An amyloid fibril pickering emulsion interfacial catalysis system, and a preparation method and use thereof

Abstract

The application discloses a kind of amyloid fiber Pickering emulsion interface catalysis system and its preparation method and purposes.A kind of composition for constructing Pickering emulsion interface catalysis system, including enzyme-loaded amyloid fiber and first amyloid fiber, the preparation raw material of the enzyme-loaded amyloid fiber includes second amyloid fiber, interface enzyme and crosslinking agent;The mass ratio of the second amyloid fiber and first amyloid fiber is 1: (0.02~0.6).The composition of the application can realize the efficient enrichment of interface enzyme in oil-water interface when constructing Pickering emulsion interface catalysis system without introducing exogenous additives;Meanwhile, the system has excellent pH stability, salt tolerance, storage stability and thermal stability.When the Pickering emulsion interface catalysis system is applied to two-phase catalytic reaction, it can significantly improve the interface catalytic efficiency, and thus has important application value in the field of biocatalysis and functional food.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a pickering emulsion interfacial catalytic system for amyloid protein fibers, its preparation method, and its uses. Background Technology

[0002] Interfacial enzymes exhibit the characteristic of "interfacial activation," leading to insufficient contact between the enzyme and substrate in homogeneous catalytic systems, resulting in low catalytic efficiency. Non-aqueous enzyme catalytic systems (reverse micelle systems) can provide a large interfacial area, but the inability to reuse the enzyme limits their application in two-phase catalysis. Two-phase catalytic systems based on Pickering emulsions stabilized by loaded interfacial enzyme particles avoid the problems associated with reverse micelles. In the chemical industry, they have proven to have significant application value in interfacial enzyme catalysis, greatly improving enzyme catalytic efficiency and exhibiting good stability. Pickering emulsions stabilized by colloidal particles are becoming an innovative platform for two-phase biocatalysis. This system combines the advantages of immobilized enzymes and reverse micelle systems. The colloidal particles in the Pickering emulsion interfacial catalytic system not only stabilize the Pickering emulsion but also have affinity for the enzyme, which helps to expand the contact area between substrate molecules and the enzyme and improve enzyme stability.

[0003] Amyloid fibers are nanofibers with a cross-sectional β-sheet structure, formed by the self-assembly of proteins under specific conditions. In recent years, amyloid fibers formed from dietary proteins (such as β-lactoglobulin and ovalbumin) have shown great application potential in food colloids and biomaterials due to their good biocompatibility, excellent interfacial activity, and abundant surface-active groups. As a Pickering emulsion stabilizer, amyloid fibers can irreversibly adsorb onto the oil-water interface to form a dense interfacial film, significantly improving emulsion stability. Simultaneously, the high specific surface area imparted by their nanofiber morphology, along with the readily accessible amino and carboxyl groups, also makes them a highly promising enzyme immobilization carrier. Currently, studies have shown that laccase and glucose oxidase can be loaded onto amyloid fibers through physical adsorption or covalent cross-linking for aqueous catalysis.

[0004] However, immobilizing interfacial enzymes on amyloid fibers often leads to adverse changes in the properties of the amyloid fibers themselves. On the one hand, the introduction of interfacial enzyme molecules may mask some hydrophobic sites and reduce the flexibility of the amyloid fiber carrier structure; on the other hand, the hydrodynamic size of the amyloid fibers loaded with these enzymes increases significantly. These changes collectively weaken the affinity of the enzyme-loaded amyloid fibers for the oil-water interface and their diffusion rate in the bulk phase. As a result, during emulsion homogenization, the enzyme-loaded amyloid fibers cannot quickly adsorb onto the newly formed oil-water interface to effectively prevent droplet coalescence, leading to larger final emulsion droplet size, decreased stability, and incomplete adsorption of the enzyme-loaded amyloid fibers onto the oil-water interface, thus failing to achieve the expected interfacial catalytic efficiency. Introducing small-molecule surfactants to improve emulsion stability violates the "clean label" requirements for clean and simple ingredients in the food and other industries.

[0005] Therefore, developing a Pickering emulsion interfacial catalysis system that does not rely on exogenous additives and can simultaneously achieve efficient emulsification and interfacial catalysis has become a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an amyloid fibrous Pickering emulsion interface catalytic system, its preparation method and uses, to solve the problems in the prior art.

[0007] To achieve the above and other related objectives, the present invention is obtained through the following technical solution.

[0008] A first aspect of the present invention provides a composition for constructing a Pickering emulsion interfacial catalytic system, comprising enzyme-loaded amyloid filaments and first amyloid filaments, wherein the raw materials for preparing the enzyme-loaded amyloid filaments comprise second amyloid filaments, an interfacial enzyme, and a crosslinking agent; the mass ratio of the second amyloid filaments to the first amyloid filaments is 1:(0.02-0.6).

[0009] A second aspect of the present invention provides a Pickering emulsion interfacial catalytic system comprising an emulsion system formed of an aqueous phase and an oil phase, wherein the aqueous phase comprises the composition described above.

[0010] A third aspect of the present invention provides a method for preparing the Pickering emulsion interfacial catalytic system as described above, comprising the following steps:

[0011] The aqueous and oil phases were homogenized to obtain the Pickering emulsion interface catalytic system.

[0012] A fourth aspect of the invention provides the use of the compositions described above or the Pickering emulsion interfacial catalytic system described above in two-phase catalytic reactions.

[0013] A fifth aspect of the present invention provides a method for catalyzing enzyme substrates, using the composition described above or the Pickering emulsion interface catalysis system described above to catalyze enzyme substrates.

[0014] As described above, the amyloid fibrous Pickering emulsion interface catalytic system of the present invention, its preparation method, and its application have the following beneficial effects:

[0015] 1) The Pickering emulsion interface catalytic system of the present invention has excellent emulsification performance and stability. It can maintain structural stability in a wide pH range (pH 1-14) from strong acid to strong base. It can also maintain a stable emulsion state in high concentration salt solutions (1-4 mol / L NaCl). At the same time, it has good thermal stability and long-term storage stability. After standing at room temperature for 1 month or being treated at 80°C for 2 hours, the emulsion droplet structure remains intact and no obvious stratification or emulsion separation occurs.

[0016] 2) The Pickering emulsion interface catalytic system of the present invention can significantly increase the total interfacial area between the oil and water phases and achieve stable and orderly anchoring of enzyme-loaded amyloid filaments at the oil-water interface, thereby promoting the efficient localization and enrichment of interfacial enzymes in the interfacial region, increasing the contact probability between enzyme substrates and interfacial enzymes, and thus significantly improving the catalytic efficiency of the two-phase reaction. Attached Figure Description

[0017] Figure 1A This is a transmission electron microscope image characterization result of β-lactoglobulin amyloid fibers (AFs) in Example 1 of the present invention.

[0018] Figure 1B In Example 1 of the present invention, B represents the fiber growth kinetic curve of β-lactoglobulin amyloid fibers (AFs).

[0019] Figure 2 This is a standard curve showing the change in fluorescence intensity with Cy5-CALB concentration in Example 2 of the present invention.

[0020] Figure 3A This is a transmission electron microscope image of enzyme-loaded amyloid fibrils (C@AFs) in Example 2 of the present invention.

[0021] Figure 3B This is the fluorescence scanning spectrum of enzyme-loaded amyloid fibrils (C@AFs) in Example 2 of the present invention.

[0022] Figure 3C The figure shows the effect of CALB concentration on immobilization efficiency and loading of enzyme-loaded amyloid fibrils (C@AFs) in Example 2 of the present invention.

[0023] Figure 4 This is a schematic diagram of the immobilization of lipase on β-lactoglobulin amyloid filaments and the Pickering emulsion interface catalytic system constructed by binary particles for interfacial catalysis in Example 3 of the present invention.

[0024] Figure 5 A schematic diagram illustrating the preparation of a Pickering emulsion interface catalytic system synergistically constructed from binary particles.

[0025] Figure 6 This study analyzes the physical stability of the Pickering emulsion interface catalytic system stabilized by different concentrations of AFs-C@AFs binary particles in Example 3 of the present invention. The AH values ​​represent 0.5% (w / v) C@AFs, 0.5% (w / v) C@AFs + 0.01% (w / v) AFs, 0.5% (w / v) C@AFs + 0.02% (w / v) AFs, 0.5% (w / v) C@AFs + 0.03% (w / v) AFs, 0.5% (w / v) C@AFs + 0.1% (w / v) AFs, 0.5% (w / v) C@AFs + 0.2% (w / v) AFs, 0.5% (w / v) C@AFs + 0.3% (w / v) AFs, and 0.3% (w / v) AFs, respectively. The evolution of permeability of the Pickering emulsion interface catalytic system stabilized by AFs; I and J are the curves of instability index change over time and the final index value of the Pickering emulsion interface catalytic system stabilized by different concentrations of AFs-C@AFs binary particles, respectively.

[0026] Figure 7 This is the particle size distribution of the Pickering emulsion interface catalytic system in Example 3 of the present invention. Wherein, A represents the particle size distribution of the Pickering emulsion interface catalytic system stabilized by different concentrations of AFs-C@AFs binary particles; B represents the average diameter (D) of the Pickering emulsion interface catalytic system stabilized by different concentrations of AFs-C@AFs binary particles. 4,3 ).

[0027] Figure 8These are optical microscope images and CLSM images of the Pickering emulsion interface catalytic system in Example 3 of the present invention. AC represents optical microscope images of the stable Pickering emulsion interface catalytic system at AFs concentrations of 0.01% (w / v), 0.02% (w / v), and 0.03% (w / v) when the C@AFs concentration is 0.5% (w / v); DF represents CLSM images corresponding to emulsions shown in A, B, and C, respectively.

[0028] Figure 9 These are optical microscope images of the Pickering emulsion interface catalytic systems formed in different oil-water systems in Example 3 of the present invention. In these images, A represents toluene-water; B represents soybean oil-water; C represents n-hexane-water; and D represents ethyl acetate-water.

[0029] Figure 10 These are optical microscope images of the Pickering emulsion interface catalytic system in Example 3 of the present invention at different pH values. Wherein, A represents pH=1.0; B represents pH=5.0; C represents pH=10.0; and D represents pH=14.0.

[0030] Figure 11 These are optical microscope images of the Pickering emulsion interface catalytic system in Example 3 of the present invention at different NaCl concentrations. Wherein, A represents 1.0 mol / L; B represents 2.0 mol / L; C represents 3.0 mol / L; and D represents 4.0 mol / L.

[0031] Figure 12 The figures show the results of the environmental stability study of the Pickering emulsion interface catalytic system in Example 3 of the present invention. In the figures, A is an optical microscope image after standing at room temperature for one month; B is an optical microscope image after standing at 80°C for 2 hours.

[0032] Figure 13 The following diagram illustrates the catalytic performance of different catalytic systems in application examples of the present invention. A is a schematic diagram of a conventional two-phase free enzyme catalytic system; B is a schematic diagram of a Pickering emulsion interface catalytic system; C shows the kinetic curves of triglyceride hydrolysis catalyzed by different catalytic systems; D is a comparison of the relative enzyme activity of lipase in the Pickering emulsion interface catalytic system stabilized by AFs-C@AFs binary particles during 5 reaction cycles; each reaction cycle lasted 11 h.

[0033] Figure 14The images show the macroscopic morphology and microstructure of Pickering emulsions stabilized by different concentrations of AFs in Comparative Example 1. In the images, A is a macroscopic photograph, and B is an optical microscope image, with concentrations of 0.1% (w / v) (first column), 0.3% (w / v) (second column), and 0.5% (w / v) (third column), respectively.

[0034] Figure 15 The images show the macroscopic morphology and microstructure of Pickering emulsions stabilized by C@AFs at different concentrations in Comparative Example 2. A is a macroscopic photograph, B is an optical microscope image, and C is a CLSM image, with concentrations of 0.1% (w / v) (first column), 0.3% (w / v) (second column), and 0.5% (w / v) (third column), respectively. Detailed Implementation

[0035] An emulsion is a heterogeneous system composed of two immiscible liquids, one of which is dispersed in the other as droplets. Due to the high surface energy at the interface, emulsion systems are thermodynamically unstable. The increase in interfacial area during emulsification leads to an increase in the system's free energy, necessitating the introduction of emulsifiers to stabilize the newly formed interface. Pickering emulsions primarily utilize the irreversible adsorption of solid particles onto the oil-water interface, forming a dense interfacial film to prevent droplet aggregation, thus achieving a highly stable emulsion. Currently, Pickering emulsions stabilized by solid particles loaded with interfacial enzymes are gradually becoming an innovative platform in the field of two-phase biocatalysis. In this reaction system, the emulsion droplets can act as microreactors, offering significant advantages such as a large oil-water interfacial area, sufficient contact between the interfacial enzyme and substrate, high catalytic efficiency, easy enzyme recovery, environmental friendliness, and convenient product separation and purification. It represents a highly promising novel interfacial enzyme two-phase catalytic system. However, current technologies often employ inorganic particles loaded with interfacial enzymes to construct such systems, and the biosafety of these inorganic particles is frequently questioned, limiting their application scope. Amyloid fibers possess advantages such as high biocompatibility, high interfacial activity, and ease of preparation, making them an emerging Pickering emulsion stabilizer. Furthermore, thanks to their nanofiber morphology, which imparts a high specific surface area, and the abundance of active groups such as amino and carboxyl groups on their surface, amyloid fibers also exhibit excellent enzyme immobilization carrier performance. Therefore, amyloid fibers show broad application prospects in constructing two-phase catalytic reaction systems based on Pickering emulsions.

[0036] However, the applicant's research revealed that the droplet size of Pickering emulsions stabilized solely by amyloid fibrous materials (AFs) is approximately 10 μm; while when AFs are loaded with an interfacial enzyme, the droplet size of the Pickering emulsion stabilized solely by AFs increases to over 150 μm, and droplet aggregation and phase separation easily occur, leading to significant emulsion separation. This indicates that the loading of the interfacial enzyme (forming enzyme-loaded amyloid fibrous materials after loading the interfacial enzyme) significantly reduces the emulsifying performance of AFs. Therefore, the Pickering emulsion interfacial catalytic system stabilized solely by enzyme-loaded amyloid fibrous materials suffers from low catalytic efficiency in enzymatic reactions in practical applications. To overcome the above-mentioned technical defects, this invention provides an amyloid fibrous material Pickering emulsion interfacial catalytic system, its preparation method, and its uses.

[0037] The first aspect of the present invention protects a composition for constructing a Pickering emulsion interfacial catalytic system, comprising enzyme-loaded amyloid filaments and first amyloid filaments, wherein the raw materials for preparing the enzyme-loaded amyloid filaments comprise second amyloid filaments, an interfacial enzyme, and a crosslinking agent; the mass ratio of the second amyloid filaments to the first amyloid filaments is 1:(0.02-0.6).

[0038] In some embodiments, the mass ratio of the second amyloid fibrous fiber to the first amyloid fibrous fiber is 1:(0.02-0.6), or it can be 1:(0.02-0.2), 1:(0.1-0.4), 1:(0.3-0.6), or 1:0.02, 1:0.04, 1:0.06, 1:0.08, 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, or 1:0.6. The applicant's research has found that as the amount of the first amyloid fibrous fiber increases, the instability index and droplet size of the final Pickering emulsion interface catalytic system show a trend of first decreasing and then stabilizing, indicating strong stability. In this invention, the mass ratio of the second amyloid fibrous fiber to the first amyloid fibrous fiber must be controlled within the range defined in this application. If the mass ratio is too high, the enzyme-carrying amyloid fibers, due to their large hydrodynamic diameter and low hydrophobicity, cannot quickly adsorb and stabilize the oil-water interface during homogenization, leading to droplet aggregation and increased particle size, thus reducing emulsion stability. If the mass ratio is too low, on the one hand, it will increase the amount of raw material (first amyloid fibers), resulting in higher costs; on the other hand, there is a risk of competitive adsorption, meaning that the first amyloid fibers, with stronger emulsifying properties, will competitively occupy a large number of interfacial sites, making it difficult for the enzyme-carrying amyloid fibers to adsorb at the oil-water interface, thereby reducing catalytic efficiency.

[0039] In some embodiments, the interfacial enzyme comprises oxidoreductases, transferases, hydrolases, lyases, isomerases, and synthases. Preferably, it is a hydrolase. More preferably, it is a lipase.

[0040] In some embodiments, the loading of interfacial enzymes in the enzyme-loaded amyloid fibers is 0.136–0.142 mg / mg, based on the second amyloid fiber in the enzyme-loaded amyloid fibers.

[0041] In some embodiments, the crosslinking agent is selected from one or more of glutaraldehyde, genipin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide, and succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester. Preferably, it is glutaraldehyde.

[0042] In some embodiments, the mass ratio of the interfacial enzyme to the cross-linking agent is 2:(1-4), or it can be 2:1, 2:2, 2:3, or 2:4. The mass ratio of the interfacial enzyme to the cross-linking agent in this invention needs to be controlled within the range defined in this application. If the mass ratio is too low, excessive cross-linking agent can easily lead to over-cross-linking between fibers, resulting in a significant increase in the hydrodynamic diameter of the enzyme-loaded amyloid fibers and a decrease in emulsification performance. Simultaneously, excessive cross-linking can also cause the interfacial enzyme to be embedded inside the amyloid fiber aggregates, making it difficult to fully contact the interfacial enzyme substrate subsequently, thereby reducing catalytic efficiency. If the mass ratio is too high, the interfacial enzyme cannot be fully immobilized on the surface of the amyloid fibers, affecting the immobilization efficiency.

[0043] In some embodiments, the mass ratio of the interfacial enzyme to the second amyloid fibrils is (1-4):3, or it can be 1:3, 2:3, 3:3, or 4:3. Preferably, it is (2-4):3. In this invention, the mass ratio of the interfacial enzyme to the second amyloid fibrils needs to be controlled within the range defined in this application. Too low a ratio will result in a low loading capacity of the interfacial enzyme per unit mass of amyloid fibrils. Too high a ratio will result in a low immobilization efficiency of the interfacial enzyme.

[0044] In some embodiments, the first or second amyloid protein fibers are prepared by heat-treating the protein in water under acidic conditions to obtain the first or second amyloid protein fibers.

[0045] In some embodiments, the protein used to prepare the first or second amyloid fibrous fiber is independently selected from one or more of β-lactoglobulin, wheat protein, lysozyme protein, whey protein, ovalbumin, α-lactoglobulin, soy globulin, soy protein isolate, pea protein, mung bean globulin, kidney bean protein isolate, rice bran protein, and β-casein. The proteins used to prepare the first or second amyloid fibrous fiber in this invention can be the same or different. For example, β-lactoglobulin can be used to prepare the first amyloid fibrous fiber, and wheat protein can be used to prepare the second amyloid fibrous fiber; alternatively, lysozyme protein can be used to prepare the first amyloid fibrous fiber, and ovalbumin can be used to prepare the second amyloid fibrous fiber. Preferably, the protein used to prepare the first or second amyloid fibrous fiber is the same. More preferably, β-lactoglobulin is used.

[0046] In some embodiments, the acid used to prepare the first or second amyloid fibrous material is independently selected from one or more of hydrochloric acid, sulfuric acid, and phosphoric acid. The acid used to prepare the first or second amyloid fibrous material in this invention can be the same or different. For example, hydrochloric acid can be used to prepare the first amyloid fibrous material, and sulfuric acid can be used to prepare the second amyloid fibrous material; alternatively, phosphoric acid can be used to prepare the first amyloid fibrous material, and hydrochloric acid can be used to prepare the second amyloid fibrous material. Preferably, the acid used to prepare the first or second amyloid fibrous material is the same. More preferably, hydrochloric acid is used.

[0047] In some embodiments, the heat treatment temperature is 70-90°C, or it can be 70°C, 72°C, 74°C, 76°C, 78°C, 80°C, 83°C, 85°C, 87°C, 88°C, or 90°C.

[0048] In some embodiments, the heat treatment time is 6 to 24 hours, or it can be 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours.

[0049] In some embodiments, the acidic conditions refer to a pH of 2 to 3, but can also be 2, 2.2, 2.4, 2.6, 2.8, or 3.

[0050] In one specific embodiment, the method for preparing the enzyme-loaded amyloid fibers is as follows: an interfacial enzyme, a second amyloid fiber, and a cross-linking agent react, followed by solid-liquid separation to obtain the enzyme-loaded amyloid fibers. The reaction temperature is 20–35°C, preferably 25°C; the reaction time is 1–10 h, preferably 5 h.

[0051] The compositions of the present invention can be stored as lyophilized powders or packaged separately; when used, they are dispersed in an aqueous phase and then mixed with an oil phase to form a Pickering emulsion.

[0052] Another aspect of the present invention protects a Pickering emulsion interfacial catalytic system, comprising an emulsion system formed of an aqueous phase and an oil phase; said aqueous phase comprising the composition described above.

[0053] This invention constructs a Pickering emulsion interfacial catalytic system synergistically stabilized by binary particles of enzyme-loaded amyloid filaments and first amyloid filaments. The droplet size of this Pickering emulsion interfacial catalytic system is 10-100 μm, significantly smaller than that of Pickering emulsions stabilized solely by enzyme-loaded amyloid filaments, effectively avoiding droplet coalescence and phase separation. This Pickering emulsion interfacial catalytic system achieves simultaneous improvement in the enrichment of interfacial enzymes at the oil-water interface and the long-term stability of the emulsion without the need for exogenous additives. Furthermore, the Pickering emulsion interfacial catalytic system of this invention exhibits excellent environmental tolerance: it maintains structural stability within a pH range of 1.0-14.0, at a sodium chloride concentration of 1-4 mol / L, under conditions of treatment at 80°C for 2 hours, and after standing at room temperature for 1 month, demonstrating excellent acid, alkali, salt, and high-temperature resistance. Furthermore, when the Pickering emulsion interface catalytic system provided by this invention is applied to a two-phase enzymatic reaction, the substrate conversion rate is increased by at least 34.3% after 7 h of reaction compared to the system that is stable by enzyme-loaded amyloid cellulose alone; after 5 cycles of use, its relative enzyme activity is still maintained above 80%, demonstrating excellent reusability.

[0054] In some embodiments, the concentration of the first amyloid fibrous material, based on the total volume of the aqueous phase, is 0.01–0.3% (w / v), or 0.01–0.07% (w / v), or 0.05–0.18% (w / v), or 0.1–0.3% (w / v), or 0.01% (w / v), 0.02% (w / v), 0.03% (w / v), 0.1% (w / v), 0.2% (w / v), or 0.3% (w / v). In this application, unless otherwise stated, the symbol "% (w / v)" represents a mass-volume percentage concentration, meaning that 1 g of solute is contained in every 100 mL of solution, i.e., 1% (w / v) = 10 mg / mL.

[0055] In some embodiments, the droplet size D of the Pickering emulsion interfacial catalytic system 4,3 The size is 10–100 μm. The liquid size D... 4,3The detection method is as follows: The solution is diluted with ultrapure water at 3000 rpm until the light-blocking rate is between 15% and 17%. Then, the average droplet size and particle size distribution are determined using a laser particle size analyzer. The volume-weighted average diameter (D) is calculated according to the following formula. 4,3 ):

[0056] , where n i Indicates the number of droplets; d i This indicates the diameter of the droplet.

[0057] In some embodiments, the volume ratio of the aqueous phase to the oil phase is not less than 1:1, and can also be 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, or 2:1.

[0058] In some embodiments, the oil phase is an oil capable of dispersing hydrophobic nanoparticles.

[0059] In some embodiments, the oil is selected from one or more of liquid paraffin, toluene, soybean oil, n-hexane, ethyl acetate, and glyceryl tributylate. The aqueous phase and oil phases such as toluene, soybean oil, n-hexane, ethyl acetate, and glyceryl tributylate of the present invention can all form stable Pickering emulsion interfacial catalytic systems, exhibiting good emulsification versatility.

[0060] Another aspect of the present invention protects a method for preparing the Pickering emulsion interface catalytic system as described above, comprising the following steps:

[0061] The aqueous and oil phases were homogenized to obtain the Pickering emulsion interface catalytic system.

[0062] In some embodiments, the homogenization speed is 10,000 to 30,000 rpm, or it can be 10,000 to 25,000 rpm, or 20,000 to 30,000 rpm, or it can be 10,000 rpm, 140,000 rpm, 16,000 rpm, 18,000 rpm, 19,000 rpm, 20,000 rpm, 21,000 rpm, 22,000 rpm, 25,000 rpm, 27,000 rpm, 29,000 rpm, and 30,000 rpm.

[0063] In some embodiments, the homogenization time is 1 to 5 min, or it can be 1 to 3.2 min, or it can be 2.5 to 5 min, or it can be 1 min, 2 min, 3 min, 4 min, or 5 min.

[0064] Another aspect of the present invention protects the use of the Pickering emulsion interface catalytic system as described above in two-phase catalytic reactions.

[0065] Two-phase catalytic reactions refer to catalytic reactions in which the catalyst and reactants are in different phases. Depending on the combination of phases, they can include various types such as gas-solid, liquid-solid, and liquid-liquid.

[0066] Enzyme-catalyzed reactions refer to reactions in which an enzyme acts as a catalyst to convert a substrate into a product under specific temperature, pressure, and time conditions.

[0067] Two-phase enzyme catalysis is a type of enzyme catalysis reaction, which refers to a reaction in which two immiscible liquid phases (usually an aqueous phase and an organic phase) exist simultaneously in the reaction system, with the enzyme mainly distributed in one phase (usually the aqueous phase) and the substrate mainly distributed in the other phase (usually the organic phase).

[0068] Another aspect of the present invention protects a method for catalyzing enzyme substrates, using the Pickering emulsion interface catalysis system as described above to catalyze enzyme substrates.

[0069] In some embodiments, the enzyme substrate is matched with the interfacial enzyme.

[0070] In some embodiments, when the interfacial enzyme is a lipase, it is because lipase is a bidirectional catalyst that can both catalyze the synthesis of esters from fatty acids and alcohols, and catalyze the hydrolysis of esters into fatty acids and alcohols.

[0071] In some embodiments, the enzyme substrate is selected from at least one of triacylglycerols, fatty acid esters, lactones, glycerol derivatives, sterol esters, phospholipids, and fatty acids and alcohols that serve as substrates for the synthesis reaction.

[0072] In some embodiments, the temperature of the catalytic reaction is 20-37°C, such as 37°C; the reaction time is 0.5-11 h; and the pH value is 6-8. These catalytic reaction conditions are conventional choices in the art, and those skilled in the art can determine them through limited conventional experiments based on the specific enzyme type, substrate characteristics, and reaction target, without requiring creative effort.

[0073] In some embodiments, the triglycerides include short-chain triglycerides (C2-C6), medium-chain triglycerides (C8-C12), and long-chain triglycerides (≥C12). The short-chain triglycerides (C2-C6) include, but are not limited to, triacetin and tripropionate; the medium-chain triglycerides (C8-C12) include, but are not limited to, tricaprylic acid and tridecanoic acid; and the long-chain triglycerides (≥C12) include, but are not limited to, trioleic acid, tristearate, and tripalmitic acid.

[0074] In some specific embodiments, the triacylglycerols are glyceryl tribanilates.

[0075] In some embodiments, the lactones include, but are not limited to, γ-butyrolactone, γ-valerolactone, γ-octyllactone, δ-decyllactone, δ-dodecyllactone, and pentadecyllactone.

[0076] In some embodiments, the glycerol ester derivatives include, but are not limited to, polyglycerol-10 stearate, polyglycerol-6 oleate, diacetyl tartrate glycerol, citrate glycerol, and lactate glycerol.

[0077] In some embodiments, the sterol esters include, but are not limited to, cholesterol stearate, cholesterol oleate, β-sitosterol ester, campesterol ester, and stigmasterol ester.

[0078] In some embodiments, the phospholipids include, but are not limited to, phosphatidylcholine (lecithin), phosphatidylethanolamine (cephalin), phosphatidylserine, and phosphatidylinositol.

[0079] In some embodiments, the fatty acids are synthesized with alcohols to form esters, including but not limited to fatty acid methyl esters (such as methyl oleate), fatty acid ethyl esters (such as ethyl stearate), mono-fatty acid glycerides (such as glyceryl monostearate), and polyol esters (such as pentaerythritol stearate).

[0080] The Pickering emulsion interfacial catalytic system of the present invention, synergistically stabilized by enzyme-loaded amyloid filaments and first amyloid filaments, not only exhibits small droplet size, effectively avoiding droplet coalescence and phase separation, but also allows the interfacial enzyme to be efficiently adsorbed at the oil-water interface. In contrast, the Pickering emulsion stabilized solely by enzyme-loaded amyloid filaments has a larger droplet size (>150 μm), exhibiting obvious emulsification, and a large number of enzyme-loaded amyloid filaments were observed failing to adsorb at the oil-water interface. Furthermore, the Pickering emulsion interfacial catalytic system of the present invention improves the catalytic efficiency of the interfacial enzyme. Taking the catalytic mode substrate glycerol tribaniate for 7 h as an example, compared with the Pickering emulsion interfacial catalytic system stabilized solely by enzyme-loaded amyloid filaments (C@AFs) (conversion rate 64.5%), the conversion rate of the Pickering emulsion interfacial catalytic system of the present invention (86.6%) is increased by 34.3%; compared with the conventional two-phase free enzyme catalytic system (conversion rate 23.4%), the conversion rate is increased by 270.1%. Furthermore, the Pickering emulsion interface catalytic system constructed in this invention still maintains a relative enzyme activity of about 80% after being reused 5 times, demonstrating excellent reusability and showing significant application prospects in the fields of biocatalysis and functional foods.

[0081] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0082] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0083] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0084] Unless otherwise specified, the raw materials used in the following embodiments and comparative examples of this application are all commercially available raw materials.

[0085] β-lactoglobulin (protein content 95%) and lipase (derived from Candida albicans) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. and Shanghai Maclean Biotechnology Co., Ltd., respectively. The CAS number of β-lactoglobulin is 9045-23-2; the CAS number of lipase is 9001-62-1, the enzyme activity is 700 U / mg, and the appearance is powder.

[0086] Example 1: Preparation and characterization of aqueous dispersion of β-lactoglobulin amyloid fibrous (AFs)

[0087] Example 1 provides a method for preparing an aqueous dispersion of β-lactoglobulin amyloid cellulose (AFs), specifically comprising the following steps:

[0088] 1.1 Preparation of AFs Aqueous Dispersion

[0089] 600 mg of β-lactoglobulin was weighed and dissolved in 30 mL of deionized water to prepare a protein solution. The pH of the system was adjusted to 2.0 with 1 M HCl solution, and the solution was incubated at 90 °C and 150 rpm for 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h, respectively. After each incubation period, an AFs aqueous dispersion with a concentration of 2% (w / v) was prepared.

[0090] 1.2 Transmission Electron Microscopy (TEM) Characterization

[0091] Take the AFs aqueous dispersion obtained from incubation for 4 h in step 1.1, dilute it to a concentration of 0.2% (w / v), and drop it onto a 300-mesh carbon support membrane copper grid for negative staining treatment.

[0092] The specific procedure for negative staining is as follows: stain with 2wt% phosphotungstic acid aqueous solution for 30 s, and after the sample is completely dried in the air, observe it under a transmission electron microscope (JEM-1400flash, Nippon Electronics Co., Ltd.), with the accelerating voltage set to 80 kV.

[0093] 1.3 Growth kinetics measurement

[0094] 8.0 mg of thiosulfate T (Th T) powder was dissolved in 10 mL of phosphate buffer (phosphate buffer contains 10 mM phosphate and 20 mM NaCl, pH=7.0), and filtered through a 0.22 μm membrane filter to obtain Th T stock solution.

[0095] Wrap the ThT stock solution in aluminum foil to protect it from light and store it at 4°C. Before use, dilute the ThT stock solution 50 times to prepare the ThT working solution.

[0096] 50 μL of 0.2% (w / v) aqueous dispersions of AFs (obtained by diluting the 2% (w / v) aqueous dispersion of AFs from step 1.1 in water) at different heat treatment times were taken and mixed thoroughly with 950 μL of ThT working solution, and incubated in the dark for 5 min. The fluorescence intensity was then measured using a fluorescence spectrophotometer (FL6500, PerkinElmer, USA), with the excitation wavelength set at 460 nm and the emission wavelength at 490 nm. Based on the measured ThT fluorescence intensity data, growth kinetic curves of β-lactoglobulin amyloid filaments were plotted.

[0097] TEM images and fiber growth kinetic curves are shown in [link to TEM image]. Figure 1A , 1B .

[0098] from Figure 1A It can be seen that AFs were successfully prepared, with a diameter of about 10 nm and a length ranging from several micrometers to tens of micrometers.

[0099] Th T fluorescence is a sensitive indicator for monitoring the process of protein fibrosis.

[0100] from Figure 1BIt was found that the Th T fluorescence intensity of the AFs aqueous dispersion showed a significant increasing trend with the extension of heat treatment time, reaching a peak after 4 h of heating. This result indicates that β-lactoglobulin reaches its maximum degree of fibrillation at 4 h.

[0101] Therefore, a 2% (w / v) amyloid cellulose aqueous dispersion, prepared by dissolving 600 mg β-lactoglobulin in 30 mL of water and heating and incubating for 4 h under acidic conditions with a pH of 2.0, was selected for subsequent experiments.

[0102] Example 2: Preparation, characterization, and determination of immobilization efficiency and loading of enzyme-loaded amyloid fibrous (C@AFs) aqueous dispersion.

[0103] In this Example 2, an enzyme-loaded amyloid fibrous protein (C@AFs) aqueous dispersion was prepared by loading lipase onto the AFs aqueous dispersion obtained in Example 1, and its microstructure, immobilization efficiency and loading amount were measured and characterized.

[0104] 2.1 Preparation of C@AFs Aqueous Dispersion

[0105] Lipase (CALB), AFs, and glutaraldehyde were mixed for a cross-linking reaction. The concentrations of glutaraldehyde (15 mM), AFs (3 mg / mL), and CALB (1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL, respectively) were based on the total volume of the reaction system.

[0106] The specific preparation methods are as follows: Take 1 mL of 10 mg / mL CALB aqueous solution, 1.5 mL of 2% (w / v) AFs aqueous dispersion from Example 1, and 57 μL of glutaraldehyde, and add water to a final volume of 10 mL to obtain a mixture with a CALB concentration of 1 mg / mL. Take 2 mL of 10 mg / mL CALB, 1.5 mL of 2% (w / v) AFs aqueous dispersion, and 57 μL of glutaraldehyde, and add water to a final volume of 10 mL to obtain a mixture with a CALB concentration of 2 mg / mL. The remaining concentration groups are prepared similarly.

[0107] The method for preparing a 10 mg / mL CALB aqueous solution is as follows: Take 100 mg of lipase powder and dilute it to 10 mL with deionized water.

[0108] After 5 h of cross-linking reaction, the reaction solution was centrifuged at 8000 rpm for 10 min, and the supernatant was discarded to remove unfixed free CALB. The precipitate was collected and redispersed with ultrapure water, and the volume was adjusted to 6 mL to obtain an aqueous dispersion of enzyme-loaded amyloid fibroblasts (C@AFs) with a concentration of 0.5% (w / v) (based on the concentration of amyloid fibroblasts AFs, i.e., 30 mg AFs divided by 6 mL).

[0109] 2.2 Characterization

[0110] 2.2.1 TEM characterization

[0111] Same as step 1.2 in Example 1.

[0112] 2.2.1 Fluorescence Spectroscopy Characterization

[0113] Cy5-labeled CALB (Cy5-CALB) was prepared by amidation reaction using Cy5 fluorescent dye to label the amino groups on the surface of CALB. The specific steps are as follows: 30 mg of CALB was dissolved in 10 mL of Tris buffer to obtain a CALB solution; 5 mg of Cy5-NHS was dissolved in 1 mL of Tris buffer to obtain a dye solution. The dye solution was added to the CALB solution, and the reaction was carried out at room temperature in the dark for 6 h. After the reaction, the reaction solution was transferred to an ultrafiltration tube and ultrafiltered at 8000 rpm for 10 min. 10 mL of Tris buffer was added to the filtrate in the ultrafiltration tube, and this washing step was repeated once. Finally, the filtrate was resuspended in pure water and brought to a final volume of 3 mL to obtain Cy5-labeled CALB (Cy5-CALB) with a concentration of 10 mg / mL.

[0114] To verify the successful immobilization of CALB on AFs, CALB was labeled with Cy5, and Cy5-labeled C@AFs (Cy5-C@AFs) were prepared according to the method described in step 2.1, followed by fluorescence scanning. To eliminate interference from the background fluorescence of the AFs carrier itself, pure AFs were used as a control for simultaneous scanning. Detection was performed using a fluorescence spectrophotometer (FL 6500, PerkinElmer, USA), with the excitation wavelength set at 605 nm and the emission spectrum scanning range set at 650-700 nm.

[0115] To determine the CALB loading and immobilization efficiency in the C@AFs aqueous dispersion obtained in step 2.1, Cy5-labeled CALB was used, and Cy5-C@AFs with different CALB addition concentrations were prepared according to the method in step 2.1.

[0116] 2.3 Determination of Loading and Immobilization Efficiency

[0117] The fluorescence intensity of a series of Cy5-CALB standard solutions (20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL, respectively) at an excitation wavelength of 605 nm and an emission wavelength of 670 nm was measured (FL 6500, Perkin Elmer, USA). A fluorescence intensity-concentration standard curve was plotted based on the results. The loading capacity and immobilization efficiency were calculated using the following formula:

[0118]

[0119]

[0120] C 固定化CALB The concentration (mg / mL) of CALB fixed on AFs is given by substituting the fluorescence intensity value of Cy5-C@AFs aqueous dispersion into the standard curve for calculation.

[0121] C 初始AFs This indicates the initial concentration of AFs (mg / mL), which is also the concentration of AFs added in step 2.1;

[0122] C 初始CALB This indicates the initial concentration of CALB (mg / mL), which is the concentration of CALB added in step 2.1.

[0123] 2.4 Results

[0124] The standard curve of fluorescence intensity versus concentration of Cy5-CALB standard solution is shown below. Figure 2 .

[0125] The TEM, fluorescence scanning spectra, immobilization efficiency, and loading of the C@AFs aqueous dispersion are shown in the figure. Figure 3A -C.

[0126] from Figure 3A It can be seen that a large amount of CALB (black) is distributed on the surface of AFs (fiber-like), indicating that CALB has been effectively fixed on the AFs support through covalent coupling with glutaraldehyde crosslinking agent. At the same time, a certain degree of physical aggregation can also be observed among the AFs.

[0127] from Figure 3B It can be seen that the standard Cy5-CALB obtained in step 2.2 has the same fluorescence signal as Cy5-C@AFs, further confirming that CALB was successfully immobilized on AFs.

[0128] The effect of CALB concentration on immobilization efficiency and loading was investigated at an AFs concentration of 3 mg / mL.

[0129] from Figure 3C It can be seen that as the concentration of CALB increases, the immobilization efficiency decreases, while the loading first increases and then levels off. Therefore, when the concentration of CALB is 2-4 mg / mL, the loading of lipase in the C@AFs aqueous dispersion is relatively high, ranging from 0.136 to 0.142 mg / mg AFs.

[0130] In summary, an aqueous dispersion of enzyme-loaded amyloid cellulose (C@AFs) with a concentration of 0.5% (w / v) obtained after cross-linking reaction was determined based on the total volume of the reaction system, with CALB concentration of 2 mg / mL, glutaraldehyde concentration of 15 mM, and AFs concentration of 3 mg / mL. This dispersion will be used for subsequent experimental studies.

[0131] Example 3: A Pickering emulsion interfacial catalytic system and its preparation method

[0132] 3.1 Preparation of Pickering emulsion interfacial catalytic system

[0133] A schematic diagram of the preparation process of the Pickering emulsion interface catalytic system is shown below. Figure 4 , 5 The 2% (w / v) amyloid cellulose aqueous dispersion (AFs aqueous dispersion) of Example 1 was freeze-dried into AFs freeze-dried powder.

[0134] Experimental groups 1-5: The aqueous phase was prepared by mixing C@AFs at a final concentration of 0.5% (w / v) with AFs at final concentrations of 0.01% (w / v), 0.02% (w / v), 0.03% (w / v), 0.1% (w / v), 0.2% (w / v), and 0.3% (w / v), respectively. Liquid paraffin was used as the oil phase, with a volume ratio of oil to water of 1:1. Subsequently, homogenization was performed at 20,000 rpm for 2 min using a homogenizer (T-18, IKA GmbH, Germany) to obtain a Pickering emulsion interfacial catalytic system synergistically stabilized by AFs-C@AFs binary particles.

[0135] The aqueous phase was prepared as follows: 10 mL of the 0.5% (w / v) C@AFs obtained in step 2.1 was added to 0.1 mg of lyophilized AFs powder to obtain a mixed aqueous dispersion with a final C@AFs concentration of 0.5% (w / v) and a final AFs concentration of 0.01% (w / v), which is the aqueous phase. Other concentrations were prepared similarly. These were labeled as 0.5%C@AFs+0.01%AFs, 0.5%C@AFs+0.02%AFs, 0.5%C@AFs+0.03%AFs, 0.5%C@AFs+0.1%AFs, 0.5%C@AFs+0.2%AFs, and 0.5%C@AFs+0.3%AFs, respectively.

[0136] Meanwhile, an emulsion obtained by homogenizing the 0.5% (w / v) C@AFs single-component dispersion obtained in Example 2 with oil-phase liquid paraffin at a volume ratio of 1:1 was used as control group 1 (labeled as 0.5% C@AFs).

[0137] In addition, an emulsion obtained by homogenizing a 0.3% (w / v) single-component dispersion of AFs and oil-phase liquid paraffin at a volume ratio of 1:1 was used as control group 2 (labeled as 0.3% AFs). The 0.3% (w / v) single-component dispersion of AFs was obtained by dispersing 30 mg of lyophilized AFs powder in 10 mL of water.

[0138] 3.2 Physical Stability Evaluation

[0139] The physical stability of the emulsions obtained in step 3.1 was evaluated using the LUMiSizer (LUM GmbH, Germany). The LUMiSizer is an instrument that uses centrifugation to accelerate the occurrence of unstable phenomena (such as sedimentation, flocculation, or emulsification stratification), providing researchers with a rapid and accurate method for assessing system stability. The separation behavior of the sample can be analyzed by tracking changes in transmittance at any location. In the transmittance curves, the red line represents the initial measurement curve, and the green line represents the final measurement curve.

[0140] The test parameters were set as follows: centrifugation speed, 200 rpm; temperature, 25℃; total test duration, 50 min.

[0141] The physical stability of the emulsion was analyzed using the "Stability Analysis" module in SEPView software. Physical stability is represented by the instability index (K), which is dimensionless and related to the stability of the dispersion system. The higher the instability index, the lower the system stability; the lower the instability index, the more stable the emulsion.

[0142] The instability index ranges from 0 to 1, where 0 represents a very stable system and 1 represents an extremely unstable system.

[0143] 3.3 Droplet Size Measurement

[0144] The average droplet size and particle size distribution of each emulsion prepared in step 3.1 were determined using a laser particle size analyzer (Mastersizer 3000, Malvern GmbH, UK). During the measurement, each emulsion was diluted with ultrapure water at 3000 rpm until the opacity was between 15% and 17%. The volume-weighted average diameter (D) was calculated using the following formula. 4,3 ):

[0145]

[0146] n i Indicates the number of droplets;

[0147] d i This indicates the diameter of the droplet.

[0148] 3.4 Microstructure Characterization

[0149] The microstructure of each emulsion obtained in step 3.1 was observed using an optical microscope and a laser confocal microscope (CLSM).

[0150] Microscopic morphology was observed using an optical microscope (Olympus Corporation, Japan). The observation environment temperature was controlled at 25℃, and a 10x objective lens was used for imaging.

[0151] Before observation using laser confocal microscopy, C@AFs need to be stained and labeled. The specific procedure is as follows: First, C@AFs are stained and labeled with Rhodamine B. Add 0.3 mL of 1 mg / mL Rhodamine B solution to 6 mL of 0.5% (w / v) C@AFs aqueous dispersion and mix for 30 min. After the reaction, centrifuge and wash three times, then redisperse the precipitate in 6 mL of pure water to obtain Rhodamine B-labeled C@AFs (Rhodamine BC@AFs). Subsequently, a Pickering emulsion is prepared using Rhodamine BC@AFs following the aforementioned steps. Finally, a small amount of the emulsion sample is dropped onto a glass slide and observed using a laser confocal microscope. The detection parameters are set as follows: excitation wavelength 550 nm, emission wavelength 590 nm, ambient temperature 25℃, and a 10x objective lens is used for observation.

[0152] 3.5 Universality and Environmental Stability Testing

[0153] To verify the versatility of AFs-C@AFs binary particles as emulsifiers, their emulsifying performance in other oil-water systems (including toluene-aqueous phase, soybean oil-aqueous phase, n-hexane-aqueous phase, and ethyl acetate-aqueous phase) was further investigated. Specifically, the aqueous phase was a mixed dispersion, prepared as shown in step 3.1, wherein the concentration of C@AFs was 0.5% (w / v) and the concentration of AFs was 0.03% (w / v); then the aqueous phase was mixed with toluene, soybean oil, n-hexane, and ethyl acetate at a volume ratio of 1:1, and homogenized to obtain the Pickering emulsion interface catalytic system.

[0154] In addition, the stability of the Pickering emulsion interface catalytic system was investigated over a wide pH range (1.0-14.0) and under conditions of high salt concentration, and its storage stability and high temperature stability were evaluated.

[0155] pH stability test: The pH values ​​of the mixed aqueous dispersion were adjusted to 1.0, 5.0, 10.0, and 14.0 using 1 M HCl or 1 M NaOH solutions, respectively; the concentration of C@AFs in the mixed aqueous dispersion was 0.5% (w / v), and the concentration of AFs was 0.03% (w / v). Using the pH-adjusted mixed dispersion as the aqueous phase, a Pickering emulsion interfacial catalytic system was prepared by mixing the aqueous phase with the oil phase liquid paraffin at a volume ratio of 1:1 to investigate its stability under different pH conditions.

[0156] Salt stability test: 0.58 g, 2.32 g, and 3.48 g of NaCl were added to 10 mL of a mixed aqueous dispersion (where the concentration of C@AFs in the mixed aqueous dispersion was 0.5% (w / v) and the concentration of AFs was 0.03% (w / v)) to prepare dispersions with final NaCl concentrations of 1 M, 4 M, and 6 M, respectively. These were used as the aqueous phase and then mixed with the oil phase liquid paraffin at a volume ratio of 1:1 to prepare the Pickering emulsion interface catalytic system to investigate its stability at high salt concentrations.

[0157] Storage stability: A Pickering emulsion interfacial catalytic system was prepared by mixing a mixed aqueous dispersion (where the concentration of C@AFs in the mixed aqueous dispersion was 0.5% (w / v) and the concentration of AFs was 0.03% (w / v)) as the aqueous phase and then mixing it with the oil phase liquid paraffin at a volume ratio of 1:1 and homogenizing. The Pickering emulsion interfacial catalytic system was then allowed to stand at 25°C for one month to investigate its storage stability.

[0158] High-temperature stability: The Pickering emulsion interfacial catalytic system, prepared using the same method as for storage stability, was allowed to stand at 80°C for 2 h to investigate its high-temperature stability.

[0159] 3.6 Results

[0160] 3.6.1 Stability evaluation of Pickering emulsion

[0161] Depend on Figure 6 It can be seen that with the increase of AFs addition, the instability index of the emulsion first decreases and then tends to stabilize; specifically, when the AFs concentration reaches 0.03% (w / v), further increases in the AFs addition do not significantly change the instability index. This indicates that the physical stability of the Pickering emulsion interfacial catalytic system first increases with the increase of AFs addition, and then tends to stabilize.

[0162] 3.6.2 Microscopic Morphology Characterization of Pickering Emulsion

[0163] from Figure 7 As shown in section A, with the gradual increase of AFs addition, the droplet size of the Pickering emulsion interface catalytic system stabilized by binary particles shows a trend of first decreasing and then stabilizing, which is consistent with the stability change pattern in step 3.6.1.

[0164] Among them, D 4,3 The average diameter is calculated based on the volume distribution of emulsion droplets, and it has high sensitivity to changes in droplet volume; when emulsions coalesce, small droplets merge to form larger droplets, leading to D... 4,3 Significantly increased, therefore D 4,3 The size can be used as a basis for judging the degree of emulsion aggregation.

[0165] Depend on Figure 7 As can be seen from B, the droplet size (D) of the Pickering emulsion stabilized solely by C@AFs particles is... 4,3 The value was 150 μm, while when the amount of AFs added was 0.03% (w / v), D 4,3 The area decreased significantly to 41.2 μm, indicating that the emulsion has a larger interfacial area at this point, which is beneficial for the two-phase catalytic reaction.

[0166] At the same time, by Figure 8 CLSM images of the DF medium show that when the amount of AFs added is 0.03% (w / v), C@AFs are almost completely adsorbed on the oil-water interface, which is conducive to improving enzyme utilization.

[0167] In summary, when the final concentrations of C@AFs and AFs in the aqueous phase are 0.5% (w / v) and 0.03% (w / v), respectively, the prepared Pickering emulsion interfacial catalytic system exhibits superior physical stability, a larger interfacial area, and higher enzyme utilization. Furthermore, compared to systems with higher AFs content, this concentration combination also saves raw materials and reduces costs. Therefore, a mixed system with final concentrations of 0.5% (w / v) C@AFs and 0.03% (w / v) AFs is preferred for subsequent experiments.

[0168] 3.6.3 Universality and Environmental Stability

[0169] The universality of AFs-C@AFs binary particles as emulsifiers and the stability of the Pickering emulsion interfacial catalytic system prepared by them under different environmental conditions were also investigated.

[0170] from Figure 9 It can be seen that the AFs-C@AFs binary particles can form a stable Pickering emulsion interface catalytic system in toluene-water, soybean oil-water, n-hexane-water and ethyl acetate-water systems, showing good emulsification versatility.

[0171] from Figure 10 It can be seen that the Pickering emulsion interface catalytic system can remain stable at pH values ​​of 1.0, 5.0, 10.0 and 14.0.

[0172] from Figure 11 It can be seen that the Pickering emulsion interface catalytic system can remain stable under 1 M, 2 M and 4 M sodium chloride concentrations.

[0173] from Figure 12 It can be seen that the Pickering emulsion interface catalytic system maintains its stability after standing at room temperature for one month and at 80°C for two hours. This indicates that the Pickering emulsion interface catalytic system of the present invention has long-term stability and is resistant to high temperatures.

[0174] Application examples

[0175] This application example evaluates the catalytic performance of a Pickering emulsion interface catalytic system.

[0176] 4.1 Enzyme-catalyzed reactions

[0177] Divided into 3 groups, including:

[0178] Group 1 (referred to as PIB system (ii)): Take 2 mL of a mixed aqueous dispersion containing 0.5% (w / v) C@AFs and 0.03% (w / v) AFs (preparation method is the same as step 3.1) as the aqueous phase, and take 4 mL of a liquid paraffin solution containing 10% (w / v) glyceryl tartrate as the oil phase. Homogenize the aqueous phase and the oil phase in a homogenizer at 20,000 rpm for 2 min to construct a Pickering emulsion interface catalytic system stabilized by binary particles.

[0179] Group 2 (referred to as PIB system (i)): 2 mL of an aqueous dispersion containing 0.5% (w / v) C@AFs (obtained from Example 2) was taken as the aqueous phase, and 4 mL of a liquid paraffin solution containing 10% (w / v) glyceryl tartrate was taken as the oil phase. The aqueous phase and the oil phase were homogenized in a homogenizer at 20,000 rpm for 2 min to construct a Pickering emulsion interface catalytic system stabilized by C@AFs alone.

[0180] Group 3 (denoted as PBC system): Mix 2 mL of 0.68 mg / mL lipase aqueous solution with 4 mL of liquid paraffin solution containing 10% (w / v) tricresyl tartrate.

[0181] The amount of lipase in each group was 0.68 mg / mL × 2 mL = 1.36 mg. As shown in step 2.4 of Example 2, the lipase loading in the 0.5% (w / v) C@AFs aqueous dispersion was 0.136 mg / mg AFs, therefore the concentration of CALB was 5 × 0.132 = 0.68 mg / mL.

[0182] Hydrolysis reaction conditions: The reaction was carried out at 37℃ and pH=7.4, and the reaction times for each group were 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 7 h, 10 h and 11 h, respectively.

[0183] After the reaction was completed, an inactivating agent (water:ethanol:acetone = 1:1:1) was added to terminate the reaction. The mixture was then centrifuged at 8000 rpm for 5 min, and 1 mL of the supernatant was titrated with NaOH solution to determine the concentration of the product fatty acid (butyric acid).

[0184] Conversion rate (%) is defined as the ratio between actual output and theoretical output within a certain period of time.

[0185] One unit of lipase activity (U) is defined as the amount of enzyme required to catalyze a reaction to produce 1 μmol of product per minute at 37°C and pH=7.4.

[0186] 4.2 Cyclic Reaction

[0187] The cycling performance of the Pickering emulsion interface catalytic system synergistically stabilized by AFs-C@AFs binary particles for the hydrolysis of glycerol tribanilate was investigated, with each cycle lasting 11 h.

[0188] After each reaction cycle, without adding an inactivating agent, the aqueous phase containing the product and the oil phase containing the substrate are directly separated by centrifugation. AFs-C@AFs are recovered and used in the next reaction cycle.

[0189] Aqueous samples were extracted and analyzed by titration with NaOH solution.

[0190] Fresh substrate solution was added to the centrifuged AFs-C@AFs, and the mixture was homogenized to prepare an emulsion for the next reaction cycle.

[0191] The reusability of Pickering emulsions was evaluated using relative enzyme activity (%), where relative enzyme activity was defined as the percentage of residual enzyme activity in the nth reaction cycle to the enzyme activity in the 1st reaction cycle.

[0192] 4.3 Results

[0193] The catalytic performance of different catalytic systems is shown in the figure. Figure 13 .

[0194] from Figure 13 As shown in Figure C, within the same reaction time, the Pickering emulsion interface catalytic system stabilized by AFs-C@AFs binary particles (PIB system (ii)) had the highest conversion rate, reaching 86.6% after 7 h; the Pickering emulsion interface catalytic system stabilized only by C@AFs (PIB system (i)) had the second highest conversion rate, at 64.5% after 7 h; and the conventional two-phase system (PBC system) had the lowest conversion rate, at only 23.4% after 7 h.

[0195] The hydrolysis of glycerol tribanilate was tested in 5 cycles using a Pickering emulsion interface catalysis system (PIB system (ii)) stabilized by AFs-C@AFs binary particles. The results are shown in the figure. Figure 13 D.

[0196] from Figure 13 As shown in Figure D, the relative enzyme activity decreased slightly with the increase of the number of cycles; after 5 cycles, the relative enzyme activity remained at about 80%, which confirms that AFs can be an effective and reusable carrier for enzyme immobilization applications.

[0197] Comparative Example 1: Pickering emulsion stabilized by AFs alone

[0198] Aqueous dispersions of AFs with concentrations of 0.1% (w / v), 0.3% (w / v), and 0.5% (w / v) were prepared as the aqueous phase, with liquid paraffin as the oil phase, and the volume ratio of the oil phase to the aqueous phase was 1:1. The oil and aqueous phases were then mixed and homogenized using a homogenizer (T-18, IKA GmbH, Germany) at 20,000 rpm for 2 min to obtain Pickering emulsions stabilized solely by AFs. The 0.1% (w / v), 0.3% (w / v), and 0.5% (w / v) AFs aqueous dispersions were prepared by diluting the 2% (w / v) AFs aqueous dispersion obtained in Example 1 proportionally.

[0199] The macroscopic morphology and optical microscopic results of the emulsion in Comparative Example 1 are shown in Figure 1. Figure 14 .

[0200] from Figure 14 It can be seen that the droplet size (approximately 10 μm) of the Pickering emulsion stabilized by AFs alone is significantly smaller than that of the Pickering emulsion stabilized by C@AFs alone in Comparative Example 2, and no emulsion separation phenomenon was observed.

[0201] Comparative Example 2: Pickering emulsion stabilized by C@AFs alone

[0202] C@AFs aqueous dispersions with concentrations of 0.1% (w / v), 0.3% (w / v), and 0.5% (w / v) were prepared as the aqueous phase, with liquid paraffin as the oil phase, and the volume ratio of the oil phase to the aqueous phase was 1:1. The oil and aqueous phases were mixed and homogenized using a homogenizer (T-18, IKA GmbH, Germany) at 20,000 rpm for 2 min to obtain Pickering emulsions stabilized by C@AFs alone. The C@AFs aqueous dispersions with concentrations of 0.1% (w / v) and 0.3% (w / v) were prepared by diluting the 0.5% (w / v) C@AFs aqueous dispersion obtained in Example 2 according to the specified ratio.

[0203] The macroscopic morphology, optical microscopy, and CLSM results of the emulsion in Comparative Example 2 are shown in the figure. Figure 15 .

[0204] like Figure 15 As shown, Pickering emulsions stabilized only by C@AFs exhibit significant emulsification. In particular, as... Figure 15As shown by the green arrow in C, the CLSM image clearly shows that a large number of C@AFs are dispersed in the external aqueous phase and are not adsorbed at the oil-water interface. The reason for this is that for Pickering emulsion systems stabilized solely by C@AFs, the droplet size is relatively large (>150 μm). In such large-droplet emulsion systems, droplet coalescence and phase separation are often more pronounced.

[0205] Comparative Examples 1 and 2 show that the emulsifying performance of AFs loaded with CALB significantly decreased. The droplet size (>150 μm) of the Pickering emulsion stabilized by C@AFs alone increased by approximately 15 times compared to the Pickering emulsion stabilized by AFs alone, and a large amount of C@AFs failed to adsorb at the oil-water interface and dispersed in the external aqueous phase. Furthermore, the Pickering emulsion interfacial catalytic system stabilized by C@AFs alone had a conversion rate of only 64.5% after 7 h; while the Pickering emulsion interfacial catalytic system synergistically stabilized by AFs and C@AFs binary particles achieved a conversion rate of 86.6% after 7 h, which was 34.3% higher than the former. Meanwhile, the Pickering emulsion interface catalytic system with synergistic stability of AFs and C@AFs binary particles described in this invention exhibits excellent environmental tolerance: it can maintain structural stability under conditions of pH 1.0-14.0, sodium chloride concentration of 1-4 mol / L, treatment at 80℃ for 2 h, and standing at room temperature for 1 month. It has excellent acid and alkali resistance, salt resistance, and high temperature resistance, and has important application value in the fields of biocatalysis and functional foods.

[0206] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A composition for constructing a Pickering emulsion interfacial catalytic system, characterized in that, The product comprises enzyme-loaded amyloid filaments and first amyloid filaments. The raw materials for preparing the enzyme-loaded amyloid filaments include second amyloid filaments, an interfacial enzyme, and a cross-linking agent. The mass ratio of the second amyloid filaments to the first amyloid filaments is 1:(0.02-0.6). The interfacial enzyme is selected from lipases. The method for preparing the first or second amyloid protein fibers is as follows: under acidic conditions, the protein is heat-treated in water to obtain the first or second amyloid protein fibers. The protein used to prepare the first or second amyloid fibrils is selected from β-lactoglobulin.

2. The composition according to claim 1, characterized in that, The crosslinking agent is selected from one or more of glutaraldehyde, genipin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide and succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester; And / or, during preparation, the ratio of the interfacial enzyme to the crosslinking agent is 2:(1-4); And / or, during preparation, the ratio of the interfacial enzyme to the second amyloid fibril is (1-4):

3.

3. The composition according to claim 1, characterized in that, Based on the second amyloid fibril in the enzyme-loaded amyloid fibril, the loading of interfacial enzymes in the enzyme-loaded amyloid fibril is 0.136–0.142 mg / mg.

4. The composition according to claim 1, characterized in that, The acid used to prepare the first or second amyloid filaments is independently selected from one or more of hydrochloric acid, sulfuric acid, and phosphoric acid; And / or, the temperature of the heat treatment is 70–90°C; And / or, the heat treatment time is 6 to 24 hours; And / or, the acidic conditions refer to a pH of 2 to 3.

5. A Pickering emulsion interface catalytic system, characterized in that, An emulsion system comprising an aqueous phase and an oil phase, wherein the aqueous phase comprises the composition as described in any one of claims 1-4.

6. The Pickering emulsion interface catalytic system as described in claim 5, characterized in that, Based on the total volume of the aqueous phase, the concentration of the first amyloid fibrils was 0.01–0.3% (w / v); And / or, the droplet size D of the Pickering emulsion interfacial catalytic system 4,3 The size is 10–100 μm; And / or, the volume ratio of the aqueous phase to the oil phase is not less than 1:1; And / or, the oil phase is an oil capable of dispersing hydrophobic nanoparticles.

7. The Pickering emulsion interface catalytic system as described in claim 6, characterized in that, The oil is selected from one or more of liquid paraffin, toluene, soybean oil, n-hexane, ethyl acetate, and glyceryl tartrate, or a mixture thereof.

8. The method for preparing the Pickering emulsion interface catalytic system according to any one of claims 5-7, characterized in that, Includes the following steps: The aqueous and oil phases were homogenized to obtain the Pickering emulsion interface catalytic system.

9. Use of the composition according to any one of claims 1-4 or the Pickering emulsion interfacial catalytic system according to any one of claims 5-7 in two-phase catalytic reactions.

10. A method for catalyzing an enzyme substrate, characterized in that, The enzyme substrate is catalyzed using the composition as described in any one of claims 1-4 or the Pickering emulsion interface catalysis system as described in any one of claims 5-7.

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