CN108486179B - Method for preparing phosphatidic acid by continuous catalysis of immobilized phospholipase D - Google Patents
Method for preparing phosphatidic acid by continuous catalysis of immobilized phospholipase D Download PDFInfo
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Abstract
The invention provides a method for continuously catalyzing and preparing phosphatidic acid by using immobilized phospholipase D, which comprises the following steps: 1. preparing immobilized phospholipase D; 2. preparing a phospholipid emulsion; 3. mixing the immobilized phospholipase D with a phospholipid emulsion, and catalyzing to prepare a phosphatidic acid crude product; 4. and removing impurities and purifying the crude product of phosphatidic acid. After the preparation is finished, the phospholipase D can be recovered for reuse. The method is completed in a pure water system, does not use any organic solvent except alcohol, is environment-friendly, simple, high in product yield, low in energy consumption and easy to operate.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for preparing phosphatidic acid by continuous catalysis of immobilized phospholipase D.
Background
Phosphatidic Acid (PA) is a basic component of animal and plant cell membranes, nuclear membranes and plastid membranes, and is a basic substance of life. From the chemical structure, phosphatidic acid is an amphoteric compound, and is the simplest diacylglycerophospholipid, and one end of a fatty acid group has strong lipophilicity, while one end of a phosphate group has strong hydrophilicity. Phosphatidic acid, although present in small amounts in the organism, is a synthetic precursor of glycerides and most phospholipids, while phosphatidic acid is an important second messenger in the cell, acting as a signaling lipid molecule. Phosphatidic acid has wide application in brain health and sports nutrition fields, and can be used in various health foods, medicines and cosmetics.
Phospholipase D (P L D) is a phospholipid hydrolase (EC3.1.4.4), which can act on the hydroxyl ester bond of organic base in phospholipid molecules to hydrolyze the phospholipid molecules into phosphatidic acid and organic base.A immobilized enzyme combines the enzyme with a proper carrier, can increase the contact area with a substrate, and forms a microenvironment with a protective effect around the enzyme to improve the tolerance and thermodynamic stability of the enzyme.
PA is mainly extracted from crude phospholipid at present, but the content of PA in plant seeds is low, and the preparation method comprises solvent extraction, chemical synthesis, enzyme conversion and the like. Although the raw materials are natural phospholipids, the process is complicated, the separation is difficult, the cost is high, various chemical organic solvents are needed, the environmental pollution is caused, and the method is not suitable for industrial production.
The enzyme method for preparing PA uses lecithin as raw material, and under the action of phospholipase D, PA and choline are decomposed, PA is weakly acidic, choline has strong water solubility, and separation is difficult.
Disclosure of Invention
The invention aims to provide a method for continuously catalyzing and preparing phosphatidic acid by using immobilized phospholipase D, which has the advantages of simple process, environmental friendliness, high separation efficiency, continuous production, low operation cost and easiness in industrialization.
The method for continuously catalyzing and preparing phosphatidic acid by using the immobilized phospholipase D comprises the following steps:
(1) preparation of immobilized enzyme:
adsorbing the phospholipase D enzyme solution by a macroporous adsorption resin column, washing the resin with deionized water after saturation adsorption, and washing away residual phospholipase D;
(2) preparing a phospholipid emulsion:
mixing lecithin and deionized water, and shearing at high speed to obtain uniform emulsion;
(3) enzymatic preparation of crude phosphatidic acid:
continuously passing the phospholipid emulsion through a chromatographic column filled with immobilized phospholipase D for catalytic preparation, and collecting effluent to obtain a phosphatidic acid crude product solution;
(4) impurity removal and purification of phosphatidic acid:
passing the crude phosphatidic acid solution through continuous ion exchange resin to remove choline and impurities, and collecting the final effluent to obtain purified phosphatidic acid solution;
(5) concentrating and drying phosphatidic acid:
concentrating and drying the purified phosphatidic acid solution to obtain a phosphatidic acid product.
According to the method, the lecithin and water are prepared into the emulsion to react with the immobilized phospholipase D, the whole process is completed in a pure water system, any organic solvent except alcohol is not used, and the method is environment-friendly.
Further, the macroporous adsorbent resin in the step (1) is selected from one of L X-1000, XAD761, SP207 and ESR 1.
XAD761 is phenolic resin, SP207 is macroporous adsorption resin with large-aperture styrene skeleton, L X-1000 and ESR1 are special immobilized enzyme resins, and the resin has good immobilization effect on phospholipase D.
Further, a preferred macroporous adsorbent resin is SP 207.
SP207 has stronger adsorption force on phospholipase D, and the phospholipase D is not easy to fall off. And the SP207 resin is beneficial to the phospholipid emulsion to pass through and react with the phospholipase D more fully. In the reaction process, parameters such as viscosity, flow rate and the like of a reaction system can also change due to the change of the proportion of the raw materials and the products, and the SP207 can better adapt to the change, so that the smooth reaction is ensured.
Further, the mode of allowing the phospholipase D enzyme solution in the step (1) to pass through a macroporous adsorption resin column is that the feeding is continuously carried out in a countercurrent mode at the bottom, and the feeding temperature is 10-20 ℃.
In the phospholipid emulsion-phospholipase D reaction system, the phospholipase D is unstable above 20 ℃, the enzyme activity is lost along with the increase of the storage time, and the viscosity of the enzyme solution begins to increase when the temperature is below 10 ℃, so that the fluidity is reduced, and the adsorption is not facilitated.
Further, in the step (2), the lecithin source is soybean, sunflower seed, egg yolk or antarctic krill, and the content of phosphatidylcholine in the lecithin source is 10% -30%.
The method of the invention can adopt lecithin with low content of phosphatidylcholine as raw material, and simultaneously obtain phosphatidic acid product with high content, low cost and high yield.
Further, the mass ratio of the lecithin to the deionized water in the step (2) is 1:30-1: 50.
in the reaction system of the invention, the performance of the phospholipid emulsion directly influences the subsequent reaction process. As the reaction of the phospholipid emulsion in the resin column progresses, phosphatidylcholine and other substances are gradually converted into phosphatidic acid, and parameters such as system viscosity, pH value and the like are gradually changed. The invention controls the ratio of lecithin and deionized water to make the reactant smoothly pass through the resin and the ultrafiltration membrane, and simultaneously ensures the yield and quality of the product.
Further, the phospholipid emulsion in the step (3) passes through a chromatographic column filled with the immobilized phospholipase D in a bottom countercurrent continuous feeding mode, the feeding temperature is 40-50 ℃, and the feeding flow rate is 5-10 BV/h. The cooperation of the temperature and the feeding speed is beneficial to the smooth reaction, the preparation efficiency of the product is improved, and the high yield is ensured.
Further, in the impurity removal and purification process of the phosphatidic acid in the step (4), choline is adsorbed by strong acid cation exchange resin, and hydrogen ions are neutralized by weak base anion exchange resin.
The strong acid cation exchange resin is selected from one of 001 × 7(732), D001, HD-8 or NKC-9, and the weak base anion exchange resin is selected from one of L X-300C, D900, D301 or D315.
Further, the preferred strong acid cation exchange resin is 001 × 7(732), the preferred weak base anion exchange resin is L X-300C, and the combination of 001 × 7(732) and L X-300C can greatly improve the yield of phosphatidic acid products.
Further, the concentration method is membrane concentration, and ultrafiltration or nanofiltration membrane with the molecular weight cutoff of 400Dalton-3000Dalton is preferably selected for concentration, so that the product of the reaction system is collected more fully; the drying method is vacuum drying or freeze drying, and the phospholipid substance is easy to be oxidized and rancid at normal temperature because the special fatty acid structure contains unsaturated double bonds, so the vacuum low temperature is selected in consideration of the drying method, and the vacuum drying or freeze drying is preferred.
The invention has the advantages and positive effects that: the process is simple and easy to operate, has low energy consumption, is easy for industrial production, does not use any organic solvent except ethanol (alcohol), and is environment-friendly; the content of the adopted raw material phosphatidylcholine can be lower than 30%, the raw material phosphatidylcholine, phosphatidylethanolamine and other phospholipids can be completely converted into phosphatidic acid by the process method, the content of the obtained phosphatidic acid product can reach more than 50%, and the method has high application value.
Detailed Description
Example 1
The procedure for the preparation of this example is as follows.
Immobilization of phospholipase D:
taking 100g of XAD761 resin, washing with water, ethanol and water in sequence, loading into a glass chromatographic column with a heat-insulating layer, and introducing 10 ℃ circulating water into the heat-insulating layer for cooling; taking 1kg of phospholipase D enzyme solution, enabling the enzyme activity to be 8U/g, and enabling the phospholipase D enzyme solution to pass through a chromatographic column from the bottom in a countercurrent mode through a peristaltic pump at the flow speed of 2 BV/h; after adsorption, the solution is washed until effluent is colorless, the enzyme activity of the immobilized phospholipase D resin is detected to be 58U/g, and the resin adsorption rate of the phospholipase D in the immobilization process is 72.5 percent.
Pretreatment of ion exchange resin:
001 × 7(732) resin 500g, soaking in 4% NaOH for 24h, washing with water to neutrality, soaking in 4% hydrochloric acid for 24h, washing with water to neutrality, and loading into chromatographic column for use, D315 resin 500g, soaking in 4% hydrochloric acid for 24h, washing with water to neutrality, soaking in 4% NaOH for 24h, washing with water to neutrality, and loading into chromatographic column for use.
Phospholipid emulsification and phosphatidic acid preparation:
taking 100g of concentrated soybean lecithin (16% of protopatitic acid, 14% of phosphatidylcholine and 24% of phosphatidylethanolamine), adding 5kg of deionized water, carrying out high-speed shearing to form phospholipid emulsion, slowly stirring the emulsion, heating to 40 ℃, carrying out countercurrent flow from the bottom through the XAD761 resin column by a peristaltic pump, carrying out circulating water temperature of a heat-insulating layer of the resin column at 40 ℃, carrying out flow rate of 10BV/h, collecting effluent liquid to obtain a crude product of the phosphatidic acid, and detecting that the content of the phosphatidic acid is 44.3% by HP L C, the content of the phosphatidylcholine is 3.2%, the content of the phosphatidylethanolamine is 4.6%, the conversion rate of the phosphatidylcholine is 77.1%, and the conversion rate.
And (3) purifying a crude phosphatidic acid product:
passing the effluent through 001 × 7(732) strong acid cation exchange resin column at flow rate of 2BV/h, collecting effluent, detecting phosphatidic acid content of 44.7%, phosphatidylcholine content of 0.4%, and phosphatidylethanolamine content of 0.6% with HP L C;
continuously passing the effluent through a D315 weak-base anion exchange resin column at the flow rate of 2BV/h, collecting the effluent, namely the phosphatidic acid purification solution, wherein the content of phosphatidic acid is 38.3 percent, the content of phosphatidylcholine is 0.4 percent, and the content of phosphatidylethanolamine is 0.5 percent by HP L C detection.
And (3) concentrating and drying a product:
the phosphatidic acid purified solution is concentrated by a 2000 Dalton ultrafiltration membrane, and then is frozen and dried to obtain 67.7g of white phosphatidic acid product, wherein the phosphatidic acid content is 38.3 percent, the phosphatidylcholine content is 0.5 percent, the phosphatidylethanolamine content is 0.6 percent through HP L C detection, and the final product quality yield is 25.93 percent (calculated by the phosphatidic acid).
Example 2
The procedure for the preparation of this example is as follows.
Immobilization of phospholipase D:
taking 100g of SP207 resin, washing with water, ethanol and water in sequence, loading into a glass chromatographic column with a heat-insulating layer, and introducing circulating water of 20 ℃ into the heat-insulating layer for cooling; taking 1kg of phospholipase D enzyme solution, enabling the enzyme activity to be 8U/g, and enabling the phospholipase D enzyme solution to pass through a chromatographic column from the bottom in a countercurrent mode through a peristaltic pump at the flow speed of 2 BV/h; after adsorption, washing with water until effluent is colorless, and detecting that the enzyme activity of the immobilized phospholipase D resin is 74U/g; the resin adsorption rate of phospholipase D during the immobilization process was 92.5%.
Pretreatment of ion exchange resin:
001 × 7(732) resin 500g, soaking in 4% NaOH for 24h, washing with water to neutrality, soaking in 4% hydrochloric acid for 24h, washing with water to neutrality, and loading into chromatographic column, L X-300C resin 500g, soaking in 4% hydrochloric acid for 24h, washing with water to neutrality, soaking in 4% NaOH for 24h, washing with water to neutrality, and loading into chromatographic column.
Phospholipid emulsification and phosphatidic acid preparation:
100g of sunflower seed lecithin (the content of protopatidic acid is 15 percent, the content of phosphatidylcholine is 26 percent, and the content of phosphatidylethanolamine is 23 percent) is taken, 4kg of deionized water is added, high-speed shearing is carried out to form phospholipid emulsion, the emulsion is slowly stirred and heated to 45 ℃, the mixture flows through the SP207 resin column from the bottom in a countercurrent way through a peristaltic pump, the circulating water temperature of a heat-insulating layer of the resin column is 45 ℃, the flow rate is 8BV/h, and effluent liquid is collected, namely a phosphatidic acid crude product, the content of phosphatidic acid is 61 percent through HP L C detection, the content of phosphatidylcholine is 2.1 percent, the content of phosphatidylethanolamine is 1.8 percent, the conversion.
And (3) purifying a crude phosphatidic acid product:
the effluent passes through a 001 × 7(732) strong acid cation exchange resin column at the flow rate of 2BV/h, the effluent is collected, and HP L C detects the phosphatidic acid content of 61.2 percent, the phosphatidylcholine content of 0.3 percent and the phosphatidylethanolamine content of 0.3 percent;
continuously passing the effluent through L X-300C weak base anion exchange resin column at flow rate of 2BV/h, collecting the effluent as purified phosphatidic acid solution, detecting phosphatidic acid content 61.3%, phosphatidylcholine content 0.3%, and phosphatidylethanolamine content 0.4% by HP L C.
And (3) concentrating and drying a product:
concentrating the purified phosphatidic acid solution with 1000 Dalton nanofiltration membrane, vacuum drying to obtain white phosphatidic acid product 90.2g, detecting phosphatidic acid content 61.4%, phosphatidylcholine content 0.3%, and phosphatidylethanolamine content 0.3% with HP L C, and obtaining final product quality yield 55.38% (calculated as phosphatidic acid).
Example 3
The procedure for the preparation of this example is as follows.
Immobilization of phospholipase D:
taking 100g of SP207 resin, washing with water, ethanol and water in sequence, loading into a glass chromatographic column with a heat-insulating layer, and introducing circulating water of 10 ℃ into the heat-insulating layer for cooling; taking 1kg of phospholipase D enzyme solution, enabling the enzyme activity to be 7U/g, and enabling the phospholipase D enzyme solution to pass through a chromatographic column from the bottom in a countercurrent mode through a peristaltic pump at the flow speed of 1 BV/h; after adsorption, the solution is washed until effluent is colorless, the enzyme activity of the immobilized phospholipase D resin is detected to be 78U/g, and the resin adsorption rate of the phospholipase D in the immobilization process is 97.5 percent.
Pretreatment of ion exchange resin:
001 × 7(732) resin 500g, soaking in 4% NaOH for 24h, washing with water to neutrality, soaking in 4% hydrochloric acid for 24h, washing with water to neutrality, and loading into chromatographic column, L X-300C resin 500g, soaking in 4% hydrochloric acid for 24h, washing with water to neutrality, soaking in 4% NaOH for 24h, washing with water to neutrality, and loading into chromatographic column.
Phospholipid emulsification and phosphatidic acid preparation:
100g of antarctic krill phospholipid (the content of protoporphyrinic acid is 27%, the content of phosphatidylcholine is 10%, the content of phosphatidylethanolamine is 17%), 3kg of deionized water is added, high-speed shearing is carried out to form phospholipid emulsion, the emulsion is slowly stirred and heated to 45 ℃, the mixture flows through the SP207 resin column from the bottom in a countercurrent mode through a peristaltic pump, the circulating water temperature of a resin column heat-insulating layer is 45 ℃, the flow rate is 5BV/h, effluent liquid is collected, namely a phosphatidic acid crude product, the content of phosphatidic acid is 51.2% by HP L C detection, the content of phosphatidylcholine is 0.7%, the content of phosphatidylethanolamine is 1.1%, the conversion rate of phosphatidylcholine is 93.
And (3) purifying a crude phosphatidic acid product:
the effluent passes through a 001 × 7(732) strong acid cation exchange resin column at the flow rate of 1BV/h, the effluent is collected, and HP L C detects the phosphatidic acid content of 51.4 percent, the phosphatidylcholine content of 0.2 percent and the phosphatidylethanolamine content of 0.3 percent;
continuously passing the effluent through L X-300C weak base anion exchange resin column at flow rate of 1BV/h, collecting effluent as purified phosphatidic acid solution, detecting phosphatidic acid content of 51.5%, phosphatidylcholine content of 0.2% and phosphatidylethanolamine content of 0.3% by HP L C.
And (3) concentrating and drying a product:
and (3) concentrating the purified phosphatidic acid solution by using a 400Dalton nanofiltration membrane, freeze-drying to obtain 93.2g of a white phosphatidic acid product, detecting the phosphatidic acid content by using HP L C to detect that the phosphatidic acid content is 51.7 percent, the phosphatidylcholine content is 0.2 percent, the phosphatidylethanolamine content is 0.2 percent, and obtaining the final product with the quality yield of 48.18 percent (calculated by the phosphatidic acid).
Example 4
The procedure for the preparation of this example is as follows.
Immobilization of phospholipase D:
the method comprises the steps of sequentially washing L X-1000 resin 100g with water, ethanol and water, loading the resin into a glass chromatographic column with a heat-insulating layer, introducing 15 ℃ circulating water into the heat-insulating layer for cooling, taking 1kg of phospholipase D enzyme solution with the enzyme activity of 7U/g, enabling the solution to flow through the chromatographic column in a countercurrent mode through a peristaltic pump from the bottom at the flow rate of 3BV/h, washing the resin with water until effluent is colorless after adsorption is finished, detecting that the enzyme activity of immobilized phospholipase D resin is 62U/g, and ensuring that the resin adsorption rate of phospholipase D in the immobilization process is 77.5%.
Pretreatment of ion exchange resin:
soaking 500g of D001 resin in 4% NaOH for 24h, washing with water to neutrality, soaking in 4% hydrochloric acid for 24h, washing with water to neutrality, and loading into a chromatographic column for later use; and (3) soaking 500g of D900 resin in 4% hydrochloric acid for 24h, washing with water to be neutral, soaking in 4% NaOH for 24h, washing with water to be neutral, and filling into a chromatographic column for later use.
Phospholipid emulsification and phosphatidic acid preparation:
100g of egg yolk lecithin (27 percent of protopatidic acid, 30 percent of phosphatidylcholine and 17 percent of phosphatidylethanolamine), adding 4.5kg of deionized water, carrying out high-speed shearing to form phospholipid emulsion, slowly stirring the emulsion, heating to 50 ℃, carrying out countercurrent flow from the bottom through the L X-1000 resin column by a peristaltic pump, carrying out circulating water temperature of a heat-insulating layer of the resin column at 50 ℃, carrying out flow rate of 5BV/h, collecting effluent liquid, namely a phosphatidic acid crude product, detecting the phosphatidic acid content by HP L C to be 62.7 percent, the phosphatidylcholine content to be 6.9 percent, the phosphatidylethanolamine content to be 4.7 percent, the phosphatidylcholine conversion rate to be 77 percent and the phosphatidylethanolamine conversion rate to be 72.
And (3) purifying a crude phosphatidic acid product:
the effluent is firstly passed through a D001 strong acid cation exchange resin column with the flow rate of 1.5BV/h, the effluent is collected, and HP L C detects that the phosphatidic acid content is 56.7 percent, the phosphatidylcholine content is 3.2 percent and the phosphatidylethanolamine content is 1.8 percent;
continuously passing the effluent through a D900 weak-base anion exchange resin column at the flow rate of 1.5BV/h, collecting the effluent, namely, the phosphatidic acid purification solution, wherein the phosphatidic acid content is 51.7 percent, the phosphatidylcholine content is 3.3 percent and the phosphatidylethanolamine content is 1.7 percent by HP L C detection.
And (3) concentrating and drying a product:
concentrating the purified phosphatidic acid solution with 3000Dalton ultrafiltering membrane, vacuum drying to obtain 59g of white phosphatidic acid product, detecting with HP L C to obtain phosphatidic acid product with phosphatidic acid content of 51.8%, phosphatidylcholine content of 3.2%, phosphatidylethanolamine content of 1.8%, and final product quality yield of 30.56% (calculated as phosphatidic acid).
Example 5
The procedure for the preparation of this example is as follows.
Immobilization of phospholipase D:
taking 100g of ESR1 resin, washing with water, ethanol and water in sequence, putting into a glass chromatographic column with an insulating layer, and introducing 15 ℃ circulating water into the insulating layer for cooling; taking 1kg of phospholipase D enzyme solution, enabling the enzyme activity to be 8U/g, and enabling the phospholipase D enzyme solution to flow through a chromatographic column from the bottom in a countercurrent mode through a peristaltic pump at the flow speed of 1.5 BV/h; after adsorption, the solution is washed until effluent is colorless, the enzyme activity of the immobilized phospholipase D resin is detected to be 51U/g, and the resin adsorption rate of the phospholipase D in the immobilization process is 63.8 percent.
Pretreatment of ion exchange resin:
001 × 7(732) resin 500g, soaking in 4% NaOH for 24h, washing with water to neutrality, soaking in 4% hydrochloric acid for 24h, washing with water to neutrality, and loading into chromatographic column, L X-300C resin 500g, soaking in 4% hydrochloric acid for 24h, washing with water to neutrality, soaking in 4% NaOH for 24h, washing with water to neutrality, and loading into chromatographic column.
Phospholipid emulsification and phosphatidic acid preparation:
100g of egg yolk lecithin (the content of protopatitic acid is 27 percent, the content of phosphatidylcholine is 30 percent, the content of phosphatidylethanolamine is 17 percent), 5kg of deionized water is added, high-speed shearing is carried out to form phospholipid emulsion, the emulsion is slowly stirred and heated to 45 ℃, the mixture flows through the ESR1 resin column from the bottom in a countercurrent way by a peristaltic pump, the circulating water temperature of the heat-insulating layer of the resin column is 45 ℃, the flow rate is 5BV/h, and effluent liquid is collected to obtain a crude product of phosphatidic acid, 60.2 percent of phosphatidic acid is detected by HP L C, 5.9 percent of phosphatidylcholine, 5.4 percent of phosphatidylethanolamine, 80.3 percent of phosphatidylcholine conversion rate, and.
And (3) purifying a crude phosphatidic acid product:
the effluent passes through a 001 × 7(732) strong acid cation exchange resin column at the flow rate of 1BV/h, the effluent is collected, and HP L C detects the phosphatidic acid content of 61.2 percent, the phosphatidylcholine content of 0.8 percent and the phosphatidylethanolamine content of 0.5 percent;
continuously passing the effluent through L X-300C weak base anion exchange resin column at flow rate of 1BV/h, collecting effluent as purified phosphatidic acid solution, detecting phosphatidic acid content 61.3%, phosphatidylcholine content 0.9%, and phosphatidylethanolamine content 0.4% by HP L C.
And (3) concentrating and drying a product:
concentrating the purified phosphatidic acid solution with 1500 Dalton ultrafiltering membrane, vacuum drying to obtain white phosphatidic acid product 67g, detecting phosphatidic acid content 61.3%, phosphatidylcholine content 0.8%, and phosphatidylethanolamine content 0.5% with HP L C, and obtaining final product quality yield 41.07% (calculated as phosphatidic acid).
Example 6
The procedure for the preparation of this example is as follows.
Immobilization of phospholipase D:
taking 100g of SP207 resin, washing with water, ethanol and water in sequence, loading into a glass chromatographic column with a heat-insulating layer, and introducing circulating water of 10 ℃ into the heat-insulating layer for cooling; taking 1kg of phospholipase D enzyme solution, enabling the enzyme activity to be 7U/g, and enabling the phospholipase D enzyme solution to pass through a chromatographic column from the bottom in a countercurrent mode through a peristaltic pump at the flow speed of 3 BV/h; after adsorption, the solution is washed until effluent is colorless, the enzyme activity of the immobilized phospholipase D resin is detected to be 77U/g, and the resin adsorption rate of the phospholipase D in the immobilization process is 96.2 percent.
Pretreatment of ion exchange resin:
500g of NKC-9 resin, soaking for 24h by using 4% NaOH, washing to be neutral, soaking for 24h by using 4% hydrochloric acid, washing to be neutral, and filling into a chromatographic column for later use, L X-300C resin, 500g of resin, soaking for 24h by using 4% hydrochloric acid, washing to be neutral, soaking for 24h by using 4% NaOH, washing to be neutral, filling into the chromatographic column for later use.
Phospholipid emulsification and phosphatidic acid preparation:
100g of sunflower seed lecithin (the content of protopatidic acid is 15%, the content of phosphatidylcholine is 26%, the content of phosphatidylethanolamine is 23%), 5kg of deionized water is added, high-speed shearing is carried out to form phospholipid emulsion, the emulsion is slowly stirred and heated to 45 ℃, the mixture flows through the SP207 resin column from the bottom in a countercurrent mode through a peristaltic pump, the circulating water temperature of a resin column heat-insulating layer is 45 ℃, the flow rate is 5BV/h, effluent liquid is collected, namely a phosphatidic acid crude product, the content of phosphatidic acid is 50.2% through detection of HP L C, the content of phosphatidylcholine is 2.1%, the content of phosphatidylethanolamine is 2.4%, the conversion rate of phosphatidylcholine is 92%, and the.
And (3) purifying a crude phosphatidic acid product:
firstly, enabling the effluent to pass through an NKC-9 strong-acid cation exchange resin column at the flow rate of 1.5BV/h, collecting the effluent, and detecting the contents of 40.2 percent of phosphatidic acid, 0.7 percent of phosphatidylcholine and 0.8 percent of phosphatidylethanolamine by using HP L C;
continuously passing the effluent through L X-300C weak base anion exchange resin column at flow rate of 1.5BV/h, collecting effluent as purified phosphatidic acid solution, HP L C detecting phosphatidic acid content of 40.3%, phosphatidylcholine content of 0.8%, and phosphatidylethanolamine content of 0.7%.
And (3) concentrating and drying a product:
concentrating the purified phosphatidic acid solution with 400Dalton ultrafiltering membrane, vacuum drying to obtain white phosphatidic acid product 72g, detecting phosphatidic acid content with HP L C of 40.2%, phosphatidylcholine content of 0.8%, phosphatidylethanolamine content of 0.7%, and final product quality yield of 28.94% (calculated as phosphatidic acid).
Although preferred embodiments of the present invention have been described above, it will be understood by those skilled in the art that this is by way of illustration only, and that the scope of the invention is defined by the appended claims. Various changes and modifications to these embodiments may be made by those skilled in the art without departing from the spirit and scope of the invention, and these changes and modifications are within the scope of the invention.
Claims (6)
1. A method for continuously catalyzing and preparing phosphatidic acid by using immobilized phospholipase D is characterized by comprising the following steps: the whole process is completed in a pure water system without using any organic solvent except alcohol, and comprises the following steps:
(1) preparation of immobilized enzyme:
adsorbing phospholipase D enzyme liquid by a macroporous adsorption resin column, wherein the adsorption mode is that the bottom is fed in a countercurrent mode continuously, the feeding temperature is 10-20 ℃, the resin is washed by deionized water after adsorption saturation, and residual phospholipase D is washed away, and the macroporous adsorption resin is selected from one of L X-1000, XAD761, SP207 or ESR 1;
(2) preparing a phospholipid emulsion:
mixing lecithin and deionized water, and shearing at high speed to prepare a uniform emulsion, wherein the mass ratio of the lecithin to the deionized water is 1:30-1:50, the lecithin source is soybean, sunflower seed, egg yolk or antarctic krill, and the content of phosphatidylcholine in the lecithin source is 10% -30%;
(3) enzymatic preparation of crude phosphatidic acid:
continuously passing the phospholipid emulsion through a chromatographic column filled with immobilized phospholipase D for catalytic preparation, and collecting effluent to obtain a phosphatidic acid crude product solution;
(4) impurity removal and purification of phosphatidic acid:
passing the crude phosphatidic acid solution through continuous ion exchange resin to remove choline and impurities, and collecting the final effluent to obtain purified phosphatidic acid solution;
(5) concentrating and drying phosphatidic acid:
concentrating and drying the purified phosphatidic acid solution to obtain a phosphatidic acid product; wherein the concentration method is membrane concentration, ultrafiltration or nanofiltration membrane with molecular weight cutoff of 400Dalton-3000Dalton is used for concentration, and the drying method is vacuum drying or freeze drying;
in the method for preparing phosphatidic acid, the content of the adopted raw material, namely, the content of the phosphatidylcholine is lower than 30%, and the content of the phosphatidic acid product is more than 50%.
2. The method for the continuous catalytic production of phosphatidic acid with immobilized phospholipase D according to claim 1, wherein: the macroporous adsorption resin is SP 207.
3. The method for the continuous catalytic production of phosphatidic acid with immobilized phospholipase D according to claim 1, wherein: and (3) allowing the phospholipid emulsion in the step (3) to pass through a chromatographic column filled with immobilized phospholipase D in a bottom countercurrent continuous feeding mode, wherein the feeding temperature is 40-50 ℃, and the feeding flow rate is 5-10 BV/h.
4. The method for the continuous catalytic production of phosphatidic acid with immobilized phospholipase D according to claim 1, wherein: and (4) in the impurity removal and purification process of the phosphatidic acid in the step (4), choline is adsorbed by strong acid cation exchange resin, and hydrogen ions are neutralized by weak base anion exchange resin.
5. The method for continuously catalyzing and preparing phosphatidic acid with immobilized phospholipase D as claimed in claim 4, wherein the strongly acidic cation exchange resin is selected from one of 001-7732, D001, HD-8 or NKC-9, and the weakly basic anion exchange resin is selected from one of L X-300C, D900, D301 or D315.
6. The method for continuously catalyzing and preparing phosphatidic acid by using immobilized phospholipase D as claimed in claim 5, wherein the strongly acidic cation exchange resin is 001-7732, and the weakly basic anion exchange resin is L X-300C.
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Denomination of invention: A method for continuous catalytic preparation of phosphatidic acid by immobilized phospholipase D Effective date of registration: 20230511 Granted publication date: 20200724 Pledgee: Fengxian Branch of Shanghai Rural Commercial Bank Co.,Ltd. Pledgor: Shanghai Yunluo Biotechnology Co.,Ltd. Registration number: Y2023310000182 |