A method for extracting lysophosphatidylcholine from phospholipids

Abstract

The invention discloses a method for extracting lysophosphatidylcholine from phospholipids, comprising the following steps: S1: using phospholipids as raw materials, using short-chain alcohols as solvents to prepare a solution with a mass concentration of 1 to 5%, and obtaining a mixed product with a PC / PE ratio greater than 10 after pretreatment; S2: subjecting the mixed product with a PC / PE ratio greater than 10 obtained in step S1 to an enzymatic hydrolysis reaction in a phospholipase reactor to obtain a product rich in lysophosphatidylcholine; S3: separating and purifying the product rich in lysophosphatidylcholine obtained in step S2 to obtain a high-purity lysophosphatidylcholine product. The method for extracting lysophosphatidylcholine of the invention not only has high enzyme activity of phospholipase A and can be reused, but also has high extraction rate and purity of the obtained lysophosphatidylcholine.

Description

Technical Field

[0001] The invention relates to the technical field of phospholipid separation and extraction, in particular to a method for extracting lysophosphatidylcholine from phospholipid. Background Art

[0002] Phospholipids are a class of lipids containing phosphoric acid. The phospholipid structure is mainly composed of a glycerol skeleton, polar groups, and fatty acid chains of different lengths and saturation. According to the different polar groups of phospholipids, phospholipids can be divided into categories such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI). Lysophosphatidylcholine (LPC) is a degradation product produced by the hydrolysis or enzymatic hydrolysis of the 1st or 2nd ester bond of phosphatidylcholine (PC) under the action of phospholipase A. Lysophosphatidylcholine is an excellent emulsifier with better wetting ability, emulsification ability, and emulsion stability than ordinary phospholipids, and it also has antioxidant and antibacterial properties. It is widely used in food, chemical, and pharmaceutical fields. Especially in the pharmaceutical field, high-purity lysophosphatidylcholine (LPC>98%) is mainly used as an emulsifier for liposomes. Its emulsification effect is better than that of phosphatidylcholine, and it is a key excipient for new targeted preparations. At the same time, high-purity lysophosphatidylcholine has broad application prospects as the starting material for synthesizing phospholipids.

[0003] Extracting high-purity lysophosphatidylcholine from phospholipids is one of the technical difficulties that currently restricts the development of the industry. At present, there are various methods for preparing and extracting lysophosphatidylcholine, including chemical synthesis, acid-base hydrolysis (alcohol), enzymatic hydrolysis (alcohol) and physical separation. However, the current methods for preparing and extracting high-purity lysophosphatidylcholine still have various shortcomings; for example, the existing enzymatic method for preparing and extracting lysophosphatidylcholine uses a phospholipase with low enzyme activity and is difficult to reuse, and the lysophosphatidylcholine obtained has a low extraction rate and purity. Summary of the invention

[0004] In view of the above shortcomings of the prior art, the present invention provides a method for extracting lysophosphatidylcholine from phospholipids to solve the problems of the existing lysophosphatidylcholine extraction method, in which the phospholipase A used not only has low enzyme activity and is difficult to reuse, but also has low extraction rate and purity of the obtained lysophosphatidylcholine.

[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0006] A method for extracting lysophosphatidylcholine from phospholipids, the method comprising the following steps:

[0007] S1: using phospholipid as raw material and short-chain alcohol as solvent to prepare a solution with a mass concentration of 1-5%, and obtaining a mixed product with a PC / PE ratio greater than 10 after pretreatment;

[0008] S2: The mixed product with a PC / PE ratio greater than 10 obtained in step S1 is subjected to an enzymatic hydrolysis reaction in a phospholipase reactor to obtain a product rich in lysophosphatidylcholine;

[0009] S3: The lysophosphatidylcholine-rich product obtained in step S2 is separated and purified to obtain a high-purity lysophosphatidylcholine product.

[0010] Regarding the method for extracting lysophosphatidylcholine from phospholipids in the present application, those skilled in the art should understand that the term "extraction" in the present application is not a physical extraction operation in a narrow sense, but is interpreted in a broad sense, that is, it includes the enzymatic reaction step of step S2 of the present application, so as to convert the components in the phospholipids into lysophosphatidylcholine as much as possible after the raw material phospholipids are enzymatically converted, thereby improving its economic value.

[0011] Preferably, the pretreatment operation comprises a solvent extraction operation, the solvent used in the solvent extraction operation is a short-chain alcohol, the extraction temperature is 25 to 55° C., and the extraction time is 1 to 5 hours.

[0012] Preferably, the pretreatment operation comprises a low-pressure chromatography column elution operation, which is performed before the solvent extraction operation, and the filler of the low-pressure chromatography column is boron oxide.

[0013] Preferably, the low-pressure chromatographic column elution operation adopts a short-chain alcohol eluent, the concentration of the eluent is greater than 75%, the elution pressure is 0.01-0.1 MPa, and the elution temperature is 20-60°C.

[0014] Preferably, the pretreatment operation comprises a high-pressure chromatography column elution operation, which is performed after the solvent extraction operation, and the high-pressure chromatography column filler is diatomaceous earth.

[0015] Preferably, the high pressure chromatography column elution operation adopts short chain alcohol eluent, the concentration of the eluent is greater than 75%, the elution pressure is 0.15-0.3 MPa, and the elution temperature is 20-60°C.

[0016] Preferably, the phospholipase reactor uses an ultrafiltration membrane with phospholipase A immobilized thereon as a reactor.

[0017] Preferably, the preparation method of the phospholipase reactor is: uniformly dispersing phospholipase A in a sodium alginate solution with a mass concentration of 1 to 5%, adding a small amount of silane coupling agent, stirring evenly, and preparing a phospholipase A solution; then immersing the hollow fiber membrane bundle in the phospholipase A solution, and after immersion for 6 to 8 hours, obtaining a hollow fiber membrane bundle with phospholipase A immobilized on the outer surface of the membrane; and then encapsulating the hollow fiber membrane bundle in a cylindrical container with epoxy resin, and after the epoxy resin is cured and cut, adding caps at both ends of the container to obtain a phospholipase reactor.

[0018] Preferably, the mass ratio of phospholipase A to sodium alginate is 1:1-3, and the immobilized amount of phospholipase A on the phospholipase reactor is 10-100 mg / g.

[0019] Preferably, the hollow fiber membrane is an asymmetric ultrafiltration membrane, the membrane material is polyvinylidene fluoride, and the molecular weight cutoff of the hollow fiber membrane is 600-700 Daltons.

[0020] Beneficial effects of the present invention:

[0021] The method for extracting lysophosphatidylcholine from phospholipids adopts a phospholipase reactor to carry out enzymatic hydrolysis reaction. The phospholipase A used has high enzyme activity and can be reused. The final extraction rate of the obtained lysophosphatidylcholine is greater than 95% and the purity is greater than 98%. DETAILED DESCRIPTION

[0022] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art may think of other obvious variations.

[0023] Example 1

[0024] The method for extracting lysophosphatidylcholine from phospholipids in this embodiment comprises the following steps:

[0025] S1: Soybean lecithin is used as raw material, mainly containing phosphatidylcholine PC (33.8wt%), phosphatidylethanolamine PE (20.5wt%), phosphatidylinositol PI (16.2wt%), phosphatidylserine PS (15.8wt%), and the remaining content is other phospholipids; short-chain alcohol ethanol is used as a solvent to prepare soybean lecithin into a solution of 500mL with a mass concentration of 1%, and a mixed product with PC / PE greater than 10 is obtained after pretreatment operation; the pretreatment operation includes low-pressure chromatographic column elution operation, solvent extraction operation and high-pressure chromatographic column elution operation in sequence. The low-pressure chromatographic column elution operation is performed before the solvent extraction operation, and the low-pressure chromatographic column filler is boron oxide. The low-pressure chromatographic column elution operation uses short-chain alcohol ethanol eluent for elution, the concentration of the eluent is 85wt%, the elution pressure is 0.05MPa, and the elution temperature is 30°C. The solvent used in the solvent extraction operation is short-chain alcohol ethanol, the extraction temperature is 35°C, and the extraction time is 3h. The high-pressure chromatographic column elution operation is performed after the solvent extraction operation, and the high-pressure chromatographic column filler is diatomaceous earth. The high-pressure chromatographic column elution operation adopts short-chain alcohol ethanol eluent elution, the concentration of the eluent is 85wt%, the elution pressure is 0.15MPa, and the elution temperature is 30°C. During the pretreatment operation of this embodiment, the low-pressure chromatographic column elution operation is first performed to utilize the strong interaction between the electron-deficient compound boron oxide and the primary amine functional group on the phosphatidylserine PS, so that the phosphatidylserine PS can be eluted and removed from the phospholipids; then, the solubility of PC in short-chain alcohol is higher than that of PE and PI to perform solvent extraction operation to remove PE and PI; and then the high-pressure chromatographic column elution operation is performed to further remove PI from the phospholipids by utilizing the hydrogen bonds formed between the hydroxyl functional groups rich in diatomaceous earth and the hydroxyl functional groups of PI.

[0026] S2: The mixed product with a PC / PE ratio greater than 10 obtained in step S1 is subjected to enzymatic hydrolysis reaction in a phospholipase reactor, the feed flow rate is 50 mL / min, the operating pressure of the phospholipase reactor is 0.25 MPa, and a product rich in lysophosphatidylcholine is obtained, and the purity of the lysophosphatidylcholine is greater than 95%; the phospholipase reactor uses an ultrafiltration membrane after phospholipase A is immobilized as a reactor. The preparation method of the phospholipase reactor is as follows: phospholipase A is uniformly dispersed in a sodium alginate solution with a mass concentration of 1%, a small amount of silane coupling agent is added (the silane coupling agent is KH560, and the mass percentage of the silane coupling agent in the phospholipase A solution is 0.2%, the same below), and after being stirred evenly, a phospholipase A solution is prepared; then a hollow fiber membrane bundle is immersed in the phospholipase A solution, and after immersion for 6 hours, a hollow fiber membrane bundle with phospholipase A immobilized on the outer surface of the membrane is obtained; then the hollow fiber membrane bundle is encapsulated in a cylindrical container with epoxy resin, and after the epoxy resin is cured and cut, caps are added at both ends of the container to obtain a phospholipase reactor. The mass ratio of phospholipase A to sodium alginate is 1:1, and the immobilized amount of phospholipase A on the phospholipase reactor is 15 mg / g. The hollow fiber membrane is an external pressure asymmetric ultrafiltration membrane, the membrane material is polyvinylidene fluoride, and the molecular weight cutoff of the hollow fiber membrane is 600-700 Daltons. The surface of the polyvinylidene fluoride membrane of the present application is irradiated with plasma to improve the immobilization efficiency of phospholipase A on the surface of the polyvinylidene fluoride membrane; the irradiation time is 60s, the irradiation power is 20w, and the vacuum degree is 30kPa. The mixed product with a PC / PE greater than 10 obtained in step S1 is pressurized and flows outside the hollow fiber membrane, and the product rich in lysophosphatidylcholine is collected on the inside of the membrane and then flows out, while the phospholipids that are not completely hydrolyzed are retained on the outside of the membrane.

[0027] S3: The product rich in lysophosphatidylcholine obtained in step S2 is separated and purified by secondary solvent extraction operation to obtain a high-purity lysophosphatidylcholine product. In this embodiment, an ultrafiltration membrane after immobilization of phospholipase A with sodium alginate and silane coupling agent is used as a reactor to obtain a phospholipase reactor for enzymatic hydrolysis. The phospholipase A used has high enzyme activity, and the final extraction rate of the obtained lysophosphatidylcholine is 96.2% and the purity is 98.3%. The extraction rate of this application refers to the percentage of phosphatidylcholine in the phospholipid raw material that is finally converted into lysophosphatidylcholine.

[0028] With the same raw materials and operating conditions, the operation of this example was repeated 5 times, each time using the same phospholipase reactor without changing the phospholipase reactor, and the final extraction rate of lysophosphatidylcholine was measured to be 95.2% and the purity was 98.1%.

[0029] Example 2

[0030] The method for extracting lysophosphatidylcholine from phospholipids in this embodiment comprises the following steps:

[0031] S1: Soybean lecithin is used as raw material, mainly containing phosphatidylcholine PC (33.8wt%), phosphatidylethanolamine PE (20.5wt%), phosphatidylinositol PI (16.2wt%), phosphatidylserine PS (15.8wt%), and the remaining content is other phospholipids; short-chain alcohol ethanol is used as a solvent to prepare soybean lecithin into a solution of 500mL with a mass concentration of 3%, and a mixed product with PC / PE greater than 10 is obtained after pretreatment operation; the pretreatment operation includes low-pressure chromatographic column elution operation, solvent extraction operation and high-pressure chromatographic column elution operation in sequence. The low-pressure chromatographic column elution operation is performed before the solvent extraction operation, and the low-pressure chromatographic column filler is boron oxide. The low-pressure chromatographic column elution operation uses short-chain alcohol ethanol eluent for elution, the concentration of the eluent is 85wt%, the elution pressure is 0.05MPa, and the elution temperature is 60°C. The solvent used in the solvent extraction operation is short-chain alcohol ethanol, the extraction temperature is 55°C, and the extraction time is 5h. The high pressure chromatographic column elution operation is performed after the solvent extraction operation, the high pressure chromatographic column filler is diatomaceous earth, the high pressure chromatographic column elution operation adopts short chain alcohol ethanol eluent, the eluent concentration is 85wt%, the elution pressure is 0.3MPa, and the elution temperature is 60℃.

[0032] S2: The mixed product with a PC / PE ratio greater than 10 obtained in step S1 is subjected to an enzymatic hydrolysis reaction in a phospholipase reactor, the feed flow rate is 50 mL / min, the operating pressure of the phospholipase reactor is 0.35 MPa, and a product rich in lysophosphatidylcholine is obtained, and the purity of the lysophosphatidylcholine is greater than 95%; the phospholipase reactor uses an ultrafiltration membrane after phospholipase A is immobilized as a reactor. The preparation method of the phospholipase reactor is: phospholipase A is uniformly dispersed in a sodium alginate solution with a mass concentration of 5%, a small amount of silane coupling agent is added, and after stirring evenly, a phospholipase A solution is prepared; then the hollow fiber membrane bundle is immersed in the phospholipase A solution, and after immersion for 8 hours, a hollow fiber membrane bundle with phospholipase A immobilized on the outer surface of the membrane is obtained; then the hollow fiber membrane bundle is encapsulated in a cylindrical container with epoxy resin, and after the epoxy resin is cured and cut, caps are added at both ends of the container to obtain a phospholipase reactor. The mass ratio of phospholipase A to sodium alginate is 1:2, and the solid loading of phospholipase A on the phospholipase reactor is 30 mg / g. The hollow fiber membrane is an asymmetric ultrafiltration membrane, the membrane material is polyvinylidene fluoride, and the molecular weight cutoff of the hollow fiber membrane is 600-700 Daltons. The surface of the polyvinylidene fluoride membrane of the present application is irradiated by plasma, and the irradiation time is 60s, the irradiation power is 30w, and the vacuum degree is 40kPa.

[0033] S3: The lysophosphatidylcholine-rich product obtained in step S2 is separated and purified by secondary solvent extraction to obtain a high-purity lysophosphatidylcholine product. In this embodiment, an ultrafiltration membrane after phospholipase A is immobilized with sodium alginate and a silane coupling agent is used as a reactor to obtain a phospholipase reactor for enzymatic hydrolysis. The phospholipase A used has high enzyme activity, and the final extraction rate of the obtained lysophosphatidylcholine is 95.8% and the purity is 98.2%.

[0034] With the same raw materials and operating conditions, the operation of this example was repeated 5 times, each operation using the same phospholipase reactor without changing the phospholipase reactor, and the final extraction rate of lysophosphatidylcholine was measured to be 95.1% and the purity was 98.0%.

[0035] Example 3

[0036] The method for extracting lysophosphatidylcholine from phospholipids in this embodiment comprises the following steps:

[0037] S1: Soybean lecithin is used as raw material, mainly containing phosphatidylcholine PC (33.8wt%), phosphatidylethanolamine PE (20.5wt%), phosphatidylinositol PI (16.2wt%), phosphatidylserine PS (15.8wt%), and the remaining content is other phospholipids; short-chain alcohol ethanol is used as a solvent to prepare soybean lecithin into a solution of 500mL with a mass concentration of 5%, and a mixed product with PC / PE greater than 10 is obtained after pretreatment operation; the pretreatment operation includes low-pressure chromatographic column elution operation, solvent extraction operation and high-pressure chromatographic column elution operation in sequence. The low-pressure chromatographic column elution operation is performed before the solvent extraction operation, and the low-pressure chromatographic column filler is boron oxide. The low-pressure chromatographic column elution operation uses short-chain alcohol ethanol eluent for elution, the concentration of the eluent is 85wt%, the elution pressure is 0.05MPa, and the elution temperature is 40°C. The solvent used in the solvent extraction operation is short-chain alcohol ethanol, the extraction temperature is 35°C, and the extraction time is 5h. The high pressure chromatographic column elution operation is performed after the solvent extraction operation, and the high pressure chromatographic column filler is diatomaceous earth. The high pressure chromatographic column elution operation adopts short chain alcohol ethanol eluent, the concentration of the eluent is 85wt%, the elution pressure is 0.2MPa, and the elution temperature is 60°C. The high pressure chromatographic column elution operation in this embodiment also includes a crystallization operation before the high pressure chromatographic column elution operation, and the extract after the solvent extraction operation is subjected to a crystallization separation operation to retain the hemolyzed PC contained in the phospholipids to the greatest extent, and prevent it from being eluted and removed during the high pressure chromatographic column elution operation. The crystal product after the crystallization separation (mainly hemolyzed PC, PC, PE) is mixed with the eluent after the high pressure chromatographic column elution operation to obtain a mixed product.

[0038] S2: The mixed product with a PC / PE ratio greater than 10 obtained in step S1 is subjected to an enzymatic hydrolysis reaction in a phospholipase reactor, the feed flow rate is 50 mL / min, the operating pressure of the phospholipase reactor is 0.25 MPa, and a product rich in lysophosphatidylcholine is obtained, and the purity of the lysophosphatidylcholine is greater than 95%; the phospholipase reactor uses an ultrafiltration membrane after phospholipase A is immobilized as a reactor. The preparation method of the phospholipase reactor is as follows: phospholipase A is uniformly dispersed in a sodium alginate solution with a mass concentration of 5%, a small amount of silane coupling agent is added, and after stirring, a phospholipase A solution is prepared; then the hollow fiber membrane bundle is immersed in the phospholipase A solution, and after immersion for 8 hours, a hollow fiber membrane bundle with phospholipase A immobilized on the outer surface of the membrane is obtained; then the hollow fiber membrane bundle is encapsulated in a cylindrical container with epoxy resin, and after the epoxy resin is cured and cut, caps are added at both ends of the container to obtain a phospholipase reactor. The mass ratio of phospholipase A to sodium alginate is 1:3, and the immobilized amount of phospholipase A on the phospholipase reactor is 50 mg / g. The hollow fiber membrane is an asymmetric ultrafiltration membrane, the membrane material is polyvinylidene fluoride, and the molecular weight cutoff of the hollow fiber membrane is 600-700 Daltons. The surface of the polyvinylidene fluoride membrane of the present application is irradiated by plasma, and the irradiation time is 30s, the irradiation power is 50w, and the vacuum degree is 50kPa.

[0039] S3: The lysophosphatidylcholine-rich product obtained in step S2 is separated and purified by secondary solvent extraction to obtain a high-purity lysophosphatidylcholine product. In this embodiment, an ultrafiltration membrane after phospholipase A is immobilized with sodium alginate and a silane coupling agent is used as a reactor to obtain a phospholipase reactor for enzymatic hydrolysis. The phospholipase A used has high enzyme activity, and the final extraction rate of the obtained lysophosphatidylcholine is 96.2% and the purity is 98.8%.

[0040] With the same raw materials and operating conditions, the operation of this example was repeated 5 times, each time using the same phospholipase reactor without changing the phospholipase reactor, and the final extraction rate of lysophosphatidylcholine was measured to be 95.4% and the purity was 98.5%.

[0041] Comparative Example 1

[0042] The method for extracting lysophosphatidylcholine from phospholipids in this comparative example has the same raw material composition and operation steps as those in Example 1, except that in the method of this comparative example, phospholipase A in step S2 is directly added to the mixed product obtained in step S1 for enzymolysis reaction, rather than being subjected to enzymolysis reaction in a phospholipase reactor. The final extraction rate of lysophosphatidylcholine was measured to be 91.3% and the purity was 93.5%.

[0043] Comparative Example 2

[0044] The method for extracting lysophosphatidylcholine from phospholipids in this comparative example has the same raw material composition and operation steps as those in Example 1, except that in the method of this comparative example, phospholipase A in step S2 is directly added to the mixed product obtained in step S1 for enzymolysis reaction, instead of being subjected to enzymolysis reaction in a phospholipase reactor. Under the same raw materials and operation conditions, the operation of this comparative example is repeated 5 times, and each operation adds the solution containing phospholipase A after the previous purification, separation and concentration to the mixed product obtained in step S1 for enzymolysis reaction (i.e., phospholipase A is reused as much as possible, instead of adding new phospholipase A), and the final extraction rate of lysophosphatidylcholine is measured to be 72.8%, and the purity is 87.3%.

[0045] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention, and these changes and improvements fall within the scope of the present invention claimed.

Claims

1. A method for extracting lysophosphatidylcholine from phospholipids, characterized in that: The method comprises the following steps: S1: using phospholipid as raw material and short-chain alcohol as solvent to prepare a solution with a mass concentration of 1-5%, and obtaining a mixed product with a PC / PE ratio greater than 10 after pretreatment operation; the pretreatment operation includes a low-pressure chromatographic column elution operation, which is performed before the solvent extraction operation, and the low-pressure chromatographic column filler is boron oxide; the pretreatment operation includes a high-pressure chromatographic column elution operation, which is performed after the solvent extraction operation, and the high-pressure chromatographic column filler is diatomaceous earth; S2: subjecting the mixed product with a PC / PE ratio greater than 10 obtained in step S1 to enzymatic hydrolysis in a phospholipase reactor to obtain a product rich in lysophosphatidylcholine; the phospholipase reactor uses an ultrafiltration membrane with phospholipase A immobilized thereon as a reactor; S3: separating and purifying the lysophosphatidylcholine-rich product obtained in step S2 to obtain a high-purity lysophosphatidylcholine product; The preparation method of the phospholipase reactor is as follows: phospholipase A is uniformly dispersed in a sodium alginate solution with a mass concentration of 1-5%, a small amount of silane coupling agent is added, and the mixture is stirred to form a phospholipase A solution; then a hollow fiber membrane bundle is immersed in the phospholipase A solution, and after immersion for 6-8 hours, a hollow fiber membrane bundle with phospholipase A immobilized on the outer surface of the membrane is obtained; then the hollow fiber membrane bundle is encapsulated in a cylindrical container with epoxy resin, and after the epoxy resin is cured and cut, caps are added to both ends of the container to obtain a phospholipase reactor; the mass ratio of the phospholipase A to the sodium alginate is 1:1-3, and the immobilized amount of the phospholipase A on the phospholipase reactor is 10-100 mg / g; the hollow fiber membrane is an asymmetric ultrafiltration membrane, the membrane material is polyvinylidene fluoride, and the molecular weight cutoff of the hollow fiber membrane is 600-700 Daltons.

2. The method for extracting lysophosphatidylcholine from phospholipids as claimed in claim 1, characterized in that: The pretreatment operation includes a solvent extraction operation, the solvent used in the solvent extraction operation is a short-chain alcohol, the extraction temperature is 25 to 55° C., and the extraction time is 1 to 5 hours.

3. The method for extracting lysophosphatidylcholine from phospholipids as claimed in claim 1, characterized in that: The low-pressure chromatographic column elution operation adopts short-chain alcohol eluent, the concentration of the eluent is greater than 75%, the elution pressure is 0.01-0.1 MPa, and the elution temperature is 20-60°C.

4. The method for extracting lysophosphatidylcholine from phospholipids according to claim 1, characterized in that: The high-pressure chromatographic column elution operation adopts short-chain alcohol eluent, the concentration of the eluent is greater than 75%, the elution pressure is 0.15-0.3MPa, and the elution temperature is 20-60°C.