CN107722051B - Method for preparing phosphatidylserine salt - Google Patents

Method for preparing phosphatidylserine salt Download PDF

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CN107722051B
CN107722051B CN201710740364.7A CN201710740364A CN107722051B CN 107722051 B CN107722051 B CN 107722051B CN 201710740364 A CN201710740364 A CN 201710740364A CN 107722051 B CN107722051 B CN 107722051B
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phosphatidylserine
solution
metal salt
salt
metal
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CN107722051A (en
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夏洪志
顾钦青
徐晓晓
朱宇雷
沈宇峰
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Nantong Licheng Biology Engineering Co ltd
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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
    • C07F9/10—Phosphatides, e.g. lecithin
    • C07F9/106—Adducts, complexes, salts of phosphatides

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Abstract

The invention discloses a method for preparing phosphatidylserine salt, which comprises the following steps: providing a phosphatidylserine first metal salt; and (3) carrying out chemical reaction on the solution of the phosphatidylserine first metal salt through the solution containing the second metal ion to obtain the phosphatidylserine second metal salt. Through the mode, the application range of the phosphatidylserine is expanded, and the content of the phosphatidylserine in the product is improved.

Description

Method for preparing phosphatidylserine salt
Technical Field
The invention relates to the field of food health products, in particular to a method for preparing phosphatidylserine.
Background
Phosphatidylserine is a naturally occurring nutrient phospholipid, exists in cell membranes, forms the basic structure of the cell membranes with other phospholipids, and plays an important role in intercellular communication and biochemical information transmission to cells. Especially in the nervous system of human body, it is one of the important components of the brain cell membrane, and plays an important role in regulating various functions of the brain.
The phosphatidylserine has the main function of improving the activity of brain cells, and has good curative effects on treating encephalatrophy, preventing senile dementia and improving the functions of the brains of the elderly; can repair brain injury and treat hyperkinetic syndrome of children; it also has effects in relieving brain fatigue, balancing mood, and relieving depression.
The preparation of phosphatidylserine is mainly an enzymatic conversion method, namely, phosphatidylserine is synthesized by natural lecithin, serine and calcium salt under the catalysis of phospholipase. The prepared phosphatidylserine exists in a calcium salt form in the prior art, the calcium salt of the phosphatidylserine is insoluble in most organic solvents such as oil, alcohol, alkane and the like, so that the application of the phosphatidylserine is limited, and in the purification process, the calcium salt forms of other phospholipids are insoluble in organic solvents such as ethanol and the like, so that the impurity removal effect of the organic solvents such as alcohol and the like is influenced, and the increase of the content of the phosphatidylserine in the product is finally influenced.
Disclosure of Invention
The invention mainly solves the technical problem of providing a method for preparing phosphatidylserine, which can enable the obtained phosphatidylserine to exist in the form of a second metal salt, expand the application range of the phosphatidylserine and improve the content of the phosphatidylserine in the product.
In order to solve the technical problems, the invention adopts a technical scheme that: provided is a method for preparing a phosphatidylserine salt, which is characterized in that the preparation method comprises the following steps: providing a phosphatidylserine first metal salt; and (3) carrying out chemical reaction on the solution of the phosphatidylserine first metal salt through the solution containing the second metal ion to obtain the phosphatidylserine second metal salt.
The invention has the beneficial effects that: in contrast to the prior art, the present invention is achieved by providing a phosphatidylserine first metal salt; and (3) carrying out chemical reaction on the solution of the phosphatidylserine first metal salt through the solution containing the second metal ion to obtain the phosphatidylserine second metal salt. The second metal salt of the phosphatidylserine can be dissolved in most organic solvents, so that the application range of the phosphatidylserine is expanded, and the second metal salt of the phosphatidylserine can be dissolved in organic solvents such as ethanol and the like in the preparation process of the phosphatidylserine, so that the impurity removal effect of the organic solvents such as ethanol and the like is improved, and the content of the phosphatidylserine in the product is finally improved.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic flow diagram of a process for preparing phosphatidylserine salts according to one embodiment of the present invention;
FIG. 2 is a schematic flow diagram of a process for preparing phosphatidylserine salts according to a second embodiment of the present invention;
FIG. 3 is a schematic flow diagram of a three embodiment method of the present invention for the preparation of phosphatidylserine salts.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, fig. 1 is a schematic flow chart of a method for preparing phosphatidylserine salt according to an embodiment of the present invention.
In this example, a method of preparing a phosphatidylserine salt is provided, which may include the steps of:
step S11: providing a phosphatidylserine first metal salt.
Alternatively, the phosphatidylserine first metal salt may be a phosphatidylserine calcium salt.
Step S12: and (3) carrying out chemical reaction on the solution of the phosphatidylserine first metal salt through the solution containing the second metal ion to obtain the phosphatidylserine second metal salt.
Alternatively, the solution containing the second metal ion may be a sodium ion salt solution. In other embodiments, the solution containing the second metal ion may be a solution of other metal ions, for example, the solution containing the second metal ion may be a solution of potassium ions.
Alternatively, the sodium ion solution may include one or more of a sodium sulfate solution, a sodium carbonate solution, a sodium bicarbonate solution, a sodium hydroxide solution, a sodium thiosulfate solution, and a potassium acetate solution. For example, the sodium ion solution may be a sodium sulfate solution, a sodium carbonate solution, or a mixture of sodium sulfate and sodium carbonate solution, and the like.
Alternatively, the potassium ion solution may include one or more of a potassium sulfate solution, a potassium carbonate solution, a potassium bicarbonate solution, a potassium hydroxide solution, a potassium thiosulfate solution, and a potassium acetate solution. For example, the potassium ion solution may be a potassium sulfate solution, a potassium carbonate solution, or a mixture of potassium sulfate and potassium carbonate solution, and the like.
Alternatively, the phosphatidylserine second metal salt may be a sodium phosphatidylserine salt. In other embodiments, the phosphatidylserine second metal salt can be other salts, for example, the phosphatidylserine second metal salt can be a potassium phosphatidylserine salt.
In the embodiment, the solution containing the second metal ion and the phosphatidylserine first metal salt are subjected to chemical reaction to obtain the phosphatidylserine second metal salt, the method is simple and easy to implement and low in cost, and the phosphatidylserine second metal salt can be dissolved in most organic solvents, so that the application range of the phosphatidylserine is expanded.
Referring to fig. 2, fig. 2 is a schematic flow chart of a method for preparing a phosphatidylserine second metal salt according to a second embodiment of the present invention.
In this example, another method for preparing phosphatidylserine salts is provided, which may include the steps of:
step S21: providing a phosphatidylserine first metal salt.
Alternatively, the phosphatidylserine first metal salt may be a phosphatidylserine calcium salt.
Step S22: dissolving a phosphatidylserine first metal salt in a first solvent to obtain a solution of the phosphatidylserine first metal salt.
Alternatively, the first solvent may be petroleum ether.
Step S23: and adding the solution containing the second metal ions into the solution of the first metal salt of the phosphatidylserine to perform chemical reaction, thereby obtaining the solution of the second metal salt of the phosphatidylserine.
Alternatively, the solution containing the second metal ion may be a sodium ion salt solution. In other embodiments, the solution containing the second metal ion can be a solution of other metal ions in salt. For example, the solution containing the second metal ion may be a potassium ion salt solution.
Alternatively, the sodium ion solution may include one or more of a sodium sulfate solution, a sodium carbonate solution, a sodium bicarbonate solution, a sodium hydroxide solution, a sodium thiosulfate solution, and a potassium acetate solution. For example, the sodium ion solution may be a sodium sulfate solution, a sodium carbonate solution, or a mixture of sodium sulfate and sodium carbonate solution, and the like.
Alternatively, the potassium ion solution may include one or more of a potassium sulfate solution, a potassium carbonate solution, a potassium bicarbonate solution, a potassium hydroxide solution, a potassium thiosulfate solution, and a potassium acetate solution. For example, the potassium ion solution may be a potassium sulfate solution, a potassium carbonate solution, or a mixture of potassium sulfate and potassium carbonate solution, and the like.
Alternatively, the mass ratio of the phosphatidylcholine-containing substance to the sodium salt in the sodium ion solution may be 1:2 to 1:0.2, for example, the mass ratio of the phosphatidylcholine-containing substance to the sodium salt in the sodium ion solution may be 1:2, 1:1, or 1: 0.2.
Alternatively, the mass ratio of the phosphatidylcholine-containing substance to the potassium salt in the potassium ion solution may be 1:2 to 1:0.2, for example, the mass ratio of the phosphatidylcholine-containing substance to the potassium salt in the potassium ion solution may be 1:2, 1:1, or 1: 0.2.
Alternatively, the chemical reaction time may be 0.5 to 2 hours, for example, the chemical reaction time may be 0.5 to 1 or 2 hours, and the chemical reaction temperature may be 20 to 60 ℃, for example, the chemical reaction temperature may be 20, 45 or 60 ℃.
Optionally, the chemical reaction is repeated at least once. In other embodiments, the number of times the chemical reaction is repeated may be determined as practical, e.g., once, twice, three times, etc.
Wherein the chemical reaction comprises:
step S231: adding the second metal ion solution to the solution of the phosphatidylserine first metal salt;
step S232: after the chemical reaction is finished, releasing the lower aqueous phase solution to obtain an upper organic phase solution;
step S233: if the content of the first metal salt of the phosphatidylserine in the organic phase solution of the upper layer is lower than the preset value, taking out the upper layer solution as a second metal salt solution of the phosphatidylserine; if the content of the phosphatidylserine first metal salt dissolved in the petroleum ether solution of the upper layer is higher than or equal to the predetermined value, the step S231 is repeated.
Through the way, the phosphatidylserine first metal salt can be fully reacted with the second metal ion to obtain the phosphatidylserine second metal salt solution with higher concentration, and further obtain the phosphatidylserine second metal salt with higher content.
Step S24: concentrating the phosphatidylserine second metal salt solution to obtain the phosphatidylserine second metal salt concentrate.
The phosphatidylserine second metal salt solution can be concentrated by recovering petroleum ether from the phosphatidylserine second metal salt solution by distillation to obtain the phosphatidylserine second metal salt concentrate.
Step S25: adding ethanol to the concentrate of the second metal salt of phosphatidylserine, and filtering to obtain insoluble substance which is solid of the second metal salt of phosphatidylserine.
Alternatively, the mass ratio of the phosphatidylcholine-containing substance to ethanol may be 1:15 to 1:10, for example, the mass ratio of the phosphatidylcholine-containing substance to ethanol may be one of 1:15, 1:13, and 1: 10.
The method comprises the steps of adding the phosphatidylserine second metal salt concentrate into ethanol, dissolving impurities which can be dissolved in the ethanol in the phosphatidylserine second metal salt concentrate into the ethanol, and removing the impurities by using the ethanol to obtain the phosphatidylserine second metal salt with higher purity.
Alternatively, the ethanol-soluble impurity may be some phospholipid second metal salt, for example the ethanol-soluble impurity may be a lecithin second metal salt. Optionally, the phospholipid second metal salt is a phospholipid sodium salt or a phospholipid potassium salt. Alternatively, the lecithin second metal salt may be a lecithin sodium salt or a lecithin potassium salt.
Step S26: and drying the phosphatidylserine second metal salt solid to obtain the phosphatidylserine second metal salt.
In the embodiment, the solution containing the second metal ions and the phosphatidylserine first metal salt are subjected to chemical reaction to obtain the phosphatidylserine second metal salt, the method is simple and easy to implement and low in cost, and the phosphatidylserine second metal salt can be dissolved in most organic solvents, so that the application range of the phosphatidylserine is expanded; the phosphatidylserine second metal salt with higher purity can be obtained by removing impurities dissolved in ethanol in the phosphatidylserine second metal salt concentrate by using ethanol.
Referring to fig. 3, fig. 3 is a schematic flow chart of a method for preparing phosphatidylserine salt according to three embodiments of the present invention.
In this example, another method for preparing phosphatidylserine salts is provided, which may include the steps of:
step S31: a reaction is generated using a phosphatidylcholine-containing material, serine, a first metal salt, and water to produce a first mixture comprising a phosphatidylserine first metal salt.
Alternatively, in other embodiments, a phosphatidylcholine-containing material, serine, a first metal salt, and water can be used to react under catalysis by a phospholipase to form a first mixture comprising a phosphatidylserine first metal salt.
Specifically, in the above reaction, phosphatidylcholine, serine, and the first metal salt may chemically react to obtain a phosphatidylserine first metal salt and a phospholipid first metal salt.
Alternatively, the phosphatidylcholine-containing material may be soy lecithin having a phosphatidylcholine content of greater than 20%. The serine can be L-serine.
Alternatively, the first metal salt may be a calcium salt, for example, the calcium salt may be calcium chloride, and in other embodiments, the calcium salt may be calcium bicarbonate, or the like.
Alternatively, the mass ratio of the phosphatidylcholine-containing substance to the L-serine may be 1:5 to 1:0.5, for example, the mass ratio of the phosphatidylcholine-containing substance to the L-serine may be 1:5, 1:3, or 1: 0.5.
Alternatively, the mass ratio of the phosphatidylcholine-containing substance to the calcium chloride may be 1:2 to 1:0.1, for example, the mass ratio of the phosphatidylcholine-containing substance to the calcium chloride may be 1:2, 1:1, or 1: 0.1.
Alternatively, the mass ratio of the phosphatidylcholine-containing substance to water may be 1:10 to 1:5, for example, the mass ratio of the phosphatidylcholine-containing substance to water may be 1:10, 1:7, or 1: 5.
Alternatively, the phospholipase may be phospholipase D. The phospholipase D may be used in an amount of 10U/g to 100U/g, for example, 10U/g, 50U/g, or 100U/g.
Alternatively, the temperature at which the reaction is generated in step S31 may be 40 ℃ to 50 ℃, for example, the temperature at which the reaction is generated in step S31 may be 40 ℃, 45 ℃, or 50 ℃. The time for the generation reaction may be 2 hours to 8 hours, for example, the time for the generation reaction may be 2 hours, 4 hours, or 8 hours.
Alternatively, the phosphatidylserine first metal salt may be a phosphatidylserine calcium salt.
Step S32: centrifuging the first mixture of phosphatidylserine first metal salts to obtain a second mixture comprising phosphatidylserine first metal salts.
The solid particles in suspension can be separated from the liquid by centrifugation to provide a second mixture comprising the phosphatidylserine first metal salt in the upper layer.
Step S33: and dissolving the second mixture by using the first solvent, and adding the second solvent into the dissolved second mixture to remove impurities, thereby obtaining a solution of the phosphatidylserine first metal salt which can be dissolved in the first solvent.
Alternatively, the first solvent may be petroleum ether and the second solvent may be a mixture of isopropanol and water.
Specifically, since the second mixture contains residual phospholipase, after isopropanol and water are added to the dissolved second mixture, the phospholipase dissolved in the first solvent can be inactivated by the isopropanol, and thus, the addition of the second mixture can not only achieve the purpose of further removing impurities, but also increase the content of phosphatidylserine in the obtained solution of the phosphatidylserine first metal salt.
Alternatively, the mass ratio of the phosphatidylcholine-containing substance to petroleum ether may be 1:10 to 1: for example, the mass ratio of the phosphatidylcholine-containing substance to the petroleum ether may be 1:10, 1:5, or 1: 1.
Alternatively, the mass ratio of the phosphatidylcholine-containing substance to isopropanol may be 1:5 to 1:1, for example, the mass ratio of the phosphatidylcholine-containing substance to isopropanol may be 1:5, 1:3, or 1: 1.
The mass ratio of the phosphatidylcholine-containing substance to water in the second solvent may be 1:5 to 1:1, for example, the mass ratio of the phosphatidylcholine-containing substance to water in the second solvent may be 1:5, 1:3, or 1: 1.
Wherein the step of adding a second solvent to the dissolved second mixture to remove impurities and further obtain a solution of the phosphatidylserine first metal salt which can be dissolved in the first solvent comprises:
step S331: and adding a second solvent into the second mixture, extracting and shaking, standing for layering to layer the liquid capable of being dissolved in the first solvent and the liquid capable of being dissolved in the second solvent.
Step S332: the liquid which is taken out of the upper layer and can be dissolved in the first solvent is a solution of the phosphatidylserine first metal salt.
Through the steps, the phospholipase in the second mixture can be removed by isopropanol in the second solvent, so that the purpose of further removing impurities is achieved, and further, the content of the phosphatidylserine in the obtained solution of the phosphatidylserine first metal salt can be higher by removing the phospholipase in the second mixture.
Step S34: and adding the solution containing the second metal ions into the solution of the first metal salt of the phosphatidylserine to perform chemical reaction, thereby obtaining the solution of the second metal salt of the phosphatidylserine.
Alternatively, the solution containing the second metal ion may be a sodium ion salt solution. In other embodiments, the solution containing the second metal ion can be a solution of other metal ions in salt. For example, the solution containing the second metal ion may be a potassium ion salt solution.
Alternatively, the sodium ion solution may include one or more of a sodium sulfate solution, a sodium carbonate solution, a sodium bicarbonate solution, a sodium hydroxide solution, a sodium thiosulfate solution, and a potassium acetate solution. For example, the sodium ion solution may be a sodium sulfate solution, a sodium carbonate solution, or a mixture of sodium sulfate and sodium carbonate solution, and the like.
Alternatively, the potassium ion solution may include one or more of a potassium sulfate solution, a potassium carbonate solution, a potassium bicarbonate solution, a potassium hydroxide solution, a potassium thiosulfate solution, and a potassium acetate solution. For example, the potassium ion solution may be a potassium sulfate solution, a potassium carbonate solution, or a mixture of potassium sulfate and potassium carbonate solution, and the like.
Alternatively, the mass ratio of the phosphatidylcholine-containing substance to the sodium salt in the sodium ion solution may be 1:2 to 1:0.2, for example, the mass ratio of the phosphatidylcholine-containing substance to the sodium salt in the sodium ion solution may be 1:2, 1:1, or 1: 0.2.
Alternatively, the mass ratio of the phosphatidylcholine-containing substance to the potassium salt in the potassium ion solution may be 1:2 to 1:0.2, for example, the mass ratio of the phosphatidylcholine-containing substance to the potassium salt in the potassium ion solution may be 1:2, 1:1, or 1: 0.2.
Alternatively, the chemical reaction time may be 0.5 to 2 hours, for example, the chemical reaction time may be 0.5 to 1 or 2 hours, and the chemical reaction temperature may be 20 to 60 ℃, for example, the chemical reaction temperature may be 20, 45 or 60 ℃.
Optionally, the chemical reaction is repeated at least once. In other embodiments, the number of times the chemical reaction is repeated may be determined as practical, e.g., once, twice, three times, etc.
Wherein the chemical reaction comprises:
step S341: adding the second metal ion solution to the solution of the phosphatidylserine first metal salt;
step S342: after the chemical reaction is finished, releasing the lower aqueous phase solution to obtain an upper organic phase solution;
step S343: if the content of the first metal salt of the phosphatidylserine in the organic phase solution of the upper layer is lower than the preset value, taking out the upper layer solution as a second metal salt solution of the phosphatidylserine; if the content of the phosphatidylserine first metal salt dissolved in the petroleum ether solution of the upper layer is higher than or equal to the predetermined value, the step S341 is repeated.
Through the way, the phosphatidylserine first metal salt can be fully reacted with the second metal ion to obtain the phosphatidylserine second metal salt solution with higher concentration, and further obtain the phosphatidylserine second metal salt with higher content.
Step S35: concentrating the phosphatidylserine second metal salt solution to obtain the phosphatidylserine second metal salt concentrate.
The phosphatidylserine second metal salt solution can be concentrated by recovering petroleum ether from the phosphatidylserine second metal salt solution by distillation to obtain the phosphatidylserine second metal salt concentrate.
Step S36: adding ethanol to the concentrate of the second metal salt of phosphatidylserine, and filtering to obtain insoluble substance which is solid of the second metal salt of phosphatidylserine.
Alternatively, the mass ratio of the phosphatidylcholine-containing substance to ethanol may be 1:15 to 1:10, for example, the mass ratio of the phosphatidylcholine-containing substance to ethanol may be one of 1:15, 1:13, and 1: 10.
The method comprises the steps of adding the phosphatidylserine second metal salt concentrate into ethanol, dissolving impurities which can be dissolved in the ethanol in the phosphatidylserine second metal salt concentrate into the ethanol, and removing the impurities by using the ethanol to obtain the phosphatidylserine second metal salt with higher purity.
Alternatively, the ethanol-soluble impurity may be some phospholipid second metal salt, for example the ethanol-soluble impurity may be a lecithin second metal salt. Optionally, the phospholipid second metal salt is a phospholipid sodium salt or a phospholipid potassium salt. Alternatively, the lecithin second metal salt may be a lecithin sodium salt or a lecithin potassium salt.
Step S37: and drying the phosphatidylserine second metal salt solid to obtain the phosphatidylserine second metal salt.
In this embodiment, by adding the second solvent to the dissolved second mixture, not only the impurities dissolved in the second solvent can be removed, but also the phospholipase remaining in the second mixture can be removed by the isopropanol in the second solvent, thereby preventing the phospholipase from damaging the stability of the product; the method is simple and easy to implement and low in cost, and the phosphatidylserine second metal salt can be dissolved in most organic solvents, so that the application range of the phosphatidylserine is expanded; the phosphatidylserine second metal salt with higher purity can be obtained by removing impurities dissolved in ethanol in the phosphatidylserine second metal salt concentrate by using ethanol.
In contrast to the prior art, the present invention is achieved by providing a phosphatidylserine first metal salt; and (3) carrying out chemical reaction on the solution of the phosphatidylserine first metal salt through the solution containing the second metal ion to obtain the phosphatidylserine second metal salt. The second metal salt of the phosphatidylserine can be dissolved in most organic solvents, so that the application range of the phosphatidylserine is expanded, and the second metal salt of the phosphatidylserine can be dissolved in organic solvents such as ethanol and the like in the preparation process of the phosphatidylserine, so that the impurity removal effect of the organic solvents such as ethanol and the like is improved, and the content of the phosphatidylserine in the product is finally improved.
The following are specific examples of phosphatidylserine salts prepared by the method of the present invention:
example 1:
to 100g of soybean lecithin having a phosphatidylcholine content of 52%, 200g of L-serine, 16g of calcium chloride and 600ml of purified water were added, and after stirring at a constant temperature of 45 ℃ for one hour, 10ml of a phospholipase D solution was added, and the mixture was reacted at a constant temperature of 45 ℃ for four hours to obtain a mixture containing a phosphatidylserine calcium salt.
The mixture containing the calcium salt of phosphatidylserine was centrifuged and the upper solid was removed.
Adding 500ml of petroleum ether into the upper-layer solid to completely dissolve the upper-layer solid in the petroleum ether, adding 300ml of isopropanol and 300ml of water, extracting, vibrating, standing, layering, and discharging the lower-layer aqueous phase solution to obtain a first petroleum ether phase solution.
Adding 300ml of aqueous solution containing 30g of sodium sulfate into the petroleum ether phase solution, reacting at the temperature of 45 ℃ for 1 hour, and taking out a second petroleum ether phase solution positioned at the upper layer;
adding 300ml of an aqueous solution containing 30g of sodium sulfate into the second petroleum ether phase solution, reacting at the temperature of 45 ℃ for 1 hour, and taking out a third petroleum ether phase solution positioned on the upper layer;
to the third petroleum ether phase solution was added 300ml of an aqueous solution containing 30g of sodium sulfate, and the reaction was carried out at 45 ℃ for 1 hour, and the fourth petroleum ether phase solution located in the upper layer was taken out.
The petroleum ether in the fourth petroleum ether phase solution was recovered in vacuo to give 192g of a solid concentrate.
The solid concentrate was put into 1L of anhydrous ethanol, and rapidly stirred at room temperature for 2 hours to separate an anhydrous ethanol-insoluble substance.
The anhydrous ethanol insoluble material was dried under vacuum to obtain 57.2g of phosphatidylserine product. Wherein the content of phosphatidylserine is 82.9%, the content of calcium is 0.48mg/g, and the content of sodium is 20.5 mg/g.
Example 2:
to 100g of soybean lecithin having a phosphatidylcholine content of 52%, 200g of L-serine, 16g of calcium chloride and 600ml of purified water were added, and after stirring at a constant temperature of 45 ℃ for one hour, 10ml of a phospholipase D solution was added, and the mixture was reacted at a constant temperature of 45 ℃ for four hours to obtain a mixture containing a phosphatidylserine calcium salt.
The mixture containing the calcium salt of phosphatidylserine was centrifuged and the upper solid was removed.
Adding 500ml of petroleum ether into the upper-layer solid to completely dissolve the upper-layer solid in the petroleum ether, adding 300ml of isopropanol and 300ml of water, extracting, vibrating, standing, layering, and discharging the lower-layer aqueous phase solution to obtain a first petroleum ether phase solution.
Adding 300ml of aqueous solution containing 30g of sodium sulfate into the petroleum ether phase solution, reacting at the temperature of 45 ℃ for 1 hour, and taking out a second petroleum ether phase solution positioned at the upper layer;
adding 300ml of an aqueous solution containing 30g of sodium sulfate into the second petroleum ether phase solution, reacting at the temperature of 45 ℃ for 1 hour, and taking out a third petroleum ether phase solution positioned on the upper layer;
the petroleum ether in the third petroleum ether phase solution was recovered in vacuo to give 211g of a solid concentrate.
The solid concentrate was put into 1L of anhydrous ethanol, and rapidly stirred at room temperature for 2 hours to separate an anhydrous ethanol-insoluble substance.
Vacuum drying the anhydrous ethanol insoluble substance to obtain 63.0g product with phosphatidylserine content. Wherein the content of phosphatidylserine is 78.1%, the content of calcium is 2.15mg/g, and the content of sodium is 13.92 mg/g.
Example 3:
to 100g of soybean lecithin having a phosphatidylcholine content of 52%, 200g of L-serine, 16g of calcium chloride and 600ml of purified water were added, and after stirring at a constant temperature of 45 ℃ for one hour, 10ml of a phospholipase D solution was added, and the mixture was reacted at a constant temperature of 45 ℃ for four hours to obtain a mixture containing a phosphatidylserine calcium salt.
The mixture containing the calcium salt of phosphatidylserine was centrifuged and the upper solid was removed.
Adding 500ml of petroleum ether into the upper-layer solid to completely dissolve the upper-layer solid in the petroleum ether, adding 300ml of isopropanol and 300ml of water, extracting, vibrating, standing, layering, and discharging the lower-layer aqueous phase solution to obtain a first petroleum ether phase solution.
Adding 300ml of aqueous solution containing 30g of sodium sulfate into the petroleum ether phase solution, reacting at the temperature of 45 ℃ for 1 hour, and taking out a second petroleum ether phase solution positioned at the upper layer;
the petroleum ether in the second petroleum ether phase solution was recovered in vacuo to give 227g of a solid concentrate.
The solid concentrate was put into 1L of anhydrous ethanol, and rapidly stirred at room temperature for 2 hours to separate an anhydrous ethanol-insoluble substance.
The anhydrous ethanol insoluble material was dried under vacuum to obtain 67.7g of phosphatidylserine product. Wherein the content of phosphatidylserine is 71.9%, the content of calcium is 6.43mg/g, and the content of sodium is 9.89 mg/g.
In order to illustrate that the content of phosphatidylserine in the product prepared by the invention is higher, the specification further provides a comparison example for preparing a phosphatidylserine product by using the prior art, wherein the comparison example 1 and the comparison example 2 are comparison examples for preparing the phosphatidylserine product by using a single aqueous phase method in the prior art, and the comparison example 3 is a comparison example for preparing the phosphatidylserine product by using a double aqueous phase method in the prior art.
Comparative example 1:
to 100g of soybean lecithin having a phosphatidylcholine content of 52%, 200g of serine, 16g of calcium chloride and 600ml of water were added, and after stirring at 45 ℃ for one hour, 10ml of a phospholipase D solution was added, and the reaction was carried out at 45 ℃ for 4 hours. And after the reaction is finished, centrifuging, taking out the upper-layer solid, adding 500ml of purified water into the upper-layer solid, washing for three times, quickly stirring the washed solid with 1L of absolute ethyl alcohol at room temperature for 2 hours, separating out absolute ethyl alcohol insoluble substances, and drying the absolute ethyl alcohol insoluble substances in vacuum to obtain 79.8g of the phosphatidylserine product, wherein the content of the phosphatidylserine is 59.5%, the content of calcium is 18.2mg/g, and the content of sodium is 0.007 mg/g.
Comparative example 2:
to 100g of soybean lecithin having a phosphatidylcholine content of 52%, 200g of serine, 16g of calcium chloride and 600ml of water were added, and after stirring at 45 ℃ for one hour, 10ml of a phospholipase D solution was added, and the reaction was carried out at 45 ℃ for 4 hours. And after the reaction is finished, centrifuging, taking out the upper-layer solid, adding 500ml of petroleum ether into the upper-layer solid for dissolving, washing the petroleum ether phase by using 300ml of isopropanol and 300ml of water, recovering the petroleum ether in vacuum to obtain 217g of solid concentrate, rapidly stirring the concentrate for 2 hours at room temperature by using 1L of absolute ethyl alcohol, separating out the absolute ethyl alcohol insoluble substance, and drying the absolute ethyl alcohol insoluble substance in vacuum to obtain 65.2g of the phosphatidylserine product. Wherein the content of phosphatidylserine is 63.1%, the content of calcium is 12.85mg/g, and the content of sodium is 0.011 mg/g.
Comparative example 3:
dissolving 100g of soybean lecithin with phosphatidylcholine content of 52% in 1000ml of petroleum ether, adding a calcium chloride aqueous solution consisting of 200g of serine, 16g of calcium chloride and 800 ml of water, adding 10ml of phospholipase D solution, reacting at 45 ℃ for 4 hours, separating out an organic phase, washing the organic phase with 1500ml of water for three times, recovering the petroleum ether in vacuum, rapidly stirring the obtained solid concentrate with 1L of absolute ethyl alcohol at room temperature for 2 hours, separating out an absolute ethyl alcohol insoluble substance, and drying the absolute ethyl alcohol insoluble substance in vacuum to obtain 67.5g of a product with the phosphatidylcholine content. Wherein the content of phosphatidylserine is 64.3%, the content of calcium is 13.72mg/g, and the content of sodium is 0.010 mg/g.
The contents of the reaction product components and the conversion in each example and comparative example are shown in Table 1 below.
TABLE 1 reaction product component contents and conversion in each example and comparative example
Figure BDA0001388498280000131
Figure BDA0001388498280000141
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims (4)

1. A method of preparing a phosphatidylserine salt, comprising:
providing a phosphatidylserine first metal salt; the first metal phosphatidylserine salt is prepared from soybean lecithin with phosphatidylcholine content of more than 20%, L-serine and calcium chloride as raw materials; wherein the mass ratio of the soybean lecithin with the content of the phosphatidylcholine being more than 20% to the L-serine is 1: 5-0.5, and the mass ratio of the soybean lecithin with the content of the phosphatidylcholine being more than 20% to the calcium chloride is 1: 2-0.1;
chemically reacting the solution of the first metal salt of phosphatidylserine with a sodium ion salt solution or a potassium ion salt solution containing a second metal ion to obtain a second metal salt of phosphatidylserine;
the mass ratio of the soybean lecithin with the content of more than 20% to the sodium and potassium ion salts is 1: 2-0.2;
wherein the method for obtaining the solution of the phosphatidylserine first metal salt comprises the following steps:
using soybean lecithin with the content of the phosphatidylcholine being more than 20%, the serine, the first metal salt and water to react to generate a first mixture containing the phosphatidylserine first metal salt;
centrifuging the first mixture of phosphatidylserine first metal salts to obtain a second mixture comprising the phosphatidylserine first metal salt;
dissolving the second mixture by using a first solvent, and adding the second solvent into the dissolved second mixture to remove impurities, thereby obtaining a solution of the phosphatidylserine first metal salt which can be dissolved in the first solvent;
the chemical reaction of the solution of the phosphatidylserine first metal salt through the solution containing the second metal ion to obtain the phosphatidylserine second metal salt comprises the following steps:
dissolving the first metal salt of the phosphatidylserine in a first solvent, and adding a second solvent to remove impurities to obtain a solution of the first metal salt of the phosphatidylserine;
adding a solution containing a second metal ion to the solution of the phosphatidylserine first metal salt to obtain the phosphatidylserine second metal salt;
the adding a solution containing a second metal ion to the solution of the phosphatidylserine first metal salt to obtain the phosphatidylserine second metal salt comprises:
adding the solution containing the second metal ions into the solution of the first metal salt of the phosphatidylserine to carry out chemical reaction, thereby obtaining a solution of the second metal salt of the phosphatidylserine;
concentrating the phosphatidylserine second metal salt solution to obtain a phosphatidylserine second metal salt concentrate;
adding ethanol to the concentrate of the second metal salt of phosphatidylserine, and filtering to obtain insoluble substances, wherein the insoluble substances are solid of the second metal salt of phosphatidylserine;
drying the phosphatidylserine second metal salt solid to obtain the phosphatidylserine salt;
the first metal phosphatidylserine salt is calcium phosphatidylserine salt;
the phosphatidylserine second metal salt is phosphatidylserine sodium salt or phosphatidylserine potassium salt;
the first solvent is petroleum ether;
the second solvent is a mixture of isopropanol and water.
2. The method of claim 1,
the sodium ion salt solution comprises one or more of a sodium sulfate solution, a sodium carbonate solution, a sodium bicarbonate solution, a sodium thiosulfate solution and a sodium acetate solution;
the potassium ion salt solution comprises one or more of potassium sulfate solution, potassium carbonate solution, potassium bicarbonate solution, potassium hydroxide solution, potassium thiosulfate solution and potassium acetate solution.
3. The method of claim 1,
the mass ratio of the substance containing phosphatidylcholine to the petroleum ether is 1: 10-1;
the mass ratio of the phosphatidylcholine-containing substance to the isopropanol is 1: 5-1.
4. The method according to claim 1, wherein the mass ratio of the phosphatidylcholine-containing substance to the ethanol is 1:15 to 10.
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CN104327114A (en) * 2014-11-06 2015-02-04 江南大学 Preparation method of phosphatidyl serine
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