Preparation method of phospholipid with enzymatic structure

Abstract

The invention provides a method for preparing phospholipid with an enzymatic structure, which comprises the following steps of: mixing a hydrophobic solvent and a hydrophilic solvent according to a certain ratio, and reasonably regulating the solubility and enzymatic reaction activity of a substrate; the phospholipids with different compositions are prepared by reacting phospholipids with fatty acids or fatty acid esters with different carbon chain lengths under the catalysis of lipase. According to the method, the problems of poor substrate solubility and low enzymatic activity of a traditional single solvent system are solved, synthesis of phospholipids with enzymatic fatty acid chain lengths from C8 to C22 can be realized by simply regulating and controlling the proportion of the hydrophobic solvent to the hydrophilic solvent, efficient preparation of the phospholipids with structures can be realized at normal temperature and normal pressure, and the method has a relatively good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biochemical engineering, and in particular relates to a method for preparing enzymatic structured phospholipids. Background Art

[0002] Structured phospholipids (SPLs) are derived from natural phospholipids through chemical structural modification, which includes the modification of fatty acid acyl groups and the replacement of polar head groups. Based on the amphiphilic properties of structured phospholipid molecules, which have both hydrophilic head groups and long hydrophobic fatty acid chains, studies have found that the bioavailability of active substances such as specific fatty acids supplemented in the form of phospholipids is higher than that in the form of triacylglycerols (TAGs). It is precisely because structured phospholipids have the potential to serve as effective carriers of bioactive compounds that they are widely used in health foods and medical health. For example, the transformation of long-chain fatty acids in natural phospholipids into medium-chain fatty acids (represented by caprylic acid C8:0 and capric acid C10:0) can obtain medium-chain structured phospholipids (MCPs), also known as low-calorie structured phospholipids, which have stronger emulsifying activity and absorption rate in the intestines, and can be used to supplement energy for patients with malabsorption syndrome. DHA is a long-chain fatty acid that accumulates in the brain and plays a key role in brain development activities such as synapse formation and neuronal differentiation. Phospholipids are one of the few lipid molecules that can cross the blood-brain barrier. The structural phospholipids formed by introducing DHA into natural phospholipid molecules are superior brain health care drugs. At the same time, the preparation of drug-structural phospholipid complexes has become a potential strategy for combating viruses (such as the new coronavirus). Studies have shown that the complex formed by quercetin (a phenolic acid drug) attached to sunflower phospholipids can improve the early symptoms of viral infection and prevent severe illness. Phosphatidylserine has been shown to be a membrane phospholipid that can be used to prevent Alzheimer's disease, but the extraction of this ingredient from animals and plants has limitations such as high cost, cumbersome operation, and low extraction rate. Its yield can be greatly increased by modifying natural phospholipids.

[0003] At present, the methods for preparing structured phospholipids are mainly divided into chemical methods and bioenzymatic methods. The chemical method is to attack the CC bond or polar head group of phospholipids through hydroxylation, acylation, hydrolysis and other reactions under the action of chemical catalysts, thereby transforming the molecular structure of phospholipids. The reaction conditions of the chemical method are harsh, often requiring high temperature, strong acid or strong alkali environment, which can easily lead to the destruction of the natural configuration of phospholipids, and the content of by-products is high, so its application in the food, medicine and cosmetics industries is very limited. The bioenzymatic method refers to the process of transforming phospholipids into hydrophobic long carbon chains or polar head groups under the catalysis of enzymes to obtain structured phospholipids. Compared with the chemical method, the reaction conditions of the bioenzymatic method are milder, which can maintain the natural molecular configuration of phospholipids and fatty acids, and the enzyme has stronger substrate specificity and catalytic site specificity, and the content of by-products is less.

[0004] At present, the main problem in the selection of solvents for enzymatic preparation of structured phospholipids is that a single solvent system is difficult to take into account the amphiphilic properties of phospholipid molecules. Because phospholipids have both non-polar fatty acid long chains and polar groups, in the common solvent n-hexane (logP = 3.5, low polarity), the polar groups will be surrounded by non-polar fatty acid long chains in the center to form an oil-in-water reverse micelle structure, which is not conducive to the binding of phospholipids and the active center of the enzyme molecule. Introducing polar solvents into the conventional hydrophobic solvent n-hexane to form a binary solvent system requires ensuring that polar molecules do not participate in the reaction of phospholipid molecules, fatty acid molecules and transition state molecules, while ensuring good substrate solubility and high enzyme reaction activity. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing enzymatic structured phospholipids.

[0006] The present invention is conceived as follows: a suitable hydrophobic solvent and a hydrophilic solvent are mixed in a certain proportion, the ratio of the two solvents is reasonably regulated, the effective dissolution of the substrate and the maintenance of the enzyme reaction activity are achieved, and the lipase catalyzes the reaction of phospholipids with fatty acids or fatty acid esters of different carbon chain lengths in a binary system to prepare structural phospholipids of different compositions. Specifically, the present invention combines polar parts such as tertiary alcohols or ketones with non-polar solvents such as n-hexane. The introduction of polar solvents not only helps dissolve the substrate, but also effectively destroys the reverse micelle structure formed by phospholipids in pure n-hexane solvents. The fatty acid acyl chain part of the phospholipid is more likely to contact the active center of the enzyme, accelerate the reaction rate, and thus effectively increase the molar ratio of new fatty acid acyl groups incorporated into phospholipids within a specific time. This process overcomes the problems of poor substrate solubility and low enzyme activity in traditional single solvent systems. The synthesis of structural phospholipids with fatty acid chain lengths from C8 to C22 can be achieved by simply regulating the ratio of hydrophobic solvents and hydrophilic solvents. The efficient preparation of structural phospholipids can be achieved at room temperature and pressure, and has good industrial application prospects.

[0007] In order to achieve the purpose of the present invention, the present invention provides a method for preparing an enzymatically structured phospholipid, in which a lipase is used to catalyze the reaction (enzymatic reaction) of phospholipid with fatty acids and / or fatty acid esters of different carbon chain lengths in the presence of a hydrophobic solvent and a hydrophilic solvent to obtain a structured phospholipid.

[0008] The hydrophobic solvent can be selected from n-hexane and / or petroleum ether and the like.

[0009] The hydrophilic solvent may be selected from at least one of acetone, butanone, tert-butanol, tert-amyl alcohol and the like.

[0010] Furthermore, the volume ratio of the hydrophobic solvent to the hydrophilic solvent is 6:1-1:1.

[0011] Furthermore, the lipase catalysis is an immobilized enzyme catalysis, and the specific method includes: adding 500-1000 enzyme activity units of immobilized lipase based on the mass of phospholipids to a mixed solution composed of a hydrophobic solvent and a hydrophilic solvent, the molar ratio of phospholipids to fatty acids and / or fatty acid esters is 1:5-1:20, and the above mixed system is reacted at a temperature of 40-60°C for 10-30 hours to obtain structured phospholipids, and the fatty acids in the structured phospholipids account for 40%-70% of the total fatty acids in the phospholipids. The reaction schematic diagram of the lipase-catalyzed reaction of phospholipids and fatty acids to prepare structured phospholipids is as follows Figure 1 The schematic diagram of the reaction of lipase-catalyzed phospholipids and fatty acid esters to prepare structured phospholipids is shown in Figure 2 shown.

[0012] Furthermore, the reaction is carried out in a single-stage or multi-stage reactor suitable for immobilized enzyme catalysis.

[0013] The lipase of the present invention includes lipases derived from yeast cells, mold cells, bacteria or other microorganisms, preferably derived from at least one of Aspergillus oryzae, Rhizomucor miehei and Candida antarctica.

[0014] Furthermore, the carbon chain length of the fatty acid and fatty acid ester is C8-C22.

[0015] Preferably, the fatty acid is a saturated fatty acid and / or unsaturated fatty acid with a carbon chain length of C8-C22, selected from at least one of polyunsaturated fatty acids such as capric acid, lauric acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, EPA, DHA, etc.

[0016] Preferably, the fatty acid ester is a methyl ester or ethyl ester of a saturated fatty acid and / or an unsaturated fatty acid with a carbon chain length of C8-C22, and is selected from at least one of methyl esters or ethyl esters of polyunsaturated fatty acids such as capric acid, lauric acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, EPA, DHA, etc.

[0017] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0018] The enzymatic structured phospholipid preparation process provided by the present invention comprises the following steps: mixing a hydrophobic solvent and a hydrophilic solvent in a certain proportion, rationally regulating the solubility of the substrate and the activity of the enzymatic reaction, and preparing structured phospholipids of different compositions by lipase-catalyzed reaction of phospholipids with fatty acids or fatty acid esters of different carbon chain lengths. The process overcomes the problems of poor substrate solubility and low enzyme activity of the traditional single solvent system, and can realize the synthesis of structured phospholipids with enzymatic fatty acid chain lengths from C8 to C22 by simply regulating the ratio of the hydrophobic solvent and the hydrophilic solvent, and can realize the efficient preparation of structured phospholipids at room temperature and pressure, and has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The schematic diagram of the reaction of preparing structured phospholipids by lipase-catalyzed reaction of phospholipids and fatty acids according to the present invention.

[0020] Figure 2 The schematic diagram of the reaction of preparing structured phospholipids by lipase-catalyzed reaction of phospholipids and fatty acid esters according to the present invention. DETAILED DESCRIPTION

[0021] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0022] The soybean lecithin and soybean lecithin used in the following examples were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0023] Example 1

[0024] 10g of soybean lecithin, 10% decanoic acid with a molar ratio of 10 to phospholipids, 1:1 volume ratio of n-hexane and tert-amyl alcohol (total volume of 20mL), and 500 standard enzyme activities of lipase from Aspergillus oryzae per unit phospholipid mass were placed in a primary or multi-stage reactor suitable for immobilized enzyme catalysis for reaction. The reaction was carried out at 40°C for 24 hours, and decanoic acid accounted for 50% of all fatty acids in the structural phospholipids.

[0025] Comparative Example 1: 10g of soybean lecithin, 10% of decanoic acid in a molar ratio of 10 to phospholipids, and lipase from Aspergillus oryzae with a standard enzyme activity of 300 per unit phospholipid mass were placed in a suitable one-stage or multi-stage reactor for reaction. The reaction was carried out at 40°C for 24 hours, and decanoic acid accounted for 15% of all fatty acids on the phospholipids.

[0026] Example 2

[0027] 10g of soybean lecithin, octanoic acid with a molar ratio of 15 to lecithin, n-hexane and tert-butanol with a volume ratio of 4:1 (total volume of 20mL), and solid lipase from Rhizomucor miehei with a standard enzyme activity of 500 per unit oil mass are placed in a primary or multi-stage reactor suitable for immobilized enzyme catalysis for reaction. The reaction is carried out at 60°C for 25 hours, and octanoic acid accounts for 55% of all fatty acids in the structural lecithin.

[0028] Example 3

[0029] 10g of soybean lecithin, oleic acid with a molar ratio of 12 to phospholipids, n-hexane and tert-butanol with a volume ratio of 2:1 (total volume of 20mL), and lipase from Candida antarctica with a standard enzyme activity of 1000 units per unit oil mass were placed in a primary or multi-stage reactor suitable for immobilized enzyme catalysis for reaction. The reaction was carried out at 55°C for 15 hours, and oleic acid on the structural phospholipids accounted for 5% of all fatty acids.

[0030] Example 4

[0031] 10g soybean lecithin, docosahexaenoic acid (DHA) with a molar ratio of 25 to lecithin, n-hexane and tert-amyl alcohol with a volume ratio of 1:1 (total volume of 20mL), and Candida antarctica lipase with a standard enzyme activity of 1500 per unit oil mass were placed in a primary or multi-stage reactor suitable for immobilized enzyme catalysis for reaction. The reaction was carried out at 45°C for 25 hours, and DHA on the structural lecithin accounted for 45% of all fatty acids.

[0032] Example 5

[0033] 10g of soybean lecithin, eicosapentaenoic acid (EPA) with a molar ratio of 5 to docosahexaenoic acid (DHA) with a molar ratio of 10 to phospholipids, petroleum ether and acetone with a volume ratio of 3:1 (total volume of 20mL), lipase from Rhizomucor miehei with a standard enzyme activity of 500 based on the unit oil mass, and lipase from Candida antarctica with a standard enzyme activity of 1000 based on the unit oil mass, are placed in a primary or multi-stage reactor suitable for immobilized enzyme catalysis for reaction. The reaction is carried out at 50°C for 15 hours, and EPA on the structural lecithin accounts for 25% of all fatty acids, and DHA on the structural lecithin accounts for 20% of all fatty acids.

[0034] Example 6

[0035] 10g of soybean lecithin, methyl octanoate with a molar ratio of 20 to phospholipids, n-hexane and tert-amyl alcohol with a volume ratio of 2:1 (total volume of 20mL), and lipase from Aspergillus oryzae with a standard enzyme activity of 1500 units per unit phospholipid mass are placed in a primary or multi-stage reactor suitable for immobilized enzyme catalysis for reaction. The reaction is carried out at 45°C for 30 hours, and octanoic acid accounts for 70% of all fatty acids on the structural phospholipids.

[0036] Comparative Example 2: 10g of soybean lecithin, methyl octanoate with a molar ratio of 20 to phospholipids, and lipase from Aspergillus oryzae with a standard enzyme activity of 1500 units per unit phospholipid mass were placed in a primary or multi-stage reactor suitable for immobilized enzyme catalysis for reaction in 20mL of n-hexane. The reaction was carried out at 45°C for 30 hours, and octanoic acid accounted for 20% of all fatty acids on the phospholipids.

[0037] Comparative Example 3: 10g of soybean lecithin, methyl octanoate with a molar ratio of 20 to phospholipids, lipase from Aspergillus oryzae with a standard enzyme activity of 1500 per unit phospholipid mass in 20mL of tert-amyl alcohol were placed in a suitable one-stage or multi-stage reactor for reaction. The reaction was carried out at 45°C for 30 hours, and octanoic acid accounted for 16% of all fatty acids on the structural phospholipids.

[0038] Example 7

[0039] 10g of soybean lecithin, methyl laurate in a molar ratio of 10 to lecithin, n-hexane and tert-butanol in a volume ratio of 6:1 (total volume of 20mL), solid lipase from Rhizomucor miehei with a standard enzyme activity of 1000 per unit oil mass, and a first-stage or multi-stage reactor suitable for immobilized enzyme catalysis are reacted at 60°C for 22 hours, and lauric acid on the structural lecithin accounts for 62% of all fatty acids.

[0040] Example 8

[0041] 10g of soybean lecithin, methyl oleate with a molar ratio of 10 to phospholipids, n-hexane and tert-butanol with a volume ratio of 2:1 (total volume of 20mL), and lipase from Candida antarctica with a standard enzyme activity of 1500 per unit oil mass are placed in a primary or multi-stage reactor suitable for immobilized enzyme catalysis for reaction. The reaction is carried out at 50°C for 20 hours, and oleic acid on the structural phospholipids accounts for 70% of all fatty acids.

[0042] Example 9

[0043] 10g soybean lecithin, ethyl docosahexaenoate with a molar ratio of 25 to phospholipids, n-hexane and acetone with a volume ratio of 1:1 (total volume of 20mL), and lipase from Candida antarctica with a standard enzyme activity of 500 per unit oil mass were placed in a primary or multi-stage reactor suitable for immobilized enzyme catalysis for reaction. The reaction was carried out at 60°C for 30 hours, and DHA on the structural lecithin accounted for 46% of all fatty acids.

[0044] Example 10

[0045] 10g of soybean lecithin, ethyl eicosapentaenoate and ethyl docosahexaenoate in a molar ratio of 5 to 5 to phospholipids, petroleum ether and acetone in a volume ratio of 1:1 (total volume of 20mL), lipase from Rhizomucor miehei with a standard enzyme activity of 1000 units per unit of oil mass and lipase from Candida antarctica with a standard enzyme activity of 500 units per unit of oil mass are placed in a primary or multi-stage reactor suitable for immobilized enzyme catalysis for reaction. After reaction at 55°C for 25 hours, EPA on the structural lecithin accounts for 35% of all fatty acids, and DHA on the structural lecithin accounts for 34% of all fatty acids.

[0046] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing an enzymatically structured phospholipid, characterized in that: In the presence of a hydrophobic solvent and a hydrophilic solvent, the phospholipids are reacted with fatty acids and / or fatty acid esters of different carbon chain lengths by lipase to obtain structured phospholipids; The hydrophobic solvent is selected from n-hexane and / or petroleum ether; The hydrophilic solvent is selected from at least one of acetone, butanone, tert-butanol and tert-amyl alcohol.

2. The method according to claim 1, characterized in that The volume ratio of the hydrophobic solvent to the hydrophilic solvent is 6:1-1:

1.

3. The method according to claim 1, characterized in that The lipase catalysis is immobilized enzyme catalysis, and the specific method includes: adding 500-1000 enzyme activity units of immobilized lipase based on the mass of phospholipids to a mixed solution composed of a hydrophobic solvent and a hydrophilic solvent, the molar ratio of phospholipids to fatty acids and / or fatty acid esters is 1:5-1:20, and reacting the above mixed system at a temperature of 40-60°C for 10-30 hours.

4. The method according to claim 3, characterized in that The reaction is carried out in one or more reactors suitable for immobilized enzyme catalysis.

5. The method according to claim 1, characterized in that The lipase includes lipase derived from yeast cells, mold cells, bacteria or other microorganisms.

6. The method according to claim 5, characterized in that The lipase is derived from at least one of Aspergillus oryzae, Rhizomucor miehei and Candida antarctica.

7. The method according to any one of claims 1 to 6, characterized in that: The carbon chain length of the fatty acid and fatty acid ester is C8-C22.

8. The method according to claim 7, characterized in that The fatty acid is a saturated fatty acid and / or unsaturated fatty acid with a carbon chain length of C8-C22, and is selected from at least one of capric acid, lauric acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, EPA, and DHA.

9. The method according to claim 7, characterized in that: The fatty acid ester is a methyl ester or ethyl ester of a saturated fatty acid and / or an unsaturated fatty acid with a carbon chain length of C8-C22, and is selected from at least one of the methyl esters or ethyl esters of capric acid, lauric acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, EPA, and DHA.

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