CN112391422B - Method for preparing glycerol phosphatidylcholine by enzyme method - Google Patents
Method for preparing glycerol phosphatidylcholine by enzyme method Download PDFInfo
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- CN112391422B CN112391422B CN202011419040.1A CN202011419040A CN112391422B CN 112391422 B CN112391422 B CN 112391422B CN 202011419040 A CN202011419040 A CN 202011419040A CN 112391422 B CN112391422 B CN 112391422B
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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 System
- 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
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 system1Catalyzing lecithin to prepare the glycerol phosphatidylcholine, and producing the glycerol phosphatidylcholine under the optimized reaction conditionThe rate reaches 100.0%, but the reaction time is as long as 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 a word, when the glycerol phosphatidylcholine is prepared by enzymatic hydrolysis at present, the yield of the glycerol phosphatidylcholine is higher, but the 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 immobilized phospholipase A 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.
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:
the enzymatic method for preparing the glycerophosphatidylcholine disclosed by the invention can promote the conversion of the sn 2-lysophospholipid to the sn 1-lysophospholipid by regulating the ratio of the sn 2-lysophospholipid and the sn 1-lysophospholipid in the product through heat treatment after the enzyme is contacted with the substrateFurther promoting the generation of the glycerol phosphatidylcholine and greatly improving the generation rate of the glycerol phosphatidylcholine; 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. It was found through previous studies 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 generation 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, mixing (total 10Kg, the concentration of lecithin in deionized water is 30mg/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(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, mixing with deionized water (total 10Kg, the concentration of lecithin in deionized water is 55mg/mL), and stirring at 600rpm for 15min, emulsifying, and delivering the reaction mixture to a packed bed reactor for reaction by a material pump, 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 and deionized water, mixing (total 10Kg, concentration of lecithin in deionized water is 45mg/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 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) 1Kg of filler, andthe temperature should be 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 ℃. 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 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. 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 (5)
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; the concentration of lecithin in deionized water is 30-55 mg/mL;
step 2: mixing the mixed substrate obtained in the step 1 with immobilized phospholipase A in a packed bed reactor1Contacting to react; immobilized phospholipase A 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 are1Temperature of contact reactionThe temperature is 35-55 ℃, 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;
and step 3: carrying out heat treatment on the reaction product obtained in the step 2, cooling to the reaction temperature of the step 2, and adding the reaction product into the mixed substrate reacted in the step 2; the heat treatment temperature is 60-70 ℃, and the heat treatment time is 20-40 min;
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 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.
3. 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 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 ℃.
4. The enzymatic method for preparing glycerophosphatidylcholine according to claim 1, wherein in step 4, the water removal is performed by vacuum concentration.
5. 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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| 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 |
| CN109265478B (en) * | 2018-10-30 | 2021-07-06 | 榆林学院 | Method for preparing glycerophosphatidylcholine based on egg shells |
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