Application of a sodium cyclamate reverse micelle system in the preparation of phospholipid DHA

Abstract

The present invention belongs to the field of bioengineering, and specifically relates to an application of a reverse micelle system composed of sodium cyclamate in the preparation of phospholipid DHA, comprising the following steps: step one: screening of surfactants; step two: preparation of reverse micelle system, dissolving the surfactant in an organic reagent to form a reverse micelle system; step three, enzyme catalysis reaction of phospholipid DHA. Step four, separation of phospholipid DHA, collecting the precipitate and supernatant after elution with acetone, vacuum concentrating to remove acetone and supernatant acetone to recover free DHA and surfactant. Compared with the surfactants used in the prior art, the present invention is safe and effective, and the incorporation rate of phospholipid DHA is increased under the action of edible surfactants, which is efficient and economical.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to the application of a cyclamate reverse micelle system in the preparation of phospholipid DHA. Background Art

[0002] Phospholipids are the main components of cell membranes and have important functions such as anti-atherosclerosis, prevention and treatment of fatty liver, and lowering blood lipids. Docosahexaenoic acid (DHA) has been proven to promote the development of the visual system, enhance the body's immunity, improve heart health, and promote the intellectual development of infants and young children. However, due to its high degree of unsaturation, DHA is easily oxidized. DHA bound to phospholipids, namely phospholipid DHA, has significantly improved oxidative stability, is easier to be absorbed by the human body, and has the dual effects of phospholipids and DHA.

[0003] Phospholipid DHA is absorbed in the body in an active way, with an absorption rate of nearly 100%. It can quickly supply the body's cerebral cortex with the required DHA. The latest research shows that phospholipid DHA has better bioavailability and biosafety than ethyl ester and glyceride forms. In addition, phospholipid DHA shows more effective biological activity in reducing liver and serum total cholesterol and triglyceride levels, increasing n-3 DHA concentration in the brain, and has significant effects on improving glucose intolerance, resisting high-fat diet-induced hyperlipidemia and obesity.

[0004] At present, in the process of preparing phospholipid DHA by enzymatic method, most of them use non-edible surfactants. For example, the Tianjin Institute of Industrial Biotechnology of the Chinese Academy of Sciences uses sodium 2-ethylhexyl sulfonate (AOT) surfactant to catalyze the synthesis of phospholipid DHA in Chinese patent CN104531790A, which has low safety. At present, in most processes of preparing phospholipid DHA by enzymatic method, AOT, cetyltrimethylammonium bromide (CTAB) and other surfactants available in non-food industry are often used for catalysis. Its food safety is low. Therefore, it is necessary to develop an edible surfactant with high catalytic efficiency and high phospholipid DHA incorporation amount for use in the preparation process of phospholipid DHA. Summary of the invention

[0005] In order to solve the above defects and shortcomings in the prior art, the present invention provides an edible surfactant sodium cyclamate, which can effectively increase the incorporation rate of DHA.

[0006] In order to solve the above technical problems, the present invention provides an application of a sodium cyclamate reverse micelle system in the preparation of phospholipid DHA.

[0007] The preparation process of the reverse micelle system is as follows: dissolving the surfactant sodium cyclamate in an organic solvent, then adding a phosphate buffer solution, and fully shaking until transparent without stratification, thereby obtaining the sodium cyclamate reverse micelle system;

[0008] The application steps are as follows: adding a substrate consisting of free fatty acids and soybean lecithin and immobilized phospholipase PLA1 into a sodium cyclamate reverse micelle system for catalytic reaction, and after the reaction, performing separation and purification to obtain phospholipid DHA.

[0009] Furthermore, the concentration of cyclamate is 0.5 g / L-150 g / L.

[0010] Furthermore, the concentration of the cyclamate is 5 g / L-50 g / L.

[0011] Furthermore, the amount of the reverse micelle system is 500%-600% of the total mass of the substrate.

[0012] Furthermore, the organic solvent is n-hexane or isooctane.

[0013] The beneficial technical effects achieved by the present invention are as follows: the present invention provides an application of a sodium cyclamate reverse micelle system in the preparation of phospholipid DHA, and establishes a reverse micelle system based on an edible surfactant sodium cyclamate, which is more environmentally friendly, safe and reliable than traditional surfactants. The phospholipid DHA is prepared using immobilized phospholipase A1 as a catalyst, and the DHA incorporation rate is as high as 53%. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a picture of the sodium cyclamate reverse micelle system prepared in an embodiment of the present invention;

[0015] Figure 2 The lecithin powder prepared in the embodiment of the present invention;

[0016] Figure 3 is the amount of DHA incorporated under different surfactants of the present invention;

[0017] Figure 4 The gas chromatogram of DHA prepared by using sodium cyclamate as a surfactant in the present invention;

[0018] Figure 5 The gas chromatogram of DHA prepared by using sucrose as a surfactant in the present invention;

[0019] Figure 6 The gas chromatogram of the present invention in which rhamnose is used as a surfactant to prepare DHA;

[0020] Figure 7 is the amount of DHA incorporated in different organic solvents of the present invention;

[0021] Figure 8The gas chromatogram of the present invention in which n-hexane is used as an organic solvent to prepare DHA;

[0022] Fig. 9 The gas chromatogram of the preparation of DHA using isooctane as an organic solvent in the present invention;

[0023] Fig.10 is the amount of DHA incorporated under different concentrations of surfactants of the present invention;

[0024] Fig.11 The gas chromatogram of DHA prepared when the concentration of sodium cyclamate in the present invention is 0.5 g / L;

[0025] Fig.12 The gas chromatogram of DHA prepared when the concentration of sodium cyclamate in the present invention is 0.75 g / L;

[0026] Fig.13 The gas chromatogram of DHA prepared when the concentration of sodium cyclamate in the present invention is 1.5 g / L;

[0027] Fig.14 The gas chromatogram of DHA prepared when the concentration of sodium cyclamate in the present invention is 15 g / L;

[0028] Fig.15 This is a gas chromatogram of DHA prepared when the concentration of cyclamate is 150 g / L. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention.

[0030] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0031] The manufacturers of the reagents used in the following examples are shown in Table 1.

[0032] Table 1 Statistics of reagent manufacturers

[0033] Reagent name Manufacturer Isooctane Shanghai Aladdin Technology Co., Ltd. n-Hexane Shanghai MacLean Biochemical Technology Co., Ltd. acetone Nanjing Wanqing Chemical Glass Instrument Co., Ltd. Potassium hydroxide Sinopharm Chemical Reagent Co., Ltd. Methanol Shanghai MacLean Biochemical Technology Co., Ltd. Boron trifluoride-ethyl ether Shanghai Jizhi Biochemical Technology Co., Ltd. Sodium chloride Shanghai Zhanyun Chemical Co., Ltd. Disodium hydrogen phosphate Shanghai MacLean Biochemical Technology Co., Ltd. Rhamnose Shanghai Myril Biochemical Technology Co., Ltd. Cyclamate Shanghai Myril Biochemical Technology Co., Ltd. Sucrose fatty acid esters Nanjing Wanqing Chemical Glass Instrument Co., Ltd. Soy lecithin Anhui Zhongchuang Lecithin Technology Co., Ltd. Free fatty acids Hunan Wanquan Yuxiang Biotechnology Co., Ltd. Phospholipase A1 Genentech Biotechnology Co., Ltd.

[0034] Example 1 Preparation of reverse micelle system

[0035] The surfactant cyclamate is dissolved in an organic solvent, and then a phosphate buffer solution is added and shaken thoroughly until it is transparent and has no stratification, which is a cyclamate reverse micelle system; wherein the mass concentration of cyclamate in the organic solvent is 15 g / L, the phosphate buffer solution is a disodium hydrogen phosphate solution, the pH value is 9, the mass of the disodium hydrogen phosphate is 1% of the total amount of the DHA reaction substrate, and the organic solvent is n-hexane. The obtained cyclamate reverse micelle system is as follows Figure 1 shown.

[0036] Example 2 Preparation of phospholipid DHA

[0037] The substrate composed of free fatty acids and soybean lecithin and immobilized phospholipase PLA1 are added to the sodium cyclamate reverse micelle system for catalytic reaction, and the phospholipid DHA is obtained by the immobilized phospholipase PLA1 catalytic reaction. After the reaction is completed, the immobilized phospholipase PLA1 is filtered, the filtrate is taken out, and acetone is added to elute, wherein the insoluble matter is phospholipid, centrifuged at 7000rpm for 5min, and the operation is repeated 5 times. Vacuum concentration and drying are performed to obtain dry phospholipid powder, such as Figure 2 shown.

[0038] Among them, the mass ratio of free fatty acids to soybean lecithin is 8:1, the weight of immobilized phospholipase PLA1 is 25% of the total substrate, the catalytic reaction is carried out on a shaker, the reaction temperature is 55°C, the reaction time is 24h, the rotation speed is 200rpm; the vacuum concentration temperature is 50°C.

[0039] Experimental Example 1 Effect of reverse micelle system prepared by different surfactants on DHA incorporation rate of phospholipid DHA

[0040] This test example uses three surfactants: sodium cyclamate, rhamnose and sucrose fatty acid esters, and detects the incorporation rate of DHA to verify the influence of different surfactants in the preparation of DHA. First, the reverse micelle system is prepared by the method of Example 1, and then the phospholipid DHA is prepared by the method of Example 2. Finally, the incorporation rate of DHA in phospholipids is calculated based on the gas phase results. Figure 3-Figure 6 shown.

[0041] Methyl esterification: weigh 0.1 g of phospholipid-DHA into a 10 mL volumetric flask, add 3 mL of 0.5 M KOH-methanol solution, react in a 65°C constant temperature water bath for 17 min, take out and cool to room temperature, add 3 mL of boron trifluoride ether-methanol (3:7, v / v), react in a 65°C constant temperature water bath for 7 min, take out and cool to room temperature; add 2 mL of saturated NaCl solution to mix thoroughly; then add 2 mL of n-hexane to extract for 30 min; take 1 mL of supernatant and filter through 0.2 µm organic membrane into a gas phase vial for testing.

[0042] Gas chromatograph: Shimadzu GC-2010; chromatographic column: model DB-23 (60 m×0.25 mm×0.25 µm); carrier gas: nitrogen; detector: FID; split ratio: 35 / 1; inlet temperature: 250 °C; detector temperature: 280 °C; column temperature program: initial temperature 100 °C, increased to 200 °C at 25 °C / min, then increased to 240 °C at 2 °C / min, and finally maintained at 240 °C for 7 min, with a total analysis time of 35 min; air flow rate 400 mL / min; hydrogen flow rate: 40 mL / min; column flow rate: 3.0 mL / min; tail flow rate: 30 mL / min; detector temperature: 280 °C; injection volume: 1 µL.

[0043] from Figure 3-Figure 6 It can be seen that when cyclamate is used as a surfactant, the incorporation rate of DHA in phospholipid DHA is 47.8%, which is much higher than the other two surfactants. This may be because the molecular structure of cyclamate may make it exhibit excellent interfacial activity at the oil-water interface, thereby more effectively promoting the combination of DHA and phospholipids. The water solubility of cyclamate may also help maintain the stability and uniformity of the reaction system during the reaction, thereby improving the incorporation efficiency of DHA.

[0044] Experimental Example 2 Effect of different organic solvents on the DHA incorporation rate in phospholipid DHA

[0045] In this test example, two organic solvents were selected: n-hexane and isooctane. The reverse micelle system was prepared by the method of Example 1, and then the phospholipid DHA was prepared by the method of Example 2. The incorporation rate of DHA in the phospholipid was calculated using the gas chromatogram. The results were as follows: Figure 7-Figure 9 shown.

[0046] from Figure 7-Figure 9 It can be seen that different organic solvents will affect the incorporation rate of DHA. When n-hexane was used as the organic solvent, the incorporation rate of DHA in the obtained phospholipid DHA reached 51.5%, and that of DPA was 12.1%. This may be because n-hexane is a non-polar solvent that can effectively dissolve fatty acids, especially long-chain polyunsaturated fatty acids (such as DHA). This non-polar property may enable n-hexane to better bind to phospholipids, thereby increasing the incorporation rate of DHA. As a solvent, n-hexane may provide a more favorable reaction environment, promoting the binding of DHA to phospholipids. The high incorporation rate of DHA indicates that n-hexane may help optimize enzyme-catalyzed transesterification or other related reactions, thereby improving the incorporation efficiency of DHA.

[0047] Experimental Example 3 Effect of Cyclamate Concentration on DHA Incorporation Rate in Phospholipid DHA

[0048] The reverse micelle system was prepared by the method of Example 1, and then phospholipid DHA was prepared by the method of Example 2. The incorporation rate of DHA in phospholipid DHA was calculated using the gas chromatogram. Figure 10-15 As shown. Among them, the mass concentrations of cyclamate in n-hexane are 0.5g / L, 0.75g / L, 1.5g / L, 15g / L, and 150g / L respectively.

[0049] from Figure 10-15 It can be seen that with the increase of cyclamate concentration, the incorporation rate of DHA in phospholipid DHA gradually increased. When the cyclamate concentration was 15g / L, the incorporation rate of DHA reached the highest, which was 53.5%. After that, with the increase of cyclamate concentration, the incorporation rate of DHA did not change significantly. Therefore, the optimal concentration of cyclamate is 15g / L.

[0050] The present invention has been disclosed above with preferred embodiments, but they are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation solutions fall within the protection scope of the present invention.

Claims

1. Application of a sodium cyclamate reverse micelle system in the preparation of phospholipid DHA, characterized in that: The preparation process of the reverse micelle system is to dissolve the surfactant sodium cyclamate in an organic solvent, then add a phosphate buffer solution, and fully shake until transparent without stratification, thereby obtaining the sodium cyclamate reverse micelle system; wherein the organic solvent is n-hexane or isooctane; The application steps are as follows: adding a substrate composed of free fatty acids and soybean lecithin and immobilized phospholipase PLA1 to a sodium cyclamate reverse micelle system for catalytic reaction, and after the reaction is completed, separating and purifying to obtain phospholipid DHA; The concentration of cyclamate is 0.5 g / L-150g / L.

2. The use according to claim 1, characterized in that: The concentration of the cyclamate is 5 g / L-50 g / L.

3. The use according to claim 1, characterized in that: The amount of the reverse micelle system used is 500%-600% of the total mass of the substrate.

Images