Method for preparing high-purity phosphatidylserine from crude phospholipid

Abstract

The invention discloses a preparation method of phosphatidylserine, which comprises the following steps: 1) hydrolyzing crude phospholipid by using broad-spectrum hydrolytic active phospholipase to generate phosphatidic acid; according to the method, the phosphatidylserine can be prepared by directly using the crude phospholipid, the process can be simplified, the production efficiency can be improved, and the production cost can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of phosphatidylserine preparation technology, specifically relating to a method for preparing phosphatidylserine by a biological enzymatic method. Background Technology

[0002] Phosphatidylserine (PS) is a phospholipid with important physiological functions, widely used to improve cognitive function, relieve anxiety, and as an adjunct therapy for neurodegenerative diseases.

[0003] Currently, the main industrial method for producing phosphatidylserine relies on the transesterification reaction of phosphatidylcholine (PC) catalyzed by phospholipase D (PLD). For example, Du Yangji's study, "Research on the Synthesis of Phosphatidylserine by Phospholipase D in a Single Aqueous Phase" (China Food Additives, 2015, Vol. 1, pp. 113-118), investigated the process of synthesizing phosphatidylserine from crude soybean phospholipids using a single aqueous phase catalysis by phospholipase D.

[0004] The existing preparation process for phosphatidylserine has the following problems.

[0005] (1) Strong dependence on raw materials. Existing technologies require the use of high-purity phosphatidylcholine as a substrate, but the extraction and purification process of phosphatidylcholine is complex and costly, which limits the large-scale production of phosphatidylserine.

[0006] (2) The process is complicated. Phosphatidylserine is synthesized by reacting high-purity phosphatidylcholine with serine under the biocatalysis of phospholipase D. The process is complicated and the yield is low.

[0007] (3) Existing phospholipases lack both broad-spectrum hydrolysis and efficient transesterification activities. In production, phospholipases with high transesterification activity (such as phospholipase D derived from Aspergillus niger) are generally used. Although some phospholipases have high transesterification efficiency for phosphatidylcholine, their hydrolytic activity for other phospholipids, such as phosphatidylinositol (PI) and phosphatidylethanolamine (PE), is extremely low. If a phospholipase with high transesterification activity is used to convert crude phospholipids, the non-phosphatidylcholine components in the crude phospholipids cannot be effectively converted into phosphatidylserine, resulting in insufficient purity of the final product (usually <80%). Among the known phospholipases, no single enzyme has been found to simultaneously possess the ability to broadly hydrolyze all phospholipids (including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, etc.) and have efficient serine transesterification activity.

[0008] In recent years, although some studies have attempted to directly convert crude phospholipids using microbial phospholipases, the limited functionality of these phospholipases has prevented the efficient conversion of all phospholipid components in crude phospholipids, thus hindering the one-step synthesis of high-purity phosphatidylserine. Using highly transesterifying phospholipases results in the underutilization of phospholipids such as phosphatidylinositol and phosphatidylethanolamine, leading to raw material waste. While broad-spectrum hydrolytic phospholipases can generate phosphatidic acid (PA), the subsequent transesterification step is inefficient, resulting in insufficient phosphatidylserine yield.

[0009] The production process of phosphatidylserine is highly dependent on raw materials, requiring the use of high-purity phosphatidylcholine. However, the separation and purification of phosphatidylcholine is energy-intensive and costly, and existing single phospholipases cannot simultaneously meet the dual requirements of broad-spectrum hydrolysis and efficient transesterification. Therefore, there is an urgent need in the field of phosphatidylserine synthesis to develop a method for preparing phosphatidylserine that can efficiently convert all phosphatidyl lipid components in crude phospholipids without purifying phosphatidylcholine. Summary of the Invention

[0010] To address the above problems, this invention provides a high-efficiency, low-cost two-step enzymatic process for preparing high-purity phosphatidylserine (PS).

[0011] This invention discloses a method for preparing high-purity phosphatidylserine from crude phospholipids. This method employs a two-step enzymatic process, utilizing the synergistic effect of two different phospholipases to sequentially complete phospholipid hydrolysis and serine esterification reactions, generating high-purity phosphatidylserine. Specifically, the first step utilizes a broad-spectrum hydrolytic phospholipase for hydrolysis, and the second step utilizes a highly transesterifying phospholipase for transesterification.

[0012] The method for preparing phosphatidylserine according to the present invention includes the following steps:

[0013] 1) Utilize broad-spectrum hydrolytic active phospholipase to hydrolyze crude phospholipids to generate phosphatidic acid;

[0014] 2) Inactivate broad-spectrum hydrolytic phospholipases;

[0015] 3) Using a highly transesterification-active phospholipase, L-serine is esterified with the phosphatidic acid generated in step 1) to generate phosphatidylserine.

[0016] Furthermore, the hydrolysis reaction of the present invention can use crude phospholipids obtained from soybeans after degreasing with acetone as raw material. A typical composition of crude phospholipids obtained from soybeans after degreasing with acetone is: 25-35% phosphatidylcholine, 20-30% phosphatidylethanolamine, 15-25% phosphatidylinositol, 10-20% phosphatidylserine, 2%-5% phosphatidic acid, and 5%-10% other phospholipids.

[0017] Furthermore, the method for preparing the crude phospholipids obtained from soybeans after acetone degreasing may include the following steps:

[0018] 1) Mix soybean oil foots, the degummed product of crude soybean oil, with acetone at a ratio of 1g:3ml, stir and heat to 45℃, and stir continuously for 10min;

[0019] 2) Centrifuge and discard the acetone supernatant;

[0020] 3) Wash the precipitate with an equal volume of acetone and collect the precipitate.

[0021] Furthermore, the hydrolysis reaction of the present invention can use phospholipase D derived from Streptomyces chromofuscus.

[0022] Furthermore, in the hydrolysis reaction system of the present invention, the crude phospholipid precipitate can be dissolved in a solution with a pH of 5.5 to 6.0, the CaCl2 concentration can be 8 to 10 mM, the enzyme addition amount can be 80 to 120 U / g substrate, the reaction temperature can be 45 to 50°C, and the reaction time can be 4 to 5 hours.

[0023] The hydrolysis reaction of the present invention has the following conversion effects: phosphatidylcholine hydrolysis rate ≥99%, phosphatidylethanolamine hydrolysis rate ≥99%, phosphatidylserine hydrolysis rate ≥98%, and phosphatidylinositol hydrolysis rate ≥97.5%.

[0024] Furthermore, after the hydrolysis reaction of the present invention is completed, the broad-spectrum hydrolytic phospholipase is inactivated.

[0025] Furthermore, the inactivation of broad-spectrum hydrolytic phospholipase can be achieved by heating to 70°C and maintaining the temperature for 15 minutes.

[0026] Furthermore, the transesterification reaction of the present invention can use lysophospholipase derived from Aspergillus niger.

[0027] Furthermore, in the transesterification reaction of the present invention, the amount of L-serine added in the reaction system can be phosphatidic acid:L-serine = 1:10 molar ratio, the amount of enzyme added can be 100-150 U / g substrate, the pH can be 5.5-6.0, the reaction temperature can be 40-45℃, and the reaction time can be 6-8 hours.

[0028] The transesterification reaction of the present invention achieves a conversion effect of phosphatidic acid conversion rate ≥95% and phosphatidylserine purity ≥85%.

[0029] The present invention also discloses phosphatidylserine prepared using the above-described preparation method.

[0030] The present invention also discloses the application of the above-mentioned phosphatidylserine in the preparation of products that improve cognitive function, relieve anxiety and neurodegenerative diseases.

[0031] Specifically, the present invention is as follows.

[0032] 1. A method for preparing phosphatidylserine, comprising the following steps:

[0033] 1) Utilize broad-spectrum hydrolytic active phospholipase to hydrolyze crude phospholipids to generate phosphatidic acid;

[0034] 2) Inactivate broad-spectrum hydrolytic phospholipases;

[0035] 3) Add highly transesterification-active phospholipase and L-serine for esterification.

[0036] 2. The method for preparing phosphatidylserine as described in item 1, characterized in that: the broad-spectrum hydrolytic active phospholipase is phospholipase D derived from Streptomyces brownii.

[0037] 3. The method for preparing phosphatidylserine as described in item 1, characterized in that: the reaction conditions in step 1) are as follows: the pH of the crude phospholipid solution is 5.5-6.0, the CaCl2 concentration is 8-10 mM, the amount of the broad-spectrum hydrolytic active phospholipase added is 80-120 U / g substrate, the reaction conditions are 45-50℃ with stirring, and the reaction time is 4-5 hours.

[0038] 4. The method for preparing phosphatidylserine as described in item 1, characterized in that: the highly transesterified phospholipase is a lysophospholipase derived from Aspergillus niger.

[0039] 5. The method for preparing phosphatidylserine as described in item 2, characterized in that: the highly transesterified phospholipase is a lysophospholipase derived from Aspergillus niger.

[0040] 6. The method for preparing phosphatidylserine as described in item 5, characterized in that: the reaction conditions in step 3) are as follows: the molar ratio of phosphatidic acid to L-serine is 1:10, the amount of enzyme added to the highly transesterified phosphatidyllipase is 100-150 U / g substrate, and then diethyl ether is added, and the reaction is carried out at 45°C with stirring for 6-8 hours.

[0041] 7. The method for preparing phosphatidylserine as described in any one of items 1 to 6, characterized in that: the crude phospholipid in step 1) is crude phospholipid obtained by degreasing soybeans with acetone.

[0042] 8. The method for preparing phosphatidylserine as described in item 7, characterized in that the method for preparing the crude phospholipid obtained by degreasing soybeans with acetone includes the following steps:

[0043] 1) Mix soybean oil foots, the degummed product of crude soybean oil, with acetone at a ratio of 1g:3ml and stir at 45℃ for 10min.

[0044] 2) Centrifuge and discard the acetone supernatant;

[0045] 3) Wash the precipitate with an equal volume of acetone and collect the precipitate.

[0046] 9. Phosphatidylserine prepared according to any one of items 1 to 8.

[0047] 10. The use of the phosphatidylserine described in item 9 in the preparation of products that improve cognitive function, relieve anxiety and neurodegenerative diseases.

[0048] The two-step enzymatic method of this invention combines highly hydrolytic phospholipases (such as Streptomyces chromofuscus phospholipase D) and highly transesterifying phospholipases (such as Aspergillus niger lysophospholipase), synergistically utilizing the broad-spectrum hydrolysis and efficient transesterification functions of the two phospholipases to prepare phosphatidylserine. It directly uses crude phospholipids as raw materials, eliminating the need for pre-purification of phosphatidylcholine, and thoroughly hydrolyzes all phospholipid components (including phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine) into phosphatidic acid, avoiding substrate waste. Efficient transesterification synthesizes phosphatidic acid into phosphatidylserine, with a phosphatidic acid conversion rate ≥95% and a phosphatidylserine product purity ≥85%.

[0049] The beneficial effects of this invention are as follows:

[0050] I. Low raw material cost. The method of this invention directly uses crude phospholipids, eliminating the phosphatidylcholine purification step, reducing production costs, simplifying the process, and improving production efficiency.

[0051] II. Simplified Process. The method of this invention achieves one-step hydrolysis and one-step transesterification through the synergistic action of two enzymes, eliminating the need for intermediate product separation. Hydrolysis and transesterification can be completed in the same reaction system, making it suitable for large-scale production of phosphatidylserine.

[0052] Third, the product has high purity. The purity of phosphatidylserine product is ≥85%, which is significantly better than the phosphatidylserine synthesis reaction that depends on a single enzyme.

[0053] The method of this invention avoids the cumbersome process of purifying phosphatidylcholine and then using phosphatidylcholine to prepare phosphatidylserine in the traditional preparation steps. It is suitable for large-scale production of phosphatidylserine, significantly reduces production costs, simplifies the process, and improves production efficiency, providing an innovative solution for the industrial production of phosphatidylserine. Attached Figure Description

[0054] Figure 1 The results are obtained from thin-layer chromatography analysis of the hydrolysis reaction products of this invention.

[0055] Figure 2 The results are obtained from the liquid chromatography detection of the hydrolysis reaction products of this invention.

[0056] Figure 3 The results show the hydrolysis rate of the hydrolysis reaction products of this invention.

[0057] Figure 4 The results are obtained from thin-layer chromatography analysis of the transesterification reaction products of this invention.

[0058] Figure 5 The results are obtained by liquid chromatography detection of the transesterification reaction products of this invention. Detailed Implementation

[0059] To better understand this invention, the following embodiments are provided in conjunction with the accompanying drawings. It should be understood that the embodiments of this invention are for illustrative purposes only and not for limiting the invention; the scope of protection of this invention is defined solely by the claims. The embodiments provided are merely preferred embodiments and are not intended to limit the invention in any way. Those skilled in the art can make changes, equivalent substitutions, or modifications based on the content of this invention to form different implementations. However, any changes and modifications, and any equivalent substitutions made to the method of this invention without departing from the inventive concept are within the scope of protection of this invention.

[0060] Example 1: Preparation of Crude Soybean Lecithin

[0061] Soybean oil residue, a degummed product of crude soybean oil, is rich in crude phospholipids but also contains some neutral lipids. First, oil removal and neutral lipid removal are necessary to preliminarily purify the soybean crude phospholipids. The soybean oil residue is mixed with acetone at a ratio of 1g:3ml, stirred, heated to 45℃, and continuously stirred for 10 minutes. The acetone supernatant is then discarded by centrifugation. The precipitate is then washed with an equal volume of acetone, collected, and the pre-oil-removed soybean crude phospholipids are obtained.

[0062] Dissolve the pre-degreased crude soybean phospholipids in 20mM acetate-sodium acetate buffer (pH=5.8), mix thoroughly, and prepare 200ml of phospholipid suspension with a concentration of 50g / L. Add CaCl2 to a final concentration of 8mM.

[0063] Example 2, Broad-spectrum hydrolysis reaction

[0064] The first step is an enzymatic reaction to carry out a broad-spectrum hydrolysis reaction. The broad-spectrum hydrolysis reaction uses phospholipase D (catalog number: BML-SE301-0025) derived from Streptomyces chromofuscus by Enzo Biochemical Company, USA.

[0065] Phospholipase D (100 U / g substrate) derived from Streptomyces chromofuscus was added to 200 ml of crude soybean phospholipids prepared in Example 1. The reaction temperature was controlled at 45°C, the stirring speed at 200 rpm, and the reaction was carried out for 5 hours.

[0066] Then heat to 70°C and maintain for 15 minutes to inactivate phospholipase D. Cool to 45°C for later use.

[0067] Example 3: Thin-layer chromatographic detection of broad-spectrum hydrolysis reaction products

[0068] Thin-layer chromatography (TLC) was performed using pre-prepared silica gel plates from Yantai Yinlong Silica Gel Co., Ltd. After the broad-spectrum hydrolysis reaction, 1 ml of the reaction solution was mixed with 1 ml of chloroform for extraction. After standing, 10 μl of the lower chloroform layer was deposited at the bottom of the TLC silica gel plate. The plate was developed for 20 minutes in a developing solvent of chloroform:methanol:acetic acid:water (V:V:V:V = 25:15:4:2). Color development was then performed in an iodine jar.

[0069] The results of thin-layer chromatography for the determination of broad-spectrum hydrolysis products of crude phospholipids are as follows: Figure 1 As shown, the first lane of the thin-layer chromatography shows the crude phospholipid before the broad-spectrum hydrolysis reaction, and the second lane shows the phosphatidic acid product after the broad-spectrum hydrolysis reaction. The results show that after the first step of the hydrolysis reaction, almost all of the phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) in the crude phospholipid were hydrolyzed into phosphatidic acid (PA).

[0070] Example 4: Liquid Chromatographic Detection of Broad-Spectrum Hydrolysis Products

[0071] After the broad-spectrum hydrolysis reaction, 1 ml of the reaction solution was mixed with 1 ml of chloroform for extraction. After standing, the lower chloroform layer was removed and dried by nitrogen blowing, and then redissolved with 200 μl of isopropanol.

[0072] Analysis was performed using an Agilent 1290 Infinity II ELSD system with evaporative light scattering detector (ELSD) from Agilent Technologies, Inc. Chromatographic separation was performed using a YMC-PackDiol-NP column (normal-phase silica-bonded diol column) from YMC Corporation, Japan (250 mm × 4.6 mm, pore size 120 Å, particle size 5 μm). The HPLC conditions were as follows: column temperature 55 °C, detector gain 50, nebulization pressure 25 psi, heated mode, and power level 60%. 20 μL of sample was injected. Mobile phase A consisted of n-hexane:isopropanol:acetic acid:triethylamine (V:V:V:V = 81.42:17:1.5:0.08), and mobile phase B consisted of isopropanol:water:acetic acid:triethylamine (V:V:V:V = 84.42:14:1.5:0.08). The flow rate was 1 mL / min. The elution program was set as follows: 0–5 min, 95%–80% A; 5–7 min, 80%–60% A; 7–17 min, 60%–30% A; 17–25 min, 30%–5% A; 25–30 min, 5%–95% A.

[0073] Figure 2 The liquid chromatography results showed that phosphatidic acid (PA) was the main hydrolysis product.

[0074] The hydrolysis rate is calculated according to the following formula 1 based on the liquid chromatography detection results.

[0075] Hydrolysis rate (%) = (1 - Remaining phospholipid peak area after reaction / Initial phospholipid peak area after reaction) × 100% (Equation 1)

[0076] The hydrolysis rate of each phospholipid was calculated based on the liquid chromatography detection results, as follows: Figure 3 As shown, the hydrolysis rates of phosphatidylcholine (PC) are ≥99%, phosphatidylethanolamine (PE) are ≥99%, phosphatidylserine (PS) are ≥98%, and phosphatidylinositol (PI) are ≥97.5%.

[0077] Example 5: Transesterification reaction

[0078] The second step involves an enzymatic transesterification reaction. The phospholipase used in the transesterification reaction is lysophospholipase (food grade) derived from Aspergillus niger from Ningxia Xiasheng Industrial Group Co., Ltd. L-serine is L-serine (product number S137887) from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0079] Phosphatidylserine (PS) was synthesized using a biphasic reaction system with phosphatidic acid (PA) and L-serine (Ser) as substrates. L-serine was added to the phosphatidic acid prepared by the broad-spectrum hydrolysis reaction in Example 2 at a ratio of PA:Ser = 1:10 (molar ratio), followed by lysophospholipase from Aspergillus niger (120 U / g substrate), and finally 200 ml of diethyl ether. The reaction was carried out at 45°C and a stirring speed of 200 rpm for 8 hours.

[0080] Example 6: Thin-layer chromatography detection of transesterification products

[0081] Thin-layer chromatography (TLC) was performed using pre-prepared silica gel plates from Yantai Yinlong Silica Gel Co., Ltd. After the transesterification reaction, 10 μl of the upper ether layer solution was directly deposited at the bottom of the TLC silica gel plate, and developed for 20 minutes in a developing solvent of chloroform:methanol:acetic acid:water (V:V:V:V = 25:15:4:2). Color development was then performed in an iodine jar.

[0082] Thin-layer chromatography detection of phospholipid transesterification results as follows Figure 4 As shown in the figure, the first lane of the thin-layer chromatography is for phosphatidic acid (PA), the product of the broad-spectrum hydrolysis reaction, and the second lane is for phosphatidylserine (PS), the product of the transesterification reaction. The results show that after the second transesterification reaction, most of the phosphatidic acid product from the first step of the broad-spectrum hydrolysis reaction is converted into phosphatidylserine.

[0083] Example 7: Liquid Chromatography Detection of Transesterification Products

[0084] After the transesterification reaction, take 1 ml of the upper ether layer solution, dry it under nitrogen, and then reconstitute it with 200 μl of isopropanol.

[0085] Analysis was performed using an Agilent 1290 Infinity II ELSD system (UHPLC-Evaporative Light Scattering Detector) from Agilent Technologies, Inc. Chromatographic separation was performed using a YMC-PackDiol-NP column (normal-phase silica-bonded diol column) (250 mm × 4.6 mm, pore size 120 Å, particle size 5 μm) from YMC Corporation, Japan. The HPLC conditions were as follows: column temperature 55 °C, detector gain 50, nebulization pressure 25 psi, heated mode, and power level 60%. The injection volume was 20 μL. Mobile phase A consisted of n-hexane:isopropanol:acetic acid:triethylamine (V:V:V:V = 81.42:17:1.5:0.08), and mobile phase B consisted of isopropanol:water:acetic acid:triethylamine (V:V:V:V = 84.42:14:1.5:0.08). The flow rate was 1 mL / min. The elution program was set as follows: 0–5 min, 95%–80% A; 5–7 min, 80%–60% A; 7–17 min, 60%–30% A; 17–25 min, 30%–5% A; 25–30 min, 5%–95% A.

[0086] Phosphatidylserine and phosphatidic acid standards were used for external standard quantification to determine the relationship between phospholipid content and peak area. The linear regression equation for the peak area of ​​phosphatidylserine and its content was y = 25.797x - 109.21, and the linear regression equation for the peak area of ​​phosphatidic acid and its content was y = 18.203x - 110.72, where y is the peak area and x is the phospholipid content.

[0087] Based on the results of liquid chromatography, the purity of phosphatidylserine (PS) and the conversion rate of phosphatidic acid (PA) were calculated according to Formulas 2 and 3 respectively.

[0088] Phosphatidylserine purity (%) = Phosphatidylserine content / Total mass of all components × 100% (Equation 2)

[0089] Phosphatidic acid conversion rate (%) = (1 - Phosphatidic acid content after reaction / Phosphatidic acid content at the beginning of reaction) × 100% (Equation 3)

[0090] The calculation results show that the phosphatidic acid conversion rate is ≥95% and the phosphatidylserine purity is ≥85%.

Claims

1. A method for preparing phosphatidylserine, comprising the following steps: 1) Utilize broad-spectrum hydrolytic active phospholipase to hydrolyze crude phospholipids to generate phosphatidic acid; 2) Inactivate broad-spectrum hydrolytic phospholipases; 3) Add highly transesterification-active phospholipase and L-serine for esterification.

2. The method for preparing phosphatidylserine according to claim 1, characterized in that: The broad-spectrum hydrolytic active phospholipase is phospholipase D derived from Streptomyces brownii.

3. The method for preparing phosphatidylserine according to claim 1, characterized in that: The reaction conditions for step 1) are as follows: the pH of the crude phospholipid solution is 5.5-6.0, the CaCl2 concentration is 8-10 mM, the enzyme addition amount of the broad-spectrum hydrolytic active phospholipid is 80-120 U / g substrate, the reaction conditions are 45-50℃ with stirring, and the reaction time is 4-5 hours.

4. The method for preparing phosphatidylserine according to claim 1, characterized in that: The highly transesterified phospholipase is a lysophospholipase derived from Aspergillus niger.

5. The method for preparing phosphatidylserine as described in claim 2, characterized in that: The highly transesterified phospholipase is a lysophospholipase derived from Aspergillus niger.

6. The method for preparing phosphatidylserine as described in claim 5, characterized in that: The reaction conditions for step 3) are as follows: the molar ratio of phosphatidic acid to L-serine is 1:10, the enzyme addition amount of the highly transesterified phospholipase is 100-150 U / g substrate, and then diethyl ether is added. The reaction is carried out at 45°C with stirring for 6-8 hours.

7. The method for preparing phosphatidylserine according to any one of claims 1 to 6, characterized in that: The crude phospholipid mentioned in step 1) is the crude phospholipid obtained by degreasing soybeans with acetone.

8. The method for preparing phosphatidylserine as described in claim 7, characterized in that, The method for preparing crude phospholipids from soybeans after acetone degreasing includes the following steps: 1) Mix soybean oil foots, the degummed product of crude soybean oil, with acetone at a ratio of 1g:3ml and stir at 45℃ for 10min. 2) Centrifuge and discard the acetone supernatant; 3) Wash the precipitate with an equal volume of acetone and collect the precipitate.

9. Phosphatidylserine prepared by the method of any one of claims 1 to 8.

10. The use of the phosphatidylserine of claim 9 in the preparation of articles for improving cognitive function, relieving anxiety and neurodegenerative diseases.

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