CN106609286B - Preparation method of phospholipid containing long-chain polyunsaturated fatty acid - Google Patents
Preparation method of phospholipid containing long-chain polyunsaturated fatty acid Download PDFInfo
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- CN106609286B CN106609286B CN201510686959.XA CN201510686959A CN106609286B CN 106609286 B CN106609286 B CN 106609286B CN 201510686959 A CN201510686959 A CN 201510686959A CN 106609286 B CN106609286 B CN 106609286B
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Abstract
The present invention relates to a process for preparing phospholipids containing long-chain polyunsaturated fatty acids. The invention provides a preparation method of phospholipid containing long-chain polyunsaturated fatty acid, which comprises the following steps: (1) contacting raw materials of phospholipid, grease and a solvent, and adding an enzyme preparation for reaction; (2) and (2) removing the solvent, and then adding an immobilized enzyme to carry out a reaction, wherein the step (1) is followed by a step of adding a carbamide. By the method, the price cost is greatly reduced, and the added value of the product is obviously improved.
Description
Technical Field
The invention provides a phospholipid rich in long-chain polyunsaturated fatty acid and a preparation method thereof.
Background
The long-chain polyunsaturated fatty acids (PUFAs) refer to straight-chain fatty acids containing more than two double bonds and having carbon chain lengths of 18-22 carbon atoms, such as EPA, DHA, DPA, ARA and the like, have important physiological activities on human bodies and have irreplaceable effects on other fatty acids. The triglyceride type PUFAs mainly come from fat and microbial fermentation of deep sea fish, and the phospholipid type PUFAs are mainly extracted from organ tissues of antarctic krill and deep sea fish. Some recent studies show that the phospholipid type PUFAs have better oxidation stability and bioavailability than the triglyceride type PUFAs, most of the phospholipid type PUFAs can be directly absorbed by cells without hydrolysis, and have double physiological activities of phospholipid and PUFAs, so the phospholipid type PUFAs have obvious advantages in maintaining the physiological functions of human bodies compared with the triglyceride type PUFAs, and the phospholipid type PUFAs can be widely used in the aspects of food additives, health care products, medicines and the like.
However, phospholipid type PUFAs are threatened by the influence of the season of krill harvesting and the possible heavy metal contamination due to the limitation of the source, and soybean phospholipids and egg yolk phospholipids rich in the source do not contain PUFAs in their natural fatty acid composition, and thus, studies on the enzymatic synthesis of phospholipids containing PUFAs structure based on general phospholipids have been gradually conducted.
CN101701229B discloses a method for preparing textured phospholipid and lecithin by mixing concentrated phospholipid and unsaturated fat and adding lipase to catalyze phospholipid for transesterification. The method takes phospholipid or concentrated phospholipid and unsaturated oil as raw materials, and lipase is used for carrying out catalytic transesterification reaction, but the method has low reaction efficiency and the reaction time is as long as about 60 hours.
CN102181498A applies high-purity seal oil free type omega-3 unsaturated fatty acid and lysophosphatidylcholine to obtain a phosphatidylcholine type omega-3 unsaturated fatty acid product under the catalysis of enzyme non-aqueous phase. The patent uses a solvent system, the reaction time is long, and meanwhile, in the catalytic reaction process, the hydrolysis side reaction is serious, so that the product yield is low.
CN101195637B provides a process for preparing polyunsaturated fatty acid-rich phospholipid in a subcritical R134a system, but the method needs a high-pressure closed reaction kettle and a related device for preparing subcritical gas, the equipment investment is large, and meanwhile, a reaction medium R134a is a chemical raw material and is not suitable for production of food.
CN104531790A discloses a method for preparing phospholipid DHA by dissolving an anionic/cationic surfactant in a hydrophobic alkane solvent to obtain a micro-reaction system and performing a catalytic reaction with phospholipase.
In addition, other domestic literature on enzymatic preparation of phospholipid-type PUFAs has mainly focused on the use of immobilized lipases or immobilized phospholipase A in solvent systems1Catalyzed by acidolysis or ester-transesterification of phospholipids, e.g. Jinhuali (research on modification of soybean phospholipids by lipase [ J)]) The soybean phospholipid is catalyzed by novozym435 to be exchanged with EPA-DHA ester, the reaction time is as long as 60 hours, and the insertion rate of EPA-DHA is about 24%; sungmega-emulsion (process for preparing n-3 polyunsaturated fatty acid phospholipid by enzyme method) using immobilized phospholipase A in solvent-free system1The soybean phospholipid and the ethyl ester type fish oil are catalyzed to carry out ester exchange reaction, the insertion rate of EPA and DHA is 25%, but the side reaction of phospholipid hydrolysis is serious.
US2005282033A provides an improved process for the enzymatic preparation of PUFAs by adding amines during the reaction, which increases the reaction rate, but does not increase the insertion rate of PUFAs and the final yield of phospholipids.
WO2005038037A2 uses phospholipase A1 and A2 to carry out esterification and transesterification reactions to prepare the structural phospholipid, and the method has long reaction time, low insertion rate and serious hydrolysis side reaction.
EP0494881B1 uses phospholipase A2 to catalyze esterification reaction in a solvent system, and the method has the defects of limited enzyme source, unstable activity after immobilization and the like besides the defects of the method.
Disclosure of Invention
An object of the present invention is to provide a method for producing a phospholipid containing a long-chain polyunsaturated fatty acid, which comprises:
step (1): contacting raw materials of phospholipid, grease and a solvent, and adding an enzyme preparation for reaction;
step (2): and (2) removing the solvent, and then adding an immobilized enzyme to carry out a reaction, wherein the step (1) is followed by a step of adding a carbamide.
The production method according to the present invention, wherein the raw material phospholipid does not contain a long-chain polyunsaturated fatty acid.
The production method according to the present invention, wherein the raw material phospholipid is a plant-derived phospholipid or an animal-derived phospholipid.
The production method according to the present invention is characterized in that the plant-derived phospholipid is at least one selected from the group consisting of soybean phospholipid, rapeseed phospholipid, sunflower phospholipid, peanut phospholipid, sesame phospholipid, and corn phospholipid.
The production method according to the present invention, wherein the animal-derived phospholipid is egg yolk phospholipid.
The preparation method comprises the step of adding 5-60 wt% of long-chain polyunsaturated fatty acid into the grease.
The production method according to the present invention is a method for producing a fat or oil, wherein the fat or oil is at least one selected from the group consisting of deep sea fish oil and microbial fat.
The preparation method according to the present invention is characterized in that the oil or fat is at least one selected from tuna oil, herring oil, salmon oil, sardine oil, docosahexaenoic acid algae oil, and arachidonic acid oil or fat.
The production method according to the present invention, wherein the solvent is an ethanol solution.
The production method according to the present invention, wherein the solvent is a 95% ethanol solution.
According to the preparation method, the ratio of the raw material phospholipid to the added amount of the oil in the step (1) is 1: 1-1: 15.
according to the preparation method, the ratio of the raw material phospholipid to the added amount of the oil in the step (1) is 1: 2-1: 10.
according to the preparation method, the ratio of the raw material phospholipid to the added amount of the oil in the step (1) is 1: 4-1: 8.
the preparation method comprises the following steps of (1) adding the total amount of the raw material phospholipid and the grease and the solvent in a weight/volume (g/mL) ratio of 1: 5-1: 80.
the preparation method comprises the following steps of (1) adding the total amount of the raw material phospholipid and the grease and the solvent in a weight/volume (g/mL) ratio of 1: 8-1: 40.
the preparation method comprises the following steps of (1) adding the total amount of the raw material phospholipid and the grease and the solvent in a weight/volume (g/mL) ratio of 1: 10-1: 30.
the preparation method comprises the following steps of (1) adding the total amount of the raw material phospholipid and the grease and the solvent in a weight/volume (g/mL) ratio of 1: 12-1: 18.
the production method according to the present invention, wherein the enzyme preparation described in the step (1) is in a free enzyme form.
The preparation method according to the present invention, wherein the enzyme preparation in the step (1) is at least one selected from phospholipase A1, phospholipase A2 and lipase.
The preparation method according to the present invention, wherein the enzyme preparation described in step (1) is derived from at least one of Aspergillus niger (Aspergillus niger), Candida antarctica (Candida antarctic), Candida rugosa (Candida rugosa), Candida cylindracea (Candida cylindracea), Thermomyces lanuginosus (Thermomyces lanuginosus), Rhizomucor miehei (Rhizomucor miehei), Mucor miehei (Mucor miehei), Rhizopus delemar (Rhizopus delemar), Rhizopus oryzae (Rhizopus oryzae), Pseudomonas pseudomonads (Pseudomonas sp.).
The preparation method comprises the following steps of (1) adding the total amount of the raw material phospholipid and the grease and the enzyme preparation in a weight/volume (g/mL) ratio of 10: 1-200: 1.
the preparation method comprises the following steps of (1) adding the total amount of the raw material phospholipid and the grease and the enzyme preparation in a weight/volume (g/mL) ratio of 20: 1-100: 1.
the preparation method comprises the following steps of (1) adding the total amount of the raw material phospholipid and the grease and the enzyme preparation in a weight/volume (g/mL) ratio of 40: 1-80: 1.
the preparation method comprises the following steps of (1) adding the total amount of the raw material phospholipid and the grease and the enzyme preparation in a weight/volume (g/mL) ratio of 50: 1-60: 1.
the production method according to the present invention is characterized in that the amount of carbamide added is 100 to 800 parts by weight based on 100 parts by weight of the total amount of the raw material phospholipid and the oil-and-fat.
According to the production method of the present invention, the amount of carbamide added is 200 to 600 parts by weight per 100 parts by weight of the total amount of the raw material phospholipid and the oil-and-fat.
According to the production method of the present invention, the amount of carbamide added is 250 to 350 parts by weight based on 100 parts by weight of the total amount of the raw material phospholipid and the oil-and-fat.
The preparation method provided by the invention is characterized in that the step (2) is carried out at the temperature of 60-80 ℃.
The preparation method according to the invention, wherein the immobilized enzyme of the step (2) is at least one of lipase or phospholipase immobilized on a macroporous adsorption resin or basic ion exchange resin carrier.
According to the preparation method of the present invention, the weight ratio of the total amount of the raw material phospholipid and the oil-and-fat to the immobilized enzyme is 10: 1-200: 1.
according to the preparation method of the present invention, the weight ratio of the total amount of the raw material phospholipid and the oil-and-fat to the immobilized enzyme is 20: 1-150: 1.
the preparation method according to the present invention, wherein the step (2) is performed under vacuum or protective gas atmosphere.
The production method according to the present invention, wherein the protective gas atmosphere of the step (2) is an inert gas atmosphere.
The production method according to the present invention, wherein the water absorbing agent is added in the step (2).
According to the production method of the present invention, the water absorbing agent is at least one selected from a molecular sieve and a high molecular water absorbent resin.
According to the preparation method of the invention, the high-molecular water-absorbing resin is at least one selected from acrylamide-acrylate copolymerization crosslinking matter and starch graft acrylate copolymerization crosslinking matter.
The preparation method of the present invention is characterized in that the raw material phospholipid has a phosphatidylcholine content of 15 to 60 wt%.
According to the preparation method, the long-chain polyunsaturated fatty acid is a straight-chain fatty acid which contains more than two double bonds and has 18-22 carbon atoms.
According to the production method of the present invention, the long-chain polyunsaturated fatty acid is at least one selected from eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid and arachidonic acid.
According to the preparation method, the content of phosphatidylcholine in the phospholipid containing long-chain polyunsaturated fatty acid is 70-85 wt%.
According to the preparation method, in the fatty acid composition of the phospholipid containing the long-chain polyunsaturated fatty acid, the content of the polyunsaturated fatty acid is 15-60 wt%.
According to the preparation method, in the fatty acid composition of the phospholipid containing the long-chain polyunsaturated fatty acid, the content of the polyunsaturated fatty acid is 25-52% by weight.
Another object of the present invention is to provide a phospholipid containing a long-chain polyunsaturated fatty acid, which is prepared by the preparation method of any one of claims 100 to 135.
The phospholipid containing long-chain polyunsaturated fatty acid has a phosphatidylcholine content of 70-85 wt%.
The phospholipid containing long-chain polyunsaturated fatty acid has a fatty acid composition in which the content of polyunsaturated fatty acid is 15-60 wt%.
The phospholipid containing long-chain polyunsaturated fatty acid has a fatty acid composition in which the content of polyunsaturated fatty acid is 25-52 wt%.
The phospholipid containing a long-chain polyunsaturated fatty acid according to the present invention is a linear fatty acid containing two or more double bonds and having 18 to 22 carbon atoms.
The phospholipid containing a long-chain polyunsaturated fatty acid according to the present invention is at least one selected from the group consisting of eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid and arachidonic acid.
The invention also aims to provide the phosphatidylcholine containing long-chain polyunsaturated fatty acid, wherein the content of the polyunsaturated fatty acid in the fatty acid composition is 20-80 wt%.
The fatty acid composition of the phosphatidylcholine containing long-chain polyunsaturated fatty acid provided by the invention contains 30-60 wt% of polyunsaturated fatty acid.
According to the phosphatidylcholine containing long-chain polyunsaturated fatty acid, the long-chain polyunsaturated fatty acid is a straight-chain fatty acid which contains more than two double bonds and has 18-22 carbon atoms.
The phosphatidylcholine containing long-chain polyunsaturated fatty acid according to the present invention, the long-chain polyunsaturated fatty acid is at least one selected from eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid and arachidonic acid.
Effects of the invention
The invention has the following advantages: (1) high-purity long-chain polyunsaturated fatty acid acyl donor and high-purity phosphatidylcholine (or lysophosphatidylcholine) are not needed as reaction raw materials, so that the price cost is greatly reduced. (2) The contents of long-chain polyunsaturated fatty acid and phosphatidylcholine in the reaction product are obviously improved, the purposes of the access of the long-chain polyunsaturated fatty acid in the phospholipid and the purification of the phosphatidylcholine are realized, and the added value of the product is obviously improved.
The method does not need to adopt high-purity Phosphatidylcholine (PC) and high-purity long-chain polyunsaturated fatty acid raw materials, has simple process, has high insertion rate of the long-chain polyunsaturated fatty acid in the reaction product, improves the content of the phosphatidylcholine to more than 70 percent, and greatly improves the added value of the product.
Detailed Description
Polyunsaturated fatty acids (PUFA) are classified into omega-3 and omega-6 polyunsaturated fatty acids, depending on the position of the 1 st double bond from the methyl end. Two of the most important unsaturated fatty acids of the omega-3 unsaturated fatty acids to the human body are DHA and EPA. EPA is an English abbreviation of eicosapentaenoic acid, has the function of clearing garbage (cholesterol and triglyceride) in blood vessels, and is commonly called as 'blood vessel scavenger'. DHA is an English abbreviation of docosahexaenoic acid, has the effects of softening blood vessels, strengthening brain, benefiting intelligence and improving eyesight, and is commonly called as brain gold.
Preparation method of phospholipid containing long-chain polyunsaturated fatty acid
The preparation method of the phospholipid containing long-chain polyunsaturated fatty acid comprises the following steps:
contacting raw materials of phospholipid, grease and a solvent, and adding an enzyme preparation for reaction;
and (2) removing the solvent, and adding an immobilized enzyme to perform a reaction, wherein the step (1) is followed by a step of adding a carbamide.
In the present invention, the long-chain polyunsaturated fatty acid is a straight-chain fatty acid having 18 to 22 carbon atoms and containing two or more double bonds, and examples thereof include eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and arachidonic acid (ARA).
In the present invention, the raw material phospholipid is a plant-derived phospholipid or an animal-derived phospholipid. The plant-derived phospholipid is selected from at least one of soybean phospholipid, rapeseed phospholipid, sunflower seed phospholipid, peanut phospholipid, sesame phospholipid and corn phospholipid. The animal-derived phospholipid is egg yolk phospholipid. The raw material phospholipid does not contain long-chain polyunsaturated fatty acid.
The phrase "free of long chain polyunsaturated fatty acids" as used herein means that no long chain polyunsaturated fatty acids are detected by the measurement method described in the present invention. The "raw material phospholipid" in the present invention refers to a phospholipid used as a raw material in a reaction. The "phospholipid containing a long-chain polyunsaturated fatty acid" of the present invention refers to a phospholipid obtained by the above-mentioned method for producing a phospholipid containing a long-chain polyunsaturated fatty acid. In the present invention, the oil or fat contains 5 to 60 wt%, preferably 10 to 55 wt%, more preferably 20 to 45 wt% of a long-chain polyunsaturated fatty acid. In a specific embodiment of the present invention, the fat or oil contains 16.2 wt%, 22.7 wt%, 27 wt%, 28.6 wt%, 40 wt%, or 50 wt% of long-chain polyunsaturated fatty acid.
The oil is at least one selected from deep sea fish oil and microbial oil. The oil is selected from at least one of tuna oil, herring oil, salmon oil, sardine oil, docosahexaenoic acid algae oil or arachidonic acid oil.
In the present invention, the solvent is an ethanol solution, preferably a 95% ethanol solution.
In a preferred embodiment of the present invention, the ratio of the raw material phospholipid and the oil-and-fat added in step (1) is 1: 1-1: 20, preferably 1: 1-1: 15, more preferably 1: 2-1: 10, more preferably 1: 4-1: 8.
in a specific embodiment of the present invention, the ratio of the raw material phospholipid and the oil-and-fat added in step (1) is 1: 1. 1: 2. 1: 4. 1: 5. 1: 8. 1: 10. 1: 15.
in a preferred embodiment of the present invention, the ratio of the total amount of the raw material phospholipid and the oil-and-fat added in step (1) to the solvent is 1: 2-1: 100, preferably 1: 5-1: 80, more preferably 1: 8-1: 40, more preferably 1: 10-1: 30, particularly preferably 1: 12-1: 20, most preferably 1: 12-1: 18.
in a specific embodiment of the present invention, the ratio of the total amount of the raw material phospholipid and the oil-and-fat added in the step (1) to the solvent is 1: 5. 1: 8. 1: 10. 1: 12. 1: 18. 1: 20. 1: 30. 1: 40.
in a preferred embodiment of the invention, the enzyme preparation described in step (1) is in the form of the free enzyme. The enzyme preparation in the step (1) is at least one selected from phospholipase A1, phospholipase A2 or lipase.
In a preferred embodiment of the invention, the enzyme preparation described in step (1) is derived from at least one of Aspergillus niger (Aspergillus niger), Candida antarctica (Candida antarctic), Candida rugosa (Candida rugosa), Candida cylindracea (Candida cylindracea), Thermomyces lanuginosus (Thermomyces lanuginosus), Rhizomucor miehei (Rhizomucor miehei), Mucor miehei (Mucor miehei), Rhizopus delemar (Rhizopus delemar), Rhizopus oryzae (Rhizopus oryzae), Pseudomonas pseudomonads (Pseudomonas sp.).
In a preferred embodiment of the present invention, the ratio of the total amount of the raw material phospholipid and the oil and fat added in the step (1) to the enzyme preparation is 10: 1-200: 1, preferably 20: 1-100: 1, more preferably 40: 1-80: 1, further 50: 1-60: 1.
in a specific embodiment of the present invention, the ratio of the total amount of the raw material phospholipid and the oil and fat added in the step (1) to the enzyme preparation is 20: 1. 30: 1. 45, and (2) 45: 1. 55: 1. 60: 1. 80: 1. 100, and (2) a step of: 1.
in a preferred embodiment of the present invention, the amount of the carbamide added is 100 to 800 parts by weight, preferably 200 to 600 parts by weight, and more preferably 250 to 350 parts by weight, based on 100 parts by weight of the total amount of the raw material phospholipid and the oil-and-fat added.
In a specific embodiment of the present invention, the amount of the carbamide added is 100 parts by weight, 200 parts by weight, 267 parts by weight, 300 parts by weight, 350 parts by weight, 400 parts by weight, or 600 parts by weight, based on 100 parts by weight of the total amount of the raw material phospholipid and the oil-and-fat.
In the present invention, the reaction conditions of the step (1) are not particularly limited as long as the object of the present invention can be achieved. For example, the reaction can be carried out at 30 to 40 ℃ such as 35 ℃. The reaction time is, for example, 1 to 10 hours, preferably 2 to 6 hours.
After the carbamide is added for reaction, crystallization treatment can be carried out at 0-4 ℃, and filtrate is separated. The time for the crystallization treatment is, for example, 1 to 6 hours, preferably 2 to 4 hours. The filtrate is then freed of the solvent by distillation (e.g. rotary evaporation).
In the invention, the step (2) is carried out at the temperature of 60-80 ℃. The reaction time is, for example, 2 to 10 hours, preferably 4 to 8 hours. And (3) carrying out reaction in the step (2) under the condition of vacuum pumping or protective gas atmosphere. The evacuation is, for example, 0.1 to 5 KPa. The protective gas atmosphere in the step (2) is an inert gas atmosphere, for example, a nitrogen atmosphere.
In a preferred embodiment of the present invention, the immobilized enzyme of step (2) is lipase or phospholipase immobilized on a macroporous adsorbent resin or basic ion exchange resin carrierAt least one of them. The lipase or phospholipase is produced by a microorganism of Mucor, Penicillium, Aspergillus, Rhizopus, Thermomyces, Pseudomonas, or Candida origin. The immobilized enzymes may be obtained commercially, for example, by novozyme 435, Lipozyme RM IM, or by CN201310307403.6 and the references Vikbjerg, Huiling Mu, Xuebing Xu, Synthesis of structured phospholipids by immobilized phospholipases A2catalyzed acidity, Journal of Biotechnology, 2007, 128: 545 to 554, for example, immobilized phospholipase A1, immobilized phospholipase A2, and the like.
In a preferred embodiment of the present invention, the weight ratio of the total amount of the raw material phospholipid and the oil-and-fat to the immobilized enzyme is 10: 1-200: 1, preferably 20: 1-150: 1, more preferably 40: 1-100: 1, further 50: 1-60: 1.
in a specific embodiment of the present invention, the weight ratio of the total amount of the raw material phospholipid and the oil-and-fat to the immobilized enzyme is 25: 1. 29: 1. 30: 1. 50: 1. 55: 1. 60: 1. 67: 1. 100, and (2) a step of: 1.
in the present invention, the water absorbing agent is added in the step (2). The amount of the water-absorbing agent added may be determined as appropriate, and the weight ratio of the total amount of the raw material phospholipid and the oil and fat to the water-absorbing agent is 10: 1-50: 1, preferably 15: 1-40: 1. for example, the weight ratio of the total amount of the raw material phospholipid and the oil to the water absorbent is 16: 1. 17: 1. 22: 1. 30: 1.
the water absorbent is at least one of molecular sieve or high molecular water absorbent resin. The molecular sieve is for example a 3A molecular sieve. The high-molecular water-absorbing resin is at least one of an acrylamide-acrylate copolymerization crosslinking substance and a starch grafted acrylate copolymerization crosslinking substance.
In the present invention, the raw material phospholipid has a phosphatidylcholine content of 15 to 60 wt%, for example, 16 wt%, 18 wt%, 22 wt%, 60 wt%.
After the reaction is completed, the immobilized enzyme is removed by filtration by a conventional method, washed by adding a solvent (e.g., acetone, etc.), and then centrifuged (e.g., at 8000 to 10000 r/min).
The phospholipid containing long-chain polyunsaturated fatty acids can be prepared by the above-mentioned preparation method of the phospholipid containing long-chain polyunsaturated fatty acids of the present invention.
The long-chain polyunsaturated fatty acid-containing phospholipid prepared by the method for producing a long-chain polyunsaturated fatty acid-containing phospholipid of the present invention has a phosphatidylcholine content of 70 to 85 wt%, for example, 70 wt%, 72 wt%, 73 wt%, 74 wt%, 78 wt%, 81 wt%, 82 wt%, 85 wt%.
The long-chain polyunsaturated fatty acid-containing phospholipid produced by the method for producing a long-chain polyunsaturated fatty acid-containing phospholipid of the present invention has a polyunsaturated fatty acid content of 20 to 80 wt%, preferably 30 to 60 wt%, for example, 24.7 wt%, 30 wt%, 38 wt%, 41.5 wt%, 44.7 wt%, 45.9 wt%, 63.3 wt%, 76.3 wt% in the fatty acid composition. Since the raw material phospholipid in the present invention does not contain a long-chain polyunsaturated fatty acid, the polyunsaturated fatty acid content of the long-chain polyunsaturated fatty acid-containing phospholipid of the present invention can also be regarded as the polyunsaturated fatty acid content increase rate.
In the phospholipid containing long-chain polyunsaturated fatty acids prepared by the preparation method of the phospholipid containing long-chain polyunsaturated fatty acids, the fatty acid composition of the phospholipid containing long-chain polyunsaturated fatty acids includes 12.9 wt% of DPA and 38.7 wt% of DHA; 5.5 weight% EPA, 21.6 weight% DHA; EPA 3.8 wt%, DHA 27.3 wt%; ARA is 40.2 wt%; 28.6% by weight of EPA and 32.4% by weight of DHA; DPA 8.9 wt%, DHA 20.7 wt%; EPA 8.6 wt%, DHA 30.2 wt%; EPA 2.7 wt%, DHA 25.2 wt%; or EPA 7.6 wt% and DHA 12.4 wt%.
Phospholipids containing long chain polyunsaturated fatty acids
The long-chain polyunsaturated fatty acid-containing phospholipid of the present invention has a phosphatidylcholine content of 70 to 85 wt%, for example, 70 wt%, 72 wt%, 73 wt%, 74 wt%, 78 wt%, 81 wt%, 82 wt%, 85 wt%.
The long-chain polyunsaturated fatty acid-containing phospholipid of the present invention has a polyunsaturated fatty acid content of 15 to 60 wt%, preferably 25 to 52 wt%, for example, 20 wt%, 27.1 wt%, 27.9 wt%, 29.6 wt%, 31.1 wt%, 38.8 wt%, 40.2 wt%, 51.6 wt%, 61 wt% in the fatty acid composition. Since the raw material phospholipid in the present invention does not contain long-chain polyunsaturated fatty acids, the polyunsaturated fatty acid content of the long-chain polyunsaturated fatty acid-containing phospholipid of the present invention can also be regarded as an increase in the polyunsaturated fatty acid content of the product phospholipid relative to the raw material phospholipid. In the present invention, the long-chain polyunsaturated fatty acid is a straight-chain fatty acid having 18 to 22 carbon atoms and containing two or more double bonds, and examples thereof include eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and arachidonic acid (ARA).
In the fatty acid composition of the phospholipid containing long-chain polyunsaturated fatty acid, the content of DPA is 12.9 wt% and the content of DHA is 38.7 wt%; 5.5 weight% EPA, 21.6 weight% DHA; EPA 3.8 wt%, DHA 27.3 wt%; ARA is 40.2 wt%; 28.6% by weight of EPA and 32.4% by weight of DHA; DPA 8.9 wt%, DHA 20.7 wt%; EPA 8.6 wt%, DHA 30.2 wt%; EPA 2.7 wt%, DHA 25.2 wt%; or EPA 7.6 wt% and DHA 12.4 wt%.
Phosphatidylcholine containing long chain polyunsaturated fatty acids
Phosphatidylcholine is isolated from the long-chain polyunsaturated fatty Acid-containing phospholipids of the invention (see Reddy, J.R. C., Vijeeta, T., Karuna, M.S. L., Rao, B.V. S., & Prasad, R.B.N. (2005), Lipase-Catalyzed Preparation of Palmitic and Stearic Acid-rich Phosphositidylcholine, Journal of the American Oil chemistry' Society, 82(10), 727-730), Phosphatidylcholine (also sometimes referred to as Phosphatidylcholine of the invention or Phosphatidylcholine containing long-chain polyunsaturated fatty acids of the invention) is isolated, and the isolated Phosphatidylcholine is then pretreated and fatty Acid assayed, respectively, with reference to national standards GB/T76 open 2008 and GB/T1734 open 2008.
In the fatty acid composition of phosphatidylcholine, the polyunsaturated fatty acid content is 20 to 80 wt%, preferably 30 to 60 wt%, for example 24.7 wt%, 30 wt%, 38 wt%, 41.5 wt%, 44.7 wt%, 45.9 wt%, 63.3 wt%, 76.3 wt%. Since the raw material phospholipid in the present invention does not contain long-chain polyunsaturated fatty acids, the polyunsaturated fatty acid content of the phosphatidylcholine in the present invention can be regarded as the increase rate of the polyunsaturated fatty acid content of the isolated phosphatidylcholine relative to the raw material phospholipid.
In the fatty acid composition of the long-chain polyunsaturated fatty acid-containing phosphatidylcholine of the invention, the content of DPA is 18.1 wt% and the content of DHA is 45.2 wt%; EPA 7.4 wt%, DHA 30.6 wt%; EPA 9.1 wt%, DHA 35.6 wt%; ARA 57.1 wt%; EPA 36.2 wt%, DHA 40.1 wt%; 12.8 wt% DPA and 28.7 wt% DHA; EPA 10.3 wt%, DHA 35.6 wt%; EPA 3.2 wt%, DHA 26.8 wt%; or 8.9% by weight of EPA and 15.8% by weight of DHA.
The features mentioned above with reference to the invention, or the features mentioned with reference to the embodiments, can be combined arbitrarily. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. All percentages, ratios, proportions, or parts are by weight unless otherwise specified.
The weight volume percentage units in the present invention are well known to those skilled in the art and refer to, for example, the weight of solute in a 100 ml solution.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Examples
The phospholipase A1, phospholipase A2, Lipase TLL, Lipase Candida cylindracea, Lipase pseudomonas sp, and Lipase Rhizopus oryzae used in the examples described below were commercial Lecitase Ultra, Lecitase 10L, Lipozyme TL 100L, Lipase AY "Amano" 30, Lipase from pseudomonas sp, Lipase F-AP 15, respectively.
The immobilized lipases Novozyme 435 used in the examples described below were purchased from Novozymes Inc., and the immobilized phospholipase A1 and immobilized phospholipase A2 were obtained by the methods of the prior art published patents, such as CN201310307403.6 and the references Vikbjerg, Huiling Mu, Xuebing Xu, Synthesis of structured phospholipids by immobilized lipase A2catalyzed acidity, Journal of Biotechnology, 2007, 128: 545 to 554, etc.
In the following examples, the sources of the raw materials used are as follows: soybean powder phospholipids and egg yolk phospholipids were purchased from beijing meiyasi phospholipids technologies ltd; DHA algal oil and ARA oil were purchased from Xiamen Congestion biology, Inc.; tuna, herring and sardine oils were purchased from Norsk Hydro A/S.
The method for measuring the content of the Phosphatidylcholine (PC) is measured according to the method in the national standard GB/T21493-2008; the method for measuring the composition of the phospholipid fatty acid is measured according to the method in the national standard GB/T24894-2008, wherein the pretreatment method is carried out according to the method in the national standard GB/T17376-2008; PC fatty Acid composition determination PC is first isolated from the product by reference to Reddy, J.R.C., Vijeeta, T., Karuna, M.S.L., Rao, B.V.S., & Prasad, R.B.N. (2005), Lipase-Catalyzed Preparation of Palmitic and Stearic Acid-rich Phosphatidholine, Journal of the American Oil chemistry' Society, 82(10), 727-730, and then pre-treated and fatty Acid determined by reference to national standards GB/T17376-.
Example 1
Weighing 15g of soybean powder phospholipid (the content of PC is 18 percent), 30g of DHA algal oil (DHA is 35 percent, DPA is 15 percent) and 450mL of 95 percent ethanol in a reaction bottle, stirring uniformly at 35 ℃, adding 0.5mL of phospholipase A1 and 1mL of lipase TL L, reacting for 4 hours, adding 90g of carbamide, after complete dissolution, placing the reaction bottle in a 0 ℃ environment for crystallization for 2 hours, filtering and separating out filtrate, removing ethanol by rotary evaporation at 70 ℃, adding 1.5g of novozyme 435, pumping vacuum to 5 KPa-0.1 KPa, and reacting for 6 hours at 70 ℃. After the reaction is finished, filtering to remove Novozyme 435 (which can be recycled), adding 30mL of acetone for washing for 3 times, and centrifuging at 10000r/min to obtain acetone insoluble substances. The results are shown in Table 1.
TABLE 1
Comparative example 1
(1) Weighing 15g of soybean powder phospholipid (the content of PC is 18%) and 150mL of 95% ethanol in a reaction bottle, uniformly stirring at 35 ℃, adding 0.5mL of phospholipase A1, reacting for 4h, and performing rotary evaporation at 70 ℃ to remove the ethanol; (2) weighing 30g of DHA algal oil (DHA 35%, DPA 15%) and 300mL of 95% ethanol in a reaction bottle, adding 1mL of lipase TL L, reacting for 4 hours, adding 90g of carbamide, after complete dissolution, placing the reaction bottle in an environment at 0 ℃ for crystallization for 2 hours, filtering and separating filtrate, and removing ethanol by rotary evaporation at 70 ℃; (3) and (3) mixing the products obtained in the step (1) and the step (2), adding 1.5g of novozyme 435, vacuumizing at 70 ℃ for 5-0.1 KPa, and reacting for 6 hours. After the reaction is finished, the novozyme 435 is removed by filtration (can be recycled), 30mL of acetone is added for washing for 3 times, acetone insoluble substances are obtained by centrifugation at 10000r/min, and the detection results are shown in the following table 2.
TABLE 2
Comparative example 2
Weighing 15g of soybean powder phospholipid (the content of PC is 18%), 30g of DHA algal oil (DHA is 35%, DPA is 15%) and 450mL of 95% ethanol in a reaction bottle, stirring uniformly at 35 ℃, adding 0.5mL of phospholipase A1 and 1mL of lipase TL L, reacting for 4h, and performing rotary evaporation at 70 ℃ to remove the ethanol; adding 1.5g of novozyme 435, vacuumizing to 5-0.1 KPa, and reacting at 70 ℃ for 6 hours. After the reaction is finished, the novozyme 435 is removed by filtration (can be recycled), 30mL of acetone is added for washing for 3 times, and the acetone insoluble substance is obtained by centrifugation at 10000 r/min. The results are shown in Table 3.
TABLE 3
Comparative example 3
Weighing 5g of soybean lecithin (with the PC content of 60%) and 15g of DHA concentrated solution (with the total content of DPA and DHA of 97.2%), adding 2g of novozyme 435, vacuumizing to 5 KPa-0.1 KPa, and reacting at 70 ℃ for 6 h. After the reaction is finished, the novozyme 435 is removed by filtration (can be recycled), 30mL of acetone is added for washing for 3 times, and the acetone insoluble substance is obtained by centrifugation at 10000 r/min. The results are shown in Table 4.
TABLE 4
Example 2
Weighing 10g of yolk phospholipid (the PC content is 60%), 100g of tuna oil (DHA 18.2%, EPA 4.5%) and 2.2L of 95% ethanol, putting the mixture into a reaction bottle, uniformly stirring the mixture at 35 ℃, adding 1mL of phospholipase A1 and 1mL of lipase Candida cylindracea, reacting for 2 hours, adding 440g of carbamide, after the carbamide is completely dissolved, putting the reaction bottle in an environment at 4 ℃ for crystallization for 4 hours, filtering and separating filtrate, removing ethanol by rotary evaporation, adding 2g of immobilized phospholipase A1 and 5g of 3A molecular sieve, and reacting for 8 hours at 60 ℃ under the nitrogen-filled condition. After the reaction is finished, filtering to remove the immobilized phospholipase A1 (which can be recycled), adding 20mL of acetone for washing for 3 times, and centrifuging at 8000r/min to obtain acetone insoluble substances. The results are shown in Table 5.
TABLE 5
Example 3
Weighing 10g of soybean powder phospholipid (the content of PC is 18 percent), 80g of menhaden fish oil (DHA is 20.1 percent and EPA is 8.5 percent) and 720mL of 95 percent ethanol into a reaction bottle, stirring the mixture evenly at 35 ℃, adding 0.8mL of phospholipase A2 and 1.2mL of lipase Pseudotonas sp, reacting for 4h, adding 270g of carbamide, placing the reaction bottle in a 0 ℃ environment for crystallization for 4h after complete dissolution, filtering and separating filtrate, removing ethanol by rotary evaporation, adding 1.8g of immobilized phospholipase A2 and 3g of high molecular water-absorbing resin (acrylamide-acrylate copolymer cross-linked polymer) and reacting for 8h at 70 ℃ under the nitrogen filling condition. After the reaction is finished, filtering to remove the immobilized phospholipase A2 (which can be recycled), adding 30mL of acetone for washing for 3 times, and centrifuging at 8000r/min to obtain acetone insoluble substances. The results are shown in Table 6.
TABLE 6
Example 4
Weighing 20g of soybean powder phospholipid (the content of PC is 16 percent), 100g of ARA grease (ARA 40 percent) and 2.16L of 95 percent ethanol into a reaction bottle, stirring uniformly at 35 ℃, adding 0.5mL of phospholipase A1 and 1mL of lipase Rhizopus oryzae, reacting for 3 hours, adding 240g of carbamide, after complete dissolution, placing the reaction bottle in a2 ℃ environment for crystallization for 3 hours, filtering to separate filtrate, removing ethanol by rotary evaporation, adding 2g of Novozyme 435, pumping vacuum to 5 KPa-0.1 KPa, and reacting for 4 hours at 80 ℃. After the reaction is finished, the novozyme 435 is removed by filtration (can be recycled), 40mL of acetone is added for washing for 3 times, and the acetone insoluble substance is obtained by centrifugation at 10000 r/min. The results are shown in Table 7.
TABLE 7
Example 5
Weighing 20g of soybean powder phospholipid (the content of PC is 22%), 160g of sardine oil (DHA is 16.8%, EPA is 10.2%) and 900mL of 95% ethanol in a reaction bottle, stirring uniformly at 35 ℃, adding 1mL of phospholipase A1 and 2mL of lipase TL L, reacting for 4 hours, adding 480g of carbamide, after complete dissolution, placing the reaction bottle in an environment at 0 ℃ for crystallization for 4 hours, filtering and separating filtrate, removing ethanol by rotary evaporation, adding 1.8g of Novozyme 435, pumping vacuum to 5 KPa-0.1 KPa, and reacting for 4 hours at 75 ℃. After the reaction is finished, the novozyme 435 is removed by filtration (can be recycled), 35mL of acetone is added for washing for 3 times, and the acetone insoluble substance is obtained by centrifugation at 8000 r/min. The results are shown in Table 8.
TABLE 8
Example 6
Weighing 15g of soybean powder phospholipid (the content of PC is 18 percent), 15g of DHA algal oil (DHA is 35 percent, DPA is 15 percent) and 360mL of 95 percent ethanol, uniformly stirring at 35 ℃, adding 0.1mL of phospholipase A1 and 0.2mL of lipase TL L, reacting for 6 hours, adding 30g of carbamide, after complete dissolution, placing the reaction bottle in a 0 ℃ environment for crystallization for 5 hours, filtering and separating filtrate, removing ethanol by rotary evaporation at 70 ℃, adding 0.45g of novozyme 435, pumping vacuum to 5 KPa-0.1 KPa, and reacting for 8 hours at 70 ℃. After the reaction is finished, the novozyme 435 is removed by filtration (can be recycled), 15mL of acetone is added for washing for 3 times, and the acetone insoluble substance is obtained by centrifugation at 10000 r/min. The results are shown in Table 9.
TABLE 9
Example 7
Weighing 10g of yolk phospholipid (the content of PC is 60%), 150g of tuna oil (DHA is 18.2%, EPA is 4.5%) and 4.8L of 95% ethanol, putting the mixture into a reaction bottle, stirring the mixture evenly at 35 ℃, adding 1mL of phospholipase A1 and 7mL of lipase TLL, reacting for 2 hours, adding 960g of carbamide, after the mixture is completely dissolved, putting the reaction bottle into an environment at 2 ℃ for crystallization for 4 hours, filtering and separating filtrate, removing ethanol by rotary evaporation, adding 5.6g of immobilized phospholipase A1 and 10g of 3A molecular sieve, and reacting for 8 hours at 60 ℃ under the condition of nitrogen filling. After the reaction is finished, filtering to remove the immobilized phospholipase A1 (which can be recycled), adding 30mL of acetone for washing for 3 times, and centrifuging at 8000r/min to obtain acetone insoluble substances. The results are shown in Table 10.
Watch 10
Example 8
Weighing 10g of soybean powder phospholipid (the content of PC is 18 percent), 40g of herring oil (DHA is 20.1 percent, EPA is 8.5 percent) and 2L of 95 percent ethanol into a reaction bottle, stirring uniformly at 35 ℃, adding 0.5mL of phospholipase A2 and 0.75mL of lipase Pseudotonas sp, reacting for 5h, adding 175g of carbamide, placing the reaction bottle in a 0 ℃ environment for crystallization for 4h after complete dissolution, filtering and separating filtrate, removing ethanol by rotary evaporation, adding 2g of immobilized phospholipase A2 and 3g of high molecular water-absorbing resin (starch graft acrylate copolymer cross-linked polymer), and reacting for 8h at 65 ℃ under the nitrogen filling condition. After the reaction is finished, filtering to remove the immobilized phospholipase A2 (which can be recycled), adding 30mL of acetone for washing for 3 times, and centrifuging at 8000r/min to obtain acetone insoluble substances. The results are shown in Table 11.
TABLE 11
Example 9
Weighing 10g of yolk phospholipid (the content of PC is 60%), 40g of salmon oil (DHA is 9.5%, EPA is 6.7%) and 2L of 95% ethanol, putting the yolk phospholipid and the salmon oil into a reaction bottle, stirring the mixture evenly at 35 ℃, adding 0.5mL of phospholipase A2 and 0.75mL of lipase Rhizopus oryzae, reacting for 3 hours, adding 175g of carbamide, after the mixture is completely dissolved, putting the reaction bottle in a 0 ℃ environment for crystallization for 4 hours, filtering and separating filtrate, removing ethanol by rotary evaporation, adding 2g of immobilized phospholipase A2, vacuumizing to 5 KPa-0.1 KPa, and reacting for 6 hours at 70 ℃. After the reaction is finished, filtering to remove the immobilized phospholipase A2 (which can be recycled), adding 30mL of acetone for washing for 3 times, and centrifuging at 8000r/min to obtain acetone insoluble substances. The results are shown in Table 11.
TABLE 12
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the scope of the invention, which is defined by the claims appended hereto, and any other technical entity or method that is encompassed by the claims as broadly defined herein, or equivalent variations thereof, is contemplated as being encompassed by the claims.
Claims (36)
1. A method for preparing a phospholipid containing long-chain polyunsaturated fatty acid, which comprises the following steps:
step (1): contacting raw materials of phospholipid, grease and a solvent, and adding an enzyme preparation for reaction;
step (2): removing the solvent, adding an immobilized enzyme to carry out a reaction, characterized in that a step of adding carbamide is provided after the step (1),
after adding carbamide for reaction, carrying out crystallization treatment at 0-4 ℃, separating filtrate,
the oil contains 5-60 wt% of long-chain polyunsaturated fatty acid,
the raw material phospholipid does not contain long-chain polyunsaturated fatty acid,
the enzyme preparation in the step (1) is a combination of lipase and at least one selected from phospholipase A1 and phospholipase A2,
the solvent is a 95% ethanol solution,
the immobilized enzyme in the step (2) is at least one of lipase or phospholipase fixed on a macroporous adsorption resin or basic ion exchange resin carrier.
2. The production method according to claim 1, wherein the raw material phospholipid is a plant-derived phospholipid or an animal-derived phospholipid.
3. The production method according to claim 2, wherein the plant-derived phospholipid is at least one selected from soybean phospholipid, rapeseed phospholipid, sunflower phospholipid, peanut phospholipid, sesame phospholipid, and corn phospholipid.
4. The method according to claim 2, wherein the animal-derived phospholipid is egg yolk phospholipid.
5. The production method according to claim 1 or 2, wherein the oil or fat is at least one selected from deep sea fish oil and microbial oil.
6. The method according to claim 1 or 2, wherein the oil or fat is at least one selected from tuna oil, menhaden oil, salmon oil, sardine oil, docosahexaenoic acid algae oil, and arachidonic acid oil or fat.
7. The production method according to claim 1 or 2, wherein the ratio of the raw material phospholipid and the oil-and-fat added in step (1) is 1: 1-1: 15.
8. the production method according to claim 1 or 2, wherein the ratio of the raw material phospholipid and the oil-and-fat added in step (1) is 1: 2-1: 10.
9. the production method according to claim 1 or 2, wherein the ratio of the raw material phospholipid and the oil-and-fat added in step (1) is 1: 4-1: 8.
10. the production method according to claim 1 or 2, wherein the ratio of the total amount of the raw material phospholipid and the oil-and-fat added in step (1) to the solvent is 1: 5-1: 80.
11. the production method according to claim 1 or 2, wherein the ratio of the total amount of the raw material phospholipid and the oil-and-fat added in step (1) to the solvent is 1: 8-1: 40.
12. the production method according to claim 1 or 2, wherein the ratio of the total amount of the raw material phospholipid and the oil-and-fat added in step (1) to the solvent is 1: 10-1: 30.
13. the production method according to claim 1 or 2, wherein the ratio of the total amount of the raw material phospholipid and the oil-and-fat added in step (1) to the solvent is 1: 12-1: 18.
14. the production method according to claim 1 or 2, wherein the enzyme preparation in the step (1) is in a free enzyme form.
15. The production method according to claim 1 or 2, wherein the enzyme preparation in step (1) is derived from at least one of Aspergillus niger (Aspergillus niger), Candida antarctica (Candida antarctic), Candida rugosa (Candida rugosa), Candida cylindracea (Candida cylindracea), Thermomyces lanuginosus (Thermomyces lanuginosus), Rhizomucor miehei (Rhizomucor miehei), Mucor miehei (Mucor miehei), Rhizopus delemar, Rhizopus oryzae (Rhizopus oryzae), Pseudomonas sp.
16. The production method according to claim 1 or 2, wherein the ratio of the total amount of the raw material phospholipid and the oil-and-fat added in step (1) to the enzyme preparation is 10: 1-200: 1.
17. the production method according to claim 1 or 2, wherein the ratio of the total amount of the raw material phospholipid and the oil-and-fat added in step (1) to the enzyme preparation is 20: 1-100: 1.
18. the production method according to claim 1 or 2, wherein the ratio of the total amount of the raw material phospholipid and the oil-and-fat added in step (1) to the enzyme preparation is 40: 1-80: 1.
19. the production method according to claim 1 or 2, wherein the ratio of the total amount of the raw material phospholipid and the oil-and-fat added in step (1) to the enzyme preparation is 50: 1-60: 1.
20. the production method according to claim 1 or 2, wherein the amount of the carbamide added is 100 to 800 parts by weight based on 100 parts by weight of the total amount of the raw material phospholipid and the oil-and-fat added.
21. The production method according to claim 1 or 2, wherein the amount of the carbamide added is 200 to 600 parts by weight based on 100 parts by weight of the total amount of the raw material phospholipid and the oil-and-fat added.
22. The production method according to claim 1 or 2, wherein the amount of the carbamide added is 250 to 350 parts by weight based on 100 parts by weight of the total amount of the raw material phospholipid and the oil-and-fat added.
23. The production method according to claim 1 or 2, wherein the step (2) is carried out at a temperature of 60 to 80 ℃.
24. The production method according to claim 1 or 2, wherein a weight ratio of the total amount of the raw material phospholipid and the oil-and-fat added to the immobilized enzyme is 10: 1-200: 1.
25. the production method according to claim 1 or 2, wherein a weight ratio of the total amount of the raw material phospholipid and the oil-and-fat added to the immobilized enzyme is 20: 1-150: 1.
26. the production method according to claim 1 or 2, wherein the step (2) is carried out under vacuum or a protective gas atmosphere.
27. The production method according to claim 26, wherein the protective gas atmosphere of the step (2) is an inert gas atmosphere.
28. The production method according to claim 1 or 2, wherein the water absorbing agent is added in the step (2).
29. The production method according to claim 28, wherein the water absorbing agent is selected from at least one of a molecular sieve or a high molecular water absorbent resin.
30. The method according to claim 29, wherein the water-absorbent polymer resin is at least one selected from the group consisting of an acrylamide-acrylate copolymer cross-linked material and a starch graft acrylate copolymer cross-linked material.
31. The method according to claim 1 or 2, wherein the raw material phospholipid has a phosphatidylcholine content of 15 to 60 wt%.
32. The method according to claim 1 or 2, wherein the long-chain polyunsaturated fatty acid is a straight-chain fatty acid having 18 to 22 carbon atoms and containing two or more double bonds.
33. The production method according to claim 1 or 2, wherein the long-chain polyunsaturated fatty acid is at least one selected from the group consisting of eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoic acid and arachidonic acid.
34. The method according to claim 1 or 2, wherein the phospholipid containing a long-chain polyunsaturated fatty acid has a phosphatidylcholine content of 70 to 85 wt%.
35. The method according to claim 1 or 2, wherein the phospholipid containing a long-chain polyunsaturated fatty acid has a fatty acid composition in which the content of the polyunsaturated fatty acid is 15 to 60% by weight.
36. The method according to claim 1 or 2, wherein the phospholipid containing a long-chain polyunsaturated fatty acid has a fatty acid composition in which the content of the polyunsaturated fatty acid is 25 to 52% by weight.
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