Process for the preparation of enzymatically structured phospholipids

Abstract

This invention provides a method for preparing enzymatically catalyzed structural phospholipids. The method involves mixing a hydrophobic solvent and a hydrophilic solvent in a specific ratio, rationally controlling the solubility of the substrate and the enzymatic activity, and using lipase to catalyze the reaction of phospholipids with fatty acids or fatty acid esters of different carbon chain lengths to prepare structural phospholipids of different compositions. This method overcomes the problems of poor substrate solubility and low enzyme activity in traditional single-solvent systems. By simply adjusting the ratio of hydrophobic and hydrophilic solvents, the enzymatic synthesis of phospholipids with fatty acid chain lengths ranging from C8 to C22 can be achieved. The method can be efficiently prepared at room temperature and pressure, showing promising prospects for industrial applications.

Description

Technical Field

[0001] This invention belongs to the field of biochemical engineering, and specifically relates to a method for preparing an enzymatically catalyzed phospholipid. Background Technology

[0002] Structured phospholipids (SPLs) are derived from natural phospholipids through chemical structural modifications, including changes to fatty acid acyl groups and the replacement of polar head groups. Based on the amphiphilic nature of structured phospholipid molecules, which possess both hydrophilic head groups and long hydrophobic fatty acid chains, studies have found that the bioavailability of specific fatty acids and other active substances is higher in phospholipid form than in triacylglycerol (TAG) form. Because of their potential as effective carriers of bioactive compounds, structured phospholipids are widely used in health foods and medical fields. For example, modifying the long-chain fatty acids in natural phospholipids into medium-chain fatty acids (represented by caprylic acid C8:0 and capric acid C10:0) yields medium-chain structured phospholipids (MCPs), also known as low-calorie structured phospholipids, which exhibit stronger emulsifying activity and higher absorption rates in the intestines, making them suitable for supplementing energy in patients with malabsorption syndrome. DHA is a long-chain fatty acid that accumulates in the brain and plays a crucial role in brain development activities such as synapse formation and neuronal differentiation. Phospholipids are among the few lipid molecules capable of crossing the blood-brain barrier. Introducing DHA into natural phospholipid molecules to form structured phospholipids presents a superior brain health product. Meanwhile, the preparation of drug-structured phospholipid complexes has become a potential strategy for combating viruses (such as COVID-19). Studies have shown that a complex formed by linking quercetin (a phenolic acid drug) to sunflower phospholipids can improve 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 extracting this component from plants and animals has limitations such as high cost, cumbersome procedures, and low extraction rates. Modifying natural phospholipids can significantly increase their yield.

[0003] Currently, the main methods for preparing structured phospholipids are divided into chemical methods and bioenzymatic methods. Chemical methods involve hydroxylation, acylation, and hydrolysis under the action of chemical catalysts to attack the C / C bonds or polar head groups of phospholipids, thereby modifying the phospholipid molecular structure. Chemical methods require harsh reaction conditions, often involving high temperatures, strong acids, or strong alkalis, which can easily destroy the native configuration of phospholipids and result in high byproduct content, thus severely limiting their application in the food, pharmaceutical, and cosmetic industries. Bioenzymatic methods refer to the process of modifying hydrophobic long carbon chains or polar head groups of phospholipids under the catalysis of enzymes to obtain structured phospholipids. Compared to chemical methods, bioenzymatic methods offer milder reaction conditions, maintain the native molecular configuration of phospholipids and fatty acids, and exhibit stronger substrate specificity and catalytic site specificity, resulting in fewer byproducts.

[0004] Currently, the main problem in solvent selection for the enzymatic preparation of structural phospholipids is that a single solvent system cannot adequately account for the amphiphilic properties of phospholipid molecules. This is because phospholipids possess both nonpolar fatty acid chains and polar groups. In a common solvent like n-hexane (logP = 3.5, relatively low polarity), the polar groups are surrounded by the nonpolar fatty acid chains, forming a water-in-oil reverse micelle structure. This is detrimental to the binding of phospholipids to the active site of enzyme molecules. Introducing a polar solvent into the conventional hydrophobic solvent n-hexane to form a binary solvent system requires ensuring that the polar molecules do not participate in the reactions of phospholipid molecules, fatty acid molecules, and transition state molecules, while simultaneously ensuring good substrate solubility and high enzyme activity. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing enzymatically catalyzed structural phospholipids.

[0006] The concept of this invention is as follows: By mixing a suitable hydrophobic solvent and a hydrophilic solvent in a certain proportion and rationally controlling the ratio of the two solvents, effective dissolution of the substrate and maintenance of enzyme activity can be achieved. In a binary system, lipase catalyzes the reaction of phospholipids with fatty acids or fatty acid esters of different carbon chain lengths to prepare structural phospholipids with different compositions. Specifically, this invention combines polar moieties such as tertiary alcohols or ketones with nonpolar solvents such as n-hexane. The introduction of polar solvents not only helps the dissolution of the substrate but also effectively destroys the reverse micelle structure formed by phospholipids in pure n-hexane solvent. The fatty acid acyl chain moieties of phospholipids can more easily contact the active site of the enzyme, accelerating the reaction rate. Thus, within a specific time, the molar ratio of new fatty acid acyl groups incorporated into the phospholipid can be effectively increased. This process overcomes the problems of poor substrate solubility and low enzyme activity in traditional single-solvent systems. By simply controlling the ratio of hydrophobic and hydrophilic solvents, the synthesis of structural phospholipids with fatty acid chain lengths from C8 to C22 can be achieved. The efficient preparation of structural phospholipids can be realized at room temperature and pressure, showing good prospects for industrial applications.

[0007] To achieve the objective of this invention, this invention provides a method for preparing enzymatically catalyzed structural phospholipids. Under the presence of hydrophobic and hydrophilic solvents, phospholipids are reacted with fatty acids and / or fatty acid esters of different carbon chain lengths by lipase catalysis (enzymatic reaction) to obtain structural phospholipids.

[0008] The hydrophobic solvent may be selected from n-hexane and / or petroleum ether, etc.

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

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

[0011] Furthermore, the lipase catalysis is immobilized enzyme catalysis. Specifically, the method includes: adding an immobilized lipase of 500-1000 enzyme activity units based on the phospholipid mass to a mixture of a hydrophobic solvent and a hydrophilic solvent; the molar ratio of phospholipid to fatty acids and / or fatty acid esters is 1:5-1:20; reacting the mixture at 40-60°C for 10-30 hours to obtain a structured phospholipid, wherein the fatty acids in the structured phospholipid account for 40%-70% of the total fatty acids in the phospholipid. A schematic diagram of the reaction of lipase catalyzing the reaction of phospholipids and fatty acids to prepare structured phospholipids is shown below. Figure 1 As shown in the diagram. A schematic diagram of the reaction catalyzed by lipase to prepare structural phospholipids from phospholipids and fatty acid esters is shown in the diagram. Figure 2 As shown.

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

[0013] The lipases of the present invention include 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 an 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, and DHA.

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

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

[0018] This invention provides an enzymatic process for preparing structural phospholipids. This process involves mixing a hydrophobic solvent and a hydrophilic solvent in a specific ratio, rationally controlling the solubility of the substrate and the enzymatic activity, and using lipase to catalyze the reaction of phospholipids with fatty acids or fatty acid esters of different carbon chain lengths to prepare structural phospholipids of various compositions. This process overcomes the problems of poor substrate solubility and low enzyme activity in traditional single-solvent systems. By simply adjusting the ratio of hydrophobic and hydrophilic solvents, the enzymatic synthesis of phospholipids with fatty acid chain lengths ranging from C8 to C22 can be achieved. The process can be efficiently prepared at room temperature and pressure, showing promising prospects for industrial applications. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the reaction of lipase catalyzing the reaction of phospholipids and fatty acids to prepare structural phospholipids according to the present invention.

[0020] Figure 2 This is a schematic diagram of the reaction of lipase catalyzing the reaction of phospholipids and fatty acid esters to prepare structural phospholipids according to the present invention. Detailed Implementation

[0021] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the 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 phospholipids used in the following examples were purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.

[0023] Example 1

[0024] 10g of soybean lecithin, 10g of caprylic acid (phospholipid molar ratio 10), and 20mL of n-hexane and tert-amyl alcohol (volume ratio 1:1) were reacted with a lipase derived from *Aspergillus oryzae* (based on 500 standard enzyme activities per unit mass of phospholipid) in a single-stage or multi-stage reactor suitable for immobilized enzyme catalysis. The reaction was carried out at 40°C for 24 hours. The resulting phospholipid contained 50% caprylic acid of all fatty acids.

[0025] Comparative Example 1: 10g of soybean lecithin and caprylic acid (phospholipid molar ratio 10) were reacted in 20mL of n-hexane with lipase derived from *Aspergillus oryzae*, containing 300 standard enzyme activities per unit mass of phospholipid. The reaction was carried out in a single-stage or multi-stage reactor at 40°C for 24 hours. The caprylic acid on the phospholipid accounted for 15% of all fatty acids.

[0026] Example 2

[0027] 10g of soybean lecithin, along with octanoic acid (at a molar ratio of 15 to 15), and n-hexane and tert-butanol (volume ratio of 4:1, total volume 20mL), were reacted with a solid lipase derived from Rhizomucor miehei, with 500 standard enzyme activities per unit oil mass. The reaction was carried out in a single-stage or multi-stage reactor suitable for immobilized enzyme catalysis. The reaction was carried out at 60℃ for 25 hours, resulting in octanoic acid comprising 55% of all fatty acids in the structural lecithin.

[0028] Example 3

[0029] 10g of soybean lecithin, oleic acid (phospholipid molar ratio 12), and n-hexane and tert-butanol (volume ratio 2:1, total volume 20mL) were reacted with a Candida antarctica lipase containing 1000 standard enzyme activities per unit oil mass in a single-stage or multi-stage reactor suitable for immobilized enzyme catalysis. The reaction was carried out at 55℃ for 15 hours, resulting in oleic acid comprising 5% of all fatty acids on the structural phospholipid.

[0030] Example 4

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

[0032] Example 5

[0033] 10g of soybean lecithin, along with eicosapentaenoic acid (EPA) in a molar ratio of 5 and docosahexaenoic acid (DHA) in a molar ratio of 10, and petroleum ether and acetone in a volume ratio of 3:1 (total volume 20mL), were reacted with lipases derived from Rhizomucor miehei (500 standard enzyme activities per unit oil mass) and Candida antarctica (1000 standard enzyme activities per unit oil mass). The reaction was carried out in a single-stage or multi-stage reactor suitable for immobilized enzyme catalysis. The reaction was carried out at 50°C for 15 hours. EPA accounted for 25% of all fatty acids on the structured phospholipids, and DHA accounted for 20% of all fatty acids.

[0034] Example 6

[0035] 10g of soybean lecithin, methyl octanoate (phospholipid molar ratio 20), and n-hexane and tert-amyl alcohol (volume ratio 2:1, total volume 20mL) were reacted with a lipase derived from *Aspergillus oryzae*, containing 1500 standard enzyme activities per unit mass of phospholipid, in a single-stage or multi-stage reactor suitable for immobilized enzyme catalysis. The reaction was carried out at 45°C for 30 hours, resulting in octanoic acid comprising 70% of all fatty acids on the structural phospholipid.

[0036] Comparative Example 2: 10g of soybean lecithin and methyl octanoate (phospholipid molar ratio 20) were reacted in 20mL of n-hexane with a lipase derived from *Aspergillus oryzae*, containing 1500 standard enzyme activities per unit mass of phospholipid. The reaction was carried out in a single-stage or multi-stage reactor suitable for immobilized enzyme catalysis. The reaction was carried out at 45°C for 30 hours, resulting in octanoic acid comprising 20% ​​of all fatty acids on the phospholipid.

[0037] Comparative Example 3: 10g of soybean lecithin and methyl octanoate (phospholipid molar ratio 20) were reacted in 20mL of tert-amyl alcohol with lipase derived from *Aspergillus oryzae*, containing 1500 standard enzyme activities per unit mass of phospholipid. The reaction was carried out in a single-stage or multi-stage reactor at 45°C for 30 hours. The octanoic acid content of the structural phospholipid was 16% of all fatty acids.

[0038] Example 7

[0039] 10g of soybean lecithin, methyl laurate (molar ratio of lecithin to methyl laurate 10), n-hexane and tert-butanol (volume ratio 6:1, total volume 20mL), and a solid lipase derived from Rhizomucor miehei with 1000 standard enzyme activities per unit oil mass were placed in a single-stage or multi-stage reactor suitable for immobilized enzyme catalysis. The reaction was carried out at 60℃ for 22 hours, resulting in lauric acid comprising 62% of all fatty acids on the structural lecithin.

[0040] Example 8

[0041] 10g of soybean lecithin, methyl oleate (phospholipid molar ratio 10), and n-hexane and tert-butanol (volume ratio 2:1, total volume 20mL) were reacted with a Candida antarctica lipase containing 1500 standard enzyme activities per unit oil mass in a single-stage or multi-stage reactor suitable for immobilized enzyme catalysis. The reaction was carried out at 50℃ for 20 hours, resulting in oleic acid comprising 70% of all fatty acids on the structural phospholipids.

[0042] Example 9

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

[0044] Example 10

[0045] 10g of soybean lecithin, along with ethyl eicosapentaenoate (EEPA) and ethyl docosahexaenoate (DHA) in a molar ratio of 5 and a volume ratio of 1:1 (20mL), and lipases derived from Rhizomucor miehei (1000 standard enzyme activities per unit oil mass) and Candida antarctica (500 standard enzyme activities per unit oil mass), were placed in a single-stage or multi-stage reactor suitable for immobilized enzyme catalysis. The reaction was carried out at 55°C for 25 hours. EPA accounted for 35% of all fatty acids on the structured phospholipids, and DHA accounted for 34% of all fatty acids.

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

Claims

1. A method for preparing enzymatically catalyzed structural phospholipids, characterized in that, Structural phospholipids are obtained by reacting phospholipids with fatty acids and / or fatty acid esters of different carbon chain lengths through lipase catalysis in the presence of hydrophobic and hydrophilic solvents. The hydrophobic solvent is n-hexane; The hydrophilic solvent is selected from at least one of acetone, tert-butanol, and tert-amyl alcohol; The volume ratio of the hydrophobic solvent to the hydrophilic solvent is 6:1 to 1:1; The lipase catalysis is immobilized enzyme catalysis. The specific method includes: adding an immobilized lipase with 500-1000 enzyme activity units based on the phospholipid mass to a mixture of hydrophobic and hydrophilic solvents, wherein the molar ratio of phospholipid to fatty acid and / or fatty acid ester is 1:5-1:20, and reacting the above mixture at a temperature of 40-60°C for 10-30 hours. The fatty acids are saturated fatty acids and / or unsaturated fatty acids with a carbon chain length of C8-C22, selected from at least one of capric acid, lauric acid, caprylic acid, EPA, and DHA. The fatty acid ester is a methyl or ethyl ester of saturated fatty acids and / or unsaturated fatty acids with a carbon chain length of C8-C22, selected from at least one of methyl or ethyl esters of capric acid, lauric acid, caprylic acid, EPA, and DHA.

2. The method according to claim 1, characterized in that, The reaction is carried out in a single-stage or multi-stage reactor suitable for immobilized enzyme catalysis.

3. The method according to claim 1, characterized in that, The lipases include those derived from yeast cells, mold cells, or bacteria.

4. The method according to claim 3, characterized in that, The lipase comes from Aspergillus oryzae , Rhizomucor miehei , Candida antarctica At least one of them.

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