Phosphatidylserine oiling agent and preparation method thereof

Abstract

The preparation method comprises the following steps: dissolving phosphatidylserine powder prepared by enzyme reaction in vegetable oil, adding pure water, stirring until the system is in a reverse micelle gel state, vacuumizing, filling nitrogen, carrying out oil bath, rapidly stirring and heating until the system is boiled to obtain upper-layer clear and transparent oil, and filtering to obtain the phosphatidylserine oil. And cooling to room temperature, and performing centrifugal separation to obtain the transparent phosphatidylserine oiling agent. According to the invention, phosphatidylserine is dissolved in grease in a path of dissociating reverse micelles by a gasification method, so that the phosphatidylserine is in a clear and transparent state, the colloidal influence of related polar substances such as sterol glycoside is eliminated, the transparent phosphatidylserine oil agent is prepared, the use of toxic solvents is avoided, and the subsequent continuous purification and preparation application are facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of separation and purification of phosphatidylserine, and more particularly to a phosphatidylserine oil agent and a preparation method thereof. BACKGROUND

[0002] Phosphatidylserine (PS) is also known as serine phospholipid, diacylglycerol acylphosphoric acid serine, and is simply referred to as PS. It is usually located in the inner layer of the cell membrane and is an important component of the brain cell membrane. It plays an important role in regulating various functions of the brain, especially in stabilizing memory and mood.

[0003] The structure of phosphatidylserine is composed of two parts: a hydrophilic glycerol skeleton as a "head" and a lipophilic hydrocarbon chain group as a "tail". This unique dual structure allows phosphatidylserine to exist in both aqueous and oily environments. Phosphatidylserine belongs to the glycerophospholipid category. Among glycerophospholipids, phosphatidic acid is the simplest form, and other glycerophospholipids are named after the hydrophilic residues attached to the phosphoric acid group, including phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and phosphatidylcholine.

[0004] The production process of phosphatidylserine is converted by phospholipase D enzyme reaction from phosphatidylcholine and L-serine. The reaction products mainly include phosphatidylserine, phosphatidic acid and a small amount of other phospholipids, but there are still related substances such as steroidal compounds, sucrose esters, and higher fatty acid esters. These compounds have intermolecular interactions with phosphatidylserine, which can form a colloidal or liquid crystal state in the water phase and the oil phase, so that the phosphatidylserine product cannot be dissolved in oil or non-polar solvents. Liquid crystal state or colloidal state is an ordered arrangement state of lipid bilayer in biological membranes. In solvents, phospholipids and other polar components form nanoscale spherical and columnar structures due to van der Waals forces and electrostatic forces, forming micelles in water and reverse micelles in oil, both of which are opaque, and have unstable phenomena such as delamination, aggregation, and demulsification during storage.

[0005] The production process of phosphatidylserine dissolves phospholipid compounds and related substances in n-hexane phase to form a reverse micelle system, which is difficult to continue to purify, and is not conducive to improving the purity. At present, a few enterprises use n-hexane and water extraction process to obtain oil-soluble phosphatidylserine product, so that the product contains n-hexane residue, which has potential accumulation toxicity. SUMMARY

[0006] Therefore, the present application provides a gasification method to dissociate reverse micelles, which dissolves phosphatidylserine in oil, presents a clear and transparent state, eliminates the colloidal effect of related polar substances such as sterol glycosides, and avoids the use of toxic solvents, which is conducive to subsequent continuous purification and preparation application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: First, this invention provides a method for preparing phosphatidylserine oil, comprising the following steps: (1) Dissolve the phosphatidylserine powder obtained by enzyme reaction in vegetable oil, then add pure water and stir at room temperature until the system is in the reverse micelle gel state. (2) Vacuum the reaction system of step (1) and fill it with nitrogen. Heat it to boiling in an oil bath at 105°C with stirring. Control the process within 30 minutes to obtain the upper clear and transparent oil. Cool it to room temperature and centrifuge to obtain the transparent phosphatidylserine oil.

[0008] Preferably, the mass ratio of phosphatidylserine powder to vegetable oil in step (1) is 1:1.

[0009] Furthermore, the vegetable oil is one or more of soybean oil, corn oil, sunflower seed oil, and MCT oil.

[0010] Furthermore, the MCT oil is caprylic / capric triglyceride.

[0011] Preferably, the amount of pure water added in step (1) is 5-10% of the total mass of phosphatidylserine powder and vegetable oil.

[0012] Preferably, the stirring speed in step (2) is 60-120 rpm.

[0013] Preferably, the centrifugation speed in step (2) is >3000 rpm.

[0014] The present invention also provides an acylserine oil preparation prepared by the method described above.

[0015] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a phosphatidylserine oil and its preparation method, which has the following beneficial effects: This invention overcomes the problem of toxic residues in the preparation of phosphatidylserine oil in the prior art. It uses a vaporization method to dissociate reverse micelles, causing polar substances to dehydrate and precipitate, resulting in an oil-soluble phosphatidylserine oil free of solvent residues. This improves product purity, and the oil is completely dissolved and transparent, exhibiting good stability and facilitating formulation applications. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0017] Figure 1 This is a reaction flow diagram of the present invention.

[0018] Figure 2 This is the HPLC detection chromatogram of the experimental example before depolarization. Figure 3 This is an HPLC detection chromatogram after depolarization of substances using the method of Example 1 of this invention in the experimental case. Detailed Implementation

[0019] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1 Phosphatidylserine was prepared by heating the reaction in a pure water system with soybean phospholipids and serine as substrates and phospholipase D as catalyst for 6 hours, followed by vacuum drying.

[0021] The PS powder obtained from the above enzymatic reaction was mixed with approximately one-fold the weight of vegetable oil. Then, 5-10% pure water (w / w) was added to the mixture, and the mixture was stirred at room temperature until it reached a reverse micelle gel state. A vacuum pump was used to evacuate the system and nitrogen was then applied to maintain the pressure in the reaction vessel. The reaction vessel was heated to 105°C in an oil bath, and the mixture was stirred while maintaining the gel at a boiling state for 30 minutes. This vaporization process released the gel, and trace amounts of insoluble substances (sterol glycolipids) and other polar substances were observed to precipitate out. After removing the water, a clear, transparent upper layer of oil was obtained. The oil was cooled to room temperature and centrifuged to obtain a transparent phosphatidylserine oil.

[0022] Experimental Example Purity test of phosphatidylserine oil prepared in Example 1 The analysis was performed using an Agilent 1290 Infinity II ELSD system with high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD), under the following chromatographic conditions: a) Column: Lichrosphere 100 Diol (4 mm x 125 mm), or equivalent column; b) Mobile phase: Mobile phase A is hexane-isopropanol-acetic acid-triethylamine (820:170:10:0.8, volume ratio), and mobile phase B is isopropanol-water-acetic acid-triethylamine (850:140:10:0.8, volume ratio). c) Flow rate: 1 mL / min; d) Measurement temperature: 30℃; e) Injection volume: 10 μL; f) Evaporation temperature: 80℃; g) Atomization temperature: 50℃; h) Nitrogen flow rate: 1 L / min; i) Gradient elution, performed according to Table 1: Table 1 Gradient elution program Step Run time / min Mobile phase A / % (v / v) Mobile phase B / % (v / v) 1 0 100 0 2 20 50 50 3 25 0 100 4 29 0 100 5 30 100 0 6 35 100 0 Measurement: The standard solution and sample solution were analyzed under chromatographic conditions. A double logarithmic standard curve was plotted, with the logarithm of the peak area of ​​the standard solution as the ordinate and the logarithm of the mass concentration of the standard solution as the abscissa. The mass concentration of the sample solution was then determined from the double logarithmic standard curve based on the peak area. The content of phosphatidylserine in the sample was calculated using the following formula:

[0023] In the formula: W： The content of phosphatidylserine in the sample; p : The mass concentration of phosphatidylserine in the sample solution, expressed in micrograms per milliliter (μg / mL); V Volume of the sample solution, in milliliters (mL); m Mass of the sample, in grams (g).

[0024] The arithmetic mean of two parallel measurements was taken as the result.

[0025] To detect the changes before and after purification, two equal masses of PS powder were weighed. One was directly dissolved in vegetable oil to form a colloid, and the other was treated with gel vaporization to remove polar impurities. The purity of the two powders was compared using the liquid chromatography method described above.

[0026] The results are as follows Figures 2-3 As shown, the purity of the PS oil product increased from 22% to 23.5% after HPLC analysis to remove glycolipid impurities, indicating the removal of approximately 1.5% of impurities. Furthermore, no n-hexane was detected by HS-GC / MS analysis, while market products contain approximately 10 mg / kg of n-hexane.

[0027] Comparative Example Phosphatidylserine was prepared by heating the reaction in a pure water system with soybean phospholipids and serine as substrates and phospholipase D as catalyst for 6 hours, followed by vacuum drying.

[0028] The PS powder obtained by the above enzymatic reaction was dissolved in about 5 times the amount of n-hexane to form a gel. About 1 times the amount of lower alcohol was added to the gel mixture, stirred and mixed evenly, and allowed to stand to separate into layers. The upper oil-soluble layer and the lower water-soluble layer were separated. The lower impurities were separated, and the upper n-hexane-dissolved substance was taken. A flocculant was added to remove trace amounts of polar substances, and the mixture was filtered to obtain a clear and transparent oil. The n-hexane was removed by vacuum drying to obtain a transparent phosphatidylserine oil.

[0029] The PS oil produced by this process was tested by HS-GC / MS and found to contain approximately 10 mg / kg of n-hexane residue, which has the potential for cumulative toxicity and is not conducive to product application, especially in the export market.

[0030] The various embodiments described in this specification are presented in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a phosphatidylserine oil, characterized in that, Includes the following steps: (1) Dissolve the phosphatidylserine powder obtained by enzyme reaction in vegetable oil, then add pure water and stir at room temperature until the system is in the reverse micelle gel state. (2) Vacuum the reaction system of step (1) and fill it with nitrogen. Heat it to boiling in an oil bath at 105°C with stirring. Control the process within 30 minutes to obtain the upper clear and transparent oil. Cool it to room temperature and centrifuge to obtain the transparent phosphatidylserine oil.

2. The method for preparing a phosphatidylserine oil according to claim 1, characterized in that, In step (1), the mass ratio of phosphatidylserine powder to vegetable oil is 1:

1.

3. The method for preparing a phosphatidylserine oil according to claim 1 or 2, characterized in that, The vegetable oil is one or more of soybean oil, corn oil, sunflower seed oil, and MCT oil.

4. The method for preparing a phosphatidylserine oil according to claim 3, characterized in that, The MCT oil is caprylic / capric glyceride.

5. The method for preparing a phosphatidylserine oil according to claim 1, characterized in that, In step (1), the amount of pure water added is 5-10% of the total mass of phosphatidylserine powder and vegetable oil.

6. The method for preparing a phosphatidylserine oil according to claim 1, characterized in that, In step (2), the stirring speed is 60-120 rpm.

7. The method for preparing a phosphatidylserine oil according to claim 1, characterized in that, In step (2), the centrifugation speed is >3000 rpm.

8. An acylserine oil preparation obtained by the method according to any one of claims 1-7.

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