An enzymatic preparation method of Antarctic krill oil lysophospholipids

Abstract

This invention provides a method for the enzymatic synthesis of lysophosphatidylcholine from Antarctic krill oil in n-hexane. The method involves selectively hydrolyzing phosphatidylcholine in krill oil to generate the desired lysophosphatidylcholine. The lysophosphatidylcholine synthesized by this method not only has a high yield but also produces less of the byproduct glycerol phosphatidylcholine, making it suitable for the enzymatic synthesis of lysophosphatidylcholine. The steps include: adding Antarctic krill oil to a 15 mL reaction tube, then adding Lipase 7000 Pro, water, and n-hexane solvent, and stirring the reaction at 60 °C and 200 r / min. After 6 hours of reaction, the yield was monitored by liquid chromatography, and the yield of lysophosphatidylcholine was 69.69%. This invention provides a new approach for the enzymatic synthesis of lysophosphatidylcholine.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically relating to a method for synthesizing lysophosphatidylcholine from Antarctic krill oil by catalyzing the hydrolysis of phosphatidylcholine in organic solvents using lipase. Background Technology

[0002] Antarctic krill oil is a marine functional lipid resource rich in phospholipid-type ω-3 polyunsaturated fatty acids, with phosphatidylcholine (PC) being its main phospholipid component. Unlike traditional fish oil, where ω-3 fatty acids mainly exist in the form of triglycerides, the EPA and DHA in Antarctic krill oil are mostly bound to phospholipids. This structural feature gives it higher bioavailability and absorption efficiency, thus showing broad application prospects in functional foods, pharmaceuticals, and nutritional supplements. However, due to its high polarity, the PC molecule does not easily cross the blood-brain barrier to exert its physiological effects. Lysophosphatidylcholine (LPC) is an important active phospholipid formed by the partial hydrolysis of phosphatidylcholine, usually obtained by hydrolyzing the S-molecule in the PC molecule. N 1 bit or S N The ester bond at position 2 is used to obtain LPC. Compared with PC, LPC is more effective as a carrier to facilitate the crossing of long-chain polyunsaturated fatty acids such as EPA and DHA across the blood-brain barrier, and has important physiological activities in neuroprotection, cognitive improvement, anti-inflammation, and lipid metabolism regulation. Therefore, the directed hydrolysis of PC from Antarctic krill oil to prepare LPC rich in ω-3 fatty acids has become an important direction in marine functional lipid research.

[0003] Currently, the main methods for preparing lysophosphatidylcholine from phosphatidylcholine are chemical and enzymatic methods. Chemical methods typically use acids, bases, or organic catalysts to break ester bonds, which suffers from poor reaction selectivity, numerous side reactions, and a tendency to cause oxidative degradation of EPA and DHA. Furthermore, the post-processing steps are complex, which is not conducive to the green preparation of high-value-added marine phospholipid products.

[0004] Enzymes, as highly efficient biocatalysts, possess characteristics such as high specificity, mild reaction conditions, environmental friendliness, and high selectivity, and have been widely used in oil processing and functional lipid preparation. In recent years, studies have reported the use of phospholipase A2 and lipases to catalyze the directed hydrolysis of egg yolk lecithin and soybean lecithin to prepare LPC. Compared with chemical methods, enzymatic methods can effectively reduce side reactions, improve product purity, and better maintain the structural stability of EPA and DHA. Hu Jie et al. used phospholipase A1 to enzymatically hydrolyze Antarctic krill phospholipids in an aqueous system, and analyzed the phospholipid components before and after enzymatic hydrolysis using high-performance liquid chromatography (HPLC), finding that S... NThe content of lysophosphatidylcholine initially increased with prolonged enzymatic hydrolysis time and then tended to reach equilibrium, with high levels of EPA and DHA in the hydrolysis products. Yasuda et al. used a high-proportion Novozym 435 lipase to catalyze the decomposition of ethanol to efficiently prepare lysophosphatidylcholine, indicating that immobilized lipase has high catalytic potential in the preparation of lysophosphatidylcholine.

[0005] However, due to the high degree of phospholipid unsaturation and easy oxidation of Antarctic krill oil, existing technologies mostly employ a stepwise processing approach: first separating the phospholipid components from the krill oil, and then catalyzing the process with immobilized lipases (such as Novozym 435). This route is not only lengthy, typically requiring 12-24 hours, but also suffers from low enzyme catalytic efficiency, insufficient substrate adaptability, and low yield of lysophosphatidylcholine (LPC). To address these limitations, this study employs a novel lipase to directly catalytically hydrolyze Antarctic krill oil without prior phospholipid separation. This enzyme can act on both phospholipid and triglyceride components simultaneously, converting triglycerides into high-value-added diglycerides while simultaneously hydrolyzing phosphatidylcholine (PC) to prepare LPC. This simultaneous catalytic strategy not only simplifies the process and improves the yield of LPC but also enables the high-value utilization of triglyceride components in krill oil, providing a new pathway for the green and efficient development of marine phospholipid resources. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for synthesizing lysophosphatidylcholine. This method features low reaction cost, fast reaction rate, environmental friendliness, safe reaction process, and high yield. Simultaneously, it hydrolyzes triglycerides in krill oil to produce more easily absorbed diglycerides, significantly reducing raw material costs, energy consumption costs, and waste treatment costs, thus enhancing environmental friendliness.

[0007] To solve the above technical problems, the proposed technical solution is as follows: Synthesize lysophosphatidylcholine using phosphatidylcholine as the raw material, organic reagents as the reaction solvent, lipase (1%-11%), and water (50-250 μL), reacting at 50-70℃ for 2-12 hours. The reaction route is shown below:

[0008]

[0009] Preferably, 0.5 g of Antarctic krill oil was dissolved in 3 ml of n-hexane, 50 μL of water was added, and 5% (0.025 g) of immobilized lipase Novozym435 was added. The reaction was carried out at 60 °C for 12 h. The reaction effect was monitored by thin-layer chromatography, and the reaction yield was monitored by HPLC-ELSD after the reaction was completed. The product lysophosphatidylcholine was 26.63 mg, with a yield of 21.35%. At the same time, the triglycerides in the krill oil were also hydrolyzed to produce more easily absorbed diglycerides.

[0010] Preferably, the immobilized lipase is selected from any one of NE-20, immobilized CRL, MASI, Chiralzyme IM-100, Novozym435, Lipase7000Pro, and RIM-03.

[0011] Preferably, the reaction solvent is selected from any one of toluene, DMSO, DMF, tetrahydrofuran, acetonitrile, ethyl acetate, n-hexane, tert-butanol, and tert-amyl alcohol.

[0012] Preferably, the amount of water added is selected from any one of 50μL, 100μL, 150μL, 200μL, and 250μL.

[0013] Preferably, the amount of enzyme added is selected from any one of 1%, 3%, 5%, 7%, 9%, and 11% (w / w).

[0014] Preferably, the reaction temperature is selected from any one of 50℃, 55℃, 60℃, 65℃, and 70℃.

[0015] Preferably, the reaction time is any one of 2h, 4h, 6h, 8h, 10h, and 12h.

[0016] Beneficial effects:

[0017] Due to the high degree of phospholipid unsaturation and easy oxidation of Antarctic krill oil, existing technologies mostly employ a stepwise processing approach: first separating the phospholipid components from the krill oil, and then catalyzing the process with immobilized lipases (such as Novozym 435). This route is not only lengthy but also suffers from low enzyme catalytic efficiency, insufficient substrate adaptability, and low yield of lysophosphatidylcholine (LPC). To address these limitations, this study employs a novel lipase to directly catalytically hydrolyze Antarctic krill oil without prior phospholipid separation. This enzyme can act on both phospholipid and triglyceride components simultaneously, converting triglycerides into high-value-added diglycerides while simultaneously hydrolyzing phosphatidylcholine (PC) to prepare LPC. This simultaneous catalytic strategy not only simplifies the process and improves the yield of LPC but also enables the high-value utilization of triglyceride components in krill oil, providing a new pathway for the green and efficient development of marine phospholipid resources.

[0018] This invention synthesizes lysophosphatidylcholine via enzymatic catalysis. For the first time, it utilizes the immobilized lipase Lipase7000Pro from Blue Ocean Biotechnology to optimize the synthesis method. It employs the low-toxicity organic reagent n-hexane as the reaction solvent, resulting in an extremely short reaction time, a safe reaction process, and a high yield. Simultaneously, it hydrolyzes the triglycerides in krill oil to produce more easily absorbed diglycerides, significantly reducing the amount of catalyst used and substantially lowering raw material costs, energy costs, and waste treatment costs, thus enhancing environmental friendliness.

[0019] The preparation method of lysophosphatidylcholine involves changing only the parameters of the immobilized lipase type, reaction solvent, water addition, enzyme addition, reaction temperature, or reaction time. Specific results are shown in Tables 1-6. In Example 1, Lipase 7000Pro was used as the immobilized lipase, n-hexane was used as the reaction solvent, the enzyme addition was 7% (w / w) (0.035 g), and the water addition was 100 μL. The optimal yield was achieved by reacting at 60°C for 6 hours, with a lysophosphatidylcholine yield reaching 69.69%. Attached Figure Description

[0020] The invention will be further described below with reference to the accompanying drawings.

[0021] Figure 1 This is a schematic diagram of the hydrolysis of phosphatidylcholine according to the present invention.

[0022] Figure 2 This is a schematic diagram of the triglyceride hydrolysis process of the present invention.

[0023] Figure 3 This is a high-performance liquid chromatogram of the reaction of the present invention.

[0024] Figure 4 The gas chromatogram of the reaction of this invention.

[0025] Figure 5 The nuclear magnetic resonance spectrum of the product of this invention. Detailed Implementation

[0026] The present invention will be further described below with reference to the embodiments. The listed embodiments are for illustrative purposes only and are intended to demonstrate that the spirit and scope of the present invention are not limited to the details and modifications herein.

[0027] Example 1:

[0028] Preparation of lysophosphatidylcholine. Krill oil mainly contains phospholipids (of which phosphatidylcholine content ≥61% (w / w)), triglycerides, and small amounts of free fatty acids and cholesterol. 0.5g of Antarctic krill oil from DeepBlue Biotechnology was dissolved in 3ml of n-hexane, 50μL of water was added, and 5% (0.025g) of immobilized lipase Novozym435 was added. The reaction was carried out at 60℃ for 8h. The reaction effect was monitored by thin-layer chromatography, and the reaction yield was monitored by HPLC-ELSD and gas chromatography after the reaction was completed.

[0029] Lipases catalyze two parallel reactions, one of which is the S-reaction of phosphatidylcholine. N - The fatty acid at position -1 is hydrolyzed to generate the target product, lysophosphatidylcholine; secondly, triglycerides at position S... N Partial hydrolysis occurred at position -1(3), resulting in a large amount of diglyceride and a small amount of free fatty acid. 26.63 mg of lysophosphatidylcholine was obtained, with a yield of 21.35%, and diglyceride was also obtained as an additional product.

[0030] Diglycerides are more beneficial to human health than triglycerides. Their digestive and metabolic pathways differ, which can reduce postprandial blood lipid peaks and lower the risk of body fat accumulation, thus enhancing the overall functional value of the product. Glycerides are readily soluble in cold acetone, while lysophosphatidylcholine is almost insoluble in cold acetone. After the reaction, the solvent is removed by rotary evaporation, and the residue is washed with cold acetone until no glyceride residue remains, thereby obtaining the product lysophosphatidylcholine.

[0031] The reaction pathway of phosphatidylcholine hydrolysis is as follows: Figure 1 As shown.

[0032] The reaction route for the hydrolysis of triglycerides is as follows: Figure 2 As shown.

[0033] Figure 3 The high-performance liquid chromatogram of the reaction of this invention shows the peak position of the product lysophosphatidylcholine at 7 min, indicating a high yield.

[0034] Figure 4 The image shows the gas chromatogram of the reaction of this invention, with the peak position of diglyceride corresponding to 14-16 min.

[0035] Figure 5 This is the nuclear magnetic resonance spectrum of the purified product of this invention.

[0036] Example 2:

[0037] The preparation method of lysophosphatidylcholine is the same as in Example 1, except that the type of immobilized lipase, reaction solvent, amount of water added, amount of enzyme added, reaction temperature, or reaction time are changed. Specific results are shown in Tables 1-6. For the preparation of lysophosphatidylcholine: 0.5g of Antarctic krill oil from DeepBlue Biotechnology was completely dissolved in 3ml of n-hexane. The immobilized lipase used was Lipase7000Pro, the reaction solvent was n-hexane, the enzyme addition was 7% (w / w) (0.035g), and the water addition was 100μL. The optimal yield was achieved by reacting at 60℃ for 6h. After the reaction, the yield was monitored by HPLC-ELSD and gas chromatography, yielding 86.9mg of lysophosphatidylcholine, with a yield of 69.69% and a diglyceride yield of 44.0%.

[0038] Table 1 compares and screens immobilized lipases:

[0039]

[0040] 0.5g of Antarctic krill oil was dissolved in 3ml of n-hexane, 50μL of water was added, and 5% (0.025g) of different commercial lipases were added. The reaction was carried out at 60℃ for 8h. The reaction effect was monitored by thin-layer chromatography, and the reaction yield was monitored by HPLC-ELSD after the reaction was completed. The results showed that the immobilized lipase Lipase7000Pro from Blue Ocean Biotechnology had the highest yield. Lipase7000Pro was used for subsequent screening of lipases.

[0041] Table 2 provides a comparison of screening solvents for the reaction:

[0042]

[0043] 0.5 g of Antarctic krill oil was dissolved in 3 ml of different organic reagents, 50 μL of water was added, and 5% (0.025 g) of immobilized lipase Lipase7000Pro was added. The reaction was carried out at 60 °C for 8 h. The reaction effect was monitored by thin-layer chromatography. After the reaction was completed, the reaction yield was monitored by HPLC-ELSD. The results showed that the yield was higher in hexane and toluene. Considering the toxicity and subsequent separation, hexane was selected as the reaction solvent.

[0044] Table 3 provides a comparison of the water addition amount:

[0045]

[0046] 0.5g of Antarctic krill oil was dissolved in 3ml of n-hexane. Different amounts of water were added, along with 5% (0.025g) of immobilized lipase Lipase7000Pro. The reaction was carried out at 60℃ for 8h. The reaction effect was monitored by thin-layer chromatography, and the reaction yield was monitored by HPLC-ELSD after the reaction was completed. The results showed that when a small amount of water was added, the yield increased with the increase of water addition, and the yield reached the highest at 100μL. However, further increasing the amount of water added would lead to excessive hydrolysis and the production of the byproduct glycerophosphatidylcholine. Therefore, the amount of water added was selected as 100μL.

[0047] Table 4 compares the results based on the amount of enzyme added:

[0048]

[0049] 0.5 g of Antarctic krill oil was dissolved in 3 ml of n-hexane, and 50 μL of water was added. Immobilized lipase Lipase 7000 Pro with different addition amounts was added, and the reaction was carried out at 60 °C for 8 h. The reaction effect was monitored by thin-layer chromatography, and the reaction yield was monitored by HPLC-ELSD after the reaction was completed. The results showed that when a small amount of enzyme was added, the yield increased with the increase of enzyme addition. When the enzyme addition was increased to 7%, the yield did not increase significantly when the enzyme addition was further increased. Considering the economic effect, the enzyme addition amount was selected to be 7%.

[0050] Table 5 provides a comparison of screening results based on reaction temperature:

[0051]

[0052] 0.5 g of Antarctic krill oil was dissolved in 3 ml of n-hexane, 50 μL of water was added, and 7% (0.035 g) of immobilized lipase Lipase7000Pro was added. The reaction was carried out at different temperatures for 8 h. The reaction effect was monitored by thin-layer chromatography, and the reaction yield was monitored by HPLC-ELSD after the reaction was completed. The results showed that the yield increased with the increase of temperature, and the yield reached the highest at 60℃. However, further increasing the temperature would lead to enzyme inactivation. Therefore, the reaction temperature was selected as 60℃.

[0053] Table 6 provides a comparison based on reaction time:

[0054]

[0055] 0.5 g of Antarctic krill oil was dissolved in 3 ml of n-hexane, 50 μL of water was added, and 7% (0.035 g) of immobilized lipase Lipase7000Pro was added. The reaction was carried out at 60 °C for different times. The reaction effect was monitored by thin-layer chromatography. After the reaction was completed, the reaction yield was monitored by HPLC-ELSD. The results showed that the yield increased with the extension of time, and the yield reached the highest at 6 h. Further delay of the reaction would lead to excessive hydrolysis and the production of the byproduct glycerophosphatidylcholine. Therefore, the reaction time was 6 h.

Claims

1. An enzymatic method for preparing Antarctic krill oil lysophospholipids, characterized in that: Lysophosphatidylcholine was synthesized using Antarctic krill oil as raw material, organic reagents as solvents, immobilized lipase (7%-11% by mass) added, and water. The reaction was carried out at 60-65℃ for 4-10 hours to obtain the product lysophosphatidylcholine. The reaction route is shown below: ； The immobilized lipase is Lipase7000Pro.

2. The enzymatic preparation method of Antarctic krill oil lysophospholipids according to claim 1, characterized in that, The reaction solvent is selected from either toluene or n-hexane.

3. The enzymatic preparation method of Antarctic krill oil lysophospholipids according to claim 1, characterized in that, The amount of water added is selected from any one of 50μL, 100μL, and 150μL.

4. The enzymatic preparation method of Antarctic krill oil lysophospholipids according to claim 1, characterized in that, The amount of enzyme added is selected from any one of the following: 7%, 9%, and 11% by mass fraction.

5. The enzymatic preparation method of Antarctic krill oil lysophospholipids according to claim 1, characterized in that, The reaction temperature is selected from any of 60°C.

6. The enzymatic preparation method of Antarctic krill oil lysophospholipids according to claim 1, characterized in that, The reaction time is selected from either 6 hours or 8 hours.

7. The enzymatic preparation method of Antarctic krill oil lysophospholipids according to claim 1, characterized in that: 0.5g of Antarctic krill oil from DeepBlue Biotechnology was dissolved in 3ml of n-hexane. The immobilized lipase was Lipase7000Pro from Blue Ocean Biotechnology. The enzyme addition amount was 7% by mass, 0.035g. The water addition amount was 100μL. The reaction temperature was 60℃ and the reaction time was 6h. The yield of lysophosphatidylcholine reached 69.9%.

8. The enzymatic preparation method of Antarctic krill oil lysophospholipids according to claim 1, characterized in that: Antarctic krill oil was dissolved in hexane, water was added, and immobilized lipase was added to catalyze the reaction to produce lysophosphatidylcholine. The reaction effect was monitored by thin-layer chromatography, and the reaction yield was monitored by HPLC-ELSD after the reaction was completed.

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