Method for preparing retinol palmitate through pH response type Pickering interfacial enzyme catalysis

Abstract

The invention belongs to the field of functional lipid enzymatic preparation, and particularly relates to a method for preparing retinol palmitate through pH response type Pickering interfacial enzyme catalysis. Aiming at the problem that the high stability of the Pickering emulsion is not beneficial to product separation and catalyst recovery and cyclic utilization, a pH response type PIB system is designed and prepared, and reversible switching between demulsification and emulsification of the O / WPickering emulsion is realized through acid-base regulation and control. The method can be widely used for transesterification of medium / long-chain fatty acid and retinol acetate to generate retinol fatty acid ester, and the obtained product has better antioxidant activity than retinol acetate and can meet the requirements of different application scenes; an emulsion enzyme catalysis system is adopted in the reaction process, the reaction condition is mild, the reaction time is short, and separation is simple; compared with a traditional chemical catalysis method, byproducts and wastes are not generated; compared with an anhydrous catalytic system, the product yield is high.

Description

Technical Field

[0001] The present invention belongs to the field of enzymatic preparation of functional lipids, and relates to a method for preparing retinyl palmitate by pH-responsive Pickering interfacial enzymatic catalysis, specifically to a method for constructing a novel pH-responsive interfacial biocatalytic system for highly efficient enzymatic esterification synthesis of retinyl palmitate. Background Art

[0002] Pickering emulsions (PEs) are stabilized by solid particles at the oil / water interface, which are highly stable, biocompatible and environmentally friendly biocatalytic reaction platforms. Compared with traditional biphasic reaction systems, the interfacial contact between the catalyst and the reactants is significantly enhanced; the lipase is adsorbed at the oil-water interface to improve its catalytic activity, and the immobilized enzyme simultaneously acts as an emulsifier and a catalyst to stabilize the emulsion. In addition, the by-product water generated in the reaction automatically transfers from the oil phase to the water phase, eliminating the need for traditional laborious water removal steps, which promotes the forward movement of the reaction. However, the demulsification of Pickering emulsions is mainly achieved by means such as centrifugation and filtration, which have problems such as filter clogging and time consumption. Secondly, due to the inherent brittleness of free biocatalysts, there are problems of leaching, denaturation and inactivation, and a decrease in biocatalytic activity. Immobilized biocatalysts can promote catalytic stability and facilitate the reuse of biocatalysts.

[0003] Stimulus-responsive Pickering interfacial biocatalytic systems (PIB) may be the best choice to solve these problems. In theory, by adjusting the response of the environment to in-situ stimuli, various state changes of the emulsion can be achieved, such as emulsion inversion or demulsification. On the other hand, the emulsifier can be recycled to support more environmentally friendly operations and adhere to the principles of green chemistry. So far, among external environmental factors, thermal triggers require more energy to overcome the high-energy barrier of adsorption energy; CO 2 / N 2 triggers require a long time for phase inversion, and light is a clean stimulus but not easy to recycle. After consulting a large number of literatures, so far, most of the studies published in the field of pH-responsive PIB have only focused on the regulation behavior of smart particles on emulsions, or only on the electrostatic interaction between free lipase and emulsifier. However, how to achieve effective immobilization of enzymes and improve the activity and stability of enzymes has always been the focus of research in the field of biocatalysis. Summary of the Invention

[0004] In view of this, the present invention provides a method for preparing retinyl palmitate by pH-responsive Pickering interfacial enzymatic catalysis in view of the above-mentioned deficiencies of the prior art.

[0005] It should be noted that Pickering emulsions can significantly increase the catalytic interface area, improve the mass transfer performance of reactants, and enhance the catalytic activity of enzymes. However, the high stability of Pickering emulsions is not conducive to the separation of products and the recycling of catalysts. The stimuli-responsive Pickering interfacial biocatalytic system (PIB) may be the best choice to solve these problems. By adjusting the response of the environment to in-situ stimuli, the inversion or demulsification of the emulsion can be achieved, and the emulsifier and catalyst can be recycled to support more environmentally friendly operations and adhere to the principles of green chemistry and sustainable development.

[0006] For this purpose, the present invention has developed surface-active particles with stimulus-induced characteristics and functional conversion. However, some pH-responsive monomers have a moderate regulation range but have the same charge as lipase (aqueous polyurethane); or have the opposite charge to lipase, but the pH value is regulated too acidic or too alkaline, which may damage the enzyme activity (amine-containing monomers, amphiphilic cellulose nanocrystals, amphiphilic charged chitin nanofibers); or carriers without mesopores cannot immobilize lipase (carboxymethyl starch nanoparticles, starch-based nanoparticles).

[0007] 2-(Dimethylamino)ethyl methacrylate (DMAEMA) is one of the pH-responsive monomers, which has advantages such as a suitable pH-responsive range (demulsification at pH = 5.0; emulsification at pH = 7.0 - 9.0), the opposite charge to lipase, biocompatibility, non-cytotoxicity, and biodegradability. It not only endows the intelligent immobilized enzyme with the environmental response characteristics of stabilizing Pickering emulsions but also provides an enzyme immobilization strategy with high stability and high activity. At the same time, due to its tertiary amine group, which is triggered in a rapid, simple, and easy-to-control manner when the pH value changes, the ammonium group of DMAEMA binds to protons in acidic conditions to form quaternary ammonium salts, becoming more hydrophilic and achieving emulsion demulsification; while in alkaline conditions, it is deprotonated, becoming more hydrophobic and facilitating the stabilization of emulsions.

[0008] The present invention first modifies the surface of hollow mesoporous silica spheres (HMSS) with vinylphosphonic acid (PVA), and then grafts it with the pH-responsive monomer 2-(dimethylamino)ethyl methacrylate (DMAEMA) to obtain pH-responsive porous carrier particles (HMSS-DMAEMA). Lipase CL is immobilized in the carrier pores and on the surface through multiple physical adsorption to obtain the immobilized enzyme CL@HMSS-DMAEMA. The immobilized enzyme particles can stabilize the pH-responsive Pickering emulsion interfacial enzyme catalytic system. The emulsion demulsifies at pH = 5.0, and when the pH is adjusted to 7.0 - 9.0, a Pickering emulsion is formed again after stirring or homogenization. It should be noted that during the pH change process, the surface charges of the carrier and lipase CL are always opposite, almost in a mirror image relationship, and electrostatic interaction is maintained throughout this process, improving the stability and immobilization amount of the immobilized enzyme. The loading amount and enzyme activity of CL@HMSS-DMAEMA are 1.8 times and 1.3 times respectively that of the immobilized enzyme (CL@HMSS) without surface modification of the pH-responsive monomer. The pH-responsive PIB system is used for the transesterification reaction of enzymatic retinol acetate and fatty acids (oleic acid, linoleic acid, linolenic acid, stearic acid, palmitic acid, myristic acid, and lauric acid) at 45°C for 1 h, and the conversion rate reaches over 90%; among them, the conversion rate of retinol palmitate reaches 96%, and the catalytic efficiency CE reaches 5.8 mmol g -1 , which is 14.8 times that of the PEs(CL@HMSS) system and the free enzyme 25.2 times that of the single-phase system. After the reaction was completed, 1 was added dropwise Adjust the pH value of the system to 5.0 with MHCl, and the emulsion demulsifies. Separate the upper organic phase containing the retinol palmitate fatty acid ester product; then add a new substrate oil phase, and add 1M NaOH dropwise to adjust the pH value of the system to 7.0 - 9.0. Stir or homogenize again to form a Pickering emulsion to start a new round of enzymatic preparation of VA fatty acid esters. The raw materials of the present invention are widely applicable, with high catalytic efficiency, high conversion rate of VA fatty acid esters, simple operation, and environmental friendliness.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] A method for preparing retinol palmitate by pH-responsive Pickering interfacial enzyme catalysis, specifically including the following steps:

[0011] 1) Prepare HMSS according to the method reported in patent CN117867041A (publication number). Take HMSS (0.5 g) and disperse it in a PBS buffer solution (pH = 7.0, 20 mL) containing VPA (1.0 g). Add NaOH solution (1 mol / L) to adjust the pH value of the solution to 6.0, and reflux at 105°C for 12 hours. Filter, wash, and dry at 60°C. The obtained material (0.5 g) is ultrasonically dispersed in ultrapure water (30 mL) at 65°C, and then add (NH 4 ) 2S 2 O 4 (2 mL, 1 wt%) and NaHSO 4 solution (1 mL, 1 wt%). Under a nitrogen protection atmosphere, the mixed system was slowly stirred for 30 min. Ultra-pure water (15 mL) containing DMAEMA (0.11 g / mL) was slowly added dropwise within 1 h, and the reflux reaction was continued for 5 h. After the reaction was completed, the product was collected by filtration, washed with absolute ethanol to remove the ungrafted monomers, and dried in an oven at 60 °C to obtain a pH-responsive carrier (HMSS-DMAEMA) for the next step. The average particle size of this carrier was 270 nm, the mesopore diameter was 4.0 nm, and the water contact angle was 78°;

[0012] 2) The free enzyme was added to the phosphate buffer to prepare an enzyme solution. Then the pH-responsive carrier HMSS-DMAEMA was dispersed in the enzyme solution, and immobilized lipase CL@HMSS-DMAEMA was obtained by freeze-drying;

[0013] 3) The mixture containing the pH-responsive carrier HMSS-DMAEMA or CL@HMSS-DMAEMA was added to the oil / water two-phase system, and a stable pH-responsive Pickering emulsion was formed by stirring or homogenization. By adding hydrochloric acid solution to adjust the pH value of the system to 5.0, the system could achieve rapid demulsification within 1 min. While by using sodium hydroxide solution to adjust the pH value of the system to 7.0 - 9.0, a Pickering emulsion could be formed again by stirring or homogenization, and this process could be cycled more than 10 times;

[0014] 4) Fatty acid and retinyl acetate were added to the n-hexane / water two-phase system containing the pH-responsive carrier HMSS-DMAEMA and CL@HMSS-DMAEMA according to a certain molar ratio, and a Pickering emulsion was formed by stirring or homogenization. Then, an ester exchange reaction was carried out within the temperature range of 35 - 50 °C to prepare VA ester. After the reaction was completed, 1 M HCl was added to adjust the pH value to 5 for demulsification, and the upper product organic phase was separated. The organic phase dissolved with the new substrate was continuously added, and after adjusting the pH = 7 - 9 with 1 M NaOH, stirring or homogenization was carried out again to form a Pickering emulsion to start a new round of enzymatic preparation of VA ester.

[0015] Preferably, in step 1), the carrier HMSS-DMAEMA has a core-shell structure, with an average particle size of 270 nm and a mesopore diameter of 4.0 nm. After being modified with the pH-responsive monomer DMAEMA, the water contact angle is 78°. During the entire pH change process, the carrier nanoparticles (HMSS-DMAEMA) always carry a charge opposite to that of the lipase CL, and maintain electrostatic attraction to the enzyme during the entire pH reaction process;

[0016] In step 2), the free enzyme is immobilized on the pH-responsive carrier by a multi-adsorption method (mainly electrostatic adsorption, supplemented by hydrogen bonding, hydrophobic and van der Waals force interactions) to obtain pH-responsive immobilized lipase; the free enzyme is one or a combination of Candida antarctica lipase, Candida rugosa lipase, Candida parapsilosis lipase, NS40086 lipase, Thermomyces lanuginosus, Candida lipolytica lipase; the pH of the enzyme solution is 5.0 - 9.0, and the concentration is 10 - 50 mg / mL; the ratio of the mass of the carrier to the volume of the enzyme solution is 1:100 - 5:100 (m / v, g / mL); the immobilization time is 10 - 50 min, and the temperature is 20 - 40 °C; the pH of the phosphate buffer solution is 5.0 - 9.0.

[0017] Preferably, in step 3), the oil-water ratio in the solution is 1:9 - 9:1, the oil phase is n-hexane, n-heptane or cyclohexane, the water phase is pure water or a buffer solution with pH = 7.0, and the addition amount of the pH-responsive carrier HMSS-DMAEMA is 1.0 - 2.5%.

[0018] Preferably, the fatty acid is one or a combination of oleic acid, linoleic acid, linolenic acid, stearic acid, palmitic acid, myristic acid and lauric acid.

[0019] Preferably, in step 4), the pH value is adjusted to 5.0 with 1M hydrochloric acid for demulsification, and demulsification is achieved within 0.5 - 2.0 min. Then, the pH is adjusted to 7.0 - 9.0 with 1M NaOH, and stirring or homogenization can be carried out again to form a Pickering emulsion to start a new round of enzymatic preparation of VA esters; this process can be alternately adjusted for 1 - 10 or more cycles.

[0020] Preferably, the prepared VA esters are one or a combination of retinol oleate, retinol linoleate, retinol linolenate, retinol stearate, retinol palmitate, retinol myristate, retinol laurate, all of which have antioxidant activity, and the antioxidant activity effect is better than that of the substrate retinol acetate.

[0021] Preferably, in step 4), the molar ratio of the fatty acid to retinol acetate is 1:1 - 1:5, the addition amount of the immobilized lipase with pH responsiveness in the synthesis of VA esters is 3.4 - 13.4 mg / mL, the transesterification reaction is carried out in a constant temperature water bath at 35 - 50 °C, the reaction time is 5 - 120 min, the emulsion particle size is between 30 - 50 μm, and the contact area reaches 1000 - 2000 cm 2 / mL.

[0022] Through the technical solution disclosed in the present invention, the conversion rate of preparing VA ester by the pH-responsive Pickering emulsion stabilized by the immobilized enzyme is as high as over 96.0%, and the conversion rate is still greater than 80.0% after 10 cycles. Under the same conditions, the conversion rate of preparing VA ester by the Pickering emulsion stabilized by CL@HMSS is only about 70%, and the conversion rate is only 2.2% after 10 cycles. Therefore, the pH-responsive PIB system constructed in the present invention has high catalytic efficiency, good stability, reusability and wide applicability.

[0023] As can be seen from the above technical solution, compared with the prior art, the beneficial effects achieved by the present invention at least include:

[0024] 1) Aiming at the problem that the high stability of Pickering emulsion is not conducive to the separation of products and the recovery and recycling of catalysts, the present invention designs and prepares a pH-responsive PIB system. The pH-responsive monomer DMAEMA (HMSS-DMAEMA) is grafted on the surface of hollow mesoporous silica spheres, which has the opposite charge to lipase CL and maintains electrostatic attraction to the enzyme throughout the pH reaction process. The carrier nanoparticles (HMSS-DMAEMA) serve as both the carrier and emulsifier of the enzyme, establishing a green and efficient responsive Pickering emulsion interfacial catalytic system, and this pH-responsive PIB system is successfully used for the continuous green preparation of VA ester.

[0025] 2) Through acid-base regulation, the present invention realizes the reversible switching between demulsification and emulsification of O / W Pickering emulsion; when the pH is regulated to 5.0 with acid, demulsification is achieved within 0.5 - 2.0 min, and then when the pH is regulated to 7.0 - 9.0 with alkali, Pickering emulsion can be reformed by stirring or homogenization within 0.5 - 1.0 min, and it can be easily regulated to achieve more than 10 cycles.

[0026] 3) The present invention can be widely used in the transesterification reaction of medium / long-chain fatty acids and retinol acetate to produce retinol fatty acid esters. The obtained products have better antioxidant activity than retinol acetate and can meet the requirements of different application scenarios;

[0027] 4) The reaction process of the present invention adopts an emulsion enzyme catalytic system, with mild reaction conditions, short reaction time and simple separation; no by-products and waste are generated compared with traditional chemical catalytic methods; and the product yield is high compared with anhydrous catalytic systems;

[0028] 5) The present invention systematically studies and emphasizes the key role of electrostatic adsorption in the stability and activity of immobilized enzymes in the pH-responsive PIB system. The results show that the thermal activity, enzyme activity and pH stability of the immobilized enzyme are significantly improved.

[0029] 6) The pH-responsive PIB system of the present invention exhibits good substrate applicability and stability, with a catalytic efficiency (CE) of 5.8 mmol g -1 h -1 , which is 14.8 times that of the unmodified pH-responsive monomer PEs (CL@HMSS) system; and 25.2 times that of the free enzyme single-phase system. After 10 cycles, the reaction yield is still higher than 80%. The present invention provides a new idea for green, sustainable and electrostatic adsorption immobilized enzyme interfacial biocatalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. The drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0031] Figure 1 SEM, TEM, BET diagrams of HMSS-DMAEMA, and FTIR diagrams of hollow mesoporous silica spheres grafted with pH-responsive monomers and lipase immobilized by electrostatic adsorption (free CL, HMSS, CL@HMSS, HMSS-DMAEMA and CL@HMSS-DMAEMA).

[0032] Figure 2 Schematic diagram of the cycle of hollow mesoporous silica spheres grafted with pH-responsive monomers and emulsion-responsive demulsification / emulsification.

[0033] Figure 3 Zeta potential of lipase CL, HMSS-DMAEMA and CL@HMSS-DMAEMA at pH = 3.0 - 11.0 (a) and zeta potential of free CL, HMSS, CL@HMSS, HMSS-DMAEMA and CL@HMSS-DMAEMA at pH = 7.0 (b).

[0034] Figure 4 Thermal stability (a) and organic solvent stability (b) of free CL, CL@HMSS and CL@HMSS-DMAEMA.

[0035] Figure 5 Optical microscope and fluorescence confocal microscope diagrams of CL@HMSS-DMAEMA stabilizing O / W Pickering emulsion (nile red labels the oil phase, FITC labels the lipase).

[0036] Figure 6 Optical microscope and photos of the cycle of pH-regulated O / W Pickering emulsion demulsification / emulsion alternation for ten times.

[0037] Figure 7 Reusability diagram of the CL@HMSS-DMAEMA-stabilized O / W pH-responsive PIB system for the transesterification of palmitic acid and retinyl acetate to produce retinyl palmitate Detailed implementation manners

[0038] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0039] The special term "embodiment" here, any embodiment described as "exemplary" does not have to be construed as superior to or better than other embodiments. For the performance index tests in the embodiments of this application, unless otherwise specified, conventional test methods in the art are adopted. It should be understood that the terms described in this application are only used to describe specific embodiments and are not used to limit the content disclosed in this application.

[0040] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the technical field to which this application belongs; other test methods and technical means not specifically noted in this application refer to the experimental methods and technical means commonly adopted by those of ordinary skill in the art.

[0041] To better illustrate the content of this application, numerous specific details are given in the following specific embodiments. Those skilled in the art should understand that this application can still be implemented without some specific details. In the embodiments, some methods, means, instruments, devices, etc. well-known to those skilled in the art are not described in detail to highlight the gist of this application. On the premise of no conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the obtained technical solutions belong to the content disclosed in the embodiments of this application.

[0042] The present invention discloses and provides a novel pH-responsive interfacial bioenzyme catalytic system for efficient enzymatic esterification synthesis of retinol Method for palmitate

[0043] It should be noted that in the following embodiments, the specific preparation process of HMSS-SP in the present invention is as follows:

[0044] (1) Prepare HMSS by the method reported in Patent CN117867041A (publication number). Take HMSS (0.5 g) and disperse it in a PBS buffer solution (pH = 7.0, 20 mL) containing VPA (0.2 - 10.0 g). Add NaOH solution (1 mol / L) to adjust the pH value of the solution to 6.0 and reflux for 12 hours. Filter, wash, and dry at 60 °C to obtain HMSS-VPA. Subsequently, ultrasonically disperse the HMSS-VPA material in ultrapure water (30 - 300 mL) at 65 °C to obtain a mixture, and add (NH 4 ) 2 S 2 O 4 (2 - 20 mL, 1 wt%) and NaHSO 4 solution (1 - 10 mL, 1 wt%). Stir slowly for 30 min under a nitrogen protection atmosphere to obtain a mixed solution. Slowly drop 15 mL, 0.1 - 3.0 g / mL DMAEMA into the mixed solution within 1 h, reflux for 5 hours, then filter, and wash with absolute ethanol to remove the ungrafted monomers. Dry in an oven at 60 °C to obtain the pH-responsive carrier HMSS-DMAEMA for the next step;

[0045] Figure 1 SEM / TEM of HMSS-DMAEMA shows that it has a core-shell mesoporous structure with a particle size of about 270 nm; BET test shows that its pore size is 4.0 nm; FT-IR spectrogram shows that HMSS-DMAEMA exhibits new characteristic peaks at 1732 cm -1 and 1470 cm -1 , corresponding to the stretching vibration of the C=O bond and the bending vibration of the N-H bond in DMAEMA respectively. It indicates that the pH-responsive monomer (DMAEMA) is successfully modified on the surface of HMSS.

[0046] Moreover, in the following examples, the specific process of preparing immobilized lipase by the adsorption method is as follows:

[0047] Dissolve 3 g of free Candida antarctica lipase (CL) in 50 mL of phosphate buffer solution (50 mM) with a pH of 7.0. Mix HMSS-DMAEMA and the enzyme solution at a solid-liquid ratio of 10 mg / mL, incubate in a shaker at 30 °C for 40 min, and then centrifuge. Freeze-dry the precipitate to obtain immobilized lipase CL@HMSS-DMAEMA.

[0048] The results are as shown in Figure 1 FT-IR of CL@HMSS-DMAEMA shows characteristic peaks of lipase CL at 1542, 1645, and 2930 cm -1 , confirming the successful immobilization of lipase CL.

[0049] To better understand the present invention, the following specific embodiments are used to further elaborate the present invention, but it should not be construed as a limitation to the present invention. For those skilled in the art, some non-essential improvements and adjustments made according to the above invention content are also considered to fall within the protection scope of the present invention.

[0050] Example 1

[0051] A novel pH-responsive Pickering interfacial enzymatic method for preparing retinol palmitate specifically includes the following steps:

[0052] Add 0.1 g of retinol acetate to a cyclohexane / water (total volume 5 mL, v:v = 3:7) containing 1.0% CL@HMSS- In n-hexane / water (total volume 5 mL, v:v = 2:8) of DMAEMA, an emulsion was formed by stirring and homogenizing liquid in a molar ratio of retinol acetate:oleic acid 1:2, and then carry out a transesterification reaction at 35 °C for 2 h to prepare retinol oleate. After the reaction is completed, demulsify with 1 M hydrochloric acid within 1 min, separate the upper product organic phase, and obtain the product.

[0053] It was determined that the conversion rate of retinol oleate was 90.4%.

[0054] Example 2

[0055] A novel pH-responsive Pickering interfacial enzymatic method for preparing retinol palmitate specifically includes the following steps:

[0056] Add 0.1 g of retinol acetate to a cyclohexane / water (total volume 5 mL, v:v = 3:7) containing 1.3% CL@HMSS-DMAEMA in a molar ratio of retinol acetate:linoleic acid 1:5, form an emulsion by stirring and homogenizing, and then carry out a transesterification reaction at 40 °C for 2 h to prepare retinol linoleate. After the reaction is completed, demulsify with 1 M hydrochloric acid within 1 min, separate the upper product organic phase, and obtain the product.

[0057] It was determined that the conversion rate of retinol linoleate was 93.2%.

[0058] Example 3

[0059] A novel pH-responsive Pickering interfacial enzymatic method for preparing retinol palmitate specifically includes the following steps:

[0060] Add 0.1 g of retinol acetate to a cyclohexane / water (total volume 5 mL, v:v = 4:6) containing 1.6% CL@HMSS-DMAEMA in a molar ratio of retinol acetate:linolenic acid 1:3, form an emulsion by stirring and homogenizing, and then carry out a transesterification reaction at 45 °C for 2 h to prepare retinol linolenate. After the reaction is completed, demulsify with 1 M hydrochloric acid within 1 min, separate the upper product organic phase, and obtain the product.

[0061] The conversion rate of retinol linolenate was determined to be 88.8%.

[0062] Example 4

[0063] A new pH-responsive Pickering interfacial enzyme-catalyzed method for preparing retinol palmitate specifically includes the following steps:

[0064] 0.1 g of retinol acetate was added to cyclohexane / water (total volume 5 mL, v:v = 5:5) containing 1.0% CL@HMSS-DMAEMA at a molar ratio of retinol acetate:stearic acid 1:1, and an emulsion was formed by stirring and homogenizing. Then, a transesterification reaction was carried out at 50 °C for 2 h to prepare retinol stearate. After the reaction, 1 M hydrochloric acid was used to demulsify within 1 min, and the upper product organic phase was separated to obtain the product.

[0065] The conversion rate of retinol stearate was determined to be 89.5%.

[0066] Example 5

[0067] A new pH-responsive Pickering interfacial enzyme-catalyzed method for preparing retinol palmitate specifically includes the following steps:

[0068] 0.1 g of retinol acetate was added to cyclohexane / water (total volume 5 mL, v:v = 1:9) containing 1.0% CL@HMSS-DMAEMA at a molar ratio of retinol acetate:palmitic acid 1:1, and an emulsion was formed by stirring and homogenizing. Then, a transesterification reaction was carried out at 45 °C for 2 h to prepare retinol palmitate. After the reaction, 1 M hydrochloric acid was used to demulsify within 1 min, and the upper product organic phase was separated to obtain the product.

[0069] Continue to add new substrates, adjust the pH = 7, 8, 9 with NaOH, and then stir or homogenize to reform the Pickering emulsion to start a new round of enzyme-catalyzed preparation of VA esters.

[0070] The conversion rate of retinol palmitate was determined to be 96.0%, and the catalytic efficiency (CE) was 5.8 mmol g -1 h -1 , and the conversion rate was still greater than 80.0% after 10 cycles.

[0071] Example 6

[0072] A new pH-responsive Pickering interfacial enzyme-catalyzed method for preparing retinol palmitate specifically includes the following steps:

[0073] Dissolve 0.1 g of retinol acetate in cyclohexane / water (total volume 5 mL, v:v = 1:9) containing 1.0% CL@HMSS-DMAEMA at a molar ratio of retinol acetate:myristic acid of 1:1. Homogenize to form an emulsion by stirring, and then carry out a transesterification reaction at 45 °C for 2 h to prepare retinol myristate. After the reaction, demulsify with 1 M hydrochloric acid within 1 min, separate the upper organic phase product to obtain the product.

[0074] It was determined that the conversion rate of retinol myristate was 94.3%.

[0075] Example 7

[0076] A novel method for preparing retinol palmitate by pH-responsive Pickering interfacial enzyme catalysis specifically includes the following steps:

[0077] Dissolve 0.1 g of retinol acetate in cyclohexane / water (total volume 5 mL, v:v = 1:9) containing 1.0% CL@HMSS-DMAEMA at a molar ratio of retinol acetate:lauric acid of 1:1. Homogenize to form an emulsion by stirring, and then carry out a transesterification reaction at 45 °C for 2 h to prepare retinol laurate. After the reaction, demulsify with 1 M hydrochloric acid within 1 min, separate the upper organic phase product to obtain the product.

[0078] It was determined that the conversion rate of retinol laurate was 95.3%.

[0079] Table 1 shows the conversion rates of the products in Examples 1-8.

[0080]

[0081] To further demonstrate the beneficial effects of the present invention for better understanding of the present invention, the following comparative examples further clarify the technical features disclosed in the present invention, but it should not be construed as a limitation to the present invention. For other improvements made by those skilled in the art without creative work based on the above-mentioned invention content, they are also considered to fall within the protection scope of the present invention.

[0082] Comparative Example 1

[0083] Free enzyme single-phase system: Dissolve 0.1 g of retinol acetate in cyclohexane / water (total volume 5 mL, v:v = 1:9) containing 1.0% free CL at a molar ratio of retinol acetate:palmitic acid of 1:1. Homogenize to form an emulsion by stirring, and then carry out a transesterification reaction at 45 °C for 2 h to prepare retinol palmitate. After the reaction, obtain the product.

[0084] It was determined that the conversion rate of retinol palmitate was 7.9%, and the catalytic efficiency (CE) was 0.23 mmol g -1 h -1The CE value of the pH-responsive Pickering interfacial enzyme-catalyzed system (Example 6) is 25.5 times that of the free enzyme single-phase system.

[0085] Comparative Example 2

[0086] Free enzyme biphasic system: 0.1 g of retinyl acetate was added to cyclohexane / water (total volume 5 mL, v:v = 1:9) containing 1.0% free CL in a molar ratio of retinyl acetate:palmic acid 1:1. An emulsion was formed by stirring and homogenizing, and then a transesterification reaction was carried out at 45 °C for 2 h to prepare retinyl palmitate. After the reaction, the upper product organic phase was separated to obtain the product.

[0087] After determination, the conversion rate of retinyl palmitate was 95%, and the catalytic efficiency (CE) was 5.7 mmol g -1 h -1 Compared with the PIB system, the lipase cannot be recovered, and the product needs to be separated by cost-consuming means such as suction filtration and centrifugation.

[0088] Comparative Example 3

[0089] Immobilized enzyme single-phase system: 0.1 g of retinyl acetate was added to cyclohexane / water (total volume 5 mL, v:v = 1:9) containing 1.0% CL@HMSS-DMAEMA in a molar ratio of retinyl acetate:palmic acid 1:1. An emulsion was formed by stirring and homogenizing, and then a transesterification reaction was carried out at 45 °C for 2 h to prepare retinyl palmitate. After the reaction, the product was obtained.

[0090] After determination, the conversion rate of retinyl palmitate was 72%, and the catalytic efficiency (CE) was 3.2 mmol g -1 h -1 , and the CE value of the pH-responsive Pickering interfacial enzyme-catalyzed system is 1.8 times that of the immobilized enzyme single-phase system. Compared with the free enzyme single-phase system of Comparative Example 1, the immobilization of lipase significantly improves the stability and enzyme activity of lipase.

[0091] Comparative Example 4

[0092] Pickering system of free enzyme: 0.1 g of retinyl acetate was added to cyclohexane / water (total volume 5 mL, v:v = 1:9) containing 1.0% free CL and a pH-responsive carrier in a molar ratio of retinyl acetate:palmic acid 1:1. An emulsion was formed by stirring and homogenizing, and then a transesterification reaction was carried out at 45 °C for 2 h to prepare retinyl palmitate. After the reaction, the emulsion was broken within 1 min by adjusting with 1 M hydrochloric acid, and the upper product organic phase was separated to obtain the product.

[0093] After determination, the conversion rate of retinyl palmitate was 95.5%, and the catalytic efficiency (CE) was 5.7 mmol g-1 h -1 Compared with the PIB system, the free enzyme has a large loss, is difficult to recover, and has poor repeatability.

[0094] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing retinol palmitate by pH-responsive Pickering interfacial enzyme catalysis, characterized in that The steps include: 1) adding 0.5 g HMSS to 20 mL of PBS buffer solution containing 0.2-10.0 g VPA at pH = 7.0, adjusting the solution pH to 6.0 with alkali and refluxing for 12 hours, then filtering, washing and drying at 60° C. to obtain HMSS-VPA, then dispersing the HMSS-VPA in 30-300 mL of ultrapure water at 65° C. to obtain a mixture, adding 2-20 mL of 1 wt% (NH4)2S2O4 and 1-10 mL of 1 wt% NaHSO4 solution to the mixture, then slowly stirring for 30 min under a nitrogen atmosphere to obtain a mixed solution, slowly adding 15 mL of 0.1-3.0 g / mL DMAEMA to the mixed solution within 1 hour, refluxing for 5 hours, filtering, washing with anhydrous ethanol to remove ungrafted monomers, and drying in an oven at 60° C. to obtain HMSS-DMAEMA; 2) adding the free enzyme into a phosphate buffer to prepare an enzyme solution; mixing the enzyme solution with the pH-responsive carrier HMSS-DMAEMA prepared in step 1), and drying to obtain a pH-responsive immobilized lipase CL@HMSS-DMAEMA; 3) adding the pH-responsive carrier HMSS-DMAEMA or CL@HMSS-DMAEMA into the oil / water phases to form a pH-responsive Pickering emulsion by stirring or homogenizing; 4) Adding fatty acids and retinol acetate in a certain proportion to n-hexane / water containing pH-responsive carrier HMSS-DMAEMA or CL@HMSS-DMAEMA, and forming a Pickering emulsion by stirring or homogenizing; performing an ester exchange reaction in a temperature range of 35-50° C. to prepare retinol fatty acid esters; after the reaction, adjusting the pH value of the system to 5.0 with 1M HCl, quickly breaking the emulsion in a short time, separating the upper organic phase, and collecting the obtained retinol fatty acid ester product; continuing to add new n-hexane, fatty acids and retinol acetate as substrates to the remaining system, adjusting the pH value of the system to 7.0-9.0 with 1M NaOH, and forming a Pickering emulsion again by stirring or homogenizing; repeating the steps to perform multiple rounds of enzyme-catalyzed reactions to prepare VA esters.

2. The method for preparing retinol palmitate by pH-responsive Pickering interfacial enzyme catalysis according to claim 1, characterized in that: In step 1), the carrier HMSS-DMAEMA has a core-shell structure, an average particle size of 270nm, a mesopore size of 4.0nm, and after the modified pH-responsive monomer DMAEMA, the water contact angle is 78°, and the charge opposite to that of the lipase CL is always maintained during the entire pH change process, and the electrostatic attraction to the enzyme is maintained during the entire pH reaction process; in step 2), the free enzyme is one or more combinations of Antarctic Candida lipase, Candida rugosa lipase, Candida rugosa lipase, NS40086 lipase, Thermomyces lanuginosus, and Candida lipolytica lipase; the pH of the enzyme solution is 5.0-9.0, and the concentration is 10-90mg / mL; the ratio of the carrier mass to the enzyme solution volume is 1:100-5:100 (m / v, g / mL); the immobilization time is 10-50min, and the temperature is 20-40°C; the pH of the phosphate buffer is 5.0-9.

0.

3. The method for preparing retinol palmitate by pH-responsive Pickering interfacial enzyme catalysis according to claim 1, characterized in that: In step 2) and step 3), pH responsiveness of the immobilized enzyme is achieved by grafting the pH responsive monomer 2-(dimethylamino)ethyl methacrylate DMAEMA. At the same time, the lipase and the smart carrier always maintain opposite charges between pH = 3 and 11, thereby improving the efficient immobilization and protection of the enzyme through electrostatic interaction. The pH responsive monomer not only gives the smart immobilized enzyme the environmental response characteristics of stabilizing the Pickering emulsion, but also provides an enzyme immobilization strategy with high stability and high activity. Among them, the free enzyme is adsorbed on the surface and pores of the pH responsive carrier through the combined effects of electrostatic adsorption, hydrogen bonding, hydrophobic interaction and van der Waals forces.

4. The method for preparing retinol palmitate by pH-responsive Pickering interfacial enzyme catalysis according to claim 1, characterized in that: The fatty acid is one or more combinations of oleic acid, linoleic acid, linolenic acid, stearic acid, palmitic acid, myristic acid, and lauric acid.

5. The method for preparing retinol palmitate by pH-responsive Pickering interfacial enzyme catalysis according to claim 1, characterized in that: In step 3), the oil-water ratio in the solution is 1:9-9:1, the oil phase is n-hexane, n-heptane or cyclohexane, the aqueous phase is pure water or a buffer solution with a pH value of 7.0, and the addition amount of the pH-responsive carrier HMSS-DMAEMA is 1.0-2.5%.

6. The method for preparing retinol palmitate by pH-responsive Pickering interfacial enzyme catalysis according to claim 1, characterized in that: In step 4), the demulsification time with 1M hydrochloric acid is 0.5-2.0min, the emulsification time when pH=7.0-9.0 is adjusted with 1M NaOH is 0.5-1.0min, and the number of alternating cycles is more than 10 times.

7. The method for preparing retinol palmitate by pH-responsive Pickering interfacial enzyme catalysis according to claim 1, characterized in that: The retinol fatty acid ester prepared in step 4) is one or more combinations of retinol oleate, retinol linoleate, retinol linolenate, retinol stearate, retinol palmitate, retinol myristate, and retinol laurate.

8. The method for preparing retinol palmitate by pH-responsive Pickering interfacial enzyme catalysis according to claim 1, characterized in that: In step 4), the molar ratio of retinol acetate to fatty acid is 1:1-1:5, the amount of immobilized lipase with pH responsiveness added in the VA ester synthesis is 3.4-13.4 mg / mL, the transesterification reaction is carried out in a constant temperature water bath at 35-50°C, the reaction time is 5-120 min, the emulsion particle size is between 30-50 μm, and the contact area is 1000-2000 cm 2 / mL.

9. The method for preparing retinol palmitate by pH-responsive Pickering interfacial enzyme catalysis according to claim 1, characterized in that: The VA ester prepared in step 4) is one or more of retinol oleate, retinol linoleate, retinol linolenate, retinol stearate, retinol palmitate, retinol myristate, and retinol laurate.

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