Method for synthesizing water-soluble phosphatidylserine

Abstract

The invention provides a method for synthesizing water-soluble phosphatidylserine, which comprises the following steps: S1, mixing and reacting a homogeneous solution of calcium-containing phosphatidylserine and water with an alkaline solution of a chelating agent to obtain a reaction solution; and S2, adding ethanol into the reaction solution, mixing, standing and splitting phases, and filtering the subnatant and the precipitate to obtain a solid, thereby obtaining the water-soluble phosphatidylserine from which the calcium ions in the phosphatidylserine are removed. The chelating agent and calcium are subjected to chelation reaction under the alkaline condition, the stability constant is large, and the calcium removal efficiency is high; the large density difference of phosphatidylserine and chelated calcium salt in an ethanol water solution system is utilized, calcium salt is removed, cross contamination is effectively avoided, the process is simple, and operation is convenient. In the whole process, except for water and ethanol, no other organic solvent is used; and along with alternate use of ethanol in the crystallization and washing processes, the impurity removal effect of ethanol is enhanced, and the content of phosphatidylserine in the product is increased.

Description

Technical Field

[0001] This invention belongs to the field of food and health product technology, and specifically relates to a method for synthesizing water-soluble phosphatidylserine. Background Technology

[0002] Phosphatidylserine (PS), also known as complex nervonic acid, is an active substance of the cell membrane, closely related to a series of cell membrane functions. It affects cell membrane fluidity and permeability, and can activate the metabolism and synthesis of various enzymes. Phosphatidylserine is particularly found in brain cells, playing an important regulatory role in brain functions such as memory and mood stability, and has significant nutritional and health benefits.

[0003] Modern lifestyles, with their changing dietary habits and increased awareness of healthy eating, have led to a reduction in the intake of phosphatidylserine-rich foods such as animal organs. Simultaneously, the natural phospholipid content in industrially produced lipids and oils has decreased, further reducing the daily recommended intake of phosphatidylserine for healthy individuals. For the elderly, particularly those with Alzheimer's disease and cognitive decline, maintaining adequate phosphatidylserine intake through supplements is crucial to ensure normal bodily functions. Therefore, phosphatidylserine supplementation is of paramount importance.

[0004] However, the poor water and oil solubility of phosphatidylserine affects its bioavailability after normal intake, limiting the development of diverse product lines. Furthermore, phosphatidylserine's stability is insufficient. For example, phosphatidylserine extracted from bovine brain denatures by approximately 0.5% daily when exposed to air at room temperature. The phosphodiester bonds at the head and the glycerol ester bonds at the tail of phosphatidylserine can be hydrolyzed and denatured, and the unsaturated fatty acids at the tail are also at risk of oxidation and denaturation. Abnormal molecular aggregation can also lead to denaturation. The poor stability of phosphatidylserine severely restricts the development of related products.

[0005] Currently, commercially available phosphatidylserine is mainly prepared by using inexpensive and readily available lecithin and L-serine as raw materials, undergoing a phosphatidyl transfer reaction under the action of phospholipase D (PLD). Because phospholipase D (PLD) typically requires calcium ions as a cofactor, sufficient calcium chloride is added during production to ensure the normal progress of the phosphatidyl transfer reaction, inevitably resulting in phosphatidylserine containing a certain amount of calcium (approximately 2.5%). US Patent 8324187 uses EDTA for decalcification, and Chinese Patent CN107722051 uses sodium sulfate or sodium carbonate for decalcification, improving product stability and water solubility. However, both processes utilize large amounts of hydrocarbon organic solvents. The former extracts PS with n-hexane after the aqueous phase reaction, while the latter first dissolves calcium phosphatidylserine in petroleum ether before treatment with sodium sulfate or sodium carbonate aqueous solution. The introduction of large amounts of organic solvents not only introduces impurities but also poses a risk of harmful substances in phosphatidylserine. Summary of the Invention

[0006] The purpose of this invention is to address the problems of low solubility, poor stability, and poor bioavailability of phosphatidylserine caused by calcium ions in calcium-containing phosphatidylserine, as well as the easy introduction of organic impurities. The invention provides a method for synthesizing water-soluble phosphatidylserine that has high calcium removal efficiency, avoids cross-contamination, and increases the content of soluble phosphatidylserine.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for synthesizing water-soluble phosphatidylserine includes the following steps:

[0009] S1. A homogenous solution of calcium-containing phosphatidylserine and water is mixed with an alkaline solution of a chelating agent to obtain a reaction solution;

[0010] S2. Add ethanol to the reaction solution, mix and let stand to separate the phases, filter the lower clear liquid and precipitate to obtain a solid, and obtain a water-soluble phosphatidylserine alkali metal salt with calcium ions removed from phosphatidylserine.

[0011] Further, in step S1, the weight ratio of calcium-containing phosphatidylserine to water in the homogenized solution is 1:0.6~1.2; preferably, the weight ratio of calcium-containing phosphatidylserine to water is 1:0.65~0.95.

[0012] Further, the mixing reaction temperature in step S1 is 5℃~50℃, and the reaction time is 0.5~5 h; preferably, the reaction temperature is 20℃~35℃, and the reaction time is 1~3 h.

[0013] Further, in step S1, the homogenized liquid and the alkaline solution of the chelating agent are mixed and reacted, and the molar ratio between phosphatidylserine and the chelating agent in the homogenized liquid is 1~5:1; preferably, the molar ratio is 2~3:1.

[0014] Further, the chelating agent in step S1 is selected from one or more of EDTA, EGTA, triacetic acid, oxalic acid, tartaric acid, malic acid, succinic acid, AKG, glutamic acid diacetic acid, citric acid, phytic acid, alginic acid, and 8-hydroxyquinoline and their corresponding alkali metal salts; preferably, the chelating agent is disodium EDTA and 8-hydroxyquinoline or disodium EDTA and citric acid, wherein the weight ratio of disodium EDTA and 8-hydroxyquinoline is 1:0.5~2.

[0015] Further, the calcium-containing phosphatidylserine in step S1 is commercially available phosphatidylserine or an intermediate in an enzymatic production process; the enzymatic production process is selected from a two-phase enzymatic production process, an organic solvent enzymatic production process, an aqueous enzymatic production process, or an immobilized enzymatic production process.

[0016] Further, in step S1, the calcium-containing phosphatidylserine contains 1% to 99% phosphatidylserine, 0.5% to 5.0% calcium, and 0.002% to 0.014% sodium; preferably, the calcium-containing phosphatidylserine contains 5% to 90% phosphatidylserine, 0.15% to 2.8% calcium, and 0.002% to 0.01% sodium.

[0017] Further, the concentration of the chelating agent in the alkaline solution in step S1 is 0.15 M to 0.25 M, preferably 0.18 M to 0.22 M, the alkaline solution is an aqueous solution of sodium hydroxide and potassium hydroxide, and the pH of the alkaline solution of the chelating agent is 8 to 13, preferably 9 to 11.

[0018] Furthermore, the solid matter described in step S2 needs to be washed and dried with ethanol. The washing process involves first washing with 50% ethanol 1-2 times, and then washing with anhydrous ethanol 1-2 times.

[0019] Further, in step S2, ethanol is added to the reaction solution, wherein the ethanol is 70%~99% ethanol, preferably 90~95% ethanol; the volume ratio of the reaction solution to ethanol is 1:1~15, and the volume ratio of the reaction solution to ethanol is 1:1~5.

[0020] Preferably, the water-soluble phosphatidylserine alkali metal salt has a phosphatidylserine content of 1%~99%, a calcium content of 0.0005%~0.005%, and a sodium content of 1.8%~5.0%; preferably, the phosphatidylserine content is 5%~90%, the calcium content is 0.0008%~0.003%, and the sodium content is 2.0%~4.0%.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) The method of synthesizing water-soluble phosphatidylserine in this invention uses a chelating agent to chelate with calcium, which has a large stability constant, high calcium removal efficiency of phosphatidylserine, and easy dissolution of phosphatidylserine.

[0023] (2) The method for synthesizing water-soluble phosphatidylserine in this invention cleverly utilizes the large density difference between phosphatidylserine and chelated calcium salt in the ethanol-water solution system after the chelation reaction is completed. The precipitated phosphatidylserine solid is suspended in the upper part of the solution, while the chelated calcium salt is precipitated in the lower part of the solution. The calcium salt can be easily removed after phase separation by releasing the lower clear liquid, effectively avoiding cross-contamination. The process is simple and easy to operate.

[0024] (2) The method for synthesizing water-soluble phosphatidylserine in this invention does not use any other organic solvents except water and ethanol; and with the alternating use of ethanol during crystallization and washing, the impurity removal effect of ethanol is enhanced, and the content of phosphatidylserine in the product is increased. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the solubility experiment of water-soluble phosphatidylserine.

[0026] Figure 1 In the figures, a and b represent the solubility of commercially available PS from Comparative Example 1 and PS from Example 1 in water after 1 min and 1 h of standing, respectively; c and d represent the solubility of commercially available PS from Comparative Example 1 and PS from Example 1 in oil after 2 h and 1 d of standing, respectively. The test tube on the left contains commercially available PS, and the test tube on the right contains PS from Example 1.

[0027] Figure 2 Results of efficacy tests for improving memory / anti-Alzheimer's disease (AD) in zebrafish experiments;

[0028] Figure 2 In the figure, a and b represent the activity of zebrafish in the MoleQ® PS and BioPS® relative blank groups and model groups, respectively.

[0029] Figure 3 Bar chart showing the efficacy of zebrafish experiments in improving memory / anti-Alzheimer's disease (AD). Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] Example 1

[0032] This embodiment provides a method for synthesizing water-soluble phosphatidylserine, comprising the following steps:

[0033] S1. A homogenous solution of calcium-containing phosphatidylserine and water is mixed with an alkaline solution of a chelating agent to obtain a reaction solution;

[0034] S2. Add ethanol to the reaction solution, mix and let stand to separate the phases, filter the lower clear liquid and precipitate to obtain a solid, and obtain water-soluble phosphatidylserine with calcium ions removed from phosphatidylserine.

[0035] The specific steps are as follows:

[0036] (1) Weigh 50 kg of sunflower seeds PS70 (containing 2.3% calcium and 0.0012% sodium) and 50 kg of purified water, and homogenize them in a homogenizer for 15 min until fully emulsified and free of lumps of solid, thus forming a homogenized liquid;

[0037] (2) Weigh 340 kg of purified water and put it into a high-level kettle. Add 1.8 kg of sodium hydroxide and 22.5 kg of chelating agent (11.25 kg of disodium EDTA and 11.25 kg of 8-hydroxyquinoline) in sequence. Stir thoroughly until completely dissolved to form an alkaline solution of the chelating agent.

[0038] (3) Transfer the two liquids (1) and (2) into the reaction vessel, heat to 35°C, stir for 4 h and then stop the reaction to become the reaction solution;

[0039] (4) Add 500 L of 95% ethanol to the reaction liquid in the reactor, stir rapidly for 15 min, let stand for phase separation, release the lower clear liquid, filter, and obtain 48.8 kg of solid.

[0040] (5) The solid obtained in (4) was washed once with 50% ethanol and once with 95% ethanol, and after being thoroughly dried, it was dried under vacuum to obtain 47.6 kg of water-soluble phosphatidylserine (containing 0.0021% calcium and 2.8% sodium).

[0041] Example 2

[0042] This embodiment provides a method for synthesizing water-soluble phosphatidylserine, comprising the following steps:

[0043] S1. A homogenous solution of calcium-containing phosphatidylserine and water is mixed with an alkaline solution of a chelating agent to obtain a reaction solution;

[0044] S2. Add ethanol to the reaction solution, mix and let stand to separate the phases, filter the lower clear liquid and precipitate to obtain a solid, and obtain a water-soluble phosphatidylserine alkali metal salt with calcium ions removed from phosphatidylserine.

[0045] The specific steps are as follows:

[0046] (1) Weigh 50 kg of soybean PS50 (containing 2.8% calcium and 0.0011% sodium) and 50 kg of purified water, and homogenize them in a homogenizer for 15 min until fully emulsified and free of lumps of solid, thus forming a homogenized liquid;

[0047] (2) Weigh 340 kg of purified water and put it into a high-level kettle. Add 4.6 kg of sodium hydroxide and 7.5 kg of chelating agent (2.5 kg of disodium EDTA and 5 kg of 8-hydroxyquinoline) in sequence. Stir thoroughly until completely dissolved to form a chelating agent solution.

[0048] (3) Transfer the two liquids (1) and (2) into the reaction vessel, heat to 35°C, stir for 4 hours and then stop the reaction to obtain the reaction solution;

[0049] (4) Add 500 L of 95% ethanol to the reaction liquid in the reactor, stir rapidly for 15 min, let stand for phase separation, release the lower clear liquid, filter, and obtain 46.7 kg of solid.

[0050] (5) The solid obtained in (4) was washed once with 50% ethanol and once with 95% ethanol, and after being thoroughly dried, it was dried under vacuum to obtain 45.6 kg of water-soluble phosphatidylserine (containing 0.0022% calcium and 3.8% sodium).

[0051] Example 3

[0052] This embodiment provides a method for synthesizing water-soluble phosphatidylserine, comprising the following steps:

[0053] S1. A homogenous solution of calcium-containing phosphatidylserine and water is mixed with an alkaline solution of a chelating agent to obtain a reaction solution;

[0054] S2. Add ethanol to the reaction solution, mix and let stand to separate the phases, filter the lower clear liquid and precipitate to obtain a solid, and obtain a water-soluble phosphatidylserine alkali metal salt with calcium ions removed from phosphatidylserine.

[0055] The specific steps are as follows:

[0056] Homemade soybean PS: Take 100 kg of soybean PC (67.42%) and fully emulsify it in 600 kg of 0.2 M acetic acid-sodium acetate aqueous solution. Then add 45 kg of L-serine, 7 kg of phospholipase D, and 15 kg of calcium chloride in sequence. After conversion at 50℃ for 2 h, filter the mixture, wash it once with 800 kg of water, and wash it once with 300 kg of 95% ethanol. Dry the mixture to obtain 59.6 kg of soybean PS (PS content 71.9%, calcium content 2.5%, sodium content 0.0013%).

[0057] The obtained PS was subjected to subsequent operations as described in Example 1 (1)-(5) to obtain 56.2 kg of water-soluble phosphatidylserine (containing 0.0018% calcium and 3.2% sodium).

[0058] Examples 4-6

[0059] This embodiment provides a method for synthesizing water-soluble phosphatidylserine. The preparation method is the same as in Example 1, except that the amount of PS remains the same. The only difference is that the mass ratio of PS to water is 1:3, 1:7 and 1:14, respectively. The phosphatidylserine obtained is the same as in Examples 4, 5 and 6.

[0060] Examples 7-11

[0061] This embodiment provides a method for synthesizing water-soluble phosphatidylserine. The preparation method is the same as in Example 1, except that the mass ratio of chelating agent and PS is 1:10, 1:5, 1:3, 2:3, and 7:10 respectively (the amount of PS remains unchanged). The phosphatidylserine obtained is the same as in Examples 7-11.

[0062] Example 12

[0063] This embodiment provides a method for synthesizing water-soluble phosphatidylserine. The preparation method is the same as in Example 1, except that the reaction temperature is 45°C, and the phosphatidylserine obtained is obtained from this method.

[0064] Example 13

[0065] This embodiment provides a method for synthesizing water-soluble phosphatidylserine. The preparation method is the same as in Example 1, except that the reaction temperature is 15°C, and the phosphatidylserine obtained is obtained from this method.

[0066] Example 14

[0067] This embodiment provides a method for synthesizing water-soluble phosphatidylserine. The preparation method is the same as in Example 1, except that a mixture of purified water and dichloromethane in a 1:1 ratio is used instead of purified water. The total volume of the liquid reaction system remains unchanged, and other steps remain unchanged. The phosphatidylserine obtained is then obtained.

[0068] Example 15

[0069] This embodiment provides a method for synthesizing water-soluble phosphatidylserine. The preparation method is the same as in Example 1, except that a mixture of purified water and acetone in a 1:1 ratio is used instead of purified water. The total volume of the liquid reaction system remains unchanged, and other steps remain unchanged. The phosphatidylserine obtained is then prepared.

[0070] Example 16

[0071] This embodiment provides a method for synthesizing water-soluble phosphatidylserine. The preparation method is the same as in Example 1, except that a mixture of purified water and ethyl acetate in a 1:1 ratio is used instead of purified water. The total volume of the liquid reaction system remains unchanged, and other steps remain unchanged. The phosphatidylserine obtained is then obtained.

[0072] Example 17

[0073] This embodiment provides a method for synthesizing water-soluble phosphatidylserine. The preparation method is the same as in Example 1, except that a mixture of purified water and chloroform in a 1:1 ratio is used instead of purified water. The total volume of the liquid reaction system remains unchanged, and other steps remain unchanged. The phosphatidylserine obtained is then obtained.

[0074] Example 18

[0075] This embodiment provides a method for synthesizing water-soluble phosphatidylserine. The preparation method is the same as in Example 1, except that the ratio of disodium EDTA to 8-hydroxyquinoline is 1:2 (7.5 kg of disodium EDTA and 15 kg of 8-hydroxyquinoline). All other steps remain unchanged, and the resulting phosphatidylserine is obtained.

[0076] Example 19

[0077] This embodiment provides a method for synthesizing water-soluble phosphatidylserine. The preparation method is the same as in Example 1, except that the ratio of disodium EDTA to 8-hydroxyquinoline is 2:1 (15 kg of disodium EDTA and 7.5 kg of 8-hydroxyquinoline). All other steps remain unchanged, and the resulting phosphatidylserine is obtained.

[0078] Example 20

[0079] This embodiment provides a method for synthesizing water-soluble phosphatidylserine. The preparation method is the same as in Example 1, except that 8-hydroxyquinoline is replaced with an equal amount of citric acid, while other steps remain unchanged. The resulting phosphatidylserine is obtained from this method.

[0080] Example 21

[0081] This embodiment provides a method for synthesizing water-soluble phosphatidylserine. The preparation method is the same as in Example 1, except that only 22.5 kg of disodium EDTA, the same chelating agent as in Example 1, is used. All other steps remain unchanged, and the resulting phosphatidylserine is obtained.

[0082] Example 22

[0083] This embodiment provides a method for synthesizing water-soluble phosphatidylserine. The preparation method is the same as in Example 1, except that only 22.5 kg of 8-hydroxyquinoline, the same amount as in Example 1, is used. All other steps remain unchanged, and the resulting phosphatidylserine is obtained.

[0084] Comparative Example 1

[0085] Commercially available sunflower seeds PS70 are processed without any treatment and are used as phosphatidylserine.

[0086] Comparative Example 2

[0087] Commercially available soybean PS50 is used as phosphatidylserine without any processing.

[0088] Test Example 1

[0089] 1. Testing Method

[0090] The calcium and sodium ion contents of calcium-containing phosphatidylserine in Examples 1-24 and Comparative Examples 1-2 were tested after treatment, respectively. The test method was based on the National Food Safety Standard GB5009.268—2025, Determination of Multiple Elements in Food, to determine the calcium and sodium ion contents. PS yield is the mass ratio of product to PS in the feed, and PS content is determined by HPLC according to (USP). <621> (CP2020) <0512> The data obtained from the test represents the mass percentage of PS in all components of the product.

[0091] 2. Test Results

[0092] As shown in Table 1, the PS yield of the methods in Examples 1-24 ranged from 49.8% to 85.9%, with calcium ion content ranging from 2.6300% to 0.0018% and sodium ion content ranging from 2.61% to 3.95%. Comparisons between various embodiments and comparative examples show that specific chelating agent types and ratios yield better results. When the weight ratio of disodium EDTA to 8-hydroxyquinoline is 1:0.5~2, the reduction in calcium ions is more significant compared to other types of chelating agents. Reaction temperatures of 15℃ or 45℃ are less effective than those at 25℃. Using pure water is more effective or comparable to using a mixture of pure water and organic solvents without introducing impurities. The mass ratio of chelating agent to PS is 1:1~1:5; preferably, a molar ratio of 1:2.0~1:3.0 is better, but exceeding this range results in relatively poor calcium ion removal. The PS to water ratio is 1:0.6~1:1.2; preferably, a range of 1:0.65~1:0.95 yields better calcium ion and PS recovery.

[0093] Table 1. Calcium and sodium ion content and PS yield in Examples 1-24

[0094] Test Example 2: Solubility Test of Water and Oil-soluble Phosphatidylserine

[0095] 1. Testing Method

[0096] (1) Solubility test in water

[0097] Take 0.1 g each of calcium-containing phosphatidylserine (PS-Ca) from Comparative Example 1 and water-soluble phosphatidylserine (PS-Na) obtained according to Examples 1-3 of this invention, and put them into a test tube containing 100 ml of water. After stirring for 1 min, observe whether there is turbidity or suspended matter; observe again after standing for 1 h to see if there is any change.

[0098] (2) Solubility test in oil

[0099] Take 5 g each of calcium-containing phosphatidylserine (PS-Ca) from Comparative Example 1 and water-soluble phosphatidylserine (PS-Na) obtained according to Examples 1-3 of this invention, and place them in test tubes containing 100 ml of MCT oil. Observe the liquid state after stirring for 2 h and after standing for 1 d.

[0100] 2. Test Results

[0101] (1) such as Figure 1 a and Figure 1As shown in b, a large amount of white flocculent matter floated in the liquid of the comparative PS-Ca test tube, while the liquid of the PS-Na test tube in Example 1 was slightly opaque but had no floating matter. After standing for 1 h, the upper part of the PS-Ca test tube was clear and transparent while the bottom had a large amount of white precipitate, while the liquid of the PS-Na test tube had neither floating matter nor precipitate and was basically transparent. The results of Examples 2 and 3 were the same as those of Example 1.

[0102] (2) such as Figure 1 c and Figure 1 As shown in d, after stirring for 2 h, the PS-Ca test tube of Comparative Example 1 was observed to be an emulsion, while the PS-Na test tube of Example 1 was a clear liquid. After standing for 1 day, the upper part of the PS-Ca test tube was clear and transparent, while a large amount of white precipitate was found at the bottom. The PS-Na test tube remained a clear liquid. The results of Examples 2 and 3 were the same as those of Example 1. The above results indicate that the solubility of water-soluble PS in water and MCT oil has been significantly improved.

[0103] Test Example 3: Zebrafish Experiment with Water-Soluble Phosphatidylserine Improving Memory / Anti-Alzheimer's Disease (AD)

[0104] 1. Testing Method

[0105] (1) Test procedure: Zebrafish experiments were conducted using water-soluble PS70 (abbreviated as MoleQ® PS) obtained in Example 1 and PS70 (abbreviated as BioPS®) of Comparative Example 1 as test samples. The zebrafish experiments used a model of memory decline and slow movement induced by aluminum trichloride. The specific method was as follows: 24 five-day-old zebrafish embryos (1 embryo per well) were exposed to 50 µM aluminum trichloride and 0.07 g / L sample solution for 48 h in 96-well plates, which were the sample test groups (MoleQ® PS and BioPS®, respectively), with 24 animals in each group; at the same time, a model control group (24 animals in each group) and a blank control group (24 animals in each group) were set up. After acclimatizing to the DanioVision zebrafish behavior tracking system for 15 minutes, the activity of each fish was recorded using EthoVision XT infrared tracking software for three cycles, including 5 minutes of light and 5 minutes of darkness. The total activity during the dark cycle was then statistically analyzed.

[0106] (2) Experimental Principle: Zebrafish exhibit neural regulation similar to that of humans, possessing the main excitatory neurotransmitter glutamate and the inhibitory neurotransmitters GABA and muscarinic choline receptors. Therefore, zebrafish have been widely used in the research and screening of drugs that inhibit or promote neuroactive activity. Excessive aluminum intake can lead to memory decline and slowed movement in animals. An aluminum chloride-induced memory improvement model was used. By measuring the activity level of zebrafish, the changes in activity level between the test substance treatment group and the model control group were compared, and the activity promotion rate was calculated to evaluate the auxiliary memory improvement effect of the raw material or product.

[0107] 2. Test Results

[0108] Based on Table 1 Figure 2 and 3 It was found that, compared to the blank control, both samples significantly improved the activity of zebrafish under both dark and light conditions, and alleviated the memory decline / dementia symptoms induced by aluminum chloride, demonstrating significant efficacy in improving memory and combating Alzheimer's disease. Among them, the MoleQ® PS sample was significantly more effective than the BioPS® sample in improving memory and combating Alzheimer's disease.

[0109] Table 2. Efficacy of PS in Improving Memory and Fighting Alzheimer's Disease in Example 1 and Comparative Example 1.

[0110] Note: [1] indicates that the test concentration is 10 times the daily intake based on an intake of 461.54 mg per day for a body weight of 70 kg; [2] indicates that the test concentration is 10 times the daily intake based on an intake of 500 mg per day for a body weight of 70 kg.

[0111] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for synthesizing water-soluble phosphatidylserine, characterized in that, Includes the following steps: S1. A homogenous solution of calcium-containing phosphatidylserine and water is mixed with an alkaline solution of a chelating agent to obtain a reaction solution; S2. Add ethanol to the reaction solution, mix and let stand to separate the phases, filter the lower clear liquid and precipitate to obtain a solid, and obtain water-soluble phosphatidylserine with calcium ions removed from phosphatidylserine.

2. The method according to claim 1, characterized in that, In step S1, the weight ratio of calcium-containing phosphatidylserine to water in the homogenized solution is 1:0.6~1.2; preferably, the weight ratio of calcium-containing phosphatidylserine to water is 1:0.65~0.

95.

3. The method according to claim 1, characterized in that, The mixing reaction temperature in step S1 is 5℃~50℃, and the reaction time is 0.5~5 h; preferably, the reaction temperature is 20℃~35℃, and the reaction time is 1~3 h.

4. The method according to claim 1, characterized in that, In step S1, the homogenized liquid and the alkaline solution of the chelating agent are mixed and reacted. The molar ratio between phosphatidylserine and the chelating agent in the homogenized liquid is 1~5:1; preferably, the molar ratio is 2~3:

1.

5. The method according to claim 1, characterized in that, The chelating agent in step S1 is selected from one or more of EDTA, EGTA, triacetic acid, oxalic acid, tartaric acid, malic acid, succinic acid, AKG, glutamic acid diacetic acid, citric acid, phytic acid, alginic acid, and 8-hydroxyquinoline and their corresponding alkali metal salts; preferably, the chelating agent is disodium EDTA and 8-hydroxyquinoline or disodium EDTA and citric acid, wherein the weight ratio of disodium EDTA and 8-hydroxyquinoline is 1:0.5~2.

6. The method according to claim 1, characterized in that, The calcium-containing phosphatidylserine mentioned in step S1 is commercially available phosphatidylserine or an intermediate produced by an enzymatic process; the enzymatic process is selected from a two-phase system enzymatic process, an organic solvent system enzymatic process, an aqueous system enzymatic process, or an immobilized enzyme process.

7. The method according to claim 1, characterized in that, The calcium-containing phosphatidylserine in step S1 has a phosphatidylserine content of 1% to 99%, a calcium content of 0.5% to 5.0%, and a sodium content of 0.002% to 0.014%; preferably, the calcium-containing phosphatidylserine has a phosphatidylserine content of 5% to 90%, a calcium content of 0.15% to 2.8%, and a sodium content of 0.002% to 0.01%.

8. The method according to claim 1, characterized in that, The concentration of the chelating agent in the alkaline solution in step S1 is 0.15 M to 0.25 M, preferably 0.18 M to 0.22 M. The alkaline solution is an aqueous solution of sodium hydroxide and potassium hydroxide. The pH of the alkaline solution of the chelating agent is 8 to 13, preferably 9 to 11.

9. The method according to claim 1, characterized in that, The solid matter described in step S2 also needs to be washed and dried with ethanol. The washing process involves first washing with 50% ethanol 1-2 times, and then washing with anhydrous ethanol 1-2 times.

10. The method according to claim 1, characterized in that, In step S2, ethanol is added to the reaction solution. The ethanol is 70% to 99% ethanol, preferably 90% to 95% ethanol. The volume ratio of the reaction solution to ethanol is 1:1 to 15, or 1:1 to 5.

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