Immobilized enzyme chitosan emulsification material and its application in catalytic esterification reaction synthesis of phytosterol acetate

Abstract

The present application relates to a kind of enzyme immobilized chitosan emulsification material and its application in catalytic esterification reaction synthesis phytosterol acetate.The technical scheme used is: lipase is immobilized to chitosan surface by crosslinking agent to prepare the chitosan particle (CSPPL) of enzyme immobilized lipase, then the prepared CSPPL is used to prepare W / O Pickering emulsion catalytic system with phytosterol (PS), and the catalytic system can be used for the synthesis of phytosterol acetate, and each drop of emulsion is used as microreactor of reactant in oil phase.CSPPL can play the role of stabilizer, and also can play the role of catalyst;PS can play the role of stabilizer, and also as reaction substrate participates in catalytic reaction.Compared with homogeneous reaction, phytosterol / chitosan composite particle stable W / O Pickering emulsion catalytic system has good conversion rate.

Description

Technical Field

[0001] This invention relates to the field of phytosterol ester preparation technology, specifically to a method for the catalytic synthesis of phytosterol acetate using a Pickering emulsion catalytic system stabilized by phytosterol / chitosan composite particles. Background Technology

[0002] Phytosterols possess lipid-lowering properties, but their insolubility in water and limited solubility in the oil phase restrict their applications. To broaden their applications, converting phytosterols into phytosterol esters is one of the most important modification methods. On the one hand, modification of phytosterols improves their lipid solubility without affecting their function, making them suitable as new raw materials for food, pharmaceuticals, and cosmetics. On the other hand, in conventional enzymatic non-aqueous homogeneous catalytic esterification reactions, the anhydrous system is beneficial for the esterification reaction in the synthesis of phytosterol acetates. However, the activity and stability of enzymes in the non-aqueous phase are generally lower than those in the aqueous phase. Therefore, proposing a two-phase interface biocatalytic system with high conversion rate and recoverable catalyst is of practical significance.

[0003] Chitosan is an important natural biodegradable polymer material. It is a homogeneous, linear, basic polysaccharide containing amino groups and one of the few naturally occurring charged products. In solution, chitosan is a positively charged polyelectrolyte with strong adsorption properties. Chitosan also exhibits excellent biocompatibility, low toxicity, and various pharmacological activities, such as antibacterial, anti-acid, lipid-lowering, and cholesterol-lowering effects. These unique properties make chitosan widely applicable in biomedicine, including controlled drug release, enzyme immobilization, artificial dialysis membranes, and artificial skin. Phytosterols are lipid-soluble substances derived from plant oils. Due to their large hydrophobic and hydroxyl groups in their molecular structure, phytosterols possess emulsifying properties and can be used as emulsifiers. Phytosterols and chitosan, with opposite charges, adsorb each other through electrostatic interactions, altering the wettability and interfacial activity of the particles. This results in both phytosterol and chitosan particles being adsorbed at the oil-water interface, thus exhibiting better surface wettability and the ability to reduce oil-water interfacial tension. Summary of the Invention

[0004] This invention immobilizes lipase onto the surface of chitosan using a cross-linking agent to obtain a composite material (CSPPL) that combines emulsifying and catalytic functions. This composite material is simple to synthesize, easy to purify, and exhibits excellent dispersibility in water, showing promising applications in the catalytic synthesis of phytosterol acetates at the water-oil interface.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: a chitosan emulsified material for immobilizing enzymes. The preparation method of the chitosan emulsified material for immobilizing enzymes includes the following steps: preparing a lipase buffer solution with phosphate buffer, mixing chitosan with the lipase buffer solution, stirring magnetically, adding glutaraldehyde, fully crosslinking, centrifuging the product, washing with water, and vacuum drying to obtain the target product, the chitosan emulsified material for immobilizing enzymes, CSPPL.

[0006] Furthermore, in the aforementioned chitosan emulsion material immobilized with an enzyme, the pH of the phosphate buffer is 7.

[0007] Furthermore, in the aforementioned chitosan emulsified material with an immobilized enzyme, the lipase is porcine pancreatic lipase.

[0008] A phytosterol / chitosan composite particle-stabilized Pickering emulsion is prepared by ultrasonically dispersing the above-mentioned enzyme-loaded chitosan emulsion in distilled water to form an aqueous phase; adding phytosterol to white oil and stirring thoroughly to dissolve it to form an oil phase; mixing the aqueous and oil phases and vortexing them at 3000 r / min for 3 min to form a W / O type Pickering emulsion.

[0009] The present invention relates to the application of the enzyme-supported chitosan emulsion material in the synthesis of phytosterol acetate via water-oil interface catalytic esterification reaction.

[0010] Further, the method is as follows: the chitosan emulsion material immobilized with enzyme is ultrasonically dispersed in an aqueous solution of the reaction substrate pyruvate and magnetically stirred to form an aqueous phase; the reaction substrate phytosterol is added to white oil and stirred thoroughly to dissolve, forming an oil phase; the aqueous phase and oil phase are mixed and vortexed at 3000 r / min for 3 min to form a W / O Pickering emulsion catalytic system; after standing at room temperature for 20-24 h, the emulsion is broken by heating, and the upper oil phase is taken to obtain the product phytosterol acetate.

[0011] Furthermore, in the aqueous phase, the concentration of the chitosan emulsion material immobilized with the enzyme is 20 mg / mL.

[0012] Furthermore, the concentration of phytosterols in the oil phase was 10 mg / mL.

[0013] Furthermore, in a molar ratio, pyruvate: phytosterols = 5:1.

[0014] Furthermore, by volume ratio, the ratio of aqueous phase to oil phase is 1:2.

[0015] Furthermore, the phytosterol is β-sitosterol.

[0016] The beneficial effects of this invention are:

[0017] 1. This invention immobilizes lipase onto the surface of chitosan using a cross-linking agent to obtain a composite material (CSPPL) that combines emulsifying and catalytic functions. This composite material is simple to synthesize, easy to purify, and exhibits excellent dispersibility in water, showing promising application prospects in the catalytic synthesis of phytosterol acetates at the water-oil interface.

[0018] 2. The phytosterol / chitosan composite particle-stabilized Pickering emulsion prepared in this invention is a temperature-switched biphase interface biocatalytic system with a large water-oil interface, protection of lipase, and simple separation and recovery.

[0019] 3. The immobilized lipase chitosan composite material prepared by the present invention can be used to catalyze the synthesis of phytosterol acetate, and has the characteristics of high conversion rate and simple separation. It has a good application prospect in the field of water-oil interface catalysis.

[0020] 4. The phytosterol / chitosan composite particle-stabilized W / O Pickering emulsion catalytic system provided by this invention can be used for the synthesis of phytosterol acetates. Each drop of emulsion is used as a microreactor for the reactants in the oil phase. CSPPL acts as both a stabilizer and a catalyst. PS acts as both a stabilizer and a substrate in the catalytic reaction. Compared to homogeneous reactions, the phytosterol / chitosan composite particle-stabilized W / O Pickering emulsion catalytic system exhibits excellent conversion rates. Attached Figure Description

[0021] Figure 1 These are SEM images of CS(a), CSPPL(b), and PS(c).

[0022] Figure 2 These are the Zeta potential diagrams for CS(a), PPL(b), and CSPPL(c).

[0023] Figure 3 This is a schematic diagram of using phytosterol / chitosan composite particles to stabilize Pickering emulsion.

[0024] Figure 4 These are optical microscope images of emulsions and emulsion droplets stained with CS (a), CSPPL (b), PS (c), and PS (d).

[0025] Figure 5 These are the contact angle diagrams for CS(a), CSPPL(b), and PS(c).

[0026] Figure 6AThe UV-Vis spectrophotometric curves of the phytosterol acetate series of standard colorimetric solutions are shown, with ag corresponding to the curves when the content of phytosterol acetate added is 0, 50, 100, 150, 200, 300 and 400 μg, respectively.

[0027] Figure 6B The curve shows the characteristic absorbance peak as a function of phytosterol acetate concentration.

[0028] Figure 6C The concentration of phytosterol acetate was 50.0 μg·mL. -1 Linear relationship between the peak intensity of characteristic absorbance within the range and the concentration of phytosterol acetate.

[0029] Figure 6D The concentration of phytosterols was 10 mg / mL. -1 UV-Vis absorption spectra of phytosterol acetate in the Pickering emulsion catalysis system. Detailed Implementation

[0030] Example 1: Enzyme-loaded chitosan emulsion material (CSPPL)

[0031] (I) Preparation method

[0032] Porcine pancreatic lipase (PPL) was prepared into a lipase buffer solution with a concentration of 5 mg / mL using phosphate buffer at pH 7. 0.2 g of chitosan (CS) was mixed with 40 mL of the lipase buffer solution and magnetically stirred at room temperature for 3 h. Then, 1.6 mL of 50% glutaraldehyde was added, and the mixture was stirred for 8 h to allow for complete cross-linking. The resulting product was centrifuged, washed with water, and vacuum dried at 65 °C to obtain the chitosan emulsion material (CSPPL) with the target product immobilized on the enzyme.

[0033] (II) Characterization

[0034] Figure 1 SEM topography images of CS, CSPPL, and PS. From Figure 1 As can be seen in image a, CS has a rough, sheet-like structure with transverse and longitudinal dimensions of approximately tens of micrometers. From... Figure 1 As can be seen in image b, CSPPL has a sheet-like structure with a diameter of approximately a few micrometers, and the shape of chitosan remained unchanged before and after enzyme immobilization. From Figure 1 As can be seen in the image, PS has a sheet-like structure with a lateral dimension of approximately a few micrometers.

[0035] Figure 2 Zeta potential plots for CS, CSPPL, and PPL. From Figure 2 As can be seen from equation (a), chitosan carries a positive charge of 4.28 mV in solution. From... Figure 2As can be seen from b, PPL carries a negative charge in the solution, which is -7.28 mV. From Figure 2 As shown in Figure c, CSPPL carries a positive charge of 16.7 mV in solution. Analysis reveals that chitosan and lipase are unstable in dispersion and prone to aggregation in solution. However, after cross-linking with the cross-linking agent glutaraldehyde, the Zeta potential of CSPPL shifts to the right by 12.42 mV, indicating enhanced anti-aggregation ability compared to chitosan molecules in solution. This results in more stable dispersion in solution and a more stable emulsion formed through emulsification.

[0036] Example 2: Pickering emulsion stabilized by phytosterol / chitosan composite particles

[0037] (I) Preparation method

[0038] The chitosan emulsion material CSPPL with enzyme immobilization prepared in Example 1 was ultrasonically dispersed in distilled water to form an aqueous phase with a concentration of 20 mg / mL. β-sitosterol (PS) was added to white oil and stirred thoroughly to dissolve, forming an oil phase with a concentration of 10 mg / mL. The aqueous and oil phases were mixed at a water-to-oil volume ratio of 1:2 and vortexed at 3000 r / min for 3 min to form a W / O Pickering emulsion catalytic system.

[0039] Figure 3 This is a schematic diagram of using phytosterol / chitosan composite particles to stabilize Pickering emulsion.

[0040] Figure 4 Optical microscope images of emulsions stained with CS, CSPPL, PS, and PS, and emulsion droplets. (See attached images.) Figure 4 As can be seen in Figure a, chitosan can emulsify water and white oil before immobilizing the enzyme, forming an O / W type Pickering emulsion with an emulsion size of approximately 300 micrometers. Figure 4 As can be seen in Figure b, chitosan, after immobilizing lipase, can emulsify water and white oil to form an O / W type Pickering emulsion with an emulsion size of approximately tens of micrometers. Figure 4 As can be seen from c and d, β-sitosterol can emulsify water and white oil to form a W / O type Pickering emulsion with an emulsion size of approximately ten micrometers.

[0041] Figure 5 The contact angle diagrams for CS, CSPPL, and PS are shown. Figure 5 As can be seen from Figure a, chitosan is relatively hydrophilic, with a contact angle of less than 90°. Emulsified water and white oil can form an O / W type Pickering emulsion, which is consistent with actual conditions. Figure 5As can be seen from Figure b, the contact angle of chitosan after enzyme immobilization is also less than 90°, and quite close to 90°, which allows for the formation of a stable O / W type Pickering emulsion. For example... Figure 5 As can be seen from c, the contact angle measured by β-sitosterol is greater than and close to 90°, which can form a stable W / O type Pickering emulsion.

[0042] Example 3: Application of enzyme-supported chitosan emulsion in heterogeneous catalytic synthesis of phytosterol acetates

[0043] The method is as follows:

[0044] 1. Plotting the standard curve:

[0045] Take 0, 50, 100, 150, 200, 300, and 400 μg of phytosterol acetate and 1 mL of ferric-phosphorus chromogenic reagent, respectively, and dilute to 6 mL with ethyl acetate. After mixing and shaking, develop the solution at room temperature for 25 min to obtain a series of standard phytosterol acetate chromogenic solutions. Measure the absorbance (A) at 630 nm using a UV-Vis spectrometer. Plot a standard curve with the concentration of phytosterol acetate on the x-axis and the absorbance at the corresponding concentration on the y-axis.

[0046] 2. Enzymatic synthesis of phytosterol acetate from chitosan emulsifiers immobilized with enzymes.

[0047] The chitosan emulsion material CSPPL, which carries the enzyme and was prepared in Example 1, was ultrasonically dispersed in an aqueous solution of pyruvate, forming an aqueous phase with a concentration of 20 mg / mL and a pyruvate concentration of 0.236 mol / L. The β-sitosterol substrate was added to white oil and stirred thoroughly to dissolve, forming an oil phase with a β-sitosterol concentration of 10 mg / mL. The aqueous and oil phases were mixed at a water-to-oil volume ratio of 1:2 (molar ratio of pyruvate to phytosterol = 5:1), and vortexed at 3000 rpm for 3 min to form a W / O Pickering emulsion catalytic system. After standing at room temperature for 24 h, the temperature was raised to 35 °C to break the emulsion. The upper oil phase was then added to a phosphorus-sulfur-iron colorimetric reagent solution, and its characteristic absorption peaks were measured using a UV-Vis spectrophotometer. The esterification rate was calculated based on the peak values.

[0048] Figure 6A This document presents the characteristic absorption curves of the UV-Vis spectrum for a series of standard colorimetric solutions. The absorbance of phytosterol acetate was measured within a specific wavelength range. The standard colorimetric solutions were placed in cuvettes, and the measurement wavelength range was selected using a Lambda 35 UV-Vis spectrophotometer to observe the absorbance at specific wavelengths. Figure 6AIn the diagram, curve a represents the UV absorption curve when the amount of phytosterol acetate is 0 μg. When 50 μg of phytosterol acetate is added, the absorbance A increases, as shown in curve b. As the amount of phytosterol acetate added gradually increases, the absorbance A gradually increases. Plotting the peak absorbance A for each group against the concentration of phytosterol acetate yields... Figure 6B .

[0049] Figure 6C This is a linear relationship between the absorbance peak value (A) of a series of standard colorimetric solutions at different concentration ranges and the concentration of phytosterol acetate. Figure 6C The medium curve represents the linear range of 0-50 μg·mL for the detection of phytosterol acetate concentration. -1 The linear equation is y = 0.012x + 0.0058, R0 2 The value is 0.9991.

[0050] Figure 6D This is a UV-Vis absorbance curve of phytosterol acetate in the oil phase after the Pickering emulsion catalytic system reaction. Based on the standard curve, the esterification rate of phytosterol acetate synthesized by the enzymatic method using chitosan emulsion material with immobilized enzyme was calculated to be 87.06%, which is approximately three times the esterification rate of the homogeneous reaction.

Claims

1. A Pickering emulsion stabilized by phytosterol / chitosan composite particles, characterized in that, The chitosan emulsion material with immobilized enzyme was ultrasonically dispersed in distilled water to form an aqueous phase; phytosterols were added to white oil and stirred thoroughly to dissolve, forming an oil phase; the aqueous and oil phases were mixed and vortexed at 3000 r / min for 3 min to form a W / O type Pickering emulsion. The preparation method of the enzyme-supported chitosan emulsion material includes the following steps: preparing a lipase buffer solution with phosphate buffer, mixing chitosan with the lipase buffer solution, stirring magnetically, adding glutaraldehyde, fully cross-linking, centrifuging the product, washing with water, and vacuum drying to obtain the enzyme-supported chitosan emulsion material.

2. The Pickering emulsion stabilized by phytosterol / chitosan composite particles according to claim 1, characterized in that, The pH value of the phosphate buffer is 7.

3. The Pickering emulsion stabilized by phytosterol / chitosan composite particles according to claim 1, characterized in that, The lipase is porcine pancreatic lipase.

4. The application of a phytosterol / chitosan composite particle-stabilized Pickering emulsion in the synthesis of phytosterol acetate via water-oil interfacial catalytic esterification, characterized in that... The preparation method of the phytosterol / chitosan composite particle-stabilized Pickering emulsion is as follows: The enzyme-loaded chitosan emulsion material is ultrasonically dispersed in an aqueous solution containing the reaction substrate pyruvate, and magnetically stirred to form an aqueous phase; the reaction substrate phytosterol is added to white oil, and after thorough stirring and dissolution, an oil phase is formed; the aqueous and oil phases are mixed and vortexed at 3000 r / min for 3 min to form a W / O Pickering emulsion catalytic system; after standing at room temperature for 20-24 h, the emulsion is broken by heating, and the upper oil phase is taken to obtain the product phytosterol acetate; wherein the preparation method of the enzyme-loaded chitosan emulsion material includes the following steps: a lipase buffer solution is prepared using phosphate buffer; chitosan is mixed with the lipase buffer solution, magnetically stirred, and glutaraldehyde is added; after thorough cross-linking, the product is centrifuged, washed with water, and vacuum dried to obtain the target product, the enzyme-loaded chitosan emulsion material.

5. The application according to claim 4, characterized in that, In the aqueous phase, the concentration of the chitosan emulsion material immobilized with the enzyme was 20 mg / mL; in the oil phase, the concentration of phytosterols was 10 mg / mL.

6. The application according to claim 4, characterized in that, The molar ratio of pyruvate to phytosterols is 5:

1.

7. The application according to claim 5, characterized in that, By volume ratio, the ratio of aqueous phase to oil phase is 1:

2.

8. The application according to any one of claims 4-7, characterized in that, The phytosterol mentioned is β-sitosterol.

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