CN112391422A - Method for preparing glycerol phosphatidylcholine by enzyme method - Google Patents
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 title claims abstract description 108
- 238000000034 method Methods 0.000 title claims abstract description 37
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 title claims abstract description 37
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 17
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims abstract description 78
- 239000000758 substrate Substances 0.000 claims abstract description 23
- 238000010438 heat treatment Methods 0.000 claims abstract description 21
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 claims description 32
- 102000015439 Phospholipases Human genes 0.000 claims description 32
- 108010064785 Phospholipases Proteins 0.000 claims description 32
- SUHOQUVVVLNYQR-MRVPVSSYSA-N choline alfoscerate Chemical compound C[N+](C)(C)CCOP([O-])(=O)OC[C@H](O)CO SUHOQUVVVLNYQR-MRVPVSSYSA-N 0.000 claims description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 claims description 22
- 229940067606 lecithin Drugs 0.000 claims description 22
- 239000000787 lecithin Substances 0.000 claims description 22
- 235000010445 lecithin Nutrition 0.000 claims description 22
- 239000008367 deionised water Substances 0.000 claims description 15
- 229910021641 deionized water Inorganic materials 0.000 claims description 15
- 238000006911 enzymatic reaction Methods 0.000 claims description 10
- 230000035484 reaction time Effects 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000004945 emulsification Methods 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 239000007795 chemical reaction product Substances 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 238000000638 solvent extraction Methods 0.000 claims description 4
- 238000011049 filling Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000011347 resin Substances 0.000 claims description 3
- 229920005989 resin Polymers 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 230000001804 emulsifying effect Effects 0.000 claims description 2
- 230000002255 enzymatic effect Effects 0.000 claims 1
- 238000000746 purification Methods 0.000 abstract description 10
- 238000000926 separation method Methods 0.000 abstract description 10
- 239000005515 coenzyme Substances 0.000 abstract description 4
- 229910021645 metal ion Inorganic materials 0.000 abstract description 4
- 239000000413 hydrolysate Substances 0.000 abstract description 3
- 230000001105 regulatory effect Effects 0.000 abstract description 2
- 239000000463 material Substances 0.000 description 19
- 239000000047 product Substances 0.000 description 17
- 239000000203 mixture Substances 0.000 description 9
- 239000011541 reaction mixture Substances 0.000 description 7
- 239000012535 impurity Substances 0.000 description 5
- 238000005303 weighing Methods 0.000 description 5
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 230000000717 retained effect Effects 0.000 description 4
- 230000003301 hydrolyzing effect Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- 108090001060 Lipase Proteins 0.000 description 2
- 102000004882 Lipase Human genes 0.000 description 2
- 239000004367 Lipase Substances 0.000 description 2
- 241000235528 Rhizopus microsporus var. chinensis Species 0.000 description 2
- 238000006136 alcoholysis reaction Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 description 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 2
- 235000019421 lipase Nutrition 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 208000020925 Bipolar disease Diseases 0.000 description 1
- 206010008025 Cerebellar ataxia Diseases 0.000 description 1
- 108010048733 Lipozyme Proteins 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- JYVHOGDBFNJNMR-UHFFFAOYSA-N hexane;hydrate Chemical compound O.CCCCCC JYVHOGDBFNJNMR-UHFFFAOYSA-N 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- FCCDDURTIIUXBY-UHFFFAOYSA-N lipoamide Chemical compound NC(=O)CCCCC1CCSS1 FCCDDURTIIUXBY-UHFFFAOYSA-N 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 201000000980 schizophrenia Diseases 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/001—Amines; Imines
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl
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- 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
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Abstract
The invention discloses a method for preparing glycerol phosphatidylcholine by an enzyme method, and belongs to the technical field of oil processing. After the enzyme is contacted with a substrate, the proportion of sn 2-lysophospholipid and sn 1-lysophospholipid in a hydrolysate is regulated and controlled through heat treatment, so that the conversion of sn 2-lysophospholipid to sn 1-lysophospholipid is promoted, the generation of the glycerol phosphatidylcholine is further promoted, and the generation rate of the glycerol phosphatidylcholine is greatly improved; in addition, any metal ion is not required to be added in the reaction process as a coenzyme factor, and the subsequent separation and purification steps are simple. The method is simple to operate, can obviously improve the generation rate of the glycerol phosphatidylcholine, does not need to add metal ions in the reaction process, has simple subsequent separation and purification steps, and has good economic benefit and industrial application prospect.
Description
Technical Field
The invention belongs to the technical field of oil processing, and particularly relates to a method for preparing glycerol phosphatidylcholine by an enzyme method.
Background
Glycerol phosphatidylcholine (L-alpha-GPC) is a choline source for the synthesis of acetylcholine and phosphatidylcholine in vivo. Earlier studies have shown that glycerophosphatidylcholine has important pharmaceutical value in the treatment of alzheimer's disease, cerebellar ataxia, schizophrenia and bipolar disorder, but higher purity glycerophosphatidylcholine is required for use as a pharmaceutical raw material and related studies. However, the natural source of the glycerophosphatidylcholine is less, so that the research and development of the preparation process of the high-purity glycerophosphatidylcholine are of great significance.
At present, the common methods for preparing the glycerophosphatidylcholine mainly comprise an enzymolysis method, a solvent extraction method, a chemical synthesis method, a chemical alcoholysis method and the like. The traditional solvent extraction method, chemical synthesis method, chemical alcoholysis method and the like all face the challenges of environmental pollution and the like. The enzymolysis method has attracted extensive attention in recent years due to the advantages of mild reaction conditions, high catalytic specificity, safety, environmental protection and the like. Blasi et al (Enzyme Microb. Tech.,2006,39:1405-1408) used Lipozyme IM and phospholipase A simultaneously2The method is characterized in that the glycerol phosphatidylcholine is prepared by catalyzing lecithin in a microemulsion system, and after the reaction is carried out for 48 hours, the yield of the glycerol phosphatidylcholine is 94%. Bang et al (Food chem.,2016,190:201-206) used phosphoipase A in a n-hexane-water two-phase aqueous system1The method catalyzes lecithin to prepare the glycerol phosphatidylcholine, and under the optimized reaction condition, the yield of the glycerol phosphatidylcholine reaches 100.0%, but the reaction time reaches 30 h. To increase the rate of the hydrolysis reaction, Zhang et al (Eur. J. Lipid Sci. Technol.,2012,114,1254-1260) used phosphoipase A1Catalyzing lecithin in a water phase system to prepare glycerol phosphatidylcholine, and adding Ca before the reaction starts2+As a coenzyme factor, the reaction is carried out for 6 hours under optimized reaction conditions, and the yield of the glycerol phosphatidylcholine is 94.5 percent; furthermore, Zhang et al (Eur. food Res. technol.,2012,234:485-491) catalyzed lecithin in an aqueous phase system by Rhizopus chinensis lipase (Rhizopus chinensis lipase) to prepare glycerophosphatidylcholine, and Ca was added before the start of the reaction2+As the coenzyme factor, the reaction is carried out for 3.5h under the optimized reaction condition, and the yield of the glycerol phosphatidylcholine is 96.8%. Despite Ca during the reaction2+The addition of the compound obviously improves the reaction rate of preparing the glycerol phosphatidylcholine by hydrolyzing the lecithin, but also increases the difficulty of subsequent separation and purification (Ca needs to be removed)2+). In conclusion, the yield of the glycerol phosphatidylcholine is higher when the glycerol phosphatidylcholine is prepared by the enzymatic hydrolysis at present, but the glycerol phosphatidylcholine is higherThe reaction time is longer; although by changing the reaction system and adding Ca2+Can obviously improve the hydrolysis reaction rate, greatly shorten the reaction time, but increase the difficulty of subsequent separation and purification. How to simultaneously consider high reaction rate and simple separation and purification steps is a key technical problem needed to be solved for preparing the glycerol phosphatidylcholine by enzyme catalysis.
Disclosure of Invention
In order to solve the above problems, it is an object of the present invention to provide an enzymatic method for preparing glycerophosphatidylcholine, which has an improved reaction rate and simplifies the subsequent separation and purification steps.
The invention is realized by the following technical scheme:
a method for preparing glycerol phosphatidylcholine by an enzyme method comprises the following steps: the method comprises the following steps:
step 1: mixing lecithin and deionized water, and emulsifying;
step 2: mixing the mixed substrate obtained in the step 1 with immobilized phospholipase A1Contacting to react;
and step 3: carrying out heat treatment on the reaction product obtained in the step 2, cooling, and adding the cooled reaction product into the mixed substrate reacted in the step 2;
and 4, step 4: and (5) circulating the step (2) and the step (3) until the reaction reaches balance, collecting reaction products, removing water, and extracting by using a solvent to obtain the glycerol phosphatidylcholine.
Preferably, in the step 1, the concentration of the lecithin in the deionized water is 30-55 mg/mL.
Preferably, in step 1, the emulsification conditions are: stirring at a rotation speed of 400-600 rpm for 15-20 min.
Preferably, in step 2, the phospholipase A is immobilized1The preparation method comprises the following steps: by physical adsorption or covalent binding according to phospholipase A1The resin is mixed with the immobilized carrier at a ratio of 20-80 mg/g, and the mixture is stirred at a rotation speed of 50-200 rpm for 1-8 hours at 30 ℃.
Preferably, the reaction in step 2 is carried out in a packed bed reactor.
Further preferably, the packed bed is invertedImmobilized phospholipase A in reactor1The filling mass of the mixed substrate is 5 to 20 percent of the total mass of the mixed substrate, and the mixed substrate and the immobilized phospholipase A are1The contact reaction temperature of the mixed substrate is 35-55 ℃, and the mixed substrate flows through a packed bed reactor and the immobilized phospholipase A1The contact reaction time is more than or equal to 10 min.
Preferably, in the step 3, the heat treatment temperature is 60-70 ℃, and the heat treatment time is 20-40 min.
Preferably, the cooling in step 3 is to the reaction temperature of step 2.
Preferably, in step 4, the water removal is performed by vacuum concentration.
Preferably, in step 4, the solvent used for solvent extraction is n-hexane or diethyl ether.
Compared with the prior art, the invention has the following beneficial technical effects:
according to the method for preparing the glycerol phosphatidylcholine by the enzyme method disclosed by the invention, after the enzyme is contacted with a substrate, the conversion of sn 2-lysophospholipid to sn 1-lysophospholipid is promoted by regulating the proportion of sn 2-lysophospholipid and sn 1-lysophospholipid in the product through heat treatment, so that the generation of the glycerol phosphatidylcholine is promoted, and the generation rate of the glycerol phosphatidylcholine is greatly improved; in addition, any metal ion is not required to be added in the reaction process as a coenzyme factor, and the subsequent separation and purification steps are simple. When the conventional single-enzyme method or double-enzyme method is used for hydrolyzing lecithin to prepare the glycerol phosphatidylcholine, Ca needs to be added2+To increase the enzymatic reaction rate, although the hydrolysis reaction rate is significantly increased, the difficulty of subsequent separation and purification is greatly increased. How to simultaneously consider high reaction rate and simple separation and purification steps is a key technical problem to be solved in the preparation of the glycerol phosphatidylcholine by enzymatic hydrolysis of lecithin. Due to phospholipase A1Substrate specificity of, phospholipase A1In the reaction process of preparing the glycerol phosphatidylcholine by hydrolyzing lecithin, the key factors for limiting the reaction rate are as follows: the rate of conversion of sn 2-lysophospholipid to sn 1-lysophospholipid. Thus, increasing the conversion rate of sn 2-lysophospholipid to sn 1-lysophospholipid in the reaction mixture directly determines the rate of production of glycerophosphatidylcholine. Preliminary studiesIt was found that the ratio of sn 2-lysophospholipid and sn 1-lysophospholipid in the hydrolysis reaction mixture can be controlled by heat treatment to promote the conversion of sn 2-lysophospholipid (main product) in the hydrolysate mixture to sn 1-lysophospholipid, thereby increasing the content of sn 1-lysophospholipid in the hydrolysate mixture and further promoting the production of glycerophosphatidylcholine. Compared with other enzyme methods for preparing the glycerol phosphatidylcholine, the method is simple to operate, can obviously improve the generation rate of the glycerol phosphatidylcholine, does not need to add metal ions in the reaction process, has simple subsequent separation and purification steps, and has good economic benefits and industrial application prospects.
Furthermore, the concentration of lecithin in deionized water is 30-55 mg/mL, which is beneficial to the generation of glycerol phosphatidylcholine and can ensure the economical efficiency of the reaction.
Further, immobilized phospholipase A1The mixed substrate is catalyzed in the packed bed reactor for reaction, the reaction effect is good, the efficiency is high, and the operation stability of the immobilized enzyme is good.
Furthermore, the reaction parameters in the packed bed reactor can effectively promote the generation of the glycerophosphatidylcholine, and the immobilized enzyme can be kept to have better operation stability.
Furthermore, the heat treatment at 60-70 ℃ for 20-40 min can effectively promote the conversion of sn 2-lysophospholipid to sn 1-lysophospholipid.
Further, cooling to the reaction temperature of step 2 after the heat treatment can more effectively promote the conversion of the sn 1-lysophospholipid to glycerophosphatidylcholine.
Further, the organic solvent used is effective in removing the by-products from the reaction mixture after water removal and avoids the loss of glycerophosphatidylcholine.
Detailed Description
The present invention will now be described in further detail with reference to specific examples, which are intended to be illustrative, but not limiting, of the invention. All percentages are by mass, unless otherwise indicated.
Example 1
Accurately weighing a certain amount of lecithin and deionized water to mix (total 10Kg, egg)The concentration of phospholipid in deionized water is 30mg/mL), stirring for 20min at the rotating speed of 400rpm for emulsification, conveying the reaction mixture to a packed bed reactor through a material pump for reaction, wherein the packed enzyme is immobilized phospholipase A1(by physical adsorption or covalent bonding according to phospholipase A)1The resin and the immobilized carrier are mixed at a ratio of 20-80 mg/g, and the mixture is stirred at a rotation speed of 50-200 rpm for 1-8 hours at 30 ℃, the filling amount is 0.5Kg, and the reaction temperature is 55 ℃. And then the reaction material enters a heat treatment device to be retained at 60 ℃ for 40min, and the reaction material after heat treatment is cooled to 55 ℃, and then is pumped into a material tank again to be subjected to circular reaction. Adjusting the flow rate of the reaction substrate to allow each cycle thereof to react with the immobilized phospholipase A1The contact reaction time of (2) was 20 min. After the reaction was circulated for 3 hours, a sample was taken to determine the composition of the product, and the yield of glycerophosphatidylcholine in the product was found to be 98.89%. After water and impurities are removed, the purity of the glycerol phosphatidylcholine in the product is 99.06%.
Example 2
Accurately weighing a certain amount of lecithin and deionized water, mixing (total 10Kg, the concentration of lecithin in deionized water is 55mg/mL), stirring at 600rpm for 15min for emulsification, delivering the reaction mixture to a packed bed reactor by a material pump for reaction, wherein the packed enzyme is immobilized phospholipase A1(preparation of immobilized phospholipase A according to the methods described in the invention1) The loading was 2Kg and the reaction temperature was 50 ℃. And then the reaction material enters a heat treatment device to be retained for 20min at 70 ℃, and the reaction material after heat treatment is cooled to 50 ℃, and then is pumped into a material tank again to be subjected to circular reaction. Adjusting the flow rate of the reaction substrate to allow each cycle thereof to react with the immobilized phospholipase A1The contact reaction time of (3) was 10 min. A sample was taken after 3 hours of cycling the reaction to determine the product composition and the product was found to have a glycerophosphatidylcholine yield of 99.16%. After water and impurities were removed, the purity of the glycerophosphatidylcholine in the product was 99.22%.
Example 3
Accurately weighing a certain amount of lecithin, mixing with deionized water (total 10Kg, concentration of lecithin in deionized water is 45mg/mL), stirring at 400rpm for 20min for emulsification, and mixing the reaction mixturePumping to a packed bed reactor for reaction, wherein the packed enzyme is immobilized phospholipase A1(preparation of immobilized phospholipase A according to the methods described in the invention1) The loading was 1.5Kg and the reaction temperature was 35 ℃. And then the reaction material enters a heat treatment device to be retained for 30min at 65 ℃, and the reaction material after heat treatment is cooled to 35 ℃, and then is pumped into a material tank again to be subjected to circular reaction. Adjusting the flow rate of the reaction substrate to allow each cycle thereof to react with the immobilized phospholipase A1The contact reaction time of (3) was 15 min. After the reaction was circulated for 3 hours, a sample was taken to determine the composition of the product, and the yield of glycerophosphatidylcholine in the product was found to be 99.02%. After water and impurities are removed, the purity of the glycerol phosphatidylcholine in the product is 99.15%.
Example 4
Accurately weighing a certain amount of lecithin and deionized water, mixing (total 10Kg, the concentration of lecithin in deionized water is 40mg/mL), stirring at 600rpm for 15min for emulsification, delivering the reaction mixture to a packed bed reactor by a material pump for reaction, wherein the packed enzyme is immobilized phospholipase A1(preparation of immobilized phospholipase A according to the methods described in the invention1) The loading was 1Kg and the reaction temperature was 45 ℃. And then the reaction material enters a heat treatment device to be retained for 20min at 70 ℃, and the reaction material after heat treatment is cooled to 45 ℃, and then is pumped into a material tank again to be subjected to circular reaction. Adjusting the flow rate of the reaction substrate to allow each cycle thereof to react with the immobilized phospholipase A1The contact reaction time of (3) was 10 min. After the reaction was circulated for 3 hours, a sample was taken to determine the composition of the product, and the yield of glycerophosphatidylcholine in the product was found to be 99.21%. After water and impurities were removed, the purity of the glycerophosphatidylcholine in the product was 99.33%.
Example 5
Accurately weighing a certain amount of lecithin and deionized water, mixing (total 10Kg, the concentration of lecithin in deionized water is 35mg/mL), stirring at 400rpm for 20min for emulsification, delivering the reaction mixture to a packed bed reactor by a material pump for reaction, wherein the packed enzyme is immobilized phospholipase A1(preparation of immobilized phospholipase A according to the methods described in the invention1) The loading was 1.5Kg and the reaction temperature was 50 ℃. The reaction mass then being fedStaying in a heat treatment device at 60 ℃ for 40min, cooling the reaction material subjected to heat treatment to 50 ℃, and pumping the reaction material into a material tank for circular reaction. Adjusting the flow rate of the reaction substrate to allow each cycle thereof to react with the immobilized phospholipase A1The contact reaction time of (3) was 10 min. After the reaction was circulated for 2.5 hours, a sample was taken to determine the product composition, and the yield of glycerophosphatidylcholine in the product was found to be 99.36%. After water and impurities are removed, the purity of the glycerol phosphatidylcholine in the product is 99.40%.
Claims (10)
1. A method for preparing glycerol phosphatidylcholine by an enzyme method is characterized by comprising the following steps:
step 1: mixing lecithin and deionized water, and emulsifying;
step 2: mixing the mixed substrate obtained in the step 1 with immobilized phospholipase A1Contacting to react;
and step 3: carrying out heat treatment on the reaction product obtained in the step 2, cooling, and adding the cooled reaction product into the mixed substrate reacted in the step 2;
and 4, step 4: and (5) circulating the step (2) and the step (3) until the reaction reaches balance, collecting reaction products, removing water, and extracting by using a solvent to obtain the glycerol phosphatidylcholine.
2. The method for preparing glycerophosphatidylcholine by using the enzymatic method according to claim 1, wherein in step 1, the concentration of lecithin in deionized water is 30-55 mg/mL.
3. The enzymatic method for preparing glycerophosphatidylcholine according to claim 1, wherein in step 1, the emulsification conditions are: stirring at a rotation speed of 400-600 rpm for 15-20 min.
4. The enzymatic method for preparing glycerophosphatidylcholine according to claim 1, wherein in step 2, the immobilized phospholipase A is phospholipase A1The preparation method comprises the following steps: by physical adsorption or covalent binding according to phospholipase A1The resin is added to the immobilized carrier at a ratio of 20 to 80mg/g, at 30 ℃ and at 50 to 200rpmStirring for 1-8 h at a rotating speed.
5. The enzymatic method for preparing glycerophosphatidylcholine according to claim 1, wherein the reaction in step 2 is carried out in a packed bed reactor.
6. The enzymatic process for preparing glycerophosphatidylcholine according to claim 5, wherein said immobilized phospholipase A is in a packed bed reactor1The filling mass of the mixed substrate is 5 to 20 percent of the total mass of the mixed substrate, and the mixed substrate and the immobilized phospholipase A are1The contact reaction temperature of the mixed substrate is 35-55 ℃, and the mixed substrate flows through a packed bed reactor and the immobilized phospholipase A1The contact reaction time is more than or equal to 10 min.
7. The method for preparing glycerophosphatidylcholine by using the enzymatic method according to claim 1, wherein in step 3, the heat treatment temperature is 60 to 70 ℃ and the heat treatment time is 20 to 40 min.
8. The enzymatic method for preparing glycerophosphatidylcholine according to claim 1, wherein the cooling in step 3 is to the reaction temperature of step 2.
9. The enzymatic method for preparing glycerophosphatidylcholine according to claim 1, wherein in step 4, the water removal is performed by vacuum concentration.
10. The enzymatic method for preparing glycerophosphatidylcholine according to claim 1, wherein in step 4, the solvent used for solvent extraction is n-hexane or diethyl ether.
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Citations (3)
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CN102041281A (en) * | 2010-08-09 | 2011-05-04 | 江南大学 | Method for preparing glycerophosphorylcholine (GPC) by phospholipase-catalyzed hydrolysis |
CN108048497A (en) * | 2017-12-29 | 2018-05-18 | 暨南大学 | A kind of method that glycerolphosphocholine is prepared using phosphatidase |
CN109265478A (en) * | 2018-10-30 | 2019-01-25 | 榆林学院 | A method of glycerolphosphocholine is prepared based on egg shell |
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Publication number | Priority date | Publication date | Assignee | Title |
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CN102041281A (en) * | 2010-08-09 | 2011-05-04 | 江南大学 | Method for preparing glycerophosphorylcholine (GPC) by phospholipase-catalyzed hydrolysis |
CN108048497A (en) * | 2017-12-29 | 2018-05-18 | 暨南大学 | A kind of method that glycerolphosphocholine is prepared using phosphatidase |
CN109265478A (en) * | 2018-10-30 | 2019-01-25 | 榆林学院 | A method of glycerolphosphocholine is prepared based on egg shell |
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