Method for catalytically synthesizing phytosterol ester by using ionic liquid modified magnetic cyclodextrin nanocomposite immobilized lipase

Abstract

The invention provides a method for catalytically synthesizing phytosterol ester by using ionic liquid modified magnetic nano composite material immobilized lipase, which comprises the following steps: taking imidazolium ionic liquid modified magnetic beta-cyclodextrin organic-inorganic nano composite material immobilized lipase as a catalyst; esterification of phytosterol and fatty acid is catalyzed in a solvent-free system to synthesize the phytosterol ester. The carrier prepared by the method combines the characteristics of good biocompatibility of the beta-cyclodextrin and the ionic liquid, capability of stabilizing the active conformation of enzyme and the like, and also combines the characteristic of easy separation of a magnetic material. The immobilized enzyme has the characteristics of convenience in separation, high catalytic activity, good stability and the like, and can be used for catalyzing esterification reaction of phytosterol and fatty acid to prepare phytosterol ester. The invention provides a novel green and efficient phytosterol ester synthesis method.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and specifically relates to a method for synthesizing phytosterol esters by using ionic liquid-modified magnetic beta-cyclodextrin-immobilized lipase to catalyze esterification reaction of phytosterol and oleic acid under solvent-free volume conditions. Background Art

[0002] Phytosterols are a type of cyclopentane polyhydrophenanthrene biological substance with a structure similar to cholesterol. They have a variety of biological activities, such as improving cholesterol levels in the body, regulating immune activity, anti-tumor and preventing cardiovascular diseases, and are widely present in many foods. However, since phytosterols are slightly soluble in hydrated oils, this limits their bioavailability in the food industry. At present, in order to improve their solubility and bioavailability, converting phytosterols into phytosterol esters can expand their commercial value.

[0003] Phytosterol esters are mainly synthesized by chemical or enzymatic synthesis by reacting phytosterols with fatty acids or their derivatives. Among them, the catalysts commonly used in the chemical synthesis of phytosterol esters include strong acids and their salts (H2SO4, p-toluenesulfonic acid, NaHSO4, etc.) or strong bases (NaOH, KOH, NaOMe) as catalysts, which have problems such as high reaction temperature, many dehydration by-products, and dark product color. Enzymatic synthesis of phytosterol esters has attracted widespread attention in the academic community due to its mild reaction conditions, few by-products, and high safety. For example, Ma Biao et al. prepared a catalyst using Ce(DS)3 as raw material to catalyze phytosterols and fatty acids. After reacting for 4 hours at 110°C, the esterification rate reached 80.7%-86.4%, but the high temperature inevitably led to the formation of a considerable amount of dehydration by-products (European Journal of Lipid Science and Technology, 2012,114(8):896-904). Compared with chemical synthesis, enzymatic synthesis can not only avoid the formation of byproduct dehydrated sterols, but also has the advantages of light product color and easy purification. However, the literature reports that the process of enzymatic synthesis of plant sterol esters has problems such as long reaction time and low yield. Cui Caixia et al. immobilized Yarrowia lipolytica lipase (Ylip) on diatomaceous earth with good biocompatibility by physical adsorption, and used the immobilized lipase to catalyze the esterification reaction of oleic acid and plant sterols. The reaction was carried out at 50°C for 78 hours, and the conversion rate of plant sterol esters reached 91.1% (Colloid Surface B, 2016, 146(5):490-497). Yu Dianyu's team used 10% Fe3O4 / SiOx-P(GMA)-CALB as a catalyst, free fatty acids and phytosterols with a molar ratio of 4:1 as substrates, and reacted at 50°C for 72 hours. The esterification rate of phytosterol esters was 85.7% (Catalysis Letters B, 2020, 150(5): 1-12). Summary of the invention

[0004] The present invention provides an ionic liquid modified magnetic β-cyclodextrin organic-inorganic composite material immobilized with lipase as a catalyst, which catalyzes the esterification of phytosterols and fatty acids in a solvent-free system to prepare phytosterol esters. The loading amount of the immobilized enzyme prepared by this method is as high as 76.5 mg / g, and its reusability and storage stability are improved. The phytosterol ester reaction can be completed in 48 hours, the phytosterol ester yield is as high as 95.0%, and there is no dehydrated sterol byproduct.

[0005] The technical solution adopted by the present invention is as follows: a method for synthesizing phytosterol esters by catalyzing lipase immobilized on a magnetic nanocomposite material, the steps of which are as follows:

[0006] (1) Preparation of magnetic β-cyclodextrin by coprecipitation method;

[0007] (2) Modification of magnetic β-cyclodextrin with carboxyl-functionalized imidazole ionic liquid;

[0008] (3) activating the magnetic β-cyclodextrin modified with the functionalized ionic liquid obtained in step (2);

[0009] (4) Preparation of ionic liquid-modified magnetic β-cyclodextrin immobilized Candida rugosa lipase: adding the activated ionic liquid-modified magnetic β-cyclodextrin obtained in step (3) to a lipase solution, wherein the amount of the lipase (Candida rugosa lipase) added is 90-150 mg / g (relative to the carrier mass), the temperature is 25-35° C., the pH is 6.5-7.5, and the oscillation is performed for 3-7 hours to carry out the lipase immobilization reaction, and then the immobilized lipase with high activity, high thermal stability and high tolerance is obtained by magnetic field separation;

[0010] (5) Adding phytosterols and oleic acid as substrates into a reactor, and then adding the immobilized lipase prepared in step (4), and performing an esterification reaction under solvent-free conditions under heating and stirring conditions.

[0011] Preferably, the conditions for preparing the immobilized lipase in step (4) are as follows: the addition amount of Candida rugosa lipase: 150 mg / g relative to the mass of the carrier; the immobilization time: 5 h; the pH: 7; and the immobilization temperature: 30° C.

[0012] Preferably, the specific preparation steps of the magnetic nanocomposite material immobilized lipase include:

[0013] (1) Synthesis of magnetic β-cyclodextrin by coprecipitation method: 4 g β-CD was dissolved in 200 ml distilled water. Next, 4 g FeCl2·4H2O and 10 g FeCl3·6H2O were added to a round-bottom flask and refluxed at 80 °C until a clear brown solution was obtained. Then a certain concentration of ammonia water was injected into the flask until the pH reached about 11-12. Then the temperature was fixed at 80 °C and stirred for 30 min. The product was filtered, washed with ethanol and water and dried in a vacuum oven to obtain Fe3O4@β-CD.

[0014] (2) Disperse 3g of Fe3O4@β-CD in 100mL of toluene and homogenize by ultrasonication. Add 4g of silane coupling agent 3-chloromethoxysilane (30mmol), use nitrogen protection, and heat under reflux for 7h. After the reaction, separate the product by magnetic field, wash with ethanol several times, and then dry in a vacuum oven at 60℃.

[0015] (3) A mixture of 25 mmol imidazole and 25 mmol sodium hydride was stirred in 100 mL toluene for 2 h, and then the product obtained in (2) was added and refluxed for 24 h. After the solid product was cooled, it was separated by a magnet and then washed with deionized water and ethanol. It was then dried at 60 °C for 10 h. It was then added to 100 mL acetonitrile for dispersion, and then 25 mmol chloroacetic acid was added. Refluxed under nitrogen protection and stirred for 24 h. The sample was washed with deionized water and ethanol, and then dried in a vacuum oven at 60 °C. Subsequently, the solid product was exchanged with excess NaBF4 anions in water for 12 h, and finally the product was washed again with water and ethanol and dried. The final product was named Fe3O4@β-CD-CIL.

[0016] (4) Activate the functionalized ionic liquid modified magnetic β-cyclodextrin obtained in step (3) by the following method: ultrasonically disperse 0.01 mol N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 0.01 mol N-hydroxysuccinimide (NHS) and 0.1 g Fe3O4@β-CD-CIL in 50 mL phosphate buffer solution, pH 6.0, and stir in a low-temperature stirrer for 1 h;

[0017] (5) Dissolve 75 mg of Candida plicata lipase in 50 mL of phosphate buffer solution at pH 7. Then add 500 mg of the ionic liquid functionalized magnetic β-cyclodextrin obtained in (4) to the lipase mixed solution, shake in a constant temperature water bath at 30 °C for 5 hours, and then obtain the immobilized lipase by washing and magnetic field separation. After freeze drying, the ionic liquid modified magnetic β-cyclodextrin immobilized lipase was obtained.

[0018] Preferably, the fatty acid in the esterification reaction catalyzed by the immobilized lipase is one of oleic acid, linoleic acid, linolenic acid or a mixed fatty acid.

[0019] Preferably, one or a mixed fatty acid of oleic acid, linoleic acid, linolenic acid is used as the reaction medium.

[0020] Preferably, the fatty acid in the esterification reaction catalyzed by the immobilized lipase is oleic acid, and serves as the reaction medium of the reaction.

[0021] Preferably, the molar ratio of phytosterols to fatty acids is 1:4-1:8.

[0022] Preferably, the amount of the immobilized lipase added in the reaction is 8.0-16.0% by mass of the phytosterols; the reaction temperature of the reaction is 35-55° C.; and the reaction time of the reaction is 18-48 hours.

[0023] Preferably, the reaction medium is oleic acid, the molar ratio of phytosterols to fatty acids is 1:6; the amount of immobilized lipase added in the reaction is 14% by mass of phytosterols, the reaction temperature is 50° C., and the reaction time is 48 hours.

[0024] Preferably, the method for synthesizing phytosterol esters comprises the following steps:

[0025] 1 mmol of pine sterol and 6 mmol of oleic acid were added to a reaction bottle, the temperature was raised to 50°C and mixed evenly, then 14% of modified magnetic β-cyclodextrin immobilized lipase was added and stirred at 50°C. TLC (n-hexane: anhydrous ether: glacial acetic acid, 90:10:1, v / v / v) was used to detect the progress of the reaction. After 48 hours of reaction, an external magnetic field was applied to separate and recover the immobilized enzyme. The esterification rate measured by gas phase was 95.0%, and there were no by-products. After the recovered immobilized lipase was subjected to 5 consecutive cycles of reaction, the yield of sterol esters could reach 81.0%.

[0026] Immobilized lipase can be quickly recovered and has good reusability, including:

[0027] (1) Magnetic separation can be used to quickly and conveniently recover lipase immobilized on ionic liquid-modified magnetic β-cyclodextrin;

[0028] (2) The activity recovery rate of immobilized lipase reached 78.9%. After the immobilized lipase was used continuously for 5 times, the yield of sterol esters could reach 81.0%.

[0029] The beneficial effects of the present invention are:

[0030] (1) The present invention uses a coprecipitation method to coat cyclodextrin on the surface of magnetic nano-ferroferric oxide, and uses ionic liquid to modify the synthesized magnetic β-cyclodextrin, thereby increasing the flexible structure of the carrier surface and its biocompatibility and stabilizing the conformation of the enzyme itself. The carrier can efficiently immobilize Candida plicata lipase on its surface by covalent interaction, thereby improving the microenvironment of the immobilized enzyme and being an excellent carrier for immobilizing lipase. The organic-inorganic magnetic nanocomposite immobilized enzyme prepared by this method has the characteristics of easy recovery and separation, high activity recovery rate and good reusability. The prepared magnetic nanocomposite immobilized lipase can be used to catalyze the esterification reaction of plant sterols and fatty acids to prepare plant sterol esters. The present invention provides a new method for synthesizing plant sterol esters in a green and efficient manner.

[0031] (2) The carboxyl functionalized ionic liquid modification of the present invention increases the flexibility between the enzyme and the carrier, reduces the diffusion restriction between the enzyme and the substrate, and improves the immobilization efficiency and enzyme activity. The loading amount and activity recovery rate of CRL-Fe3O4@β-CD-CIL reached 76.5 mg / g and 78.9%, respectively, while the loading amount and activity recovery rate of CRL-Fe3O4@β-CD were 66.5 mg / g and 53.7%.

[0032] (3) The ionic liquid-modified magnetic β-cyclodextrin immobilized enzyme of the present invention has good thermal stability and storage stability, and an external magnetic field can be used to achieve rapid and effective recovery of the immobilized enzyme.

[0033] (4) In previous studies, our research group found that Candida plicatilis lipase (CRL) has good catalytic performance in the esterification reaction of pine sterol and oleic acid. On this basis, we tried to develop a new method of magnetic modification combined with ionic liquid modified cyclodextrin to immobilize CRL. The ionic liquid modification was used to achieve flexible immobilization of the enzyme, stabilize the active conformation of the enzyme molecule, and improve the stability of the enzyme-catalyzed synthesis of plant sterol esters. The immobilized enzyme was applied to the esterification reaction of plant sterols and fatty acids and achieved satisfactory results. The modified magnetic β-cyclodextrin immobilized lipase with a lipase addition of 14% was completely reacted in 48 hours. The reaction temperature was 50°C, the reaction conditions were mild, and the yield of plant sterol esters was as high as 95.0%, without dehydrated sterol by-products. After the immobilized lipase after magnetic separation was used for 5 consecutive times, the esterification rate could still reach 81.0%.

[0034] (5) As can be seen from Table 1, the immobilized enzyme loading and activity recovery rate of the immobilized reaction conditions of Candida rugosa lipase (CRL) in step (5) of Example 1 are the best. That is, 75 mg of lipase was dissolved in 50 mL of pH 7 phosphate buffer solution, 500 mg of the obtained functionalized magnetic β-cyclodextrin was added to the lipase mixed solution, and after shaking in a constant temperature water bath at 30°C for 5 hours, the immobilized lipase was obtained by washing and magnetic field separation. After freeze drying, the ionic liquid-modified magnetic β-cyclodextrin immobilized lipase was obtained.

[0035] (6) As can be seen from Table 2, the reaction effect is optimal when the molar ratio of sterol to oleic acid is 1:6; adding the modified magnetic β-cyclodextrin immobilized lipase prepared in Example 1 with a mass fraction of 14% of phytosterol, the reaction time is 48 hours, and the reaction temperature is 50°C, which are the optimal reaction conditions for the synthesis of phytosterol esters catalyzed by the ionic liquid modified magnetic cyclodextrin nanocomposite material immobilized lipase. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1The infrared spectra of the products Fe3O4@β-CD-CIL in the synthesis process of the magnetic β-cyclodextrin and the ionic liquid-modified magnetic β-cyclodextrin prepared in Example 1.

[0037] Figure 2 This is the thermogravimetric characterization diagram of ferrosoferric oxide, magnetic β-CD and ionic liquid modified magnetic β-CD prepared in Example 2.

[0038] Figure 3 This is the gas chromatogram of phytosterols (A) and phytosterol esters (B) in Example 2. DETAILED DESCRIPTION

[0039] The present invention is further described below with reference to the following examples. The examples listed are for illustration only and show that the spirit and scope of the present invention are not limited to the details and modifications thereof.

[0040] The biological enzymes and other reagents involved in the present invention are all purchased from the market, and none of the reagents are further purified; infrared is used for characterization of the immobilized carrier; and gas chromatograph Agilent 6890N is used for quantitative and qualitative determination of phytosterol esters.

[0041] Embodiment 1:

[0042] (1) Synthesis of magnetic β-cyclodextrin by coprecipitation method: 4 g β-CD was dissolved in 200 ml distilled water. Next, 4 g FeCl2·4H2O and 10 g FeCl3·6H2O were added to a round-bottom flask and refluxed at 80 °C until a clear brown solution was obtained. Then 25% by mass ammonia water was injected into the flask until the pH reached about 11-12. Then the temperature was fixed at 80 °C and stirred for 30 min. The product was filtered, washed with ethanol and water and dried in a vacuum oven to obtain Fe3O4@β-CD.

[0043] (2) Disperse 3g of Fe3O4@β-CD in 100mL of toluene and homogenize by ultrasonication. Add 4g of silane coupling agent 3-chloromethoxysilane (30mmol), use nitrogen protection, and heat under reflux for 7h. After the reaction, separate the product by magnetic field, wash with ethanol several times, and then dry in a vacuum oven at 60℃.

[0044] (3) A mixture of 25 mmol imidazole and 25 mmol sodium hydride was stirred in 100 mL toluene for 2 h, and then the product obtained in step (2) was added and refluxed for 24 h. After the solid product was cooled, it was separated by a magnet and then washed with deionized water and ethanol. It was then dried at 60 ° C for 10 h. It was then added to 100 mL acetonitrile for dispersion, and then 25 mmol chloroacetic acid was added. Refluxed under nitrogen protection and stirred for 24 h. The sample was washed with deionized water and ethanol, and then dried in a vacuum oven at 60 ° C. Subsequently, the solid product was exchanged with excess NaBF4 anions in water for 12 h, and finally the product was washed again with water and ethanol and dried. The final product was named Fe3O4@β-CD-CIL.

[0045] The infrared characterization of the prepared Fe3O4@β-CD-CIL is as follows Figure 1 As shown, 3395 cm -1 , 2922 cm -1 and 1037cm -1 The stretching vibrations of -OH, CH and CO on the surface of β-cyclodextrin are related. The infrared spectrum of magnetic β-cyclodextrin prepared by coprecipitation method showed a peak at 570 cm -1 The Fe-O characteristic peak at 2927 cm -1 The additional peak at 1106 cm -1 and 1450 cm -1 Two peaks were also found at 1028 cm-1, which were attributed to the CN stretching vibration of the imidazole ring of the ionic liquid and the COOH vibration of the ionic liquid. In addition, the in-plane asymmetric stretching vibration of the Si-O group appeared at 1028 cm-1. -1 The results showed that the ionic liquid successfully modified the magnetic β-CD.

[0046] like Figure 2 , the total weight of Fe3O4 nanoparticles decreased by 4.6% during the whole process, which was due to the evaporation of water on the surface. The total weight loss of Fe3O4@β-CD was 13.7%, which was due to the continuous oxidative decomposition of β-CD at 250-400 °C. However, the total weight loss of Fe3O4@β-CD-CIL was 20.7%, which was attributed to the decomposition of ionic liquid. Thermogravimetric analysis results showed that Fe3O4@β-CD nanoparticles were successfully modified by ionic liquid.

[0047] (4) The activation method of carboxyl functionalized ionic liquid modified magnetic β-cyclodextrin Fe3O4@β-CD-CIL is as follows: 0.01 mol N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 0.01 mol N-hydroxysuccinimide (NHS) and 0.1 g Fe3O4@β-CD-CIL are ultrasonically dispersed in 50 mL phosphate buffer solution pH 6.0 and stirred in a low-temperature stirrer for 1 h;

[0048] (5) At room temperature, 75 mg of Candida rugosa lipase was dissolved in 50 mL of pH 7 phosphate buffer solution. 500 mg of the obtained ionic liquid-modified magnetic β-cyclodextrin was added to the lipase mixed solution, and after shaking in a constant temperature water bath at 30°C for 5 hours, the immobilized lipase was obtained by washing and magnetic field separation. After freeze drying, the ionic liquid-modified magnetic β-cyclodextrin immobilized lipase was obtained.

[0049] (6) In order to measure the thermal stability of the immobilized lipase, the Candida plicata lipase and the immobilized Candida plicata lipase prepared in step (5) were subjected to thermal stability test. After treatment at 60°C for 2 hours, the residual activities of the Candida plicata lipase and the immobilized Candida plicata lipase reached 27% and 81% of the initial activity, respectively. In addition, after storage at 4°C for 30 days, the Candida plicata lipase retained only 25% of the initial activity, while the immobilized lipase retained 73% of the initial activity after storage for 30 days.

[0050] (7) In order to better highlight the advantages of ionic liquids, Fe3O4@β-CD-CIL was used to immobilize lipase under optimized conditions (enzyme addition: 150 mg / g; immobilization time: 5 h; pH: 7; immobilization temperature: 30°C). The loading amount and activity recovery rate of immobilized lipase CRL-Fe3O4@β-CD-CIL were 76.5 mg / g and 78.9%, respectively. Compared with the immobilized enzyme CRL-Fe3O4@β-CD, the loading amount increased by 10.0 mg / g; the activity recovery rate increased by 25.2%.

[0051] Embodiment 2:

[0052] The synthesis process of the immobilized enzyme was similar to that of Example 1, except that the reaction conditions for immobilizing the lipase in step (5) were different. The loading amount and activity recovery rate of the immobilized lipase Fe3O4@β-CD-CIL under different reaction conditions are shown in Table 1

[0053] Table 1 Effect of different immobilization conditions on the immobilization results of Fe3O4@β-CD-CIL

[0054] As can be seen from Table 1, the immobilized enzyme loading and activity recovery rate of the immobilized reaction conditions of Candida rugosa lipase (CRL) in step (5) of Example 1 are the best. That is, 75 mg of lipase is dissolved in 50 mL of pH 7 phosphate buffer solution, 500 mg of the obtained functionalized magnetic β-cyclodextrin is added to the lipase mixed solution, and after shaking in a constant temperature water bath at 30°C for 5 hours, the immobilized lipase is obtained by washing and magnetic field separation. After freeze drying, the ionic liquid-modified magnetic β-cyclodextrin immobilized lipase is obtained.

[0055] Embodiment 3:

[0056] The preparation process of magnetic immobilized enzyme is based on the optimal conditions in Reference Example 1.

[0057] Add 1 mmol of pine sterol and 4-8 mmol of oleic acid into the reaction bottle, heat to 50℃ and mix well, then

[0058] Then, 12% of the mass fraction of phytosterols was added to the modified magnetic β-cyclodextrin immobilized lipase, stirred at 50°C, and the reaction progress was detected by TLC (n-hexane: anhydrous ether: glacial acetic acid, 90:10:1, v / v / v). After 48 hours of reaction, the immobilized enzyme was separated and recovered by an external magnetic field, and the esterification rate was detected by gas phase detection, as shown in Table 2.

[0059] The reaction effect is optimal when the molar ratio of pine sterol to oleic acid is 1:6.

[0060] Table 2 Effect of different substrate molar ratios on reaction performance

[0061] Embodiment 4:

[0062] The preparation process of magnetic immobilized enzyme is shown in Example 1.

[0063] 1mmol of pine sterol and 6mmol of oleic acid were added to the reaction bottle, heated to 50°C and mixed evenly, then the modified magnetic β-cyclodextrin immobilized lipase prepared in Example 1 with a mass fraction of 14% of phytosterol was added, stirred at 50°C, and the reaction progress was detected by TLC (n-hexane: anhydrous ether: glacial acetic acid, 90:10:1, v / v / v). After reacting for 48 hours, the immobilized enzyme was separated and recovered by an external magnetic field, and the esterification rate was 95.0% using gas phase detection.

[0064] Embodiment 5:

[0065] The preparation process of magnetic immobilized enzyme is shown in Example 1.

[0066] Add 1 mmol of pine sterol and 6 mmol of oleic acid into the reaction bottle, heat to 50℃ and mix well, then

[0067] The modified magnetic β-cyclodextrin immobilized lipase prepared in Example 1 with a mass fraction of 14% of phytosterol was added, stirred at 50°C, and the reaction progress was detected by TLC (n-hexane: anhydrous ether: glacial acetic acid, 90:10:1, v / v / v). After 18, 24, 36, and 48 hours of reaction, the immobilized enzyme was separated and recovered by applying an external magnetic field, and the esterification rate was detected by gas phase detection, as shown in Table 3. The reaction was not complete in 24 hours. The reaction was complete in 48 hours.

[0068] Table 3 Effect of reaction time on esterification of phytosterols

[0069] 1 mmol of pine sterol and 6 mmol of oleic acid were added to a reaction bottle, heated to 50°C and mixed evenly, then the modified magnetic β-cyclodextrin immobilized lipase prepared in Example 1 was added with a mass fraction of 14% of phytosterol, and stirred at 50°C. TLC (n-hexane: anhydrous ether: glacial acetic acid, 90:10:1, v / v / v) was used to detect the progress of the reaction. After reacting for 48 hours, an external magnetic field was applied to separate and recover the immobilized enzyme. The esterification rate measured by gas phase was 95.0%. The results are as follows: Figure 3 As shown, there are no by-products. The immobilized lipase recovered by magnetic field separation can be directly used in the next round of reaction. After repeated use for 5 times, the esterification rate of sterol still reached 81.0%.

[0070] Embodiment 6:

[0071] The preparation process of magnetic immobilized enzyme is shown in Example 1.

[0072] 1 mmol of pine sterol and 6 mmol of oleic acid were added to the reaction bottle, heated to 55°C and mixed evenly, and then the modified magnetic β-cyclodextrin immobilized lipase prepared in Example 1 with a mass fraction of 14% of phytosterol was added, stirred at 55°C, and the reaction progress was detected by TLC (n-hexane: anhydrous ether: glacial acetic acid, 90:10:1, v / v / v). After 24 hours of reaction, the immobilized enzyme was separated and recovered by an external magnetic field, and the esterification rate was 92.3% using gas phase detection. After the immobilized lipase after the reaction was recovered with a magnet, the esterification rate was only 38% after 5 consecutive cycles. This example shows that increasing the temperature can accelerate the reaction rate, but increasing the temperature will lead to the inactivation of the enzyme, thereby reducing the reusability of the immobilized enzyme.

[0073] Embodiment 7:

[0074] The preparation process of magnetic immobilized enzyme is shown in Example 1.

[0075] 1 mmol of pine sterol and 6 mmol of linolenic acid were added to the reaction bottle, heated to 50°C and mixed evenly, then the modified magnetic β-cyclodextrin immobilized lipase prepared in Example 1 with a mass fraction of 14% of phytosterol was added, stirred at 50°C, and the reaction progress was detected by TLC (n-hexane: anhydrous ether: glacial acetic acid, 90:10:1, v / v / v). After 48 hours of reaction, the immobilized enzyme was separated and recovered by an external magnetic field, and the esterification rate was 92.6% using gas phase detection.

[0076] Embodiment 8:

[0077] The preparation process of magnetic immobilized enzyme is referred to Example 1.

[0078] 1mmol of pine sterol and 6mmol of linoleic acid were added to the reaction bottle, heated to 50°C and mixed evenly, and then the modified magnetic β-cyclodextrin immobilized lipase prepared in Example 1 with a mass fraction of 14% of phytosterol was added, stirred at 50°C, and the reaction progress was detected by TLC (n-hexane: anhydrous ether: glacial acetic acid, 90:10:1, v / v / v). After 48 hours of reaction, the immobilized enzyme was separated and recovered by an external magnetic field, and the esterification rate was 93.9% using gas phase detection.

[0079] Embodiment 9:

[0080] The preparation process of magnetic immobilized enzyme is as in Reference Example 1.

[0081] 1mmol of pine sterol and 2mmol of oleic acid, 2mmol of linoleic acid, and 2mmol of linolenic acid (simulated mixed fatty acids) were added to a reaction bottle, heated to 50°C and mixed evenly, and then the modified magnetic β-cyclodextrin immobilized lipase prepared in Example 1 with a mass fraction of 14% of phytosterol was added, stirred at 50°C, and the reaction progress was detected by TLC (n-hexane: anhydrous ether: glacial acetic acid, 90:10:1, v / v / v). After 48 hours of reaction, the immobilized enzyme was separated and recovered by an external magnetic field, and the esterification rate was 94.5% using gas phase detection.

[0082] The present invention is not limited to the specific technical solutions described in the above embodiments, and all technical solutions formed by equivalent replacement are within the protection scope required by the present invention.

Claims

1. A method for synthesizing phytosterol esters by catalyzing lipase immobilized on an ionic liquid-modified magnetic cyclodextrin nanocomposite material, characterized in that: (1) Preparation of magnetic β-cyclodextrin by coprecipitation method; (2) Modification of magnetic β-cyclodextrin with carboxyl-functionalized imidazole ionic liquid; (3) activating the magnetic β-cyclodextrin modified with the functionalized imidazole ionic liquid obtained in step (2); (4) Preparation of ionic liquid-modified magnetic β-cyclodextrin immobilized Candida plicata lipase: adding the activated ionic liquid-modified magnetic β-cyclodextrin obtained in step (3) to a lipase solution, wherein the amount of the Candida plicata lipase added is 90-150 mg / g relative to the carrier mass, the temperature is 25-35° C., the pH is 6.5-7.5, and the lipase is shaken for 3-7 hours to perform a lipase immobilization reaction, and then the immobilized lipase with high activity, high thermal stability and high tolerance is obtained by magnetic field separation; (5) Adding phytosterols and fatty acids as substrates into a reactor, followed by adding the immobilized lipase prepared in step (4), and performing an esterification reaction under solvent-free conditions under heating and stirring conditions.

2. The method for synthesizing phytosterol esters by catalyzing lipase using an ionic liquid-modified magnetic cyclodextrin nanocomposite material according to claim 1, characterized in that: The conditions for preparing the immobilized lipase in step (4) are as follows: the addition amount of Candida plicatilis lipase: 150 mg / g relative to the mass of the carrier; the immobilization time: 5 h; pH: 7; Immobilization temperature: 30°C.

3. The method for synthesizing phytosterol esters by catalyzing lipase using an ionic liquid-modified magnetic cyclodextrin nanocomposite material according to claim 1, characterized in that: The specific preparation steps include: (1) Synthesis of magnetic β-cyclodextrin by coprecipitation method: 4 g β-CD was dissolved in 200 ml distilled water, 4 g FeCl2·4H2O and 10 g FeCl3·6H2O were added to a round-bottom flask and refluxed at 80 °C until a clear brown solution was obtained; then 25% ammonia water was injected into the flask until the pH reached 11-12, and then the temperature was fixed at 80 °C and stirred for 30 min; the product was filtered, washed with ethanol and water and dried in a vacuum oven to obtain Fe3O4@β-CD; (2) Disperse 3 g of Fe3O4@β-CD in 100 mL of toluene, homogenize with ultrasound, add 4 g of silane coupling agent 3-chloromethoxysilane, use nitrogen protection, heat and reflux for 7 h, separate the product by magnetic field after the reaction, wash with ethanol several times, and then dry in a vacuum drying oven at 60 °C; (3) A mixture of 25 mmol imidazole and 25 mmol sodium hydride was stirred in 100 mL toluene for 2 h, and then the product obtained in step (2) was added and refluxed for 24 h. After the solid product was cooled, it was separated by a magnet, and then washed with deionized water and ethanol, and then dried at 60 ° C for 10 h. It was then added to 100 mL acetonitrile for dispersion, and then 25 mmol chloroacetic acid was added. The mixture was refluxed under nitrogen protection and stirred for 24 h. The sample was washed with deionized water and ethanol, and then dried in a vacuum oven at 60 ° C. Subsequently, the solid product was exchanged with excess NaBF4 anions in water for 12 h. Finally, the product was washed again with water and ethanol and dried. The final product was named Fe3O4@β-CD-CIL; (4) Activate the functionalized ionic liquid modified magnetic β-cyclodextrin Fe3O4@β-CD-CIL obtained in step (3) by the following method: ultrasonically disperse 0.01 mol N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 0.01 mol N-hydroxysuccinimide (NHS) and 0.1 g Fe3O4@β-CD-CIL in 50 mL phosphate buffer solution, pH 6.0, and stir in a low-temperature stirrer for 1 h; (5) 75 mg of Candida rugosa lipase was dissolved in 50 mL of pH 7 phosphate buffer solution, and then 500 mg of the ionic liquid functionalized magnetic β-cyclodextrin Fe3O4@β-CD-CIL obtained in step (4) was added to the lipase mixed solution. After shaking in a constant temperature water bath at 30°C for 5 hours, the immobilized lipase was obtained by washing and magnetic field separation, and then freeze-dried to obtain the ionic liquid modified magnetic β-cyclodextrin immobilized lipase.

4. The method for synthesizing phytosterol esters in a solvent-free system using lipase immobilized in an ionic liquid-modified magnetic nanocomposite material according to claim 1, characterized in that: The fatty acid in the esterification reaction catalyzed by the immobilized lipase is one of oleic acid, linoleic acid and linolenic acid or a mixed fatty acid.

5. The method for synthesizing phytosterol esters in a solvent-free system using lipase immobilized in an ionic liquid-modified magnetic nanocomposite material according to claim 4, characterized in that: The oleic acid, linoleic acid, linolenic acid or a mixed fatty acid is used as the reaction medium.

6. The method for synthesizing phytosterol esters in a solvent-free system using lipase immobilized in an ionic liquid-modified magnetic nanocomposite material according to claim 1, characterized in that: The molar ratio of the phytosterol to oleic acid is 1:4-1:

8.

7. The method for synthesizing phytosterol esters in a solvent-free system using lipase immobilized in an ionic liquid-modified magnetic nanocomposite material according to claim 1, characterized in that: The molar ratio of the phytosterol to oleic acid is 1:6, and the amount of the immobilized lipase added in the reaction is 14% by mass of the phytosterol; the reaction temperature of the reaction is 50° C.; and the reaction time of the reaction is 48 hours.

8. The method for synthesizing phytosterol esters in a solvent-free system by catalyzing lipase immobilized on an ionic liquid-modified magnetic cyclodextrin nanocomposite material according to claim 1, characterized in that: The following steps are involved: (1) Synthesis of magnetic β-cyclodextrin by coprecipitation method: 4 g β-CD was dissolved in 200 ml distilled water, 4 g FeCl2·4H2O and 10 g FeCl3·6H2O were added to a round-bottom flask and refluxed at 80 °C until a clear brown solution was obtained. A certain concentration of ammonia water was then injected into the flask until the pH reached 11-12. The temperature was then fixed at 80 °C and stirred for 30 min. The product was filtered, washed with ethanol and water and dried in a vacuum oven to obtain Fe3O4@β-CD. (2) Disperse 3 g of Fe3O4@β-CD in 100 mL of toluene and mix them evenly with ultrasound. Add 4 g of silane coupling agent 3-chloromethoxysilane (30 mmol) and heat under reflux for 7 h under nitrogen protection. After the reaction, separate the product by magnetic field, wash it with ethanol several times, and then dry it in a vacuum oven at 60 °C. (3) A mixture of 25 mmol imidazole and 25 mmol sodium hydride was stirred in 100 mL toluene for 2 h, then the product obtained in step (2) was added and refluxed for 24 h. After the solid product was cooled, it was separated by a magnet, then washed with deionized water and ethanol, and then dried at 60 ° C for 10 h. It was then added to 100 mL acetonitrile for dispersion, and then 25 mmol chloroacetic acid was added. It was refluxed under nitrogen protection and stirred for 24 h. The sample was washed with deionized water and ethanol, and then dried in a vacuum oven at 60 ° C. Subsequently, the solid product was exchanged with excess NaBF4 anions in water for 12 h, and finally the product was washed again with water and ethanol and dried. The final product was named Fe3O4@β-CD-CIL; (4) Activate the functionalized ionic liquid modified magnetic β-cyclodextrin Fe3O4@β-CD-CIL obtained in step (3) by the following method: ultrasonically disperse 0.01 mol N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 0.01 mol N-hydroxysuccinimide (NHS) and 0.1 g Fe3O4@β-CD-CIL in 50 mL phosphate buffer solution, pH 6.0, and stir in a low-temperature stirrer for 1 h; (5) Dissolve 45-75 mg of Candida rugosa lipase in 50 mL of pH 7 phosphate buffer solution; Then, 500 mg of the ionic liquid functionalized magnetic β-cyclodextrin obtained in step (4) was added to the lipase mixed solution, and the solution was shaken in a constant temperature water bath at 30° C. for 5 hours, followed by washing and magnetic field separation to obtain the immobilized lipase; and after freeze drying, the ionic liquid modified magnetic β-cyclodextrin immobilized lipase was obtained; 1 mmol pine sterol and 6 mmol oleic acid were added to the reaction bottle, heated to 50 ℃ and mixed evenly, then modified magnetic β-cyclodextrin immobilized lipase with a mass fraction of 14% of phytosterol was added, and stirred at 50 ℃. TLC (n-hexane: anhydrous ether: glacial acetic acid, 90:10:1, v / v / v) was used to detect the reaction progress. After 48 hours of reaction, an external magnetic field was applied to separate and recover the immobilized enzyme. The esterification rate measured by gas phase was 95.0%, and there were no by-products. After the recovered immobilized lipase was reacted for 5 consecutive cycles, the yield of sterol esters could reach 81.0%.

9. The method for synthesizing phytosterol esters in a solvent-free system using lipase immobilized in an ionic liquid-modified magnetic nanocomposite material according to claim 1, characterized in that: Immobilized lipase can be quickly recovered and has good reusability, including: (1) Magnetic separation can be used to quickly and conveniently recover lipase immobilized on ionic liquid-modified magnetic β-cyclodextrin; (2) The activity recovery rate of immobilized lipase reached 78.9%. After the immobilized lipase was used continuously for 5 times, the yield of sterol esters could reach 81.0%.

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