A food-grade emulsion enzyme catalytic system, a construction method and application in functional lipid synthesis

Abstract

The application belongs to the technical field of food engineering, and particularly relates to a food-grade emulsion enzyme catalysis system, a construction method and application in functional lipid synthesis. The food-grade Pickering emulsion is formed by taking water-insoluble protein-chitin nanoparticles and a lipase CRL compound as the water phase and taking a reaction substrate fatty acid as the oil phase, and is used for preparing phytosterol esters. The water-insoluble protein-chitin nanoparticles have excellent dispersibility, stability and high surface positive charge, the lipase is adsorbed on the surface of the nanoparticles through electrostatic and hydrogen bond interaction, has reduced interfacial tension and enhanced interfacial activity, the lipase in the Pickering emulsion stabilized by the nanoparticles is distributed on the oil-water interface, the catalytic efficiency of the lipase is effectively improved, and the food-grade Pickering emulsion interface catalysis is used to efficiently synthesize phytosterol esters.

Description

Technical Field

[0001] This invention belongs to the field of food engineering technology, specifically relating to a food-grade emulsion enzyme catalytic system, its construction method, and its application in functional lipid synthesis. Background Technology

[0002] Phytosterols are natural active ingredients found in various vegetable oils, nuts, and seeds, possessing multiple functions such as lowering cholesterol levels, preventing cardiovascular disease, reducing inflammation, and preventing cancer. However, their poor solubility and bioavailability limit their application in the food industry. Studies have shown that oil-soluble phytosterol esters can significantly improve bioavailability while retaining the functional characteristics of phytosterols. Since sterol esters can be converted into sterols and fatty acids in the human body, their physiological functions include those of both phytosterols and fatty acids. Therefore, synthesizing phytosterol esters through the esterification reaction of fatty acids and phytosterols can effectively improve their utilization efficiency.

[0003] Currently, phytosterol esters are mainly synthesized using chemical or enzymatic methods. Chemical synthesis is carried out at relatively high temperatures, which easily leads to various side reactions, including unsaturated fatty acid oxidation, phytosterol dehydration, and phytosterol oxidation. This results in increased impurities in the sterol ester product, reduced yield due to byproduct formation, and complex separation and purification processes, including the challenge of removing toxic chemical catalysts. Compared to chemical synthesis, the enzymatic synthesis of sterol esters using fatty acids and phytosterols has attracted widespread attention due to its mild conditions, high specificity, high conversion rate, and minimal byproducts. However, many lipase substrates exhibit poor water solubility, while lipases inherently require an aqueous environment to achieve optimal activity. In industrial applications, lipases are typically located in immiscible two-phase systems, and their small interfacial area severely inhibits the reaction rate.

[0004] Pickering emulsions are emulsion systems stabilized by solid particles rather than traditional surfactants. These solid particles are precisely anchored at the oil-water interface, forming stable, dispersed droplets that significantly increase the interfacial area. Lipases can be distributed in the aqueous phase (the inner phase) or combined with the solid particles at the oil-water interface, while the substrate dissolves in the oil phase. This significantly enhances enzyme-substrate interactions and increases the reaction rate. Currently reported solid particles used in the Pickering emulsion interface catalysis for the preparation of phytosterol esters are mostly modified / unmodified silica spheres and carbon nanoparticles. These particles are primarily derived from inorganic materials dependent on non-renewable resources, and their production involves complex, energy-intensive processes, resulting in high manufacturing costs and often requiring toxic chemical reagents. Because these materials typically do not meet food-grade standards, their application in the food and pharmaceutical industries remains severely limited. Therefore, developing food-grade natural biopolymer nanoparticles for the Pickering interface biocatalysis of phytosterol esters is crucial. Summary of the Invention

[0005] In view of this, the present invention provides a food-grade emulsion enzyme catalytic system, a method for its construction, and its application in the synthesis of functional lipids.

[0006] This invention first successfully prepared well-dispersible, water-insoluble protein-chitosan nanoparticles, then used them to adsorb lipase and stabilize Pickering emulsions. Finally, phytosterol esters were prepared using fatty acids and phytosterols as substrates (oil phase). The food-grade emulsion enzyme catalytic system provided by this invention offers an increased interfacial area for the reaction, and the water byproduct generated during the reaction can migrate into the aqueous phase through the oil-water interface, thereby greatly improving the reaction efficiency. The water-insoluble protein-chitosan nanoparticles involved in this method are natural high-molecular-weight biopolymers, possessing advantages such as simple preparation method, safety and environmental friendliness, low cost, and renewability. Furthermore, they can be separated for the next cycle of reaction by simple centrifugation after the reaction is completed.

[0007] Furthermore, this invention is the first to utilize the electrostatic interaction between chitosan and water-insoluble protein to prepare nanoparticles with excellent dispersibility and stability and enhanced positive charge. It also effectively adsorbs negatively charged lipase through electrostatic interaction to form a water-insoluble protein-chitosan-lipase complex. This complex is used to stabilize food-grade Pickering emulsions and for the efficient preparation of phytosterol esters via interfacial biocatalysis.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for constructing a food-grade emulsion enzyme catalytic system and its application in the synthesis of phytosterol esters includes the following steps: (1) Preparation of water-insoluble protein-chitosan nanoparticles: Water-insoluble protein-chitosan nanoparticles were prepared by antisolvent precipitation method. The protein powder was dissolved in 70% ethanol solution, and then the solution was slowly added to 3 times the volume of chitosan aqueous solution while magnetically stirring. After removing the ethanol by rotary evaporation, nanoparticles were formed.

[0010] It is worth noting that water-insoluble proteins such as corn protein, wheat gliadin, and sorghum gliadin are natural amphiphilic plant proteins with renewability and biocompatibility, making them safe biomaterials. The amphiphilic nature of these water-insoluble proteins allows them to spontaneously self-assemble into micro / nano structures, making them particularly valuable in nanoparticle preparation and Pickering emulsion stabilization. However, Pickering emulsions stabilized solely by nanoparticles assembled from water-insoluble proteins exhibit poor stability. Chitosan, with its high-density positive charge, can co-stabilize Pickering emulsions with the aforementioned water-insoluble proteins.

[0011] (2) Adsorption of lipase CRL by water-insoluble protein-chitosan nanoparticles: Lipase CRL was dissolved in distilled water, and after centrifugation, the supernatant was combined with water-insoluble protein-chitosan nanoparticles to form a water-insoluble protein-chitosan-lipase complex.

[0012] (3) Construction of a stable Pickering emulsion system using a water-insoluble protein-chitosan-lipase complex: Pickering emulsion was prepared by homogenization using a water-insoluble protein-chitosan-lipase CRL complex as the aqueous phase and fatty acids as the oil phase.

[0013] (4) Preparation and separation of phytosterol esters: Phytosterols were dissolved in fatty acids and used as the oil phase. The emulsion system was prepared according to the method in step (3) and heated in a constant temperature water bath to prepare phytosterol esters. The oil phase was separated by centrifugation, and the aqueous phase and water-insoluble protein-chitosan-lipase complex were used for the next cycle reaction.

[0014] According to the above scheme, the fatty acids include oleic acid, linoleic acid, α-linolenic acid, nervonic acid, DHA, EPA, and ARA; and in step (1), the concentration of water-insoluble protein dissolved in ethanol solution is 4.0-6.0 wt%, and the concentration of chitosan aqueous solution is 0.02-0.3 wt%.

[0015] According to the above scheme, the temperature of the rotary evaporator is 35-50℃ and the rotation speed is 60-120rpm.

[0016] According to the above scheme, the water-insoluble protein-chitosan nanoparticles have a particle size of 100-300 nm, a zeta potential of 20-50 mV, and a polydispersity index (PDI) of 0.05-0.4.

[0017] According to the above scheme, in step (2), the lipase CRL is dissolved by magnetic stirring in an ice-water bath, the lipase concentration is 10-60 mg / mL, and the protein concentration in the supernatant after centrifugation is 1.0-10.0 mg / mL.

[0018] According to the above scheme, in step (2), the centrifugation temperature of lipase CRL is 4℃, the centrifugation speed is 5000-8000rpm, and the centrifugation time is 5-10min.

[0019] According to the above scheme, water is added after rotary evaporation to make the final concentration of water-insoluble protein in the dispersion system 1.0-3.0 wt% and the final concentration of chitosan 0.01-0.2%.

[0020] According to the above scheme, in step (2), the water-insoluble protein-chitosan nanoparticles and lipase supernatant are combined in an ice-water bath with magnetic stirring. The volume ratio of the solutions is 1:1, and the combination time is 2-3 hours.

[0021] According to the above scheme, in step (3), the Pickering emulsion system is prepared using the water-insoluble protein-chitosan nanoparticle dispersion obtained in step (1) as the aqueous phase and fatty acids as the oil phase. Pickering emulsions with different oil-water ratios are formed through homogenized mixing. The emulsion particle size ranges from 30 to 100 µm, and the specific surface area is 718 to 1196 cm². 2 / mL.

[0022] According to the above scheme, in step (3), the homogenizing instrument is a handheld homogenizer with a rotation speed of 8000-15000 rpm and a homogenization time of 1-3 min.

[0023] According to the above scheme, in step (4), the phytosterol is added to the fatty acid and dissolved under magnetic stirring, accounting for 8-10% of the fatty acid by mass, and the magnetic stirring speed is 1000-1500 rpm.

[0024] According to the above scheme, in step (4), the preparation conditions for the emulsion system of phytosterol esters are the same as in step (3), and the specific surface area of ​​the resulting emulsion is 718-1196 cm². 2 / mL.

[0025] According to the above scheme, in step (4), the reaction temperature is 50-60℃ and the reaction time is 2-4h.

[0026] According to the above scheme, in step (4), the rotation speed of the centrifugation separation of oil phase, water-insoluble protein-chitosan-lipase complex and aqueous phase is 8000-10000 rpm, and the centrifugation time is 5-10 min.

[0027] Compared with the prior art, the beneficial effects of the present invention are: 1. The water-insoluble protein used in this invention is a natural amphiphilic plant protein material. As a biocompatible excipient, it exhibits significant advantages in constructing food-grade emulsion enzyme catalytic systems. Compared to traditionally used synthetic materials such as silica and carbon spheres, this technical solution has the following outstanding features: (1) Raw material sustainability: It avoids dependence on non-renewable resources such as tetraethyl orthosilicate and quartz sand as raw materials; (2) Environmental protection of process: It overcomes the defects of existing inorganic nanoparticle synthesis process which is complicated and requires the use of toxic chemical reagents; (3) Ease of operation: The preparation method used is simple and efficient, and the preparation steps are simplified.

[0028] 2. This invention is the first to propose using water-insoluble protein-chitosan nanoparticles and lipase as a Pickering emulsion stabilizer and catalyst. The prepared reaction system has a significantly increased interfacial area, and the lipase is distributed at the oil-water interface. The reaction byproduct water can be transferred to the aqueous phase through the interface, thereby greatly improving the reaction efficiency and yield.

[0029] 3. After the reaction described in this invention is completed, the oil phase, aqueous phase, nanoparticles and lipase can be separated by simple centrifugation. By retaining the aqueous phase, nanoparticles and lipase, a new oil phase and phytosterols are added and homogenized to form an emulsion for the next cycle reaction. The emulsion can still maintain high catalytic activity in a certain number of cycles reaction, thereby improving the utilization efficiency of lipase. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0031] Figure 1 This is a schematic diagram of the synthesis of phytosterol esters using the food-grade emulsion enzyme catalytic system of this invention. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in the embodiments of this application, unless otherwise specified, employs conventional testing methods in the art. It should be understood that the terminology used in this application is merely for describing particular implementations and is not intended to limit the scope of this disclosure.

[0034] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; other experimental methods and technical means not specifically mentioned herein refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0035] To better illustrate the content of this application, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this application can be implemented even without certain specific details. In the embodiments, some methods, means, instruments, and devices well-known to those skilled in the art are not described in detail in order to highlight the main points of this application.

[0036] Without conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the resulting technical solution belongs to the content disclosed in the embodiments of this application.

[0037] This invention discloses a food-grade emulsion enzyme catalytic system, its construction method, and its application in functional lipid synthesis.

[0038] In this invention, water-insoluble protein-chitosan nanoparticles are prepared using an antisolvent precipitation method. The specific steps are as follows: the water-insoluble protein is dissolved in a 70% ethanol solution at a concentration of 4.0-6.0 wt%. After complete dissolution, it is slowly added to three times the volume of chitosan aqueous solution of different concentrations (0.02-0.3 wt%) using a syringe, and the solution is magnetically stirred for 30 min. The resulting solution is then transferred to a round-bottom flask and evaporated under reduced pressure using a rotary evaporator at a temperature of 35-50℃ and a rotation speed of 60-120 rpm until the volume of the remaining solution is less than the original volume. Distilled water is then added to the resulting solution to bring the protein concentration to the required level, thus obtaining water-insoluble protein-chitosan nanoparticles.

[0039] The water-insoluble protein-chitosan nanoparticles obtained by the above method have a particle size of 100-300 nm, a zeta potential of 20-50 mV, and a polydispersity index (PDI) of 0.05-0.4.

[0040] Furthermore, a water-insoluble protein-chitosan-lipase complex was prepared by electrostatic adsorption. The specific steps were as follows: lipase was dissolved in distilled water, and after removing impurities by centrifugation, it was added to the above-obtained water-insoluble protein-chitosan nanoparticle solution at a volume ratio of 1:1. The complex was then magnetically stirred in an ice-water bath for 2-3 hours to obtain the water-insoluble protein-chitosan-lipase complex.

[0041] The following examples are only used to illustrate the content of the present invention in detail, so as to facilitate a better understanding of the present invention, and are included within the scope of protection of the present invention, but do not limit the present invention.

[0042] It should be noted that the phytosterol ester products synthesized by the water-insoluble protein-chitosan-lipase complex-stabilized food-grade Pickering emulsion were detected by gas chromatography.

[0043] The gas chromatography detection conditions are as follows: The chromatographic column was a DB-5HT (15m × 0.320 mm, 0.10 μm), and the detector was an FID detector. The injection port temperature was 380 ℃, the split ratio was 50:1, and the injection volume was 1 μL. The carrier gas was high-purity helium, with a flow rate of 2 mL / min. The hydrogen flow rate was 32 mL / min, and the air flow rate was 200 mL / min. The column oven temperature program was: initial temperature 170 ℃, hold for 2 min, then increase to 380 ℃ at a rate of 5 ℃ / min and hold for 6 min.

[0044] To better understand the present invention, the following embodiments are provided for further detailed description of the present invention, but they should not be construed as limiting the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also considered to fall within the protection scope of the present invention.

[0045] The preparation method of the sorghum prolysin-chitosan-lipase complex used in the embodiments of the present invention is as follows: Sorghum prolysin was dissolved in a 70% ethanol solution to a concentration of 4.0 wt%. After complete dissolution, it was slowly added to an aqueous solution containing 0.05 g of chitosan using a syringe and magnetically stirred for 30 min. The resulting solution was then transferred to a round-bottom flask and evaporated under reduced pressure using a rotary evaporator at a temperature of 45 °C and a rotation speed of 100 rpm until the volume of the remaining solution was less than the original volume. Distilled water was then added to the resulting solution to bring the protein concentration to the desired level, yielding sorghum prolysin-chitosan nanoparticles.

[0046] Lipase CRL was dissolved in distilled water, and after removing impurities by centrifugation, it was added to the above-obtained sorghum prolysin-chitosan nanoparticle solution at a volume ratio of 1:1. The mixture was then stirred magnetically in an ice-water bath at a speed of 600 rpm for 2 hours to obtain the sorghum prolysin-chitosan-lipase complex.

[0047] Example 1 2.0 mL of the sorghum alcohol-chitosan-lipase complex prepared above was added to a 10 mL glass reaction flask, with a lipase concentration of 10 mg / mL. Then, 3.0 mL of oleic acid containing 0.3 g of phytosterols was added. The mixture was homogenized at 12000 rpm for 1 min using a handheld homogenizer to form a Pickering emulsion. The emulsion was an oil-in-water emulsion. The mixture was then quickly placed in a constant temperature water bath and reacted at 55 °C for 3 h. 100 µL of the reaction product was taken with a pipette and dissolved in 1 mL of n-hexane. After filtration through a 0.22 μm pore size filter membrane, the product was analyzed by gas chromatography. The sterol ester yield reached 60.6%.

[0048] Example 2 2.0 mL of the sorghum alcohol-chitosan-lipase complex prepared above was added to a 10 mL glass reaction flask, with a lipase concentration of 20 mg / mL. Then, 3.0 mL of oleic acid containing 0.3 g of phytosterols was added. The mixture was homogenized at 12000 rpm for 1 min using a handheld homogenizer to form a Pickering emulsion. The emulsion was an oil-in-water emulsion. The mixture was then quickly placed in a constant temperature water bath and reacted at 55 °C for 3 h. 100 µL of the reaction product was taken with a pipette and dissolved in 1 mL of n-hexane. After filtration through a 0.22 μm pore size filter membrane, the product was analyzed by gas chromatography. The sterol ester yield reached 85.9%.

[0049] Example 3 2.0 mL of the sorghum alcohol-chitosan-lipase complex prepared above was added to a 10 mL glass reaction flask, with a lipase concentration of 30 mg / mL. Then, 3.0 mL of oleic acid containing 0.3 g of phytosterols was added. The mixture was homogenized at 12000 rpm for 1 min using a handheld homogenizer to form a Pickering emulsion. The emulsion was an oil-in-water emulsion. The mixture was then quickly placed in a constant temperature water bath and reacted at 55 °C for 3 h. 100 µL of the reaction product was taken with a pipette and dissolved in 1 mL of n-hexane. After filtration through a 0.22 μm pore size filter membrane, the product was analyzed by gas chromatography. The sterol ester yield reached 89.8%.

[0050] Example 4 2.0 mL of the sorghum alcohol-chitosan-lipase complex prepared above was added to a 10 mL glass reaction flask, with a lipase concentration of 40 mg / mL. Then, 3.0 mL of oleic acid containing 0.3 g of phytosterols was added. The mixture was homogenized at 12000 rpm for 1 min using a handheld homogenizer to form a Pickering emulsion. The emulsion was an oil-in-water emulsion. The mixture was then rapidly placed in a constant temperature water bath and reacted at 55 °C for 3 h. 100 µL of the reaction product was taken using a pipette and dissolved in 1 mL of n-hexane. After filtration through a 0.22 μm pore size filter membrane, the product was analyzed by gas chromatography. The sterol ester yield reached 95.2%.

[0051] Example 5 2.0 mL of the sorghum alcohol-chitosan-lipase complex prepared above was added to a 10 mL glass reaction flask, with a lipase concentration of 50 mg / mL. Then, 3.0 mL of oleic acid containing 0.3 g of phytosterols was added. The mixture was homogenized at 12000 rpm for 1 min using a handheld homogenizer to form a Pickering emulsion. The emulsion was an oil-in-water emulsion. The mixture was then rapidly placed in a constant temperature water bath and reacted at 55 °C for 3 h. 100 µL of the reaction product was taken using a pipette and dissolved in 1 mL of n-hexane. After filtration through a 0.22 μm pore size filter membrane, the product was analyzed by gas chromatography. The sterol ester yield reached 90.0%.

[0052] Example 6 2.0 mL of the sorghum alcohol-chitosan-lipase complex prepared above was added to a 10 mL glass reaction flask, with a lipase concentration of 40 mg / mL. Then, 3.0 mL of EPA containing 0.3 g of phytosterol was added. The mixture was homogenized at 12000 rpm for 1 min using a handheld homogenizer to form a Pickering emulsion of oil-in-water type. The emulsion was then rapidly placed in a constant temperature water bath and reacted at 55 °C for 3 h. 100 µL of the reaction product was taken with a pipette and dissolved in 1 mL of n-hexane. After filtration through a 0.22 μm pore size filter membrane, the product was analyzed by gas chromatography, and the sterol ester yield reached 89.6%.

[0053] Example 7 2.0 mL of the sorghum alcohol-chitosan-lipase complex prepared above was added to a 10 mL glass reaction flask, with a lipase concentration of 40 mg / mL. Then, 3.0 mL of ARA containing 0.3 g of phytosterols was added. The mixture was homogenized at 12000 rpm for 1 min using a handheld homogenizer to form a Pickering emulsion. The emulsion was an oil-in-water emulsion. The mixture was then quickly placed in a constant temperature water bath and reacted at 55 °C for 3 h. 100 µL of the reaction product was taken with a pipette and dissolved in 1 mL of n-hexane. After filtration through a 0.22 μm pore size filter membrane, the product was analyzed by gas chromatography. The sterol ester yield reached 82.0%.

[0054] Example 8 2.0 mL of the sorghum alcohol-chitosan-lipase complex prepared above was added to a 10 mL glass reaction flask, with a lipase concentration of 40 mg / mL. Then, 3.0 mL of nervonic acid containing 0.3 g of phytosterol was added. The mixture was homogenized at 12000 rpm for 1 min using a handheld homogenizer to form a Pickering emulsion. The emulsion was an oil-in-water emulsion. The mixture was then rapidly placed in a constant temperature water bath and reacted at 55 °C for 3 h. 100 µL of the reaction product was taken using a pipette and dissolved in 1 mL of n-hexane. After filtration through a 0.22 μm pore size filter membrane, the product was analyzed by gas chromatography. The sterol ester yield reached 84.8%.

[0055] Example 9 2.0 mL of the sorghum alcohol-chitosan-lipase complex prepared above was added to a 10 mL glass reaction flask, with a lipase concentration of 40 mg / mL. Then, 3.0 mL of DHA containing 0.3 g of phytosterols was added. The mixture was homogenized at 12000 rpm for 1 min using a handheld homogenizer to form a Pickering emulsion. The emulsion was an oil-in-water emulsion. The mixture was then quickly placed in a constant temperature water bath and reacted at 55 °C for 3 h. 100 µL of the reaction product was taken with a pipette and dissolved in 1 mL of n-hexane. After filtration through a 0.22 μm pore size filter membrane, the product was analyzed by gas chromatography. The sterol ester yield reached 81.7%.

[0056] Example 10 2.0 mL of the sorghum alcohol-chitosan-lipase complex prepared above was added to a 10 mL glass reaction flask, with a lipase concentration of 40 mg / mL. Then, 3.0 mL of α-linolenic acid containing 0.3 g of phytosterol was added. The mixture was homogenized at 12000 rpm for 1 min using a handheld homogenizer to form a Pickering emulsion. The emulsion was an oil-in-water emulsion. The mixture was then rapidly placed in a constant temperature water bath and reacted at 55 °C for 3 h. 100 µL of the reaction product was taken with a pipette and dissolved in 1 mL of n-hexane. After filtration through a 0.22 μm pore size filter membrane, the product was analyzed by gas chromatography. The sterol ester yield reached 85.9%.

[0057] Example 11 2.0 mL of the sorghum alcohol-chitosan-lipase complex prepared above was added to a 10 mL glass reaction flask, with a lipase concentration of 40 mg / mL. Then, 3.0 mL of linoleic acid containing 0.3 g of phytosterols was added. The mixture was homogenized at 12000 rpm for 1 min using a handheld homogenizer to form a Pickering emulsion. The emulsion was an oil-in-water emulsion. The mixture was then quickly placed in a constant temperature water bath and reacted at 55 °C for 3 h. 100 µL of the reaction product was taken with a pipette and dissolved in 1 mL of n-hexane. After filtration through a 0.22 μm pore size filter membrane, the product was analyzed by gas chromatography. The sterol ester yield reached 91.9%.

[0058] To further demonstrate the beneficial effects of the present invention and to better understand it, the technical features disclosed in the present invention are further illustrated by the following comparative examples, but these should not be construed as limiting the present invention. Other improvements made by those skilled in the art based on the above description of the invention, without inventive effort, are also considered to fall within the protection scope of the present invention.

[0059] Comparative Example 1 2.0 mL of free lipase at a concentration of 40 mg / mL was added to a 10 mL glass reaction flask. Then, 3.0 mL of oleic acid containing 0.3 g of phytosterols was added. The mixture was homogenized at 12,000 rpm for 1 min using a handheld homogenizer. The mixture was then quickly placed in a constant temperature water bath and reacted at 55 °C for 3 h. 100 µL of the reaction product was taken with a pipette and dissolved in 1 mL of n-hexane. After filtration through a 0.22 μm pore size filter, the product was analyzed by gas chromatography. The yield of sterol esters was 50.2%.

[0060] Comparative Example 2 2.0 mL of free lipase at a concentration of 40 mg / mL was added to a 10 mL glass reaction flask, followed by 3.0 mL of oleic acid containing 0.3 g of phytosterols. The flask was then placed in a constant temperature water bath and reacted at 55 °C for 3 h. 100 µL of the reaction product was taken with a pipette and dissolved in 1 mL of n-hexane. After filtration through a 0.22 μm pore size filter, the product was analyzed by gas chromatography. The yield of sterol esters was 18.8%.

[0061] Table 1. Yields of phytosterol esters in different embodiments

[0062] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing a food-grade emulsion enzyme catalytic system, characterized in that, The construction method specifically includes the following steps: Water-insoluble protein-chitosan nanoparticles were prepared using an antisolvent precipitation method. Lipase CRL was then combined with the nanoparticles to adsorb onto the nanoparticle surface, forming a water-insoluble protein-chitosan-lipase complex. The complex was used as the aqueous phase, and fatty acids as the oil phase, and a Pickering emulsion was formed through homogenization. After catalytic reaction, the oil phase, aqueous phase, and water-insoluble protein-chitosan-lipase complex were separated by centrifugation, and the aqueous phase and water-insoluble protein-chitosan-lipase complex were used in the next cycle reaction. The water-insoluble proteins include corn protein, wheat prolysin, or sorghum prolysin. The antisolvent precipitation method is as follows: for any water-insoluble protein, it is dissolved in 70% ethanol, and then added dropwise to 3 times the amount of chitosan solution with different masses. The ethanol is removed by rotary evaporation to obtain water-insoluble protein-chitosan nanoparticles. The concentration of the water-insoluble protein dissolved in 70% ethanol is 4.0-6.0 wt%, and the concentration of the chitosan solution is 0.02-0.3 wt%. The water-insoluble protein-chitosan nanoparticles have a particle size of 100-300 nm, a zeta potential of 20-50 mV, and a polydispersity index (PDI) of 0.05-0.

4.

2. The method for constructing the food-grade emulsion enzyme catalytic system according to claim 1, characterized in that, The fatty acids include oleic acid, linoleic acid, alpha-linolenic acid, nervonic acid, DHA, EPA, and ARA.

3. The method for constructing the food-grade emulsion enzyme catalytic system according to claim 1, characterized in that, The rotary evaporation temperature is 35-50℃, the rotation speed is 60-120 rpm, and the final concentration of water-insoluble protein in the dispersion system after rotary evaporation is 1.0-3.0 wt%, and the final concentration of chitosan is 0.01-0.2%.

4. The method for constructing the food-grade emulsion enzyme catalytic system according to claim 1, characterized in that, Lipase CRL was dissolved in distilled water, and after centrifugation to remove impurities, the supernatant was added dropwise to a water-insoluble protein-chitosan nanoparticle dispersion and magnetically stirred to form a water-insoluble protein-chitosan-lipase complex.

5. The method for constructing the food-grade emulsion enzyme catalytic system according to claim 4, characterized in that, The concentration of the lipase CRL solution was 10-60 mg / mL, and the protein concentration of the supernatant after centrifugation was 1.0-10.0 mg / mL. The complexation of the lipase with the nanoparticles was carried out by magnetic stirring in an ice-water bath at a speed of 400-800 rpm for 2-3 hours.

6. The method for constructing the food-grade emulsion enzyme catalytic system according to claim 1, characterized in that, Pickering emulsion systems are prepared by using the complex as the aqueous phase and fatty acids as the oil phase, and forming Pickering emulsions with different oil-water ratios through homogenization. The resulting emulsion has a particle size of 30-100µm and an oil-water ratio of 8:2-3:

7.

7. The application of the food-grade emulsion enzyme catalytic system obtained by the construction method according to any one of claims 1-6 in the synthesis of functional lipids.

8. The application according to claim 7, characterized in that, Phytosterol esters were prepared by homogenizing a Pickering emulsion using a water-insoluble protein-chitosan-lipase complex as the aqueous phase and fatty acids containing phytosterols as the oil phase. Phytosterols are soluble in fatty acids, accounting for 8-10% of the fatty acid mass. The reaction temperature is 50-60℃, and the reaction time is 2-4 hours.

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