-purity phospholipid can be obtained.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0038] 以下に実施例を示して本発明を更に詳細に説明するが、本発明はこれらの実施例 に限定されるものではない。 [0038] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
実施例 1  Example 1
不飽和脂肪酸含有レシチンとしてイカ由来レシチン（日本ィ匕学飼料社製、ホスファチ ジルコリン（PC) 49. 1重量0 /0、ホスファチジルエタノールァミン（PE) 8. 7重量0 /0)を 用い、 20% (重量 Z容量）になるように、ヘプタンおよびアセトンの混合液に溶解した 。ヘプタンおよびアセトンの混合比は、ヘプタン:アセトン =6 :4 (容量:容量）とした。 さらに理研 Eオイル 600 (理研ビタミン社製ビタミン E)をレシチン 100重量部に対して 、表 1に示す濃度で 0〜10重量部添加して、レシチン溶液とした。以後、この溶液を レシチン溶液という。 Squid-derived lecithin (Nippon I匕学feed Co., Hosufachi phosphatidylcholine (PC) 49. 1 wt 0/0, phosphatidylethanolamine § Min (PE) 8. 7 wt 0/0) was used as the unsaturated fatty acid-containing lecithin, 20 It was dissolved in a mixed solution of heptane and acetone so as to be% (weight Z volume). The mixing ratio of heptane and acetone was heptane: acetone = 6: 4 (volume: volume). Furthermore, Riken E Oil 600 (Vitamin E manufactured by Riken Vitamin Co., Ltd.) was added at 0 to 10 parts by weight at a concentration shown in Table 1 with respect to 100 parts by weight of lecithin to obtain a lecithin solution. Hereinafter, this solution is referred to as a lecithin solution.
次に Lーセリン (協和発酵社製)を 25% (重量 Z容量）になるように、 0. 6M酢酸ナトリ ゥム緩衝液 (PH4. 0)に溶解した。以後、この溶液をセリン溶液という。  Next, L-serine (manufactured by Kyowa Hakko) was dissolved in 0.6 M sodium acetate buffer (PH 4.0) to a concentration of 25% (weight Z volume). Hereinafter, this solution is referred to as a serine solution.
上記の通り調製したレシチン溶液およびセリン溶液を、レシチンとセリンのモル比力 レシチン：セリン = 1 : 10となる比率で、全量を lmlになるように、 2mlエツペンドルフチ ユーブに分注した。レシチン溶液およびセリン溶液の容量比は、レシチン溶液:セリン 溶液 = 1 : 1. 12であった。さらに、ホスホリパーゼ D (ナガセケムテックス社製）をレシ チン lg当り 90Uとなるように添カロし、 TITEC社製の Bio Shaker M— BR— 022U Pを用い、 30°Cで 16時間攪拌した。反応後、反応液を遠心分離にて分離したレシチ ン溶液に 10倍量 (容量比）のアセトンをカ卩えて、不溶ィ匕したレシチン（ホスファチジル セリン (PS) )を回収し、減圧下でアセトンを除去した。このようにして得られたレシチン の DHA量を測定した。 The lecithin solution and the serine solution prepared as described above were used as a molar ratio of lecithin and serine. In a ratio of lecithin: serine = 1: 10, the whole amount was dispensed into 2 ml Eppendorf tube. The volume ratio of the lecithin solution and the serine solution was lecithin solution: serine solution = 1: 1.12. Further, phospholipase D (manufactured by Nagase ChemteX) was added to 90 U per liter of lecithin and stirred for 16 hours at 30 ° C. using Bio Shaker M—BR-022UP manufactured by TITEC. After the reaction, 10-fold volume (volume ratio) of acetone is added to the lecithin solution separated by centrifugation, and the insoluble lecithin (phosphatidylserine (PS)) is recovered. Was removed. The DHA content of the lecithin thus obtained was measured.
リン脂質組成は、反応液 50 μ 1をクロ口ホルム:メタノール = 1： 1 (容量:容量)の混合 液に溶解し、遠心分離にて不溶物を除去した後、下記の条件の HPLCにて分析した 使用カラム： GL Sciences製 Unisil Q NH2 (内径 4. 6mm X 25cm) 移動相：ァセトニトリル：メタノール： 1 OmMリン酸ニ水素アンモ-ゥム = 619 : 291 : 90 Phospholipid composition was determined by dissolving 50 μ1 of the reaction solution in a mixed solution of chloroform: methanol = 1: 1 (volume: volume), removing insolubles by centrifugation, and then using HPLC under the following conditions. Analyzed column: Unisil Q NH2 manufactured by GL Sciences (inner diameter 4.6 mm x 25 cm) Mobile phase: Acetonitrile: Methanol: 1 OmM Ammonium dihydrogen phosphate = 619: 291: 90
(v/ V/ v) (v / V / v)
流速： 1. 3ml/ mm  Flow rate: 1. 3ml / mm
カラム温度： 37°C  Column temperature: 37 ° C
検出： UV 205nm  Detection: UV 205nm
また、 PSの生成率は、反応前の PCおよび PE量に対する生成した PS量を表した。 PS生成率。 /0 = (生成した PS量 Z反応前の PC + PE量） X 100 The PS production rate represents the amount of PS produced relative to the amount of PC and PE before the reaction. PS generation rate. / 0 = (Amount of generated PS Z amount of PC + PE before reaction) X 100
脂肪酸組成の分析は GCにて行った。下記に分析条件を記す。  Analysis of fatty acid composition was performed by GC. The analysis conditions are described below.
[0039] (試料調製） [0039] (Sample preparation)
試料 5mg/mlのクロ口ホルム溶液 lmlに 10%塩化水素含メタノール lml混合し、 80 °Cで 3時間処理を行った。さらにへキサン lml、蒸留水 6mlを加え、よく混合した後、 遠心分離にて分離したへキサン'クロ口ホルム層を採取して、 GCに供試した。  The sample was mixed with 1 ml of a 5 mg / ml black mouth form solution and mixed with 1 ml of methanol containing 10% hydrogen chloride, and treated at 80 ° C. for 3 hours. Further, 1 ml of hexane and 6 ml of distilled water were added and mixed well, and then the hexane'form layer separated by centrifugation was collected and used for GC.
[0040] (GC分析） [0040] (GC analysis)
カラム： Thermon— 3000B HRシリーズ（0. 25mm X 30m)  Column: Thermon—3000B HR series (0.25 mm x 30m)
キャリアガス：ヘリウム  Carrier gas: helium
検出器: FID スプリット比： 1 : 1000 Detector: FID Split ratio: 1: 1000
試料注入量：: l  Sample injection volume: l
温度：カラム温度 150から 220°Cまで、 2°CZminで上昇、  Temperature: Column temperature from 150 to 220 ° C, rising at 2 ° CZmin,
試料注入口 220。C、  Sample inlet 220. C,
検出器 220°C  Detector 220 ° C
ガス圧力：ヘリウム（キャリアガス） 150kPa、窒素（メイクアップガス） 75kPa、水素 60 kPa、空気 50kPa  Gas pressure: Helium (carrier gas) 150 kPa, nitrogen (make-up gas) 75 kPa, hydrogen 60 kPa, air 50 kPa
これらの結果を表 1に示す。  These results are shown in Table 1.
[0041] [表 1] [0041] [Table 1]
Figure imgf000012_0001
Figure imgf000012_0001
[0042] 表 1の結果から、抗酸化剤を反応系に添加することにより、収率を下げることなく反応 後のレシチン溶液とセリン溶液とが分離しやすくなることが分力つた。また、抗酸化剤 の添加量が 5. 0重量部以上であると、レシチン溶液とセリン溶液との分離が良好とな り、またリン脂質中の DHAの割合が反応前と同程度となることが分力つた。  [0042] From the results in Table 1, it was found that the addition of an antioxidant to the reaction system facilitates separation of the lecithin solution and the serine solution after the reaction without lowering the yield. In addition, if the added amount of the antioxidant is 5.0 parts by weight or more, the separation between the lecithin solution and the serine solution will be good, and the proportion of DHA in the phospholipid will be about the same as before the reaction. However, it was divided.
[0043] 実施例 2  [0043] Example 2
不飽和脂肪酸含有レシチンとしてイカ由来レシチン（日本ィ匕学飼料社製、 PC49. 1 重量％、 PE8. 7重量％)を用い、 20% (重量 Z容量）になるように、ヘプタンおよび アセトンの混合液に溶解した。ヘプタンおよびアセトンの混合比は、ヘプタン:ァセト ン = 6： 4 (容量:容量）とした。さらに理研 Eオイル 600 (理研ビタミン社製)をレシチン 100重量部に対して、 10重量部添加して、レシチン溶液とした。また比較のため、理 研 Eオイル 600 (理研ビタミン社製)を添加しな 、ものも用意した。  Use lecithin derived from squid as an unsaturated fatty acid-containing lecithin (PC49.1% by weight, PE8.7% by weight, manufactured by Nihon Kagaku Feed Co., Ltd.) and mix heptane and acetone to 20% (weight Z volume). Dissolved in the liquid. The mixing ratio of heptane and acetone was heptane: acetone = 6: 4 (volume: volume). Furthermore, 10 parts by weight of RIKEN E Oil 600 (manufactured by Riken Vitamin Co., Ltd.) was added to 100 parts by weight of lecithin to obtain a lecithin solution. For comparison, a sample without RIKEN E Oil 600 (Riken Vitamin Co., Ltd.) was also prepared.
セリン溶液は実施例 1と同様に調製した。  A serine solution was prepared in the same manner as in Example 1.
上記の通り、調製したレシチン溶液およびセリン溶液を、レシチンとセリンのモル比が 、レシチン：セリン = 1 : 10となる比率で、全量を 100ml〖こなるよう〖こ、 500ml栓付ビン に分注し (レシチン溶液およびセリン溶液の容量比は、レシチン溶液:セリン溶液 = 1 : 1. 12)、さらに、ホスホリパーゼ D (ナガセケムテックス社製）をレシチン lg当り 90U となるように添加し、 30°Cの水浴中で、 5時間、マグネットスターラーを用いて攪拌し、 反応を行った。 As described above, the prepared lecithin solution and serine solution were mixed with lecithin and serine in molar ratio. , Lecithin: serine = 1:10 in a ratio of 100ml and dispense into a 500ml bottle with a stopper (the volume ratio of lecithin solution and serine solution is lecithin solution: serine solution = 1: 1. 12) Furthermore, add phospholipase D (manufactured by Nagase ChemteX) to 90 U per lecithin and stir in a 30 ° C water bath for 5 hours using a magnetic stirrer to carry out the reaction. It was.
反応後、反応液を遠心分離にて分離したレシチン溶液に 10倍量 (容量比）のァセト ンを加えて、不溶ィ匕したレシチン PSを回収し、減圧下でアセトンを除去した。  After the reaction, 10-fold volume (volume ratio) of acetonitrile was added to the lecithin solution separated from the reaction solution by centrifugation to recover insoluble lecithin PS, and acetone was removed under reduced pressure.
PSの生成率、脂肪酸組成は実施例 1記載の方法で実施した。また日本油化学会制 定の過酸化物価は基準油脂分析法参 2. 4に基づいて実施した。結果を表 2に示す  The production rate of PS and the fatty acid composition were carried out by the methods described in Example 1. The peroxide value established by the Japan Oil Chemists' Society was based on the standard method for analyzing fats and oils 2.4. The results are shown in Table 2.
[0044] [表 2] [0044] [Table 2]
Figure imgf000013_0001
Figure imgf000013_0001
[0045] 表 2の結果から、抗酸化剤を反応系に添加することにより、リン脂質の収率を下げるこ となぐリン脂質中の多価不飽和脂肪酸の酸ィ匕が抑制されることが分力つた。また今 回実施し、得られたリン脂質の n— 3系多価不飽和脂肪酸と n— 6系多価不飽和脂肪 酸の割合（n— 3Zn— 6)は、 37. 7〜53. 6であった。  [0045] From the results in Table 2, the addition of an antioxidant to the reaction system suppresses the acidity of polyunsaturated fatty acids in the phospholipid, which decreases the yield of phospholipid. I was divided. The ratio of n-3 polyunsaturated fatty acids to n-6 polyunsaturated fatty acids (n-3Zn-6) in the phospholipids obtained this time was 37.7-53.6. Met.
[0046] 調剤例 1  [0046] Formulation Example 1
実施例 2で得られた PSを含む下記処方を V型混合機で混合し、打錠機にて打錠し、 直径 7. 5mm、 200mgの錠剤を得た。  The following formulation containing PS obtained in Example 2 was mixed with a V-type mixer and tableted with a tableting machine to obtain tablets with a diameter of 7.5 mm and 200 mg.
ホスファチジノレセリン 150mg  Phosphatidinoreserin 150mg
イチヨウ葉エキス 300mg  Yewyo Leaf Extract 300mg
ビタミン C lOOmg  Vitamin C lOOmg
結晶セノレロース 750mg  Crystalline cellulose 750mg
部分 α化デンプン 480mg  Partial pregelatinized starch 480mg
微粒二酸化ケイ素 20mg 合計 1800mgZday Fine silicon dioxide 20mg Total 1800mgZday
[0047] 調剤例 2 [0047] Formulation Example 2
実施例 2で得られた PSを含む下記処方を攪拌造粒機で混合し、粉末重量の 5%の 精製水を添加し攪拌造粒を行った。造粒物を流動層乾燥機で乾燥して良好な細粒 を得た。  The following formulation containing PS obtained in Example 2 was mixed with a stirring granulator, and purified water of 5% of the powder weight was added to perform stirring granulation. The granulated product was dried with a fluid bed dryer to obtain good fine granules.
ホスファチジノレセリン 200mg  Phosphatidinoreserin 200mg
イチヨウ葉エキス 200mg  Ichiyo leaf extract 200mg
ビタミン E2倍散 200mg  Vitamin E2 powder 200mg
結晶セノレロース 650mg  Crystalline cellulose 650mg
トレハロース 470mg  Trehalose 470mg
メチルセルロース 30mg  Methylcellulose 30mg
合計 1750mgZday  Total 1750mgZday
産業上の利用可能性  Industrial applicability
[0048] 本発明の多価不飽和脂肪酸含有リン脂質の製造方法では、非極性溶媒と極性溶媒 との配合比、多価不飽和脂肪酸含有リン脂質とヒドロキシル基含有化合物との配合 比を限定することにより、反応系に水が多く含まれていても効率よく塩基交換反応が 進むので、ヒドロキシル基含有ィ匕合物を多く配合することができ、目的とするリン脂質 の収率を上げることができる。更に、反応系に抗酸化剤を添加することにより、多価不 飽和脂肪酸の酸化が抑制され、また、反応後も水相と有機溶媒相が良好に分離する ので、生成するリン脂質の分離精製が簡便となり、高純度のリン脂質が得られる。 [0048] In the method for producing a polyunsaturated fatty acid-containing phospholipid of the present invention, the blending ratio of the nonpolar solvent and the polar solvent and the blending ratio of the polyunsaturated fatty acid-containing phospholipid and the hydroxyl group-containing compound are limited. As a result, even if the reaction system contains a large amount of water, the base exchange reaction proceeds efficiently, so that a large amount of the hydroxyl group-containing compound can be added, and the yield of the target phospholipid can be increased. it can. Furthermore, by adding an antioxidant to the reaction system, oxidation of polyunsaturated fatty acids is suppressed, and the aqueous phase and organic solvent phase are well separated after the reaction. Becomes simple and high-purity phospholipid is obtained.

Claims

請求の範囲 The scope of the claims
[1] 多価不飽和脂肪酸含有リン脂質とヒドロキシル基含有化合物とを、抗酸化剤とホスホ リパーゼ Dの存在下、非極性溶媒、極性溶媒及び水からなる二相系中で反応させる 工程を含み、極性溶媒の含有量が、非極性溶媒及び極性溶媒の合計量に対して、 容積基準で 20%を超え、かつ 80%以下の量であり、リン脂質に対するヒドロキシル 基含有ィ匕合物のモル比が 4以上 30以下であることを特徴とする多価不飽和脂肪酸 含有リン脂質の製造方法。  [1] A step of reacting a polyunsaturated fatty acid-containing phospholipid and a hydroxyl group-containing compound in the presence of an antioxidant and phospholipase D in a two-phase system comprising a nonpolar solvent, a polar solvent and water. The content of the polar solvent is more than 20% on a volume basis and 80% or less with respect to the total amount of the nonpolar solvent and the polar solvent, and the moles of the hydroxyl group-containing compound relative to the phospholipid. A method for producing a polyunsaturated fatty acid-containing phospholipid, wherein the ratio is 4 or more and 30 or less.
[2] 抗酸化剤が脂溶性抗酸化剤である請求項 1記載の製造方法。 [2] The production method according to claim 1, wherein the antioxidant is a fat-soluble antioxidant.
[3] 脂溶性抗酸化剤がビタミン E又はその誘導体である請求項 2記載の製造方法。 [3] The production method according to claim 2, wherein the fat-soluble antioxidant is vitamin E or a derivative thereof.
[4] 水の含有量が、非極性溶媒及び極性溶媒の合計量に対して、容積基準で 30〜200[4] The water content is 30 to 200 on a volume basis with respect to the total amount of nonpolar solvent and polar solvent.
%である請求項 1〜3いずれか 1項記載の製造方法。 The method according to any one of claims 1 to 3, wherein the production method is%.
[5] 塩基交換反応を開始する前に抗酸化剤を添加する請求項 1〜4 ヽずれか 1項記載の 製造方法。 [5] The production method according to claim 1, wherein an antioxidant is added before the base exchange reaction is started.
[6] 多価不飽和脂肪酸含有リン脂質がイカ由来のレシチンである請求項 1〜5いずれか 1 項記載の製造方法。  6. The production method according to any one of claims 1 to 5, wherein the polyunsaturated fatty acid-containing phospholipid is squid-derived lecithin.
[7] 請求項 1〜6いずれか 1項記載の製造方法により得られた多価不飽和脂肪酸含有リ ン脂質。  [7] Polyunsaturated fatty acid-containing phospholipid obtained by the production method according to any one of claims 1 to 6.
[8] 多価不飽和脂肪酸含有リン脂質を構成する n— 3系多価不飽和脂肪酸と n— 6系多 価不飽和脂肪酸の構成比 (n— 3Zn— 6)が 25以上である請求項 7記載の多価不飽 和脂肪酸含有リン脂質。  [8] The composition ratio (n-3Zn-6) of n-3 polyunsaturated fatty acid and n-6 polyunsaturated fatty acid constituting the polyunsaturated fatty acid-containing phospholipid is 25 or more. 7. The polyunsaturated fatty acid-containing phospholipid according to 7.
[9] 請求項 7又は 8記載の多価不飽和脂肪酸含有リン脂質を 10〜100質量％含む油脂  [9] An oil or fat comprising 10 to 100% by mass of the polyunsaturated fatty acid-containing phospholipid according to claim 7 or 8
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JPH09249688A (en) * 1996-01-11 1997-09-22 Cci Corp Chromanol glycoside, its production and antioxidant using the same
JPH11514887A (en) * 1995-11-14 1999-12-21 ロダース・クロックラーン・ビー・ブイ Method for preparing a material having a high content of long-chain polyunsaturated fatty acids
JP2001149087A (en) * 1989-11-17 2001-06-05 Omegatech Inc HETEROTROPHIC PRODUCTION OF MICROBIAL PRODUCT CONTAINING ω-3 HIGHLY UNSATURATED FATTY ACID AT HIGH CONCENTRATION
JP2001186898A (en) * 1999-10-19 2001-07-10 Bizen Kasei Kk Method for producing phosphatidyl serine having polyvalent unsaturated fatty acid residue
JP2003502035A (en) * 1999-06-15 2003-01-21 イサム・リサーチ・デベロツプメント・カンパニー・オブ・ザ・ヘブルー・ユニバーシテイ・オブ・エルサレム Enzymatic production of phospholipids in aqueous media
JP2003304814A (en) * 2002-04-17 2003-10-28 Fancl Corp Composition with high phosphatidyl serine content
JP2003319793A (en) * 2002-02-28 2003-11-11 Nagase Chemtex Corp Phospholipid base exchange method

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Publication number Priority date Publication date Assignee Title
JPS6336791A (en) * 1986-08-01 1988-02-17 Nippon Oil & Fats Co Ltd Production of phospholipid by enzyme
JP2001149087A (en) * 1989-11-17 2001-06-05 Omegatech Inc HETEROTROPHIC PRODUCTION OF MICROBIAL PRODUCT CONTAINING ω-3 HIGHLY UNSATURATED FATTY ACID AT HIGH CONCENTRATION
JPH0471497A (en) * 1990-07-09 1992-03-06 Kao Corp Preparation of monoacylglycerophospholipid
JPH11514887A (en) * 1995-11-14 1999-12-21 ロダース・クロックラーン・ビー・ブイ Method for preparing a material having a high content of long-chain polyunsaturated fatty acids
JPH09249688A (en) * 1996-01-11 1997-09-22 Cci Corp Chromanol glycoside, its production and antioxidant using the same
JP2003502035A (en) * 1999-06-15 2003-01-21 イサム・リサーチ・デベロツプメント・カンパニー・オブ・ザ・ヘブルー・ユニバーシテイ・オブ・エルサレム Enzymatic production of phospholipids in aqueous media
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JP2003304814A (en) * 2002-04-17 2003-10-28 Fancl Corp Composition with high phosphatidyl serine content

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