Stabilizer based on natural macromolecular self-assembled micelles, temperature-sensitive Pickering emulsion, preparation method and application

Abstract

The present invention provides a stabilizer, a temperature-sensitive Pickering emulsion, a preparation method and an application based on natural macromolecular self-assembled micelles; first, lipase and negatively charged natural macromolecules are added to positively charged natural macromolecule chitosan, and the electrostatic interaction between cationic natural macromolecule chitosan and anionic natural macromolecules is used to self-assemble micelles to form micelles, and lipase is loaded in the hydrophobic micro-regions in the micelles; with phase change material as the oil phase, the self-assembled micelles are secondary assembled to the oil-water interface under high-speed homogenization, and a temperature-sensitive Pickering emulsion is constructed, which is applied to the interfacial catalysis of lipase; by changing the temperature, the oil phase is induced to solidify and dissolve to achieve demulsification, and the self-assembled micelles are recovered to improve the cyclic performance of lipase. The temperature-sensitive emulsion prepared by the present invention has a simple preparation process, mild conditions, high catalytic activity, and good cyclic stability of lipase.

Description

Technical Field

[0001] The invention relates to macromolecular self-assembly and biocatalysis, and in particular to a stabilizer based on natural macromolecular self-assembly micelles, a temperature-sensitive Pickering emulsion, a preparation method and application in biocatalysis. Background Art

[0002] Lipase is one of the most important industrial biocatalysts and is widely used in various industries. However, most of the substrates of lipase are oil-soluble and need to be in contact with hydrophobic substances to activate the catalytic activity. Using the oil-water interface to activate lipase activity is a common method. However, the traditional oil-water interface area is small, which limits the catalytic rate of lipase on the oil phase. In order to overcome this difficulty, surfactants or particles are added to form water-in-oil (O / W) or oil-in-water (W / O) emulsions to increase the oil-water interface area. Emulsions stabilized by solid particles are called Pickering emulsions, and Pickering emulsions have potential application prospects in the field of biocatalysis. Immobilizing lipase on the Pickering emulsion interface can not only provide a short mass transfer distance, but also facilitate the contact between lipase and substrate. The application of Pickering emulsions in the field of biocatalysis has the following advantages: high catalytic activity, easy separation of enzymes, and recyclable enzymes.

[0003] In practical applications, in order to separate the product and reuse the lipase, the Pickering emulsion can be destabilized by simple centrifugation to achieve the purpose of recovery. However, the thermal effect generated during the centrifugation process may lead to the inactivation of the lipase, and the centrifugation will also cause the lipase to be enriched in a certain place, which is not conducive to the subsequent uniform dispersion of the lipase in the solution, affecting the catalytic activity; some Pickering emulsions cannot be recovered by centrifugation due to their small particle size and stable properties. Constructing stimulus-responsive Pickering emulsions (N2 / CO2, pH, temperature, light, magnetism, ionic strength, etc.) and adjusting the emulsion stability to achieve demulsification by environmental stimulation is currently an effective means, among which temperature response is the most widely studied. The construction of traditional temperature stimulus-responsive Pickering emulsions is to modify the emulsifier with temperature sensitivity, and change the aggregation state of the stabilizer at the oil-water interface through temperature stimulation to achieve the stability and instability of the emulsion. However, this method has clear restrictions on the structure of the emulsifier, and it is necessary to contain groups that interact with the thermosensitive monomer; in addition, the thermosensitive group must be bound to the precursor through chemical reaction. Summary of the invention

[0004] The purpose of the present invention is to provide a stabilizer based on natural macromolecular self-assembled micelles. The stabilizer is prepared by first preparing a natural macromolecular solution and then loading lipase on the natural macromolecular self-assembled micelles.

[0005] Another object of the present invention is to provide a thermosensitive Pickering emulsion and a preparation method thereof, wherein a phase change material is used as an oil phase and nanoparticles based on self-assembled natural macromolecules are used as an emulsifier to construct a thermosensitive Pickering emulsion; the oil phase is induced to undergo a coagulation and dissolution process by temperature change, coagulating below 18° and melting above 18° to achieve the purpose of oil-water separation and recovery of lipase.

[0006] Another object of the present invention is to provide an application of the above-mentioned temperature-sensitive Pickering emulsion, which is applied to the fixation of lipase on the emulsion interface and its application in the field of biocatalysis.

[0007] The specific technical solutions of the present invention are as follows:

[0008] The invention provides a stabilizer based on natural macromolecular self-assembled micelles, which is a natural macromolecular self-assembled micelle loaded with lipase.

[0009] The preparation method of the stabilizer based on natural macromolecular self-assembled micelles is:

[0010] The lipase is placed in an anionic natural macromolecular solution and stirred at room temperature to obtain a mixed solution; the mixed solution is added dropwise into the chitosan solution under stirring conditions, the pH is adjusted to 3.0-10.0, and stirring is continued to obtain the product.

[0011] In the preparation method of the stabilizer, the preparation method of the chitosan solution is: placing cationic natural macromolecular chitosan in an acidic solution, stirring and dissolving at room temperature to obtain the solution. The acidic solution is an acetic acid solution or a hydrochloric acid solution, and the concentration of the acidic solution is 1%-5% by volume; the concentration of chitosan in the acidic solution is 0.01-10 mg·mL -1 .

[0012] In the preparation method of the stabilizer, the preparation method of the anionic natural macromolecule solution is: placing the anionic natural macromolecule in ultrapure water and stirring and dissolving at room temperature to obtain the anionic natural macromolecule. The anionic natural macromolecule is sodium hyaluronate, sodium γ-polyglutamate or sodium alginate; the concentration of the anionic natural macromolecule in the anionic natural macromolecule solution is 0.01-10 mg·mL -1 .

[0013] In the preparation method of the stabilizer, the concentration of the lipase in the anionic natural macromolecular solution is 0.01-10 mg·mL -1 .

[0014] In the preparation method of the stabilizer, the volume ratio of the chitosan solution to the mixed solution is 4:1-1:4;

[0015] The solution used for adjusting pH is potassium hydroxide solution or sodium hydroxide solution; the pH value of the solution is adjusted to 3.0-10.0.

[0016] In the preparation method of the stabilizer, the pH value of the solution is adjusted to induce natural macromolecules to self-assemble to form micelles, and lipase is loaded in the hydrophobic microregion of the micelle to obtain natural macromolecule self-assembled micelles loaded with lipase.

[0017] The present invention provides a method for preparing a temperature-sensitive Pickering emulsion:

[0018] The solution of the stabilizer based on the natural macromolecular self-assembled micelle is the water phase, and the phase change material is the oil phase, and the temperature-sensitive Pickering emulsion is prepared under the action of high-speed homogenization.

[0019] Furthermore, in the preparation method of the thermosensitive Pickering emulsion, the concentration of the stabilizer solution based on the natural macromolecular self-assembled micelle is 0.01-10 mg·mL -1 , the solvent is water (calculated by the mass of lipase); the pH range of the solution of the stabilizer based on the natural macromolecular self-assembled micelle is 3.0-10.0, and the volume ratio of the solution of the stabilizer based on the natural macromolecular self-assembled micelle to the phase change material solution is 10:1-1:10; the phase change material includes but is not limited to fatty acid esters, paraffins, and polymer materials (polyethylene glycol, polyolefins). The fatty acid is selected from lauric acid and octadecanoic acid; the paraffin is selected from n-hexadecane, n-octadecane, and n-eicosane; the polymer material is selected from polyethylene glycol and polyolefin;

[0020] The high-speed homogenization means: a homogenization speed of 1000-20000 rpm, a homogenization time of 0.1-4 min, and a homogenization temperature of 10-50°C.

[0021] The invention provides a temperature-sensitive Pickering emulsion, which is prepared by adopting the above-mentioned preparation method.

[0022] The application of the temperature-sensitive Pickering emulsion provided by the present invention is used for biocatalysis, including but not limited to catalyzing the hydrolysis of oils and fats, catalyzing the synthesis of biodiesel, and catalyzing the synthesis of organic molecules;

[0023] The application method of catalyzing the hydrolysis of oil is as follows: the solution of the stabilizer based on the natural macromolecular self-assembled micelle is the water phase, and the phase change material containing oil is the oil phase, and high-speed homogenization is performed; the concentration of oil in the phase change material is 1%-85%.

[0024] The oil includes olive oil or soybean oil;

[0025] The application of the temperature-sensitive Pickering emulsion provided by the present invention is used to catalyze the organic phase-water interface reaction, and can achieve oil-water separation by controlling the temperature. Olive oil (triglyceride) is hydrolyzed into glycerol and fatty acids by lipase.

[0026] The present invention first adds lipase and negatively charged natural macromolecules to positively charged natural macromolecule chitosan, utilizes the electrostatic interaction between cationic natural macromolecule chitosan and anionic natural macromolecules to self-assemble to form micelles, and loads lipase in the hydrophobic micro-regions in the micelles; uses phase change material as the oil phase, and re-assembles the self-assembled micelles to the oil-water interface under high-speed homogenization to construct a thermosensitive Pickering emulsion, which is applied to the interfacial catalysis of lipase; by changing the temperature, the oil phase is induced to coagulate-dissolve to achieve demulsification, the self-assembled micelles are recovered, and the recycling performance of lipase is improved. The thermosensitive emulsion prepared by the present invention has the advantages of simple preparation process, mild conditions, high catalytic activity, good cyclic stability of lipase, and is expected to be applied to the fields of food, medicine, cosmetics and fine chemicals.

[0027] Compared with the prior art, the main advantages of the present invention are:

[0028] 1) The preparation process of the temperature-sensitive Pickering emulsion based on natural macromolecular self-assembled micelles used in the present invention is simple, the reaction conditions are mild, and it is conducive to maintaining the activity of lipase.

[0029] 2) The thermosensitive Pickering emulsion based on natural macromolecular self-assembled micelles prepared by the present invention realizes oil-water separation by controlling the temperature, and the recovery process is simple and mild, which is beneficial to improving the circulation stability of lipase.

[0030] 3) The temperature-sensitive Pickering emulsion based on natural macromolecular self-assembled micelles prepared by the present invention has the advantages of high catalytic efficiency, good recovery efficiency, low cost, green environmental protection, etc.

[0031] 4) The temperature-sensitive Pickering emulsion based on natural macromolecular self-assembled micelles prepared by the present invention has good biocompatibility, which is beneficial to its application in the fields of food, medicine, cosmetics and fine chemicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Scanning electron microscopy (SEM) image of the prepared natural macromolecular self-assembled micelles loaded with lipase;

[0033] Figure 2 Scanning electron microscopy images (SEM) of Pickering emulsions stabilized by natural macromolecular self-assembled micelles loaded with lipase at different magnifications;

[0034] Figure 3 Optical microscopy (a) and fluorescence microscopy (b) of the thermosensitive Pickering emulsion stabilized by the natural macromolecular self-assembled micelles loaded with lipase;

[0035] Figure 4 Cyclic stability of lipase-catalyzed olive hydrolysis in a thermosensitive Pickering emulsion. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Unless otherwise specified, the experimental materials and reagents used in the following examples can be obtained from commercial sources. If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0038] Example 1

[0039] A stabilizer based on natural macromolecular self-assembled micelles, comprising the following steps:

[0040] 1) Preparation of natural macromolecular solution: 0.2 g of chitosan was added with 100 mL of 1% acetic acid aqueous solution, and stirred at room temperature for 8 h to obtain a chitosan solution; 0.08 g of sodium hyaluronate was placed in 100 mL of ultrapure water, and stirred to dissolve to obtain a sodium hyaluronate solution.

[0041] 2) Natural macromolecular self-assembled micelles loaded with lipase: 0.2 g of lipase was placed in 100 mL of sodium hyaluronate solution in step 1), and stirred at room temperature for 8 h to obtain a mixed solution. 100 mL of the prepared mixed solution was added dropwise to 50 mL of chitosan solution in step 1) under stirring, and the solution pH was adjusted to 4.5 with potassium hydroxide solution (KOH), and stirring was continued for 1 h to induce natural macromolecules to self-assemble to form micelles, and lipase was loaded in the hydrophobic microregions of the micelles to obtain natural macromolecular self-assembled micelles loaded with lipase.

[0042] Figure 1 This is a scanning electron microscope (SEM) image of the natural macromolecular self-assembled micelles loaded with lipase. It can be seen from the figure that the micelles are spherical, with a particle size range of 180-640nm and a wide distribution. Sample detection steps: a drop of 2mg·mL -1 The micelle solution at pH 4.5 was dropped on a clean silicon wafer. After drying, it was gold-sprayed and its morphology was characterized using SEM.

[0043] The preparation method of the thermosensitive Pickering emulsion prepared by the stabilizer prepared in Example 1 is as follows:

[0044] Take 3 mL (pH 4.5) of the natural macromolecular self-assembled micelle solution loaded with lipase in step 2) above, add 3 mL of n-hexadecane, and high-speed shear at 10000 rpm for 0.8 min under a high-speed homogenizer. The water bath temperature is maintained at 25° C. during the homogenization process.

[0045] Figure 2 Scanning electron microscope images (SEM) of Pickering emulsions stabilized by natural macromolecular self-assembled micelles loaded with lipase at different ratios. Sample detection steps: Take 3 mL of the micelle solution in step 2), add 3 mL of azobisisobutyronitrile styrene, and shear at 10,000 rpm for 0.8 min under a high-speed homogenizer. The water bath temperature is maintained at 25°C during the homogenization process. React at 60°C for 72 hours. After the styrene solidifies, the product is washed with water, freeze-dried, and the surface of the product is sprayed with gold. The surface is characterized by SEM, and it can be seen that many micelles are attached to the emulsion, as well as the location where the micelles are attached to the emulsion. This shows that the micelles adsorbed on the surface of the oil droplets play a role in stabilizing the emulsion, which is a typical Pickering emulsion; it can also be concluded that the average particle size of the emulsion droplets is 220 μm.

[0046] Figure 3 Optical microscope image (a) and fluorescence microscope image (b) of the thermosensitive Pickering emulsion stabilized by the natural macromolecular self-assembled micelles loaded with lipase. It can be seen from the figure that the stable emulsion formed is of O / W type. Optical microscope image sample detection steps: the emulsion after the above homogenization is observed using an optical microscope; the fluorescence microscope observation is as follows: take 3mL of the micelle solution in step 2), add 3mL of n-hexadecane containing Nile red, and high-speed shear at 10000rpm for 0.8min under a high-speed homogenizer. The water bath temperature during the homogenization process is maintained at 25°C. Take the emulsion after homogenization and observe it using a fluorescence microscope. Figure 3 In a, by looking at the microscope, we can find that the particle size of the emulsion is 200-250μm. The particle size is related to stability. Then the height of the emulsion in the bottle and the height of the water phase can characterize the emulsification properties of the micelles. Figure 3 In b, fluorescence microscopy shows that the emulsion is of O / W type.

[0047] Application of the temperature-sensitive Pickering emulsion prepared in Example 1: catalyzing the hydrolysis of olive oil:

[0048] Take 3 mL of the natural macromolecular self-assembled micelle solution loaded with lipase in step 2), add 3 mL of n-hexadecane containing 5% olive oil by volume, and shear at 10000 rpm for 0.8 min under a high-speed homogenizer. The water bath temperature during the homogenization process is maintained at 25°C. The emulsion after homogenization is reacted on a shaker at 37°C for 1 hour. The reaction is completed. The emulsion is placed in ice water, and after the emulsion solidifies, it is placed at 30°C to melt the solidified emulsion, and the upper oil phase after the reaction is completed is taken, and the fatty acid content is measured by ultraviolet spectrophotometer, and the hydrolysis rate of olive oil is determined according to the amount of fatty acids generated.

[0049] Figure 4 The cyclic stability of olive hydrolysis catalyzed by lipase in thermosensitive Pickering emulsion. Sample detection steps: Take 3mL of natural macromolecular micelle solution loaded with lipase in step 2), add 3mL of hexadecane containing 5% olive oil, and shear at 10000rpm for 0.8min under a high-speed homogenizer. The water bath temperature during the homogenization process is maintained at 25°C. The emulsion after homogenization is shaken at 37°C for 120min, and the reaction is completed. The emulsion is placed in ice water, and after the emulsion solidifies, it is placed at 30°C to melt the solidified emulsion, and the oil phase at the end of the upper reaction is taken. The fatty acid content is measured, and the hydrolysis rate of different cycles is determined according to the amount of fatty acids generated; the upper oil phase is removed, a new oil phase is added, and the above operation is repeated to test the cyclic stability of lipase. It shows that with the increase of the number of cycles, the catalytic performance gradually decreases. The possible reasons are: the destruction of the spatial structure of lipase during the homogenization process causes the enzyme to lose activity. The relative catalytic activity after 2 cycles is 96%, and the catalytic activity is still 57% after 10 cycles, indicating that the system has good cyclic performance.

[0050] Determine the fatty acid content, take 100 μL of the oil phase, add 1 ml of pyridine-copper acetate, add 4.9 ml of n-hexane, and compare the ultraviolet absorbance at 714 nm with the standard curve to obtain the amount of fatty acid generated and the activity of lipase. The data of cyclic stability can show that the activity of lipase is good. If the activity decreases, the cyclic stability will be poor. The above results of the present invention also show that the cyclic stability is good.

[0051] Example 2

[0052] A stabilizer based on natural macromolecular self-assembled micelles, comprising the following steps:

[0053] 1) Preparation of natural macromolecular solution: 0.2 g of chitosan was added to 50 mL of 5% acetic acid aqueous solution, and stirred at room temperature for 6 h to obtain a chitosan solution. 0.06 g of sodium γ-polyglutamate was placed in 50 mL of ultrapure water, and stirred to dissolve to obtain a sodium γ-polyglutamate solution.

[0054] 2) Natural macromolecular self-assembled micelles loaded with lipase: 0.2 g of lipase was placed in 50 mL of sodium γ-polyglutamate solution in step 1), and stirred at room temperature for 6 h to obtain a mixed solution. 50 mL of the prepared mixed solution was slowly added dropwise to 50 mL of chitosan solution in step 1) under stirring, and potassium hydroxide (KOH) was used to adjust the solution pH to 5.0, and stirring was continued for 45 min to induce natural macromolecules to self-assemble to form micelles, and lipase was loaded in the hydrophobic microregions of the micelles to obtain natural macromolecular self-assembled micelles loaded with lipase.

[0055] The preparation method of the thermosensitive Pickering emulsion prepared by the stabilizer prepared in Example 2 is as follows:

[0056] Take 4 mL of the natural macromolecular micelle solution loaded with lipase in step 2), add 2 mL of n-octadecane, and high-speed shear at 8000 rpm for 1 min in a high-speed homogenizer. The water bath temperature is maintained at 30° C. during the homogenization process.

[0057] The stabilizer prepared in Example 2 is used to prepare a thermosensitive Pickering emulsion, catalyzing the hydrolysis of olive oil: 4 mL of the natural macromolecular self-assembled micelle solution loaded with lipase in step 2) is taken, 2 mL of n-octadecane containing 10% olive oil is added, and high-speed shearing is carried out at 8000 rpm for 1 min under a high-speed homogenizer, and the water bath temperature during the homogenization process is maintained at 30°C. The emulsion after homogenization is reacted on a shaker at 37°C for 2h. The reaction is completed. The emulsion is placed in ice water, and after the emulsion solidifies, it is placed at 30°C to melt the solidified emulsion, and the upper oil phase at the end of the reaction is taken, and the fatty acid content is measured. The reaction hydrolysis rate is determined according to the amount of fatty acids generated. Through experiments, the hydrolysis rate of the present invention is relatively high.

[0058] Example 3

[0059] A stabilizer based on natural macromolecular self-assembled micelles, comprising the following steps:

[0060] 1) Preparation of natural macromolecular solution: 0.1 g of chitosan was added to 50 mL of 2% hydrochloric acid solution, and stirred at room temperature for 4 h to obtain a chitosan solution. 0.01 g of sodium alginate was placed in 50 mL of ultrapure water, and stirred to dissolve to obtain a sodium alginate solution.

[0061] 2) Natural macromolecule self-assembled micelles immobilize lipase: 0.1 g of lipase is placed in the sodium alginate solution of step 1), stirred at room temperature for 4 hours to obtain a mixed solution. 50 mL of the prepared mixed solution is slowly added dropwise to 50 mL of chitosan solution of step 1) under stirring, and the solution pH is adjusted to 6.0 with sodium hydroxide (NaOH), and stirring is continued for 60 minutes to induce natural macromolecules to self-assemble to form micelles, and lipase is loaded in the hydrophobic microregions of the micelles to obtain natural macromolecule self-assembled micelles loaded with lipase.

[0062] The preparation method of the thermosensitive Pickering emulsion prepared by the stabilizer prepared in Example 3 is as follows:

[0063] Take 2 mL of the natural macromolecular micelle solution loaded with lipase in step 2), add 4 mL of n-eicosane, and high-speed shear at 6000 rpm for 2.0 min under a high-speed homogenizer. The water bath temperature is maintained at 37° C. during the homogenization process.

[0064] Application of the stabilizer prepared in Example 3 in preparing a temperature-sensitive Pickering emulsion to catalyze the hydrolysis of olive oil:

[0065] Take 2 mL of the natural macromolecular self-assembled micelle solution loaded with lipase in step 2), add 4 mL of n-eicosane containing 5% olive oil, and high-speed shear at 6000 rpm for 2.0 min under a high-speed homogenizer. The water bath temperature during the homogenization process is maintained at 37°C. The emulsion after homogenization is reacted on a 37°C shaker for 2 hours. The reaction is completed. The emulsion is placed in ice water, and after the emulsion solidifies, it is placed at 37°C to melt the solidified emulsion, and the oil phase at the end of the upper reaction is taken, and the oleic acid content is measured. The reaction hydrolysis rate is determined according to the amount of oleic acid generated. Through experiments, the hydrolysis rate of the present invention is relatively high.

Claims

1. A temperature-sensitive Pickering emulsion, characterized in that: The temperature-sensitive Pickering emulsion comprises an aqueous phase and an oil phase; The aqueous phase is a solution of a stabilizer based on natural macromolecular self-assembled micelles, and the oil phase is a phase change material n-hexadecane; The preparation method of the stabilizer based on natural macromolecular self-assembled micelles is: The lipase is placed in an anionic natural macromolecular solution and stirred at room temperature to obtain a mixed solution; the mixed solution is added dropwise to the chitosan solution under stirring, the pH is adjusted to 3.0-10.0, and stirring is continued to obtain; The preparation method of the chitosan solution is as follows: placing cationic natural macromolecular chitosan in an acidic solution, stirring and dissolving, and obtaining the chitosan solution; the acidic solution is an acetic acid solution or a hydrochloric acid solution, and the volume concentration of the acidic solution is 1%-5%; the concentration of chitosan in the acidic solution is 0.01-10 mg·mL -1 ; The preparation method of the anionic natural macromolecule solution is as follows: placing anionic natural macromolecules in ultrapure water and stirring to dissolve; the anionic natural macromolecules are sodium hyaluronate, sodium γ-polyglutamate or sodium alginate; the concentration of the anionic natural macromolecules in the anionic natural macromolecule solution is 0.01-10 mg·mL -1 ; The concentration of the lipase in the anionic natural macromolecular solution is 0.01-10 mg·mL -1 ; The volume ratio of the chitosan solution to the mixed solution is 4:1-1:

4.

2. A method for preparing the temperature-sensitive Pickering emulsion according to claim 1, characterized in that: The preparation method comprises the following steps: using a stabilizer solution based on natural macromolecular self-assembled micelles as a water phase, using a phase change material n-hexadecane as an oil phase, and preparing a temperature-sensitive Pickering emulsion under high-speed homogenization.

3. The preparation method according to claim 2, characterized in that: The volume ratio of the stabilizer solution based on natural macromolecular self-assembled micelles to the phase change material solution is 10:1-1:

10.

4. An application of a temperature-sensitive Pickering emulsion, characterized in that: The temperature-sensitive Pickering emulsion according to claim 1 is used to catalyze the hydrolysis of oils and fats.

Images