WO2011004794A1 - リン脂質の製造方法 - Google Patents
リン脂質の製造方法 Download PDFInfo
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- WO2011004794A1 WO2011004794A1 PCT/JP2010/061414 JP2010061414W WO2011004794A1 WO 2011004794 A1 WO2011004794 A1 WO 2011004794A1 JP 2010061414 W JP2010061414 W JP 2010061414W WO 2011004794 A1 WO2011004794 A1 WO 2011004794A1
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- phospholipid
- glycerin
- solvent
- phospholipase
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- 239000003960 organic solvent Substances 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical group CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 235000010384 tocopherol Nutrition 0.000 description 1
- 229960001295 tocopherol Drugs 0.000 description 1
- 229930003799 tocopherol Natural products 0.000 description 1
- 239000011732 tocopherol Substances 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
- C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
- C11C3/04—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils
- C11C3/10—Ester interchange
-
- 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
- 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
- C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
-
- 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
Definitions
- the present invention relates to a method for producing phospholipids, and particularly to a method for producing phospholipids using phospholipase A2.
- DHA docosahexaenoic acid
- EPA eicosapentaenoic acid
- DHA docosahexaenoic acid
- EPA eicosapentaenoic acid
- the method for producing phospholipids to which functional fatty acids such as DHA are bound is divided into a method for extracting naturally occurring ones and a method for synthesizing soybean phospholipids as raw materials.
- specific examples of the former include a method of extracting DHA-bound phospholipids from marine animals' eggs (Patent Document 1), a method of extracting from marine products such as squid using an organic solvent (Patent Document 2), and the like.
- the raw materials are expensive, the supply is not necessarily stable, and the composition of phospholipids depends on the raw materials, so that a functional fatty acid other than DHA is produced. I can't.
- Patent Document 3 As a method for introducing a desired fatty acid regardless of the composition of the raw material, there is a method in which an arbitrary fatty acid is added to a culture solution of a microorganism and a phospholipid bound to the microorganism is produced (Patent Document 3). In this method, only a small amount of phospholipid can be obtained from a large amount of culture solution, and the production efficiency is not good.
- Patent Document 4 As a method for binding DHA to soybean phospholipid or the like, there is a method of adding a high dielectric constant hydrogen bond-forming product to a reaction system of lipase and phospholipase (Patent Document 4).
- a high reaction rate was obtained by the reaction of lysophospholipid and fatty acid by lipase, but a high reaction rate was not obtained by the reaction by phospholipase A2.
- Patent Document 5 As a method for efficiently binding a desired fatty acid to the 2-position of phospholipid, several methods using phospholipase A2 in glycerin have been reported (Patent Document 5, Non-Patent Document 1). However, in these reports, since the extraction method after the reaction uses harmful chloroform-methanol, the solvent cannot be used depending on the use of phospholipid, or an equipment for removing the solvent is required. Since phospholipase A2 is expensive, significant cost reduction is required.
- the present invention provides a method for producing a phospholipid in which an arbitrary fatty acid is bonded to the 2-position of a phospholipid by using an esterification reaction with phospholipase A2 in glycerin. It aims at providing the method of manufacturing a phospholipid.
- the present inventors have extracted phospholipids with a solvent immiscible with glycerin after esterification of lysophospholipid with phospholipase A2 in glycerin, and then the solvent is retained. Finally, by adding lysophospholipids and acyl donors, it was found that phospholipase A2 remaining in glycerin was utilized to enable esterification reaction between lysophospholipids and acyl donors, and the present invention was completed.
- a solvent immiscible with glycerin is added to form a glycerin layer and a solvent layer, Extracting the phospholipid into the solvent layer, transferring phospholipase A2 to the glycerin layer, and then adding the lysophospholipid and acyl donor to the glycerin solution obtained by distilling off the residual solvent from the collected glycerin layer, thereby the glycerin solution
- the present invention relates to a method for producing a phospholipid, characterized in that an esterification reaction is carried out using phospholipase A2 remaining in the above.
- a ketone solvent may be added as a solvent immiscible with the glycerin, or after adding an alcohol having 4 or less carbon atoms after the esterification reaction, You may add the solvent which consists of at least 1 sort (s) chosen from the group which consists of a hydrogen solvent, a ketone solvent, and an ester solvent.
- an amino acid and / or a peptide having 3 or less amino acids may be added to the reaction system.
- the amino acid is preferably at least one selected from the group consisting of glycine, alanine, asparagine, glutamine, isoleucine, leucine, serine, threonine, valine, phenylalanine and tyrosine.
- the peptide is preferably composed of a combination of glycine, alanine and serine.
- the phospholipase A2 in a method for producing a phospholipid in which an arbitrary fatty acid is bonded to the 2-position of a phospholipid using an esterification reaction with phospholipase A2 in glycerin, the phospholipase A2 can be reused at low cost.
- a method for producing the phospholipid can be provided.
- a phospholipid is produced by esterification reaction between lysophospholipid and acyl donor by phospholipase A2 in glycerin, and then a solvent immiscible with glycerin is added to form a glycerin layer and a solvent layer.
- a solvent immiscible with glycerin is added to form a glycerin layer and a solvent layer.
- Forming phospholipids into the solvent layer transferring phospholipase A2 to the glycerin layer, and then adding lysophospholipid and acyl donor to the glycerin solution obtained by distilling off the residual solvent from the separated glycerin layer.
- another esterification reaction is performed using phospholipase A2 remaining in the glycerin solution.
- the lysophospholipid in the present invention refers to a product obtained by removing the fatty acid at the 2-position from the phospholipid, and means a lipid different from the phospholipid.
- a phospholipid-modified one can be used. From the viewpoint of availability, those derived from soybean, rapeseed, and egg yolk are preferable, and those derived from soybean from the price aspect. More preferably, other plant-derived lysophospholipids can also be used.
- the method for modifying the phospholipid to remove the fatty acid residue at the 2-position is not particularly limited, and examples thereof include a method of hydrolyzing the fatty acid residue at the 2-position of phospholipid using phospholipase A2 or the like. It is done.
- the phospholipid that can be used in this case is a molecule having a glycerin skeleton, a phosphate group, and two fatty acid esters, which can be a substrate for phospholipase A2, and does not include those having a sphingosine skeleton. Specific examples include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and the like. Since the method using phospholipase A2 can be carried out without using harmful substances, it is effective, for example, in producing phospholipids for food use.
- the fatty acid that is an acyl donor introduced into the 2-position of lysophospholipid by the esterification reaction of lysophospholipid with phospholipase A2 in glycerin is not particularly limited, and a free fatty acid may be used, or an ethyl ester or triglyceride However, it is preferable to use a free fatty acid from the viewpoint of reactivity, although it can be hydrolyzed with an enzyme such as lipase in the reaction system. Specific examples include highly unsaturated fatty acids such as DHA, EPA, arachidonic acid, and conjugated linoleic acid from the viewpoint of functionality.
- the DHA and EPA include oils and fats obtained mainly from marine animal oils and algae. Can be used in the form of free fatty acids.
- the acyl donor used in the present invention is preferably used in an amount of 30 to 1000 parts by weight with respect to 100 parts by weight of lysophospholipid in terms of reaction efficiency and cost.
- a mixture of fatty acids containing a desired fatty acid can be used.
- the content of the desired fatty acid in the fatty acid mixture is desirably approximately 20% by weight or more.
- the concentration of DHA in the DHA-containing fatty acid is preferably 20% by weight or more, and more preferably having a DHA concentration of 45% by weight or more.
- the concentration of the phospholipid as a reaction product may be increased by performing solvent fractionation after the esterification reaction according to the present invention.
- the origin of the phospholipase A2 used in the present invention is not particularly limited, but those that can be generally used for food are preferable, and examples include those derived from porcine pancreas and those derived from microorganisms.
- the phospholipase A2 used in the present invention is preferably used in an amount of 1000 to 100,000 U per 1 g of lysophospholipid from the viewpoint of reaction efficiency and cost.
- an esterification reaction is performed in glycerin.
- glycerin has high polarity and is effective as a field for esterification reaction, and can be used for food. It is also effective in that it can dissolve an amino acid described later, which is an optional component.
- Glycerin is preferably used in an amount of 500 to 10,000 parts by weight with respect to 100 parts by weight of lysophospholipid in consideration of reactivity and the like.
- the esterification reaction is performed in glycerin as described above, the phospholipid is extracted using a solvent that is not miscible with glycerin in order to extract the phospholipid to be produced.
- a solvent composed of at least one selected from the group consisting of a hydrocarbon solvent, a ketone solvent and an ester solvent can be used. Can be extracted.
- the reason why the addition of the alcohol is unnecessary is that the solubility in glycerin is better than that of the hydrocarbon solvent or the ester solvent.
- the solvent is preferably added in an amount of 20 to 300 parts by weight with respect to 100 parts by weight of glycerin in consideration of phospholipid recovery efficiency and the like.
- the hydrocarbon solvent refers to a compound that can be used as a solvent among compounds composed only of carbon and hydrogen. Specifically, pentane, hexane, heptane and the like are preferable, but hexane is more preferable because it has a low boiling point and can be easily distilled off and used as a food additive.
- the ketone solvent refers to a compound that can be used as a solvent among compounds having a keto group in the molecule. Specific examples include acetone and butanone, but acetone is preferable because it has a low boiling point and can be easily distilled off and used as a food additive.
- the ester solvent refers to a compound that can be used as a solvent among compounds having an ester bond in the molecule, specifically, methyl acetate, ethyl acetate, etc., but ethyl acetate is preferable because it is used for food applications. .
- an antioxidant is used to prevent acyl donor oxidation, or a calcium source such as calcium chloride, or an amino acid or amino acid is used to activate phospholipase A2.
- a calcium source such as calcium chloride
- an amino acid or amino acid is used to activate phospholipase A2.
- the antioxidant is not particularly limited as long as it can be expected to have an antioxidant power against fatty acids such as DHA.
- fatty acids such as DHA.
- polyphenols such as catechin, tocopherol, ascorbic acid and derivatives thereof And dibutylhydroxytoluene (BHT).
- BHT dibutylhydroxytoluene
- the esterification reaction of lysophospholipid may be performed in a nitrogen atmosphere while blocking oxygen.
- the amino acid is preferably a compound that mainly serves as a component of a protein and has a carboxyl group and an amino group in the molecule and can be used for food.
- neutral amino acids are preferable because phospholipase A2 can be activated without relatively affecting the charge state of phospholipase A2, and glycine, alanine, asparagine, glutamine, isoleucine, leucine, serine, threonine, valine, phenylalanine, tyrosine, etc.
- at least one selected from these groups can be used.
- the above peptide having 3 or less amino acids refers to a peptide in which amino acids are mainly dimerized or trimerized by an amide bond, and is synthesized from amino acids or decomposes a protein with an enzyme or the like.
- it is preferably a peptide composed of glycine, alanine, and serine because of its relatively high solubility in glycerin, and examples thereof include glycylglycine.
- a peptide having few amino acid residues a high molar concentration can be obtained when dissolved in glycerin, and phospholipase A2 can be activated efficiently.
- the calcium source is used to activate phospholipase A2 as described above, but it is preferable that the calcium source can exist as calcium ions in the reaction system. Therefore, as a calcium source, what has comparatively high solubility and is used also as a food raw material is preferable.
- a preferred example thereof is calcium chloride as described above.
- the amount of the antioxidant, calcium source, amino acid, and peptide having 3 or less residues of amino acids may be any amount that is suitable for achieving the above objects.
- amino acids and peptides having 3 or less amino acids are preferably added in an amount of 10 to 2000 parts by weight, more preferably 50 to 500 parts by weight, per 100 parts by weight of lysophospholipid. If it is less than 10 parts by weight, the reaction efficiency may be deteriorated, and if it exceeds 2000 parts by weight, the cost may be disadvantageous and the reaction efficiency may be deteriorated.
- amino acids or peptides having 3 or less amino acids may be added in combination of two or more in order to increase the total dissolution amount in the esterification reaction system.
- lysophospholipid and acyl donor are dissolved in glycerin and obtained by adding phospholipase A2, and optionally, an amino acid for activation of phospholipase A2, a peptide having 3 or less amino acids, and calcium chloride.
- the glycerin reaction solution is stirred to carry out esterification reaction between lysophospholipid and acyl donor. Further, if necessary, in order to prevent oxidation of fatty acids, the esterification reaction may be performed in a nitrogen atmosphere while blocking oxygen.
- the esterification reaction is preferably performed in the temperature range of 35 ° C. to 80 ° C., and in the range of 45 ° C. to 70 ° C., from the viewpoint of the optimum temperature of phospholipase A2 and the oxidation of fatty acid which is an acyl donor. It is more preferable.
- a decompression operation may be performed in order to remove water generated by the esterification reaction with phospholipase A2 and promote the esterification reaction.
- the conditions may be, for example, that the temperature is 35 to 80 ° C. and 150 torr (20 kPa) or less for 12 to 24 hours.
- a ketone solvent to the glycerin reaction solution containing the phospholipid produced by the esterification reaction as described above, or an alcohol having 4 or less carbon atoms to reduce the viscosity of glycerin and perform an extraction treatment
- at least one solvent selected from the group consisting of a hydrocarbon solvent, a ketone solvent and an ester solvent is added to the reaction solution to form a glycerin layer and a solvent layer.
- generated by esterification reaction can be extracted to a solvent layer, phospholipase A2 can be migrated to a glycerol layer, and the target phospholipid can be extracted.
- the solvent layer (upper layer) contained the target phospholipid / lysophospholipid, the solvent, and an acyl donor, and the glycerin layer (lower layer) was mainly unable to extract glycerin, the solvent, and phospholipase A2. Phospholipid / lysophospholipid and other additives are included.
- the solvent layer (upper layer) and the glycerin layer (lower layer) are separated (sorted). At this time, the addition of the predetermined solvent and the separation operation may be repeated as appropriate in consideration of the improvement in the purity of the phospholipid and the working efficiency.
- the target phospholipid can be efficiently extracted from the glycerin reaction solution.
- phospholipase A2 may be contained in a trace amount in a solvent layer so that it may accompany the target phospholipid. In this case, the target phospholipid and phospholipase A2 may be mixed even after the treatment exemplified below. Even if phospholipase A2 is contained in this way, there is no particular problem for food use. However, if necessary, phospholipase A2 may be decomposed and inactivated by appropriately using a degrading enzyme such as protease.
- a degrading enzyme such as protease.
- the alcohol having 4 or less carbon atoms refers to methanol, ethanol, propanol and butanol, and ethanol is more preferable because it has low toxicity and can be used as a food additive.
- the alcohol is preferably added in an amount of 10 to 150 parts by weight per 100 parts by weight of glycerin.
- the acyl donor fatty acid may be removed from the solvent layer (upper layer) containing the target phospholipid as described above.
- the method for removing the fatty acid as the acyl donor from the separated upper layer is not particularly limited, and examples thereof include a method of degreasing with a ketone solvent, an ethanol-hexane mixed solvent, a method of removing the fatty acid using silica gel, and the like. .
- a new ketone solvent is added separately and cooled to 5 ° C. or lower to precipitate phospholipids,
- the ketone solvent added after the solvent is distilled off is preferably acetone which has a low boiling point, can be easily distilled off and can be used as a food additive.
- the upper layer is passed through a column filled with silica gel to adsorb phospholipids and then removed by causing the fatty acid to flow out.
- Pour effluent solvent such as methanol through the column to desorb phospholipids adsorbed on silica gel, fractionate only the desired phospholipid fraction, and then recrystallize the phospholipid, which is desired at the 2nd position of lysophospholipid It is possible to obtain a phospholipid into which the fatty acid is introduced.
- the activity of phospholipase A2 may be inhibited by a solvent such as ethanol
- the upper layer and the lower layer are separated (sorted) as described above, and the lower layer glycerin layer is subjected to reduced pressure treatment and remains.
- the solvent can be distilled off.
- a glycerin solution containing phospholipase A2 the remainder of phospholipid / lysophospholipid, other optional additives, and the like is obtained.
- phospholipase A2 is re-regenerated by performing esterification reaction again using phospholipase A2 remaining in the glycerin solution. It can be used.
- the lysophospholipid and the acyl donor may be added simultaneously to the glycerin solution, the acyl donor is added after adding the lysophospholipid, or vice versa. May be.
- the amount added is equivalent to that of the esterification reaction performed previously, depending on the degree of progress and extraction of the reaction, taking into account that unreacted lysophospholipid remains in the glycerin solution. May be adjusted as appropriate.
- the amount reduced by extracting phospholipids is reduced by lysophospholipids and acyl donors. When adding, it can add suitably.
- the conditions for the esterification reaction again may be the same as those for the esterification reaction described above, and the phospholipid extraction process may be performed in the same manner as described above.
- phospholipase A2 maintains activity, it is possible to produce the desired phospholipid at a lower cost by repeating the above-described operation and repeatedly using phospholipase A2. Become. At this time, if the productivity of phospholipid is reduced, phospholipase A2, amino acids, peptides with 3 or less amino acids, arbitrary additives such as calcium chloride, glycerin, etc. may be partially reduced by the extraction operation. Since phospholipase A2 and the like are considered to have a reduced activity, they may be added as necessary. In addition, as in the previous esterification reaction, in order to prevent fatty acid oxidation, if necessary, an antioxidant is added, or oxygen is blocked and the reaction system is changed to a nitrogen atmosphere, thereby esterifying. You may react.
- the phospholipid production method of the present invention can be suitably used as a method for producing a phospholipid in which a desired fatty acid is introduced at the 2-position.
- a raw material that is not edible in the production process or a solvent such as chloroform is used. Since it can be performed without using it, it can be suitably used particularly for the production of edible phospholipids.
- the phospholipid thus obtained, in particular, a product in which a highly unsaturated fatty acid is introduced at the 2-position thereof can be suitably used as a high-functional edible phospholipid.
- the lower layer subjected to the above treatment containing phospholipid and fatty acid is developed with the above solvent by TLC (thin layer chromatography), phospholipid and lysophospholipid fractions are separated, and methyl esterified with sodium methylate.
- TLC thin layer chromatography
- the fatty acid composition of fatty acids bound to phospholipids and lysophospholipids was analyzed by gas chromatography (“GC-14B” manufactured by Shimadzu Corporation). The area% of each fatty acid in gas chromatography was regarded as the weight% of fatty acid.
- Example 1 35 mg of lysophosphatidylcholine (“SLP-LPC70” manufactured by Sakai Oil Co., Ltd.) and 105 mg of DHA-containing fatty acid prepared by hydrolyzing DHA-50G (manufactured by Nippon Chemical Feed Co., Ltd., DHA content: 51.8 wt%) and glycerin 1 g of Sakamoto Pharmaceutical Co., Ltd. was added, and 37.5 mg of glycine (Showa Denko) and 37.5 mg of alanine (Musashino Chemical Laboratory) were added.
- SLP-LPC70 lysophosphatidylcholine
- DHA-50G manufactured by Nippon Chemical Feed Co., Ltd., DHA content: 51.8 wt
- glycerin 1 g of Sakamoto Pharmaceutical Co., Ltd. was added, and 37.5 mg of glycine (Showa Denko) and 37.5 mg of alanine (Musashino Chemical Laboratory) were added.
- the separated (prepared) glycerin layer (lower layer) was depressurized at 0.6 torr (80 Pa) for 15 minutes to distill off the solvent.
- 18 mg of the lysophosphatidylcholine (SLP-LPC70) and 105 mg of the DHA-containing fatty acid were added to the reduced-pressure glycerin layer (glycerin solution), and 40 ⁇ l of water was further added and reacted at 50 ° C. for 24 hours.
- the DHA content in the phospholipid and lysophospholipid contained in the reaction solution was 13.6% by weight.
- Example 2 30 mg of DHA-containing fatty acid prepared by hydrolyzing Incromega DHA-J46 (manufactured by Croda Japan, DHA content: 49.7 wt%) to 35 mg of lysophosphatidylcholine (“SLP-LPC70” manufactured by Sakai Oil Co., Ltd.), glycerin ( 1 g of Sakamoto Pharmaceutical Co., Ltd.) was added, and 25 mg of glycine (Showa Denko) and 25 mg of alanine (Musashino Chemical Laboratories) were added.
- SLP-LPC70 lysophosphatidylcholine
- glycerin 1 g of Sakamoto Pharmaceutical Co., Ltd.
- This reaction solution was extracted twice with 1 ml of acetone, and an acetone layer (upper layer) and a glycerin layer (lower layer) were separated (sorted) by a conventional method.
- the DHA content is 17.6% by weight for the acetone layer and 14.1 for the glycerin layer, respectively. % By weight.
- the separated (prepared) glycerol layer was depressurized at 0.6 torr (80 Pa) for 15 minutes to distill away acetone.
- 30 mg of lysophosphatidylcholine (SLP-LPC70) was added to the reduced glycerin layer (glycerin solution) and dissolved by stirring at 50 ° C. for 30 minutes.
- the phospholipid contained in the reaction solution and the DHA content in the lysophospholipid were determined. As a result of measurement, it was 4.8% by weight.
- 30 mg of DHA-containing fatty acid was added and reacted at 50 ° C. for 24 hours while reducing the pressure at 50 torr (6.7 kPa).
- the DHA content in the phospholipid and lysophospholipid contained in the reaction solution after the reaction for 24 hours was 11.2% by weight.
- Example 3 50 mg of lysophosphatidylcholine (“SLP-white lyso” manufactured by Sakai Oil Co., Ltd.) and 30 mg of DHA-containing fatty acid prepared by hydrolyzing Incromega DHA-J46 (manufactured by Croda Japan Co., Ltd., DHA content: 49.7 wt%) and glycerin 1 g (manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.) was added, and 25 mg glycine (manufactured by Showa Denko KK) and 25 mg alanine (manufactured by Musashino Chemical Laboratory) were further added.
- SLP-white lyso manufactured by Sakai Oil Co., Ltd.
- DHA-containing fatty acid prepared by hydrolyzing Incromega DHA-J46 (manufactured by Croda Japan Co., Ltd., DHA content: 49.7 wt%) and glycerin 1 g (manu
- the separated (prepared) glycerol layer was depressurized at 0.6 torr (80 Pa) for 15 minutes to distill off the solvent.
- 30 mg of lysophosphatidylcholine (SLP-white lyso) was added to this reduced glycerin layer (glycerin solution) and dissolved by stirring at 50 ° C. for 30 minutes.
- the phospholipid contained in the reaction solution and the DHA content in the lysophospholipid As a result, it was 6.6% by weight.
- 30 mg of DHA-containing fatty acid was added and reacted at 50 ° C. for 24 hours while reducing the pressure at 50 torr (6.7 kPa).
- the DHA content in the phospholipid and lysophospholipid contained in the reaction solution after the reaction for 24 hours was 12.3% by weight.
- Example 4 50 mg of lysophosphatidylcholine (“SLP-white lyso” manufactured by Sakai Oil Co., Ltd.) and 30 mg of EPA-containing fatty acid prepared by hydrolyzing EPA-45G (manufactured by Nippon Chemical Feed Co., Ltd., EPA content: 45.7% by weight), glycerin 1 g (manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.) was added, and 25 mg glycine (manufactured by Showa Denko KK) and 25 mg alanine (manufactured by Musashino Chemical Laboratory) were further added.
- SLP-white lyso manufactured by Sakai Oil Co., Ltd.
- EPA-45G manufactured by Nippon Chemical Feed Co., Ltd., EPA content: 45.7% by weight
- glycerin 1 g manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.
- 25 mg glycine
- the separated (prepared) glycerol layer was depressurized at 0.6 torr (80 Pa) for 15 minutes to distill off the solvent.
- 36 mg of lysophosphatidylcholine (SLP-white lyso) was added to this reduced glycerin layer (glycerin solution) and dissolved by stirring at 50 ° C. for 30 minutes.
- the phospholipid contained in the reaction solution and the EPA content in the lysophospholipid As a result, it was 11.2% by weight.
- 30 mg of EPA-containing fatty acid was added thereto, and the mixture was reacted at 50 ° C. for 24 hours while reducing the pressure at 50 torr (6.7 kPa).
- the EPA content in the phospholipid and lysophospholipid contained in the reaction solution after the reaction for 24 hours was 16.3% by weight.
- Example 5 3 g of DHA-containing fatty acid prepared by hydrolyzing Incromega DHA-J46 (Chrode Japan, DHA content: 49.7 wt%) to 7.5 g of lysophosphatidylcholine (“SLP-white lyso” manufactured by Sakai Oil Co., Ltd.) Then, 100 g of glycerin (manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.) was added, and 3 g of glycine (manufactured by Showa Denko KK) and 3 g of alanine (manufactured by Musashino Chemical Laboratory Co., Ltd.) were added.
- glycerin manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.
- 3 g of glycine manufactured by Showa Denko KK
- 3 g of alanine manufactured by Musashino Chemical Laboratory Co., Ltd.
- the reaction solution was mixed with 50 ml of ethanol, stirred, extracted twice with 50 ml of hexane, and a hexane layer (upper layer) and a glycerin layer (lower layer) were separated (sorted) by a conventional method.
- the solvent of the separated hexane layer (upper layer) was distilled off, 50 ml of acetone was added, and the mixture was cooled at 0 ° C. for 1 hour to obtain 6.3 g of the target phospholipid as a precipitate.
- the DHA content was 17.0% by weight.
- the DHA content was 8.3 wt%.
- lysophosphatidylcholine SLP-white lyso
- DHA-containing fatty acid Incromega DHA-J46
- the reaction was carried out at 50 ° C. for 24 hours while reducing the pressure at (0.40 kPa).
- extraction with ethanol and hexane and purification with acetone were performed in the same manner as described above to obtain 6.0 g of the target phospholipid.
- the DHA content was 15.8% by weight.
- phospholipase A2 (“Lecitase 100S” manufactured by Novozymes Japan, 130 U / mg) and 2.5 ⁇ l of 1.0 mol / l calcium chloride (manufactured by Tomita Pharmaceutical) were added and reacted at 60 ° C. for 24 hours.
- a phospholipid (phosphatidylcholine) having an acid bonded to the 2-position was obtained.
- the oleic acid content was 39.2% by weight.
- phospholipase A2 (“Lecitase 100S” manufactured by Novozymes Japan, 130 U / mg) and 2.5 ⁇ l of 1.0 mol / l calcium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) were added, and dibutylhydroxy as an antioxidant.
- 3 mg of toluene (manufactured by Wako Pure Chemical Industries, Ltd.) was added and reacted at 60 ° C. for 48 hours to obtain a phospholipid (phosphatidylcholine) having a highly unsaturated fatty acid (DHA) bonded to the 2-position.
- DHA highly unsaturated fatty acid
- phospholipase A2 manufactured by Sanyo Fine, powder lysonase, 53 U / mg
- 2.5 ⁇ l of 1.2 mol / l calcium chloride manufactured by Wako Pure Chemical Industries, Ltd.
- sancatel containing catechin as an antioxidant.
- Phospholipid phosphatidylcholine
- NO1 highly unsaturated fatty acid
- ascorbic acid manufactured by Wako Pure Chemical Industries, Ltd.
- phospholipase A2 (“Powder Lysonase” manufactured by Sanyo Fine Co., Ltd.) was added and the water was removed by reducing the pressure at 0.6 torr (80 Pa) for 10 minutes, and then 10 ⁇ l of 0.3 mol / l calcium chloride (manufactured by Tomita Pharmaceutical Co., Ltd.) solution. And reacted at 60 ° C. for 48 hours to obtain a phospholipid (phosphatidylcholine) in which a highly unsaturated fatty acid (DHA) was bonded to the 2-position.
- DHA highly unsaturated fatty acid
- phospholipase A2 (“Powder Lysonase” manufactured by Sanyo Fine Co., Ltd.) was added and the water was removed by reducing the pressure at 0.6 torr (80 Pa) for 10 minutes, and then 10 ⁇ l of 0.3 mol / l calcium chloride (manufactured by Tomita Pharmaceutical Co., Ltd.) solution. And reacted at 60 ° C. for 48 hours to obtain a phospholipid (phosphatidylcholine) in which a highly unsaturated fatty acid (DHA) was bonded to the 2-position.
- DHA highly unsaturated fatty acid
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Abstract
Description
また、脂肪酸の酸化を防止するため、リゾリン脂質のエステル化反応は酸素を遮断し、窒素雰囲気下で行ってもよい。
まず、リゾリン脂質およびアシルドナーを、グリセリンに溶解し、ホスホリパーゼA2、及び必要に応じて酸化防止剤、ホスホリパーゼA2を活性化するためのアミノ酸やアミノ酸が3残基以下のペプチド、塩化カルシウムを加えて得られるグリセリン反応液を攪拌することによりリゾリン脂質とアシルドナーとのエステル化反応を行う。また、必要に応じ、脂肪酸の酸化防止のため、酸素を遮断して窒素雰囲気下でエステル化反応を行ってもよい。
その後、当該グリセリン溶液に、反応基質であるリゾリン脂質及びアシルドナーである脂肪酸を更に添加すれば、グリセリン溶液中に残存するホスホリパーゼA2を利用して再度のエステル化反応をさせることにより、ホスホリパーゼA2を再利用することができることとなる。
この際、リゾリン脂質及びアシルドナーの添加の仕方に特に限定はなく、グリセリン溶液にリゾリン脂質とアシルドナーを同時に添加しても良いし、リゾリン脂質を添加した後にアシルドナーを添加し、あるいは、その逆であっても良い。また添加量としては、グリセリン溶液に未反応のリゾリン脂質が残留していることも考慮しながら、反応の進行度合いや抽出度合いに応じて、先に行ったエステル化反応の場合と同等となるように適宜調整すればよい。また、リゾリン脂質とアシルドナー以外の原料である、カルシウム源、アミノ酸又は/及びアミノ酸が3残基以下のペプチド、グリセリンなどについても、リン脂質を抽出することにより減少した分は、リゾリン脂質およびアシルドナーを添加する際に適宜追加することができる。また、再度のエステル化反応の条件は前述のエステル化反応の場合と同様にすればよいし、リン脂質の抽出処理も前述と同様にして行えばよい。
<リン脂質の脂肪酸組成の測定>
実施例および比較例などにおいて、反応終了後、反応溶液50μlにクロロホルム:メタノール=2:1(容量比)の溶媒200μlと飽和塩化ナトリウム溶液500μlとを加えて攪拌し、13000rpmで1分間遠心分離して下層を抽出し、再度クロロホルム:メタノール=2:1(容量比)の溶媒200μlで同様に遠心分離して下層を抽出した。リン脂質と脂肪酸を含有する前記処理を施した下層をTLC(薄層クロマトグラフィー)で前記溶媒にて展開し、リン脂質及びリゾリン脂質画分を分取して、ナトリウムメチラートでメチルエステル化を行い、リン脂質及びリゾリン脂質に結合している脂肪酸の脂肪酸組成をガスクロマトグラフィー(島津製作所製「GC-14B」)で分析した。ガスクロマトグラフィーにおける各脂肪酸の面積%を脂肪酸の重量%とみなした。
リゾホスファチジルコリン(辻製油社製「SLP-LPC70」)35mgに、DHA-50G(日本化学飼料社製、DHA含量:51.8重量%)を定法により加水分解して調製したDHA含有脂肪酸105mg、グリセリン(阪本薬品工業社製)1gを加えて、さらにグリシン(昭和電工社製)37.5mgとアラニン(武蔵野化学研究所社製)37.5mgを加えた。さらにホスホリパーゼA2(サンヨーファイン社製「粉末リゾナーゼ」、53U/mg)20mgを加えて0.6torr(80Pa)で10分間減圧して水分を除去した後、0.3mol/l塩化カルシウム(富田製薬社製)溶液10μl、及び水30μlを加えて50℃で24時間反応させた。反応液中に含まれるリン脂質及びリゾリン脂質中のDHA含量は13.4重量%であった。
リゾホスファチジルコリン(辻製油社製「SLP-LPC70」)35mgにインクロメガDHA-J46(クローダジャパン社製、DHA含量:49.7重量%)を定法により加水分解して調製したDHA含有脂肪酸30mg、グリセリン(阪本薬品工業社製)1gを加えて、さらにグリシン(昭和電工社製)25mgとアラニン(武蔵野化学研究所社製)25mgを加えた。さらにホスホリパーゼA2(サンヨーファイン社製「粉末リゾナーゼ」、53U/mg)20mgを加えて0.6torr(80Pa)で10分間減圧して水分を除去した後、0.3mol/l塩化カルシウム(富田製薬社製)溶液10μlを加えて50torr(6.7kPa)で減圧しながら50℃で24時間反応させた。反応液中に含まれるリン脂質及びリゾリン脂質中のDHA含量は16.9重量%であった。
リゾホスファチジルコリン(辻製油社製「SLP-ホワイトリゾ」)50mgにインクロメガDHA-J46(クローダジャパン社製、DHA含量:49.7重量%)を定法により加水分解して調製したDHA含有脂肪酸30mg、グリセリン(阪本薬品工業社製)1gを加えて、さらにグリシン(昭和電工社製)25mgとアラニン(武蔵野化学研究所社製)25mgを加えた。さらにホスホリパーゼA2(サンヨーファイン社製「粉末リゾナーゼ」、53U/mg)20mgを加えて0.6torr(80Pa)で10分間減圧して水分を除去した後、0.3mol/l塩化カルシウム(富田製薬社製)溶液10μlを加えて50torr(6.7kPa)で減圧しながら50℃で24時間反応させた。反応液中に含まれるリン脂質及びリゾリン脂質中のDHA含量は15.3重量%であった。
リゾホスファチジルコリン(辻製油社製「SLP-ホワイトリゾ」)50mgにEPA-45G(日本化学飼料社製、EPA含量:45.7重量%)を定法により加水分解して調製したEPA含有脂肪酸30mg、グリセリン(阪本薬品工業社製)1gを加えて、さらにグリシン(昭和電工社製)25mgとアラニン(武蔵野化学研究所社製)25mgを加えた。さらにホスホリパーゼA2(サンヨーファイン社製「粉末リゾナーゼ」、53U/mg)20mgを加えて0.6torr(80Pa)で10分間減圧して水分を除去した後、0.3mol/l塩化カルシウム(富田製薬社製)溶液10μlを加えて50torr(6.7kPa)で減圧しながら50℃で24時間反応させた。反応液中に含まれるリン脂質及びリゾリン脂質中のEPA含量は19.5重量%であった。
リゾホスファチジルコリン(辻製油社製「SLP-ホワイトリゾ」)7.5gにインクロメガDHA-J46(クローダジャパン社製、DHA含量:49.7重量%)を定法により加水分解して調製したDHA含有脂肪酸3g、グリセリン(阪本薬品工業社製)100gを加えて、さらにグリシン(昭和電工社製)3gとアラニン(武蔵野化学研究所社製)3gを加えた。さらにホスホリパーゼA2(サンヨーファイン社製「粉末リゾナーゼ」、53U/mg)3gを加え、2mol/l塩化カルシウム(富田製薬社製)溶液0.5mlを加えて、3torr(0.40kPa)の減圧下、50℃で24時間反応させた。
下記に示すように、各種のアミノ酸、アミノ酸が3残基以下のペプチド等を用いることで、所望のリン脂質を効率よく製造することが可能であることが分かる。従って、これらを用いてホスホリパーゼA2を再利用した場合も、所望のリン脂質の効率の良い製造が期待できる。
リゾホスファチジルコリン(辻製油社製「SLP-LPC70H」)35mgにオレイン酸(東京化成工業社製)97mg、グリセリン(阪本薬品工業社製)1gを加えて、さらにグリシン(和光純薬工業社製)50mgを加えた。さらにホスホリパーゼA2(ノボザイムズジャパン社製「レシターゼ100S」、130U/mg)10mgと1.0mol/l塩化カルシウム(富田製薬社製)溶液2.5μlを加えて60℃で24時間反応させ、オレイン酸が2位に結合したリン脂質(ホスファチジルコリン)を得た。得られたリン脂質及びリゾリン脂質中の脂肪酸組成において、オレイン酸含量は39.2重量%であった。
リゾホスファチジルコリン(辻製油社製「SLP-LPC70」)35mgにDHA(東京化成工業社製)113mg、グリセリン(和光純薬工業社製)1gを加えて、さらにグリシン(和光純薬工業社製)50mgを加えた。さらにホスホリパーゼA2(ノボザイムズジャパン社製「レシターゼ100S」、130U/mg)10mgと1.0mol/l塩化カルシウム(和光純薬工業社製)溶液2.5μlを加え、さらに抗酸化剤としてジブチルヒドロキシトルエン3mg(和光純薬工業社製)を加えて60℃で48時間反応させ、高度不飽和脂肪酸(DHA)が2位に結合したリン脂質(ホスファチジルコリン)を得た。得られたリン脂質及びリゾリン脂質中の脂肪酸組成において、DHA含量は34.2重量%であった。
リゾホスファチジルコリン(辻製油社製SLP-LPC70)35mgにDHA(東京化成工業社製)113mg、グリセリン(和光純薬工業社製)1gを加えて、さらにグリシルグリシン(和光純薬工業社製)60mgを加えた。さらにホスホリパーゼA2(サンヨーファイン社製、粉末リゾナーゼ、53U/mg)20mgと1.2mol/l塩化カルシウム(和光純薬工業社製)溶液2.5μlを加え、さらに抗酸化剤としてカテキンを含有するサンカトールNO1(太陽化学社製)3mg、及びアスコルビン酸3mg(和光純薬工業社製)を加えて60℃で48時間反応させ、高度不飽和脂肪酸(DHA)が2位に結合したリン脂質(ホスファチジルコリン)を得た。得られたリン脂質及びリゾリン脂質中の脂肪酸組成において、DHA含量は30.7重量%であった。
DHA113mgの代わりにEPA(ナカライテスク社製)104mgを用い、さらにグリシルグリシン60mgの代わりにグリシン60mgを用いた以外は実施例2と同様にして高度不飽和脂肪酸(EPA)が2位に結合したリン脂質を得た。得られたリン脂質及びリゾリン脂質中の脂肪酸組成において、EPA含量は28.5重量%であった。
DHA113mgの代わりにアラキドン酸(シグマアルドリッチジャパン社製)103mgを用い、さらにグリシルグリシン60mgの代わりにグリシン40mgを用いた以外は実施例2と同様にして高度不飽和脂肪酸(アラキドン酸)が2位に結合したリン脂質(ホスファチジルコリン)を得た。得られたリン脂質及びリゾリン脂質中の脂肪酸組成において、アラキドン酸含量は32.3重量%であった。
リゾホスファチジルコリン(辻製油社製「SLP-LPC70」)35mgにDHA-50G(日本化学飼料社製、DHA51.8重量%含有)を定法により加水分解して調製したDHA含有脂肪酸105mg、グリセリン(阪本薬品工業社製)1gを加えて、さらにグリシン(昭和電工社製)75mgを加えた。さらにホスホリパーゼA2(サンヨーファイン社製「粉末リゾナーゼ」)20mgを加えて0.6torr(80Pa)で10分減圧して水分を除去した後、0.3mol/l塩化カルシウム(富田製薬社製)溶液10μlを加えて60℃で48時間反応させ、高度不飽和脂肪酸(DHA)が2位に結合したリン脂質(ホスファチジルコリン)を得た。得られたリン脂質及びリゾリン脂質中の脂肪酸組成において、DHA含量は15.5重量%であった。
リゾホスファチジルコリン(辻製油社製「SLP-LPC70」)35mgにDHA-50G(日本化学飼料社製、DHA51.8重量%含有)を定法により加水分解して調製したDHA含有脂肪酸105mg、グリセリン(阪本薬品工業社製)1gを加えて、さらにグリシン(昭和電工社製)37.5mgとアラニン(武蔵野化学研究所社製)37.5mgを加えた。さらにホスホリパーゼA2(サンヨーファイン社製「粉末リゾナーゼ」)20mgを加えて0.6torr(80Pa)で10分減圧して水分を除去した後、0.3mol/l塩化カルシウム(富田製薬社製)溶液10μlを加えて60℃で48時間反応させ、高度不飽和脂肪酸(DHA)が2位に結合したリン脂質(ホスファチジルコリン)を得た。得られたリン脂質及びリゾリン脂質中の脂肪酸組成において、DHA含量は17.2重量%であった。
リゾホスファチジルコリン(辻製油社製「SLP-LPC70」)35mgにDHA-50G(日本化学飼料社製、DHA51.8重量%含有)を定法により加水分解して調製したDHA含有脂肪酸30mg、グリセリン(阪本薬品工業社製)1gを加えて、さらにグリシン(昭和電工社製)37.5mgとアラニン(武蔵野化学研究所社製)37.5mgを加えた。さらにホスホリパーゼA2(サンヨーファイン社製「粉末リゾナーゼ」)20mgを加え、さらに0.5mol/l塩化カルシウム(富田製薬社製)溶液6μlを加え、0.6torr(80Pa)で10分減圧して水分を除去し、50℃で24時間反応させ、ホスファチジルコリン純度向上用のリン脂質含有エステル化反応溶液を得た。該反応液中に含まれるリン脂質、及びリゾリン脂質中のDHA含量は15.3重量%であった。
グリシンを用いなかった以外は参考例1と同様にしてオレイン酸が2位に結合したリン脂質を得た。得られたリン脂質及びリゾリン脂質中の脂肪酸組成において、オレイン酸含量は19.4重量%であった。
グリシルグリシンを用いなかった以外は参考例3と同様にして高度不飽和脂肪酸(DHA)が2位に結合したリン脂質を得た。得られたリン脂質及びリゾリン脂質中の脂肪酸組成において、DHA含量は12.9重量%であった。
グリシンを用いず、水を60μl加えた以外は参考例4と同様にして高度不飽和脂肪酸(EPA)が2位に結合したリン脂質を得た。得られたリン脂質の及びリゾリン脂質中の脂肪酸組成において、EPA含量は8.9重量%であった。
グリシンを用いなかった以外は参考例5と同様にして高度不飽和脂肪酸(アラキドン酸)が2位に結合したリン脂質を得た。得られたリン脂質及びリゾリン脂質中の脂肪酸組成において、アラキドン酸含量は5.5重量%であった。
グリシンを用いなかったこと以外は参考例6と同様にして高度不飽和脂肪酸(DHA)が2位に結合したリン脂質を得た。得られたリン脂質の、及びリゾリン脂質中の脂肪酸組成において、DHA含量は4.4重量%であった。
グリシン及びアラニンを用いなかったこと以外は参考例8と同様にして高度不飽和脂肪酸(DHA)が2位に結合したリン脂質を得た。得られたリン脂質の、及びリゾリン脂質中の脂肪酸組成において、DHA含量は7.6重量%であった。
Claims (6)
- グリセリン中でのホスホリパーゼA2によるリゾリン脂質とアシルドナーとのエステル化反応によりリン脂質を生成させた後、グリセリンと混和しない溶剤を添加してグリセリン層と溶剤層を形成させて、前記リン脂質を前記溶剤層に抽出し、ホスホリパーゼA2を前記グリセリン層に移行させ、その後分取したグリセリン層から残留溶剤を留去したグリセリン溶液にリゾリン脂質及びアシルドナーを更に添加することにより、前記グリセリン溶液に残存するホスホリパーゼA2を利用して再度のエステル化反応を行うことを特徴とするリン脂質の製造方法。
- グリセリンと混和しない溶剤として、ケトン溶剤を添加する請求項1に記載のリン脂質の製造方法。
- エステル化反応後に炭素数4以下のアルコールを加えてから、グリセリンと混和しない溶剤として、炭化水素溶剤、ケトン溶剤及びエステル溶剤からなる群から選ばれる少なくとも1種からなる溶剤を添加する請求項1に記載のリン脂質の製造方法。
- 前記エステル化反応において、当該反応系にアミノ酸及び/又はアミノ酸が3残基以下のペプチドを添加する請求項1~3の何れかに記載のリン脂質の製造方法。
- 前記アミノ酸が、グリシン、アラニン、アスパラギン、グルタミン、イソロイシン、ロイシン、セリン、トレオニン、バリン、フェニルアラニン、チロシンからなる群より選ばれる少なくとも1種である請求項4に記載のリン脂質の製造方法。
- 前記ペプチドが、グリシン、アラニン、セリンの組み合わせからなる請求項4又は5に記載のリン脂質の製造方法。
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/382,815 US8530208B2 (en) | 2009-07-06 | 2010-07-05 | Method for producing phospholipid |
JP2011521910A JP4978751B2 (ja) | 2009-07-06 | 2010-07-05 | リン脂質の製造方法 |
ES10797105.3T ES2468799T3 (es) | 2009-07-06 | 2010-07-05 | Procedimiento para producir fosfol�pido |
EP10797105.3A EP2453020B1 (en) | 2009-07-06 | 2010-07-05 | Method for producing phospholipid |
CN201080030159.8A CN102471788B (zh) | 2009-07-06 | 2010-07-05 | 磷脂的制造方法 |
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WO2013187328A1 (ja) * | 2012-06-13 | 2013-12-19 | 株式会社カネカ | リン脂質含有組成物の製造方法及びリン脂質含有組成物 |
CN114934080A (zh) * | 2022-04-06 | 2022-08-23 | 西北大学 | 一种磷脂型dha的制备方法 |
JP7297135B1 (ja) | 2022-10-06 | 2023-06-23 | キユーピー株式会社 | リゾリン脂質含有組成物の製造方法、およびこれを用いた水中油型乳化組成物の製造方法 |
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US8658401B2 (en) * | 2011-03-24 | 2014-02-25 | Jiannan University | Method for preparing high purity L-α glycerylphosphorylcholine |
CN104561156B (zh) * | 2013-10-28 | 2020-07-07 | 丰益(上海)生物技术研发中心有限公司 | 制备饱和型磷脂的方法 |
CN109759123B (zh) * | 2019-02-18 | 2021-09-24 | 大连工业大学 | 一种负载铁sba-15的制备方法及其在合成结构磷脂中的应用 |
CN111733193B (zh) * | 2020-07-22 | 2021-12-07 | 河北德嵩生物科技有限公司 | 一种水溶性好耐高温磷脂的生产方法 |
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Cited By (7)
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WO2013187328A1 (ja) * | 2012-06-13 | 2013-12-19 | 株式会社カネカ | リン脂質含有組成物の製造方法及びリン脂質含有組成物 |
JP5477521B1 (ja) * | 2012-06-13 | 2014-04-23 | 株式会社カネカ | リン脂質含有組成物の製造方法及びリン脂質含有組成物 |
US10160984B2 (en) | 2012-06-13 | 2018-12-25 | Kaneka Corporation | Method for producing phospholipid-containing composition, and phospholipid-containing composition |
CN114934080A (zh) * | 2022-04-06 | 2022-08-23 | 西北大学 | 一种磷脂型dha的制备方法 |
JP7297135B1 (ja) | 2022-10-06 | 2023-06-23 | キユーピー株式会社 | リゾリン脂質含有組成物の製造方法、およびこれを用いた水中油型乳化組成物の製造方法 |
WO2024075335A1 (ja) * | 2022-10-06 | 2024-04-11 | キユーピー株式会社 | リゾリン脂質含有組成物の製造方法、およびこれを用いた水中油型乳化組成物の製造方法 |
JP2024054894A (ja) * | 2022-10-06 | 2024-04-18 | キユーピー株式会社 | リゾリン脂質含有組成物の製造方法、およびこれを用いた水中油型乳化組成物の製造方法 |
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CN102471788B (zh) | 2015-01-14 |
US20120100580A1 (en) | 2012-04-26 |
JP4978751B2 (ja) | 2012-07-18 |
CL2012000020A1 (es) | 2012-08-17 |
EP2453020A4 (en) | 2013-06-05 |
JPWO2011004794A1 (ja) | 2012-12-20 |
ES2468799T3 (es) | 2014-06-17 |
EP2453020A1 (en) | 2012-05-16 |
CN102471788A (zh) | 2012-05-23 |
US8530208B2 (en) | 2013-09-10 |
EP2453020B1 (en) | 2014-03-12 |
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