A method for the enzymatic catalytic preparation of diester oils

Abstract

The application relates to the technical field of oil processing, and discloses a method for preparing diester oil through enzyme catalysis, which comprises the following steps: adding plant raw oil, glycerol, tert-butyl alcohol and cross-linked gelatin immobilized whole-cell enzyme into a reactor to mix, performing constant-temperature mass transfer reaction under normal pressure by heating, stopping heating when the reaction reaches the end point, and performing vacuumization on the reactor under reduced pressure, using part of the tert-butyl alcohol in the system to absorb latent heat through reduced-pressure boiling vaporization, so that the system is rapidly cooled; then, the vaporized tert-butyl alcohol is condensed and refluxed, and the constant-temperature quenching reaction is continuously performed at a lower temperature; finally, the cross-linked gelatin immobilized whole-cell enzyme is separated, the supernatant is collected, and the tert-butyl alcohol is removed, so that the diester oil is obtained. The method utilizes reduced-pressure flashing to realize in-volume synchronous heat absorption, eliminates the lag phenomenon of conventional physical heat transfer, makes the reaction system rapidly drop below the activation energy threshold of consecutive side reactions, effectively blocks the conversion generation of by-products, and guarantees the overall yield of the target product.

Description

Technical Field

[0001] This invention relates to the field of oil processing technology, specifically to a method for preparing diester oil by enzymatic catalysis. Background Technology

[0002] The core active component of diglyceride oil is diglyceride. In industrial preparation, compared with traditional chemical catalysis, enzymatic catalysis has the advantages of mild reaction conditions and high specificity of the target product. In the transesterification or alcoholysis system for the enzymatic synthesis of diglycerides, the plant raw material oil is nonpolar, while glycerol exhibits strong polarity, and the natural miscibility between the two is very low. In order to reduce the mass transfer resistance of substrate mixing and increase the rate of enzymatic reaction, it is usually necessary to add an organic co-solvent and carry out the reaction at a higher temperature in actual production.

[0003] While this high-temperature environment promotes the positive main reaction, it also provides the thermodynamic conditions for the system to overcome the activation energy barrier of side reactions. The already formed diglycerides readily react with glycerol at high temperatures to form monoglycerides, or undergo intermolecular acyl transfer, recombining into monoglycerides and triglycerides. To suppress the consumption of the target product by this series of side reactions, the process requires a rapid reduction in system temperature when the main reaction reaches a high conversion rate. Existing industrial reactors commonly use a jacketed cooling medium for physical cooling. This heat transfer method, relying on the reactor wall, is limited by the thermal resistance of the heat transfer boundary layer, resulting in a significant lag in the overall cooling process. During the slow cooling transition period, the reaction system remains in a high-temperature range prone to side reactions, leading to a large amount of diglycerides being converted into byproducts, severely restricting the final yield of the target product.

[0004] Furthermore, industrial-scale enzymatic production relies on immobilization technology to recycle biocatalysts. Gelatin, due to its excellent biocompatibility and gelling properties, is often used as an embedding carrier for immobilized whole-cell enzymes. However, in the high-temperature organic co-solvent system required for the preparation of diester oils, conventionally physically embedded gelatin carriers are prone to phase transition and melting from gel to sol. Damage to the carrier structure directly leads to the loss of embedded cytoplasm, and the enzyme protein, deprived of steric protection, is also prone to unfolding and inactivation at high temperatures. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing diester oil by enzymatic catalysis, which solves the technical problem that the conventional cooling process for preparing diester oil leads to increased side reactions and reduced yield of the target product.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing diester oil by enzymatic catalysis, comprising the following steps:

[0007] Vegetable raw material oil, glycerin, tert-butanol, and cross-linked gelatin-immobilized whole-cell enzymes were added to the reactor and mixed. Under normal pressure and stirring conditions, the temperature of the system inside the reactor is raised to 60-70°C to carry out an isothermal mass transfer reaction; When the isothermal mass transfer reaction reaches its endpoint, heating is stopped, and the reactor is depressurized and evacuated to reduce the pressure inside the reactor. The latent heat is absorbed by the depressurized boiling vaporization of some tert-butanol in the system, thereby cooling the system. The tert-butanol vaporized in the vacuum stage is condensed and refluxed back into the reactor, where it continues to undergo isothermal quenching reaction at 45–55°C with continuous stirring. After the reaction is complete, the cross-linked gelatin-immobilized whole-cell enzyme at the bottom is separated, the supernatant is collected and tert-butanol is removed, and the remaining unreacted glycerol is separated to obtain the diester oil.

[0008] By adopting the above technical solution, the following effects are achieved: This invention divides the enzymatic synthesis of diglycerides into two stages: high-temperature mass transfer and low-temperature quenching, and introduces a reduced-pressure flash cooling operation between the two. The reactions within the synthesis system mainly involve the alcoholysis of fats and oils. The main reaction involves the transesterification and alcoholysis of triglycerides with added glycerol, resulting in the target product, diglycerides, and some monoglycerides. Accompanying this process are a series of side reactions and acyl transfer phenomena; specifically, the already generated diglycerides continue to react with glycerol, converting into more monoglycerides and free fatty acids. Simultaneously, acyl transfer occurs between diglyceride molecules, causing them to recombine into monoglycerides and triglycerides. Because the activation energy barriers for these side reactions are relatively high, the system achieves thermodynamic conditions that allow it to overcome these barriers at temperatures between 60 and 70°C. While high temperatures can increase the miscibility of plant-based oils with glycerol and reduce mass transfer resistance, thereby accelerating the forward alcoholysis process, they can also easily lead to a significant conversion of already synthesized diglycerides into monoglycerides.

[0009] To address this contradiction, this scheme stops heating and switches to a vacuum system when the mass transfer reaction reaches its endpoint. Based on the phase equilibrium mechanism, when the absolute pressure of the system is reduced to below the saturated vapor pressure of tert-butanol, the tert-butanol deep in the liquid phase will directly boil and vaporize. Since vaporization requires the absorption of latent heat of phase change, this heat is directly provided by the sensible heat of the main body of the reaction system. Conventional jacket cooling is often limited by the heat conduction of the reactor wall, which can easily lead to cooling lag. Vacuum-reduced flash evaporation is essentially an endothermic process that occurs simultaneously throughout the entire volume, avoiding the thermal resistance caused by the heat transfer boundary layer. This allows the system temperature to drop rapidly, and the total system energy immediately falls below the activation energy threshold required for a series of side reactions. This endothermic phase change induced by physical decompression effectively cuts off the thermodynamic pathway for the continued conversion of diglycerides, locking the content of the target product at a high level.

[0010] Preferably, in the step of adding the ingredients to the reactor for mixing, the plant raw material oil, glycerol, tert-butanol, and cross-linked gelatin-immobilized whole-cell enzyme are added to the reactor and mixed in the following parts by weight: 100 parts by weight of vegetable oil; 4.0 to 8.0 parts by weight of glycerin; 50-150 parts by weight of tert-butanol; Cross-linked gelatin immobilized whole-cell enzymes, 1.0–5.0 parts by weight.

[0011] The plant-based oil is selected from at least one of rapeseed oil, soybean oil, peanut oil, sunflower oil, and corn oil.

[0012] By adopting the above technical solution, the added tert-butanol mainly acts as a homogeneous cosolvent in the system, which helps to improve the miscibility between nonpolar vegetable oil and polar glycerol. In addition, it also acts as a volatile medium to provide latent heat of vaporization in the subsequent depressurization stage, thereby completing the phase change and cooling of the system.

[0013] Preferably, the isothermal mass transfer reaction takes 3 to 5 hours, and the stirring speed of the isothermal mass transfer reaction is 150 to 250 rpm. The isothermal quenching reaction takes 5 to 9 hours, the stirring speed of the isothermal quenching reaction is 150 to 250 rpm, and the total reaction time of the isothermal mass transfer reaction and the isothermal quenching reaction is controlled to be 8 to 14 hours.

[0014] The pressure reduction and vacuuming operation involves rapidly reducing the absolute pressure to 10-40 kPa within 1-5 minutes and rapidly reducing the temperature of the mixed liquid in the reactor to 45-55°C within 3-8 minutes.

[0015] By employing the above technical solution, the absolute pressure is controlled within the range of 10–40 kPa. This effectively ensures that the boiling point of tert-butanol falls precisely within the temperature range of 45–55 °C, thus controlling the final cooling endpoint. This pressure change, completed within minutes, rapidly removes the system temperature from the high-temperature sensitive zone that is prone to side reactions, thereby achieving a quenching effect.

[0016] Preferably, the cross-linked gelatin-immobilized whole-cell enzyme is prepared by a method comprising the following steps: Recombinant Escherichia coli expressing the Candida antarcticis lipase B gene was cultured, and wet cells were collected by centrifugation after the expression was induced. The wet bacterial cells are mixed into a gelatin aqueous solution that is heated and dissolved, and stirred evenly. Then, they are dripped into a cooling liquid at a uniform rate, where they undergo a phase change upon cooling to form spherical gelatin particles. The spherical gelatin particles were chemically cross-linked by immersing them in an aqueous solution of glutaraldehyde, and after washing and drying, the cross-linked gelatin-immobilized whole-cell enzyme was obtained.

[0017] In the process of culturing recombinant Escherichia coli, after the recombinant Escherichia coli is cultured in fermentation medium to the logarithmic growth phase, isopropyl-β-D-thiogalactoside is added as an inducer at a final concentration of 0.05–0.2 mmol / L. Then the fermentation temperature is lowered to 16–30 °C and cultured for 16–24 hours.

[0018] By employing the above technical solution, for the catalyst portion, the gelatin carrier treated with glutaraldehyde undergoes a Schiff base reaction with the aldehyde groups of glutaraldehyde, forming a three-dimensional covalent network. This network structure alters the original thermodynamic state of the gelatin to some extent, restricting the relative slippage between polypeptide chains and allowing the gel particles to maintain their original solid shape even in high-temperature organic solvent environments. This modification treatment prevents the physical detachment of embedded cells and provides a steric barrier for lipases, making the enzyme protein less prone to unfolding and inactivation at high temperatures, ultimately maintaining the catalyst's operational lifespan through multiple cycles.

[0019] Preferably, the specific implementation of forming spherical gelatin particles is as follows: Gelatin is heated and dissolved at 50-60°C to prepare a gelatin aqueous solution with a mass concentration of 5%-15%, and then cooled and kept constant at 35-40°C. The wet bacterial cells were added to a gelatin aqueous solution at a mass ratio of 1:2 to 1:5 to obtain an embedding mixture. Granulation is achieved by dripping the encapsulation mixture into cold water or cold liquid paraffin at 0–5°C using a pelletizing device.

[0020] The specific method for performing chemical cross-linking is as follows: Prepare a glutaraldehyde aqueous solution with a mass fraction of 0.1% to 2.0%, and adjust the pH to 6.5 to 7.5; Gelatin particles were soaked in glutaraldehyde aqueous solution at a solid-liquid ratio of 1:5 to 1:10, and the mixture was shaken and reacted at 0 to 10°C for 1 to 4 hours.

[0021] By adopting the above technical solution, the control of parameters such as glutaraldehyde concentration and cross-linking time is to ensure that the carrier has the necessary structural strength, while avoiding the blockage of the mass transfer channels for substrate entry and exit due to excessive cross-linking, or adverse changes in the conformation of the enzyme active site.

[0022] Preferably, in the step of separating the product after the reaction, the cross-linked gelatin immobilized whole-cell enzyme is recovered by low-speed centrifugation at 2000-4000 rpm. The supernatant was subjected to reduced pressure rotary evaporation or thin-film evaporation under vacuum conditions to remove tert-butanol, and the remaining unreacted glycerol at the bottom was separated by centrifugation.

[0023] By adopting the above technical solution, the separation step is mainly used for the recycling of solid-phase catalysts and the purification of liquid-phase final products, which can control the overall material consumption cost in multi-batch continuous production.

[0024] This invention provides a method for preparing diester oil by enzymatic catalysis. It has the following beneficial effects: 1. This invention introduces a depressurization and vacuum operation into the reaction system. The tert-butanol in the system undergoes boiling vaporization when the pressure decreases, absorbing the latent heat of phase change to achieve overall cooling of the reaction liquid. Conventional jacket cooling suffers from boundary layer thermal resistance, easily leading to cooling lag. The depressurization flash evaporation method in this invention is a simultaneous intravolume endothermic process, capable of rapidly reducing the system temperature below the activation energy threshold required for a series of side reactions. This blocks the pathway for the already formed diglyceride to undergo further acyl transfer and transform into byproducts, thereby ensuring the yield of the target product.

[0025] 2. The enzymatic synthesis process of this invention is divided into a heating mass transfer stage and a cooling quenching stage. The initial heating operation aims to improve the miscibility of nonpolar vegetable oil and polar glycerol, thereby reducing the mass transfer resistance of substrate mixing and promoting the alcoholysis process of the main reaction. The subsequent low-temperature quenching operation avoids the sensitive temperature range that easily triggers side reactions by changing the thermodynamic state of the system. This segmented control method controls the formation of byproducts while maintaining substrate conversion efficiency.

[0026] 3. This invention employs chemical cross-linking to treat a gelatin carrier to prepare immobilized whole-cell enzymes. The covalent network structure formed after cross-linking alters the thermodynamic state of the gelatin carrier, restricting the relative slippage of the internal polypeptide chains. This allows the gel particles to maintain their solid shape even in heated organic solvent environments, avoiding the phase transition melting phenomenon that easily occurs with conventional physical embedding carriers. This modification treatment not only prevents the physical detachment of embedded cells but also protects the active conformation of the enzyme, extending the catalyst's lifespan in continuous cyclic feeding. Attached Figure Description

[0027] Figure 1 The graph shows the change in mass retention rate of the cross-linked gelatin immobilized whole-cell carrier of the present invention under high temperature and mechanical shear conditions over time. Figure 2 This is a comparison diagram of the cooling trajectory of the micro-negative pressure in-situ flash evaporation temperature-changing operation of the present invention and the traditional jacketed cooling in an industrial scale-up reactor. Figure 3 A dot-line trend diagram showing the composition distribution of the final product under different process conditions of the present invention and that of the comparative example. Figure 4 This is a graph showing the continuous cycle life decay of the cross-linked gelatin-immobilized whole-cell catalyst of the present invention under different process conditions. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the preparation examples, comparative examples, and test examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Preparation Examples 1-4: Preparation Example 1: This preparation example provides a method for preparing cross-linked gelatin-immobilized whole-cell enzymes, including the following steps: The recombinant expression vector pET-21a(+) carrying the Candida antarcticis lipase B gene was transformed into Escherichia coli BL21 competent cells to construct a recombinant expression strain. The recombinant expression strain was inoculated into fermentation medium and cultured at 37°C to the logarithmic growth phase. Isopropyl-β-D-thiogalactoside was added to the fermentation broth to a final concentration of 0.1 mmol / L. The fermentation temperature was lowered to 20°C and cultured for 20 hours. After fermentation, the recombinant E. coli cells were collected by centrifugation at 6000 rpm for 10 minutes and washed twice with physiological saline.

[0030] Weigh out gelatin and add it to purified water. Heat and stir in a 55°C water bath to dissolve the gelatin, preparing a 10% (w / w) gelatin aqueous solution. Cool the solution and maintain the temperature at 40°C. Mix the collected wet bacterial cells with the 40°C gelatin aqueous solution at a mass ratio of 1:3 and stir at low speed until homogeneous to obtain the embedding mixture.

[0031] The embedding mixture was dripped at a constant rate into ice water at 0°C using a syringe pump to form spherical gel particles, which were then collected by filtration.

[0032] Prepare a 0.5% (w / w) glutaraldehyde aqueous solution and adjust the pH to 7.0 with phosphate buffer. Immerse gelatin particles in the glutaraldehyde crosslinking solution, with a particle mass to crosslinking solution volume solid-liquid ratio of 1:5 (g / mL). React with shaking at 4°C for 2 hours. After the reaction, filter out the particles, wash with sterile water until no free glutaraldehyde residue remains, and dehydrate and dry in a vacuum drying oven to obtain crosslinked gelatin-immobilized whole-cell enzyme.

[0033] Preparation Example 2: This preparation example provides a method for preparing cross-linked gelatin-immobilized whole-cell enzymes, including the following steps: The recombinant expression vector pET-21a(+) carrying the Candida antarcticis lipase B gene was transformed into Escherichia coli BL21 competent cells to construct a recombinant expression strain. The recombinant expression strain was inoculated into fermentation medium and cultured at 37°C to the logarithmic growth phase. Isopropyl-β-D-thiogalactoside was added to the fermentation broth to a final concentration of 0.05 mmol / L. The fermentation temperature was lowered to 16°C and cultured for 24 hours. After fermentation, the recombinant E. coli cells were collected by centrifugation at 4000 rpm for 15 minutes and washed twice with physiological saline.

[0034] Weigh out gelatin and add it to purified water. Heat and stir in a 50°C water bath to dissolve the gelatin, preparing a 5% (w / w) gelatin aqueous solution. Cool the solution and maintain the temperature at 35°C. Mix the collected wet bacterial cells with the 35°C gelatin aqueous solution at a mass ratio of 1:2 and stir at low speed until homogeneous to obtain the embedding mixture.

[0035] The embedding mixture was dripped at a constant rate into ice water at 0°C using a syringe pump to form spherical gel particles, which were then collected by filtration.

[0036] Prepare a 0.1% (w / w) glutaraldehyde aqueous solution and adjust the pH to 6.5 with phosphate buffer. Immerse gelatin particles in the glutaraldehyde crosslinking solution, with a particle mass to crosslinking solution volume solid-liquid ratio of 1:5 (g / mL). React with shaking at 0°C for 1 hour. After the reaction, filter out the particles, wash with sterile water until no free glutaraldehyde residue remains, and dehydrate and dry in a vacuum drying oven to obtain crosslinked gelatin-immobilized whole-cell enzyme.

[0037] Preparation Example 3: This preparation example provides a method for preparing cross-linked gelatin-immobilized whole-cell enzymes, including the following steps: The recombinant expression vector pET-21a(+) carrying the Candida antarcticis lipase B gene was transformed into Escherichia coli BL21 competent cells to construct a recombinant expression strain. The recombinant expression strain was inoculated into fermentation medium and cultured at 37°C to the logarithmic growth phase. Isopropyl-β-D-thiogalactoside was added to the fermentation broth to a final concentration of 0.2 mmol / L. The fermentation temperature was lowered to 30°C and cultured for 16 hours. After fermentation, the recombinant E. coli cells were collected by centrifugation at 8000 rpm for 10 minutes and washed twice with physiological saline.

[0038] Weigh out gelatin and add it to purified water. Heat and stir in a 60°C water bath to dissolve the gelatin, preparing a 15% (w / w) gelatin aqueous solution. Cool the solution and maintain the temperature at 40°C. Mix the collected wet bacterial cells with the 40°C gelatin aqueous solution at a mass ratio of 1:5 and stir at low speed until homogeneous to obtain the embedding mixture.

[0039] The embedding mixture was dripped at a constant rate into cold liquid paraffin at 5°C using a syringe pump to form spherical gel particles. The particles were then collected by filtration and surface impurities were washed away.

[0040] A 2.0% (w / w) glutaraldehyde aqueous solution was prepared, and the pH was adjusted to 7.5 with phosphate buffer. Gelatin particles were immersed in the glutaraldehyde crosslinking solution, with a particle mass to crosslinking solution volume solid-liquid ratio of 1:10 (g / mL). The reaction was carried out with shaking at 10°C for 4 hours. After the reaction was completed, the particles were filtered out, washed with sterile water until no free glutaraldehyde residue remained, and dehydrated in a vacuum drying oven to obtain crosslinked gelatin-immobilized whole-cell enzymes.

[0041] Preparation Example 4: This preparation example provides a method for preparing cross-linked gelatin-immobilized whole-cell enzymes, including the following steps: The recombinant expression vector pET-21a(+) carrying the Candida antarcticis lipase B gene was transformed into Escherichia coli BL21 competent cells to construct a recombinant expression strain. The recombinant expression strain was inoculated into fermentation medium and cultured at 37°C to the logarithmic growth phase. Isopropyl-β-D-thiogalactoside was added to the fermentation broth to a final concentration of 0.15 mmol / L. The fermentation temperature was lowered to 25°C and cultured for 18 hours. After fermentation, the recombinant E. coli cells were collected by centrifugation at 5000 rpm for 12 minutes and washed twice with physiological saline.

[0042] Weigh out gelatin and add it to purified water. Heat and stir in a 55°C water bath to dissolve the gelatin, preparing a 12% (w / w) gelatin aqueous solution. Cool the solution and maintain the temperature at 38°C. Mix the collected wet bacterial cells with the 38°C gelatin aqueous solution at a mass ratio of 1:4 and stir at low speed until homogeneous to obtain the embedding mixture.

[0043] The embedding mixture was dripped at a constant rate into ice water at 2°C using a syringe pump to form spherical gel particles, which were then collected by filtration.

[0044] Prepare a 1.0% (w / w) glutaraldehyde aqueous solution and adjust the pH to 7.0 with phosphate buffer. Immerse gelatin particles in the glutaraldehyde crosslinking solution, with a particle mass to crosslinking solution volume solid-liquid ratio of 1:8 (g / mL). React with shaking at 4°C for 3 hours. After the reaction, filter out the particles, wash with sterile water until no free glutaraldehyde residue remains, and dehydrate and dry in a vacuum drying oven to obtain crosslinked gelatin-immobilized whole-cell enzymes.

[0045] Examples 1-5: Example 1: This embodiment provides a method for preparing diester oil by enzymatic catalysis, including the following steps: In a reactor equipped with a variable frequency stirrer, a jacketed temperature control system and a top vacuum reflux condenser, 1000 g of rapeseed oil, 62.5 g of glycerol, 1000 g of tert-butanol and 30 g of cross-linked gelatin-immobilized whole-cell enzyme prepared in Preparation Example 1 were added.

[0046] Turn on the stirrer and control the speed at 200 rpm. Heat the system inside the reactor to 65°C using the jacket heating system and maintain the temperature at a constant temperature for 4 hours under normal pressure.

[0047] When the reaction time reached the 4-hour endpoint, the jacket heating source was shut off, and the vacuum pump system connected to the top of the reactor was instantly activated, rapidly reducing the absolute pressure inside the reactor to 20 kPa within 3 minutes. Utilizing the latent heat absorbed by the reduced-pressure boiling vaporization of some tert-butanol within the system, the temperature of the mixed liquid inside the reactor was rapidly and uniformly reduced to 50°C within 5 minutes. The tert-butanol released during flash vaporization was liquefied via the condenser at the top of the reactor and refluxed back into the reactor. The reaction was then continued at a constant temperature of 50°C with continuous stirring at 200 rpm for 6 hours to quench the reaction.

[0048] After the reaction was complete, stirring was stopped, and the cross-linked gelatin-immobilized whole-cell enzyme at the bottom was separated and collected by low-speed centrifugation at 3000 rpm. The supernatant was collected and sent to a vacuum rotary evaporator to remove tert-butanol under vacuum conditions. The trace amounts of unreacted glycerol remaining at the bottom were then separated by centrifugation, finally yielding the diester oil product.

[0049] Example 2: This embodiment provides a method for preparing diester oil by enzymatic catalysis, including the following steps: In a reactor equipped with a variable frequency stirrer, a jacketed temperature control system and a top vacuum reflux condenser, 1000 g of soybean oil, 62.5 g of glycerol, 1000 g of tert-butanol and 30 g of cross-linked gelatin-immobilized whole-cell enzyme prepared in Preparation Example 1 were added.

[0050] Turn on the stirrer and control the speed at 200 rpm. Heat the system inside the reactor to 65°C using the jacket heating system and maintain the temperature at a constant temperature for 4 hours under normal pressure.

[0051] When the reaction time reached the 4-hour endpoint, the jacket heating source was shut off, and the vacuum pump system connected to the top of the reactor was instantly activated, rapidly reducing the absolute pressure inside the reactor to 20 kPa within 3 minutes. Utilizing the latent heat absorbed by the reduced-pressure boiling vaporization of some tert-butanol within the system, the temperature of the mixed liquid inside the reactor was rapidly and uniformly reduced to 50°C within 5 minutes. The tert-butanol released during flash vaporization was liquefied via the condenser at the top of the reactor and refluxed back into the reactor. The reaction was then continued at a constant temperature of 50°C with continuous stirring at 200 rpm for 6 hours to quench the reaction.

[0052] After the reaction was complete, stirring was stopped, and the cross-linked gelatin-immobilized whole-cell enzyme at the bottom was separated and collected by low-speed centrifugation at 3000 rpm. The supernatant was collected and sent to a vacuum rotary evaporator to remove tert-butanol under vacuum conditions. The trace amounts of unreacted glycerol remaining at the bottom were then separated by centrifugation, finally yielding the diester oil product.

[0053] Example 3: This embodiment provides a method for preparing diester oil by enzymatic catalysis, including the following steps: In a reactor equipped with a variable frequency stirrer, a jacketed temperature control system and a top vacuum reflux condenser, 1000 g of rapeseed oil, 62.5 g of glycerol, 1000 g of tert-butanol and 30 g of cross-linked gelatin-immobilized whole-cell enzyme prepared in Preparation Example 1 were added.

[0054] Turn on the stirrer and control the speed at 200 rpm. Heat the system inside the reactor to 70°C using the jacket heating system and maintain the temperature at a constant temperature for 3 hours under normal pressure.

[0055] When the reaction time reaches the 3-hour endpoint, the jacket heating source is shut off, and the vacuum pump system connected to the top of the reactor is instantly activated, rapidly reducing the absolute pressure inside the reactor to 20 kPa within 3 minutes. Utilizing the latent heat absorbed by the reduced-pressure boiling vaporization of some tert-butanol within the system, the temperature of the mixed liquid inside the reactor is rapidly and uniformly reduced to 50°C within 5 minutes. The tert-butanol released during flash vaporization is liquefied via the condenser at the top of the reactor and refluxed back into the reactor. The reaction is then quenched at a constant temperature of 50°C with continuous stirring at 200 rpm for 7 hours.

[0056] After the reaction was complete, stirring was stopped, and the cross-linked gelatin-immobilized whole-cell enzyme at the bottom was separated and collected by low-speed centrifugation at 3000 rpm. The supernatant was collected and sent to a vacuum rotary evaporator to remove tert-butanol under vacuum conditions. The trace amounts of unreacted glycerol remaining at the bottom were then separated by centrifugation, finally yielding the diester oil product.

[0057] Example 4: This embodiment provides a method for preparing diester oil by enzymatic catalysis, including the following steps: In a reactor equipped with a variable frequency stirrer, a jacketed temperature control system and a top vacuum reflux condenser, 1000 g of rapeseed oil, 62.5 g of glycerol, 1000 g of tert-butanol and 30 g of cross-linked gelatin-immobilized whole-cell enzyme prepared in Preparation Example 1 were added.

[0058] Turn on the stirrer and control the speed at 200 rpm. Heat the system inside the reactor to 65°C using the jacket heating system and maintain the temperature at a constant temperature for 4 hours under normal pressure.

[0059] When the reaction time reached the 4-hour endpoint, the jacket heating source was shut off, and the vacuum pump system connected to the top of the reactor was instantly activated, rapidly reducing the absolute pressure inside the reactor to 10 kPa within 3 minutes. Utilizing the latent heat absorbed by the reduced-pressure boiling vaporization of some tert-butanol within the system, the temperature of the mixed liquid inside the reactor was rapidly and uniformly reduced to 45°C within 5 minutes. The tert-butanol released during flash vaporization was liquefied via the condenser at the top of the reactor and refluxed back into the reactor. The reaction was then continued at a constant temperature of 45°C with continuous stirring at 200 rpm for 8 hours to quench the reaction.

[0060] After the reaction was complete, stirring was stopped, and the cross-linked gelatin-immobilized whole-cell enzyme at the bottom was separated and collected by low-speed centrifugation at 3000 rpm. The supernatant was collected and sent to a vacuum rotary evaporator to remove tert-butanol under vacuum conditions. The trace amounts of unreacted glycerol remaining at the bottom were then separated by centrifugation, finally yielding the diester oil product.

[0061] Example 5: This embodiment provides a method for preparing diester oil by enzymatic catalysis, including the following steps: In a reactor equipped with a variable frequency stirrer, a jacketed temperature control system and a top vacuum reflux condenser, 1000 g of rapeseed oil, 62.5 g of glycerol, 1000 g of tert-butanol and 30 g of cross-linked gelatin-immobilized whole-cell enzyme prepared in Preparation Example 1 were added.

[0062] Turn on the stirrer and control the speed at 200 rpm. Heat the system inside the reactor to 65°C using the jacket heating system and maintain the temperature at a constant temperature for 4 hours under normal pressure.

[0063] When the reaction time reached the 4-hour endpoint, the jacket heating source was shut off, and the vacuum pump system connected to the top of the reactor was instantly activated, rapidly reducing the absolute pressure inside the reactor to 40 kPa within 5 minutes. Utilizing the latent heat absorbed by the reduced-pressure boiling vaporization of some tert-butanol within the system, the temperature of the mixed liquid inside the reactor was rapidly and uniformly reduced to 55°C within 8 minutes. The tert-butanol released from flash vaporization was liquefied via the condenser at the top of the reactor and refluxed back into the reactor. The reaction was then quenched at a constant temperature of 55°C with continuous stirring at 200 rpm for 6 hours.

[0064] After the reaction was complete, stirring was stopped, and the cross-linked gelatin-immobilized whole-cell enzyme at the bottom was separated and collected by low-speed centrifugation at 3000 rpm. The supernatant was collected and sent to a vacuum rotary evaporator to remove tert-butanol under vacuum conditions. The trace amounts of unreacted glycerol remaining at the bottom were then separated by centrifugation, finally yielding the diester oil product.

[0065] Comparative Examples 1-3: Comparative Example 1: The difference from Example 1 is that the catalyst used is pure gelatin that has not undergone chemical cross-linking treatment with glutaraldehyde to physically embed whole cells; all other aspects are the same.

[0066] Comparative Example 2: Compared with Example 1, the difference is that the micro-negative pressure flash evaporation and temperature change operation are not performed. Instead, the reaction is carried out at a constant temperature of 60°C for 10 hours under normal pressure. All other aspects are the same.

[0067] Comparative Example 3: Compared with Example 1, the difference is that after the first stage reaction is completed, the vacuum pump system is not turned on for depressurization flash cooling. Instead, the reactor jacket is circulated with cooling water for conventional slow cooling to 50°C. All other aspects are the same.

[0068] Test Examples 1-4: Test Example 1: High Temperature Resistance and Shear Stability Test of Cross-linked Gelatin Carrier Multiple immobilized whole-cell catalyst samples with an oven-dry weight of 10.0 g were weighed. The test samples were obtained from the cross-linked gelatin catalysts prepared in Preparation Example 1, Preparation Example 2, and Preparation Example 3, as well as the uncross-linked pure gelatin catalyst prepared in Comparative Example 1.

[0069] Catalyst samples from different sources were added to multiple parallel reactors containing 1000g of tert-butanol solvent. The variable frequency stirrer of the reactor was set to 200rpm to simulate the fluid shear force in a real industrial catalytic process.

[0070] Turn on the temperature control system of the reactor jacket and keep the temperature of the system inside the reactor constant at two test gradients of 65℃ and 70℃ respectively. Start timing to carry out continuous tolerance simulation experiments.

[0071] At the 2-hour, 4-hour, 8-hour, 12-hour, and 24-hour mark of the experiment, samples were taken through the discharge valve at the bottom of the reactor to obtain a suspension containing the catalyst.

[0072] The extracted suspension was centrifuged at 3000 rpm to separate the solid phase. The surface residue of the solid phase was washed twice with tert-butanol at room temperature, and then the solvent was removed in a vacuum drying oven until constant weight was achieved.

[0073] Accurately weigh the remaining solid carrier after drying and calculate its percentage of the initial input weight to obtain the mass retention rate at the corresponding time point.

[0074] Table 1: Mass retention rate data of different immobilized carriers under high temperature and mechanical shear conditions

[0075] Reference Appendix Figure 1 The data includes the loss curve of the uncrosslinked pure gelatin carrier prepared in Comparative Example 1 at 65°C, and the mass retention curves of gelatin carriers with different degrees of crosslinking prepared in Preparation Examples 1, 2 and 3 of the present invention under test environments of 65°C or 70°C.

[0076] According to the data in Table 1, the gelatin physical encapsulation technique undergoes a gel-to-sol phase transition when simulated in an organic solvent system at 65°C. In actual tests, it was observed that the uncrosslinked pure gelatin particles in Comparative Example 1 began to melt upon initial contact with the hot solvent. The reaction solution became emulsified within 2 hours, at which point the mass loss exceeded 85%, and the solid support completely dissolved by the 4-hour mark. The liquefaction of the solid support not only caused leakage of the internally encapsulated cells but also made product separation and purification difficult. In contrast, the glutaraldehyde post-crosslinking treatment altered the thermodynamic properties of the support. The support synthesized in Preparation Example 1 under moderate crosslinking parameters maintained a 97.1% mass retention after 24 hours of continuous stirring at 65°C; in the 70°C test, the retention rate stabilized at 95.4%. The Schiff base covalent network restricted the relative slippage of the polypeptide chains, and the slight decrease in support mass was mainly due to mechanical wear caused by liquid scouring. Preparation Example 2 showed a decrease in network density but still retained 91.8% of the solid mass after 24 hours; Preparation Example 3 achieved a retention rate of 98.9%. The cross-linking process enables the carrier to maintain its particle morphology within the 60-70℃ range, providing a material basis for reducing substrate mass transfer resistance by utilizing high temperatures.

[0077] Test Example 2: Thermodynamic Efficiency Test of In-situ Flash Evaporation and Rapid Cooling under Micro-Negative Pressure The test object was a mixed reaction liquid in a 10L pilot-scale variable frequency stirred reactor at the end of the first stage of atmospheric pressure and high temperature mass transfer. The comparative groups included Example 1, Example 4, and Example 5, which used different flash pressure parameters, and Comparative Example 3, which used conventional jacket cooling water cooling.

[0078] When the reaction system in each parallel reactor reaches the specified high-temperature reaction endpoint at 65°C and 200 rpm under stirring, it is taken as the zero point of the cooling test (T=0).

[0079] For Examples 1, 4 and 5, the jacket heating source valve is instantly closed at T=0, and the vacuum pump system connected to the top of the vessel is started simultaneously. By adjusting the self-regulating vacuum control valve, the absolute pressure inside the vessel is rapidly reduced and maintained at 20 kPa, 10 kPa and 40 kPa respectively.

[0080] For Comparative Example 3, after the heating valve is closed at T=0, no vacuuming operation is performed. Instead, the inlet and outlet valves of the jacket cooling medium are fully opened, and industrial chilled water at 15°C is introduced at a constant flow rate for conventional heat transfer and cooling.

[0081] Using Pt100 high-precision resistance temperature detectors (RTDs) that are pre-inserted deep below the center liquid level inside each reactor, the temperature changes of the main liquid phase inside the reactor are collected in real time.

[0082] Transient temperatures were recorded every minute for the first 10 minutes, and then at the 15th, 20th, 30th and 40th minute points, and the cooling trajectory data of each system over time were summarized.

[0083] Table 2: Temperature changes of the bulk liquid phase inside the reactor over time under different cooling operation modes

[0084] Reference Appendix Figure 2 The data includes the conventional temperature decay curve of Comparative Example 3, which relies on external cooling water for sensible heat exchange, and the rapid cliff-like cooling curves of Examples 1, 4, and 5, which are generated by the internal solvent boiling and endothermic reaction under different micro-negative pressure gradients.

[0085] According to the data in Table 2, jacketed heat transfer exhibits significant heat transfer lag during the cooling of large-volume reaction liquids. In the pilot-scale operation, in Comparative Example 3, due to the reduced surface area of ​​the reactor, heat conduction solely through the reactor wall and 15°C cold water resulted in a slow temperature drop at the center of the system. The prolonged high-temperature transition period, lasting tens of minutes, allowed diglycerides to continue undergoing acyl transfer reactions at high temperatures, generating monoglycerides and affecting the conversion rate of the final product. The vacuum flash evaporation mechanism used in the examples utilized the volatility of tert-butanol. When the system pressure dropped below the saturated vapor pressure, the deep liquid phase boiled. In Example 1, the latent heat of vaporization was used to lower the main body temperature to around 50°C within 5 minutes at a pressure of 20 kPa. In Example 4, the vacuum was set to 10 kPa, increasing the heat absorption, and the system took approximately 4 minutes to cool to 45°C. In Example 5, cooling was completed within 10 minutes at 40 kPa. The rapid cooling method relying on the latent heat of phase change can shorten the high-temperature residence time, providing an industrial-grade temperature control scheme for the retention of enzymatic reactions within reactors.

[0086] Test Example 3: Comparison Test of Target Product Conversion Rate and Reaction Specificity The final residues of Examples 1 to 5 and Comparative Examples 1 to 3 after the completion of the entire reaction process, after centrifugation to separate the solid catalyst and vacuum rotary evaporation to remove the co-solvent tert-butanol, were collected as oil samples to be tested. For Comparative Example 1, which failed prematurely due to gelatinization, the turbid liquid in its terminated state was directly subjected to rotary evaporation and the upper oil phase was extracted.

[0087] Accurately weigh 0.05 g of the oil sample to be tested from each group and place it in a 10 mL volumetric flask. Dissolve the sample in a mixed solvent of n-hexane and isopropanol at a volume ratio of 9:1. Then, perform injection filtration using a 0.22 μm polytetrafluoroethylene organic microporous membrane and collect the filtrate as the chromatographic injection solution.

[0088] The testing instrument used was a high-performance liquid chromatograph equipped with an evaporative light scattering detector. The chromatographic column used was a conventional silica gel normal-phase analytical column (4.6 mm × 250 mm, particle size 5 μm).

[0089] The flow rate was set to a constant 1.0 mL / min, and the column oven temperature was maintained at 35 °C. The mobile phase system adopted gradient elution mode, in which mobile phase A was chromatographic grade n-hexane, and mobile phase B was a solution of n-hexane, isopropanol and glacial acetic acid mixed in a volume ratio of 100:10:0.1.

[0090] Under the same test conditions, standard solutions of triglycerides, diglycerides, and monoglycerides with a purity greater than 99% were pre-injected, and the peak area integrals at different concentrations were recorded to plot the standard curves for each component.

[0091] The injection solutions of each experimental group were injected into the chromatograph in sequence, and the peak elution of the detection chromatograms were recorded. The peak positions of the components were confirmed by using the external standard method and combined with the retention time. The mass fractions of triglycerides, diglycerides and monoglycerides in the mixture were calculated and normalized.

[0092] Table 3: Compositional distribution data of the final oil phase products under different process conditions and comparative examples

[0093] Figure 3 This is a line graph showing the compositional distribution of the final products under different process conditions and comparative examples of the present invention. The graph illustrates the relative changes in the mass fractions of residual substrate triglycerides, target product diglycerides, and deep alcoholysis byproduct monoglycerides in Examples 1 to 5 and Comparative Examples 1 to 3 after the reaction, intuitively reflecting the intervention effect of different cooling modes on the selectivity of the series of reaction products.

[0094] According to the data in Table 3, different process routes affected the distribution of the target product. The enzymatic alcoholysis of triglycerides is a series reaction system. Comparative Example 2 maintained a constant temperature of 60°C for 10 hours. The reaction system had sufficient thermodynamic conditions to overcome the activation energy barrier of the side reactions, and the generated diester underwent acyl transfer at this stage, ultimately achieving a monoglyceride mass fraction of 41.36%. Comparative Example 3 used jacketed water cooling, and the longer cooling time extended the window of the system in the high-temperature range. The measured monoglyceride content was 28.71%, and the diester yield was less than 50%. Examples 1 to 5 employed a vacuum flash evaporation coupled with a variable temperature mechanism. The rapid decrease in the main body temperature of the reactor kept the system energy below the activation energy threshold of the side reactions. This process slowed the conversion of diesters to monoesters. The diglyceride content in the example groups remained within the range of 58% to 63%, and the monoglyceride content was controlled below 6%. Example 4 used a lower quenching temperature, resulting in a byproduct formation rate of 3.14%. In Comparative Example 1, due to the lack of physical support from the covalent network, the gelatin carrier melted and leaked, leading to the loss of enzyme activity, with 85.62% of the substrate remaining in an unreacted form. Chromatographic analysis showed that the reduced-pressure flash evaporation cooling operation interrupted a series of side reaction pathways and played a practical role in controlling the product composition.

[0095] Test Example 4: Industrial Continuous Cycle Life Test of Catalysts The test subjects were the cross-linked gelatin-immobilized whole-cell catalysts used in Examples 1, 3, and 5, and the uncross-linked pure gelatin-encapsulated whole-cell catalysts used in Comparative Example 1. The initial dry weight of the catalyst in each group was strictly uniformly 30.0 grams.

[0096] In a parallel reactor equipped with a jacketed temperature control and vacuum reflux system, the entire process of single-batch esterification reaction was carried out according to the process parameters (including temperature gradient, micro-negative pressure flash evaporation setting and stirring speed) corresponding to the aforementioned embodiments and comparative examples.

[0097] After the set total reaction time for a single batch is reached, the stirring system is turned off, and the mixed liquid is transported to the settling tank using the discharge pump at the bottom of the reactor. The mixture is then centrifuged at 3000 rpm for 10 minutes to achieve solid-liquid phase separation.

[0098] After pouring out the clear liquid rich in ester oil and tert-butanol from the upper layer, add 200 grams of room temperature tert-butanol to the solid catalyst retained at the bottom of the settling tank for washing, resuspend at low speed and centrifuge again to remove the viscous grease adhering to the surface, and then briefly dry the solvent under vacuum conditions at room temperature.

[0099] The recovered catalyst was directly fed back into a new reactor containing fresh rapeseed oil, glycerol, and tert-butanol substrates to start the next batch of reaction. For minor catalyst loss observed during testing, no fresh catalyst was added to examine the ultimate service capability of the original batch.

[0100] The chromatographic peak area or conversion rate of diglycerides in the final product of the first batch of reaction was recorded as 100% relative enzyme activity. At the end of the 5th, 10th, 20th, and 30th batches of continuous operation, samples were taken for HPLC chromatographic analysis, and the relative enzyme activity retention rate of the corresponding batches was calculated and recorded.

[0101] Table 4: Relative enzyme activity retention rate of catalysts under different reaction systems during continuous recycling

[0102] Figure 4 This is a continuous cycle lifetime decay graph of the cross-linked gelatin-immobilized whole-cell catalyst of the present invention under different process conditions. The graph specifically shows the long-cycle enzyme activity tracking curves of the chemically cross-linked carriers under different process stresses used in Examples 1, 3, and 5, and also shows the failure data point of the uncross-linked physical embedding carrier in Comparative Example 1, which experienced structural collapse in the first batch.

[0103] According to the data in Table 4, the physically embedded gel carrier could not maintain its structural strength in a high-temperature organic solvent system. In the initial centrifugation recovery operation of Comparative Example 1, the pure gelatin particles swelled and gelatinized after contacting the 65°C reaction solution, and no macroscopically shaped solid material could be collected from the bottom of the centrifuge. The conversion rate of the residual system was 12.1%, and continuous recycling of the catalyst could not be carried out. Mechanical stirring and thermodynamic expansion and contraction affect the conformation of the enzyme protein and the stability of the carrier channels. After introducing glutaraldehyde-induced crosslinking and in-situ flash evaporation mechanisms in the Example groups, the catalysts exhibited different cycle lifetimes. After 30 batches of use, the crosslinked carrier of Example 1 maintained 84.2% of the relative catalytic activity. In the high-temperature mass transfer test at 70°C, the final enzyme activity retention rate of Example 3 was 77.6%. The three-dimensional backbone network not only restricts the physical shedding of the whole cell, but also provides a steric barrier for the internal lipase, reducing protein unfolding caused by thermal stress accumulation. Combined with the reduced pressure flash evaporation technology, the system removes the excess heat exposure area during the conventional cooling process. Cross-linked materials combined with rapid temperature control extend the continuous service life of whole-cell catalysts in oil-modified systems.

Claims

1. A method for preparing diester oil by enzymatic catalysis, characterized in that, Includes the following steps: Vegetable raw material oil, glycerin, tert-butanol, and cross-linked gelatin-immobilized whole-cell enzymes were added to the reactor and mixed. Under normal pressure and stirring conditions, the temperature of the system inside the reactor is raised to 60-70°C to carry out an isothermal mass transfer reaction; When the isothermal mass transfer reaction reaches its endpoint, heating is stopped, and the reactor is depressurized and evacuated to reduce the pressure inside the reactor. The latent heat is absorbed by the depressurized boiling vaporization of some tert-butanol in the system, thereby cooling the system. The tert-butanol vaporized in the vacuum stage is condensed and refluxed back into the reactor, where it continues to undergo isothermal quenching reaction at 45–55°C with continuous stirring. After the reaction is complete, the cross-linked gelatin-immobilized whole-cell enzyme at the bottom is separated, the supernatant is collected and tert-butanol is removed, and the remaining unreacted glycerol is separated to obtain the diester oil.

2. The method for preparing diester oil by enzymatic catalysis according to claim 1, characterized in that, In the step of adding the ingredients to the reactor for mixing, the plant raw material oil, glycerol, tert-butanol, and cross-linked gelatin-immobilized whole-cell enzyme are added to the reactor and mixed in the following parts by weight: 100 parts by weight of vegetable oil; 4.0 to 8.0 parts by weight of glycerin; 50-150 parts by weight of tert-butanol; Cross-linked gelatin immobilized whole-cell enzymes, 1.0–5.0 parts by weight.

3. The method for preparing diester oil by enzymatic catalysis according to claim 2, characterized in that, The plant-based oil is selected from at least one of rapeseed oil, soybean oil, peanut oil, sunflower oil, and corn oil.

4. The method for preparing diester oil by enzymatic catalysis according to claim 1, characterized in that, The isothermal mass transfer reaction takes 3 to 5 hours, and the stirring speed of the isothermal mass transfer reaction is 150 to 250 rpm. The isothermal quenching reaction takes 5 to 9 hours, the stirring speed of the isothermal quenching reaction is 150 to 250 rpm, and the total reaction time of the isothermal mass transfer reaction and the isothermal quenching reaction is controlled to be 8 to 14 hours.

5. The method for preparing diester oil by enzymatic catalysis according to claim 1, characterized in that, The pressure reduction and vacuuming operation involves rapidly reducing the absolute pressure to 10-40 kPa within 1-5 minutes and rapidly reducing the temperature of the mixed liquid in the reactor to 45-55°C within 3-8 minutes.

6. The method for preparing diester oil by enzymatic catalysis according to claim 1, characterized in that, The cross-linked gelatin-immobilized whole-cell enzyme was prepared by a method comprising the following steps: Recombinant Escherichia coli expressing the Candida antarcticis lipase B gene was cultured, and wet cells were collected by centrifugation after the expression was induced. The wet bacterial cells are mixed into a gelatin aqueous solution that is heated and dissolved, and stirred evenly. Then, they are dripped into a cooling liquid at a uniform rate, where they undergo a phase change upon cooling to form spherical gelatin particles. The spherical gelatin particles were chemically cross-linked by immersing them in an aqueous solution of glutaraldehyde, and after washing and drying, the cross-linked gelatin-immobilized whole-cell enzyme was obtained.

7. The method for preparing diester oil by enzymatic catalysis according to claim 6, characterized in that, In the process of culturing recombinant Escherichia coli, after the recombinant Escherichia coli is cultured in fermentation medium to the logarithmic growth phase, isopropyl-β-D-thiogalactoside is added as an inducer at a final concentration of 0.05–0.2 mmol / L. Then the fermentation temperature is lowered to 16–30 °C and cultured for 16–24 hours.

8. The method for preparing diester oil by enzymatic catalysis according to claim 6, characterized in that, The specific method for forming spherical gelatin particles is as follows: Gelatin is heated and dissolved at 50-60°C to prepare a gelatin aqueous solution with a mass concentration of 5%-15%, and then cooled and kept constant at 35-40°C. The wet bacterial cells were added to a gelatin aqueous solution at a mass ratio of 1:2 to 1:5 to obtain an embedding mixture. Granulation is achieved by dripping the encapsulation mixture into cold water or cold liquid paraffin at 0–5°C using a pelletizing device.

9. The method for preparing diester oil by enzymatic catalysis according to claim 6, characterized in that, The specific method for performing chemical cross-linking is as follows: Prepare a glutaraldehyde aqueous solution with a mass fraction of 0.1% to 2.0%, and adjust the pH to 6.5 to 7.5; Gelatin particles were soaked in glutaraldehyde aqueous solution at a solid-liquid ratio of 1:5 to 1:10, and the mixture was shaken and reacted at 0 to 10°C for 1 to 4 hours.

10. The method for preparing diester oil by enzymatic catalysis according to claim 1, characterized in that, In the step of separating the products after the reaction, the cross-linked gelatin immobilized whole-cell enzyme is recovered by low-speed centrifugation at 2000-4000 rpm. The supernatant was subjected to reduced pressure rotary evaporation or thin-film evaporation under vacuum conditions to remove tert-butanol, and the remaining unreacted glycerol at the bottom was separated by centrifugation.

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