Core-shell structured lysozyme nanoparticles and preparation method and application thereof

Abstract

The present invention provides core-shell structured lysozyme nanoparticles and a preparation method and application thereof, belonging to the technical field of nanomaterials. The core-shell structured lysozyme nanoparticles provided by the present invention include an outer shell and a core wrapped in the outer shell, wherein the outer shell is formed by self-assembly of lysozyme, and the core is a gas core formed by hydrogen or a liquid core formed by an aqueous solution. The core-shell structured lysozyme nanoparticles of the present invention are based on physical methods to modify lysozyme, which only changes the secondary structure of lysozyme, has higher safety and higher nutritional value retention, and can also effectively improve the enzyme activity of lysozyme.

Description

Technical Field

[0001] The invention relates to the technical field of nano materials, in particular to core-shell structured lysozyme nanoparticles and a preparation method and application thereof. Background Art

[0002] Lysozyme is an alkaline enzyme that can hydrolyze mucopolysaccharides in bacteria. It is widely used in food, medicine, bioengineering, and daily chemical industries. For example, it is used as a "green" preservative in the food industry, used for anti-toxicity and anti-inflammatory in the pharmaceutical industry, used to assist in the extraction of cell extracts in bioengineering, and used in daily chemical industries to prevent dental caries. Lysozyme is derived from organisms, is safe and non-toxic, and has a broad market scope. At a time when the problem of microbial resistance is becoming increasingly prominent, the development and utilization of lysozymes with natural activity have become increasingly important.

[0003] Lysozyme activity can be improved by modifying it. Currently, lysozyme modification methods are mostly chemical and biological methods, which can easily damage the structure of lysozyme molecules and pose a risk of physiological toxicity. Summary of the invention

[0004] The object of the present invention is to provide core-shell structured lysozyme nanoparticles and a preparation method and application thereof. The core-shell structured lysozyme nanoparticles provided by the present invention have high safety and can also effectively improve the enzyme activity of lysozyme.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a core-shell structured lysozyme nanoparticle, comprising an outer shell and a core wrapped in the outer shell, wherein the outer shell is formed by self-assembly of lysozyme, and the core is a gas core formed by hydrogen or a liquid core formed by an aqueous solution.

[0007] Preferably, the particle size of the core-shell structured lysozyme nanoparticles is 200-550 nm.

[0008] Preferably, the lysozyme comprises hen egg white lysozyme.

[0009] The present invention provides a method for preparing the core-shell structured lysozyme nanoparticles described in the above technical solution, comprising the following steps:

[0010] Mixing hydrogen gas with the first buffer solution, and sequentially pressurizing and depressurizing the solution under closed conditions to obtain a hydrogen gas phase nanobubble solution;

[0011] mixing lysozyme with a second buffer solution and performing hydration treatment to obtain a lysozyme solution;

[0012] The lysozyme solution is mixed with a hydrogen gas phase nanobubble solution to perform self-assembly to obtain a dispersion of lysozyme nanoparticles with a core-shell structure containing a gas core;

[0013] The dispersion of the core-shell structured lysozyme nanoparticles containing a gas core is freeze-dried, and the obtained residue is mixed with water to obtain the core-shell structured lysozyme nanoparticles containing a liquid core.

[0014] Preferably, the first buffer solution and the second buffer solution independently include Tris-HCl buffer solution, Na2HPO4-NaH2PO4 buffer solution, Na2HPO4-C6H8O7 buffer solution or C6H8O7-NaOH-HCl buffer solution.

[0015] Preferably, the hydrated particle size of the hydrogen nanobubbles in the hydrogen gas phase nanobubble solution is 200-750 nm, and the concentration of the hydrogen nanobubbles is ≥10 9 / mL; the concentration of the lysozyme solution is 0.25~2.00mg / mL; the volume ratio of the lysozyme solution to the hydrogen gas phase nanobubble solution is 3:1~1:3.

[0016] Preferably, the pressurization includes: increasing the pressure from atmospheric pressure to 400-500 kPa within 2 seconds; the decompression includes: restoring the pressure to atmospheric pressure within 2 seconds; one pressurization and one decompression is counted as one cycle, and the number of cycles is 20-500 times.

[0017] Preferably, the temperature of the hydration treatment is 2-8°C and the time is 12-36 hours; the hydration treatment is carried out under static conditions.

[0018] Preferably, the self-assembly conditions include: pH value of 2-9, temperature of 4-30° C., and time of 3-10 min; and the self-assembly is performed under stirring conditions.

[0019] The present invention provides the use of the core-shell structure lysozyme nanoparticles described in the above technical solution or the core-shell structure lysozyme nanoparticles prepared by the preparation method described in the above technical solution in preparing enzyme preparations with improved enzymatic activity or preparing nanocarrier delivery systems.

[0020] Beneficial effects: The core-shell structured lysozyme nanoparticles provided by the present invention include an outer shell and a core wrapped in the outer shell, wherein the outer shell is formed by self-assembly of lysozyme, and the core is a gas core formed by hydrogen or a liquid core formed by an aqueous solution. The core-shell structured lysozyme nanoparticles of the present invention are based on physical methods to modify lysozyme, which only changes the secondary structure of lysozyme, has high safety and high nutritional value retention, and can also effectively improve the enzyme activity of lysozyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a transmission electron microscopy image of the nanoparticles (numbered A5) prepared in Example 5;

[0022] Figure 2 This is a transmission electron microscopy image of the nanoparticles prepared in Comparative Example 5 (numbered C5). DETAILED DESCRIPTION

[0023] The invention provides a core-shell structured lysozyme nanoparticle, comprising an outer shell and a core wrapped in the outer shell, wherein the outer shell is formed by self-assembly of lysozyme, and the core is a gas core formed by hydrogen or a liquid core formed by an aqueous solution.

[0024] In an embodiment of the present invention, the particle size of the core-shell structured lysozyme nanoparticles (specifically, the particle size displayed in a transmission electron microscope image) can be 200-550 nm, for example, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm or 550 nm, specifically 281.34±7.09 nm and 496.61±5.94 nm.

[0025] In an embodiment of the present invention, the lysozyme may include hen egg white lysozyme.

[0026] In an embodiment of the present invention, the aqueous solution is specifically a buffer solution with water as solvent, for example, it can be a Tris-HCl buffer solution, a Na2HPO4-NaH2PO4 buffer solution, a Na2HPO4-C6H8O7 buffer solution or a C6H8O7-NaOH-HCl buffer solution; the concentration of the Tris-HCl buffer solution, the Na2HPO4-NaH2PO4 buffer solution, the Na2HPO4-C6H8O7 buffer solution or the C6H8O7-NaOH-HCl buffer solution can independently be 5~50mM, specifically 5mM, 8mM, 10mM, 15mM, 20mM, 30mM, 40mM or 50mM.

[0027] The present invention provides a method for preparing the core-shell structured lysozyme nanoparticles described in the above technical solution, comprising the following steps:

[0028] Mixing hydrogen gas with the first buffer solution, and sequentially pressurizing and depressurizing the solution under closed conditions to obtain a hydrogen gas phase nanobubble solution;

[0029] mixing lysozyme with a second buffer solution and performing hydration treatment to obtain a lysozyme solution;

[0030] The lysozyme solution is mixed with a hydrogen gas phase nanobubble solution to perform self-assembly to obtain a dispersion of lysozyme nanoparticles with a core-shell structure containing a gas core;

[0031] The dispersion of the core-shell structured lysozyme nanoparticles containing a gas core is freeze-dried, and the obtained residue is mixed with water to obtain the core-shell structured lysozyme nanoparticles containing a liquid core.

[0032] In the present invention, unless otherwise specified, the raw materials and reagents used are commercially available products well known to those skilled in the art or are prepared by methods well known to those skilled in the art.

[0033] The present invention mixes hydrogen with a first buffer solution, and sequentially pressurizes and depressurizes under closed conditions to obtain a hydrogen gas phase nanobubble solution. In an embodiment of the present invention, the first buffer solution may include a Tris-HCl buffer solution, a Na2HPO4-NaH2PO4 buffer solution, a Na2HPO4-C6H8O7 buffer solution, or a C6H8O7-NaOH-HCl buffer solution; the concentration of the first buffer solution may be 5 to 50 mM, specifically 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, or 50 mM.

[0034] In an embodiment of the present invention, the pressurization may include: increasing the pressure from atmospheric pressure to 400-500 kPa within 2 seconds, for example, it may be increased to 400 kPa, 430 kPa, 450 kPa, 455 kPa, 460 kPa, 480 kPa or 500 kPa; the decompression may include: restoring the pressure to atmospheric pressure within 2 seconds; one pressurization and one decompression is recorded as one cycle, and the number of cycles may be 20-500 times, specifically 20 times, 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times or 500 times.

[0035] In an embodiment of the present invention, a first buffer solution is placed in a sealed container, hydrogen is introduced into the sealed container, and then the pressure in the sealed container is repeatedly increased and decreased according to the above conditions, hydrogen gas phase nanobubbles are generated in the first buffer solution, and a hydrogen gas phase nanobubble solution is obtained. In an embodiment of the present invention, the hydrated particle size of the hydrogen nanobubbles in the hydrogen gas phase nanobubble solution can be 200-750nm, for example, 200nm, 220nm, 240nm, 260nm, 300nm, 400nm, 500nm, 600nm, 700nm or 750nm, specifically 219.7±0.6nm, 241.0±8.5nm or 726.8±26.8nm; the concentration of the hydrogen nanobubbles can be ≥10 9 / mL, for example, it can be (0.1~5)×10 10 / mL, further can be (0.5~3)×10 10 / mL, specifically (1.54±0.12)×10 10 Pieces / mL.

[0036] The present invention mixes lysozyme with a second buffer solution and performs a hydration treatment to obtain a lysozyme solution. In an embodiment of the present invention, the second buffer solution may include a Tris-HCl buffer solution, a Na2HPO4-NaH2PO4 buffer solution, a Na2HPO4-C6H8O7 buffer solution or a C6H8O7-NaOH-HCl buffer solution; the second buffer solution may be the same as the first buffer solution or may be different, and the present invention has no special limitation on this; the concentration of the second buffer solution may be 5 to 50 mM, specifically 5 mM, 8 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM or 50 mM.

[0037] In an embodiment of the present invention, the mixing of the lysozyme and the second buffer solution can be carried out under stirring conditions; the mixing temperature can be 4-30°C, specifically 4°C, 10°C, 15°C, 20°C, 25°C or 30°C; the mixing time can be 1-6h, specifically 1h, 2h, 3h, 4h, 5h or 6h; the stirring speed can be 300-800rpm, specifically 400rpm. In an embodiment of the present invention, the temperature of the hydration treatment can be 2-8°C, specifically 2°C, 4°C, 6°C or 8°C; the hydration treatment time can be 12-36h, specifically 12h, 16h, 20h, 24h, 28h, 32h or 36h; the hydration treatment can be carried out under static conditions. In an embodiment of the present invention, during the hydration treatment, lysozyme interacts with water molecules such as hydrogen bonds, and water molecules form a hydration shell on the surface of the lysozyme molecules, so that the lysozyme can exist stably in the solution.

[0038] In an embodiment of the present invention, the hydration treatment may further include filtration, and the filtrate is collected as the lysozyme solution. In an embodiment of the present invention, the filtration may be membrane filtration, and the filter membrane used for the membrane filtration may specifically be a 0.22 μm water filter membrane; the present invention removes insoluble aggregates by filtration. In an embodiment of the present invention, the concentration of the lysozyme solution may be 0.25 to 2.00 mg / mL, specifically 0.25 mg / mL, 0.50 mg / mL, 1.00 mg / mL, 1.50 mg / mL or 2.00 mg / mL.

[0039] After obtaining the lysozyme solution and the hydrogen gas phase nanobubble solution, the present invention mixes the lysozyme solution with the hydrogen gas phase nanobubble solution, performs self-assembly, and obtains a dispersion of lysozyme nanoparticles with a core-shell structure containing a gas core. In an embodiment of the present invention, the volume ratio of the lysozyme solution to the hydrogen gas phase nanobubble solution can be 3:1~1:3, specifically 3:1, 2:1, 1:1, 1:2 or 1:3. In an embodiment of the present invention, the conditions for the self-assembly may include: the temperature may be 4~30°C, 4°C, 10°C, 15°C, 20°C, 25°C or 30°C; the time may be 3~10min, specifically 3min, 5min, 7min or 10min; the pH value may be 2~9, specifically 2, 3, 4, 5, 6, 7, 8 or 9. In an embodiment of the present invention, the self-assembly may be carried out under stirring conditions, and the stirring rate may be 300~800rpm, specifically 400rpm.

[0040] In an embodiment of the present invention, the hydrogen gas phase nanobubble solution is specifically added dropwise to a continuously stirred lysozyme solution, and after the addition is completed, a pH regulator is used to adjust the pH value of the system to within the above range, and then the self-assembly is performed to obtain a dispersion of lysozyme nanoparticles with a core-shell structure containing a gas core. In an embodiment of the present invention, the dropwise acceleration rate of the hydrogen gas phase nanobubble solution can be 1-5 mL / min, specifically 1 mL / min, 2 mL / min, 3 mL / min, 4 mL / min or 5 mL / min; the stirring rate can be 300-800 rpm, specifically 400 rpm. In an embodiment of the present invention, the pH regulator can include hydrochloric acid, H3PO4 aqueous solution, C6H8O7 aqueous solution or NaOH aqueous solution; the concentration of the pH regulator can be 0.01-1 mol / L, for example, 0.1 mol / L. In an embodiment of the present invention, during the self-assembly process, lysozyme is specifically self-assembled at the gas-liquid interface of the hydrogen nanobubble, and finally a dispersion of lysozyme nanoparticles with a core-shell structure containing a gas core is obtained.

[0041] After obtaining the dispersion of the core-shell structured lysozyme nanoparticles containing a gas core, the present invention can further separate and obtain the core-shell structured lysozyme nanoparticles containing a gas core, or can directly use the dispersion of the core-shell structured lysozyme nanoparticles containing a gas core to further prepare the core-shell structured lysozyme nanoparticles containing a liquid core without any post-treatment. The following is a specific description.

[0042] The present invention freeze-dries the dispersion of the core-shell structure lysozyme nanoparticles containing the gas core, mixes the obtained residue with water, and obtains the core-shell structure lysozyme nanoparticles containing the liquid core. In an embodiment of the present invention, the freeze-drying may include sequentially performing the first stage freeze-drying and the second stage freeze-drying. The first stage freeze-drying may be carried out under atmospheric pressure, and the temperature of the first stage freeze-drying may be -75~-85°C, specifically -80°C; the time may be 6~24h, specifically 6h, 12h or 24h. The second stage freeze-drying may be vacuum freeze-drying, and the vacuum degree may be 0.01~1Pa, specifically 0.05Pa; the temperature of the second stage freeze-drying may be -60~-80°C, specifically -70°C; the time may be 12~48h, specifically 36h. In an embodiment of the present invention, the purpose of the first stage freeze-drying is to freeze the system, and the second freeze-drying may remove all the water in the system and the hydrogen is removed together.

[0043] In an embodiment of the present invention, after the freeze-drying, the obtained residue is mixed with water to obtain core-shell structured lysozyme nanoparticles containing a liquid core; the water may be ultrapure water, and the conductivity of the ultrapure water may be ≥18.2MΩ·cm, specifically 18.2MΩ·cm. In an embodiment of the present invention, water enters the cavity of the outer shell during the mixing process to form a liquid core. The present invention does not have any special restrictions on the amount of water used, as long as it can ensure that the cavity of the outer shell is fully filled; in an embodiment of the present invention, after the obtained residue is mixed with water, a dispersion of core-shell structured lysozyme nanoparticles containing a liquid core is obtained; the amount of water used is sufficient to ensure that the volume of the dispersion of the core-shell structured lysozyme nanoparticles containing a gas core is consistent before freeze-drying.

[0044] In the embodiment of the present invention, hydrogen gas phase nanobubbles are used to regulate the self-assembly of lysozyme to form nanoparticles and enhance its enzyme activity. Bulk phase nanobubbles are quasi-spherical bubbles with a size range of 50 to 1000 nm that are free in the bulk phase solution. They can perform stable Brownian motion in the bulk phase solution and maintain their size and concentration without significant changes during the static process of dozens of days to several months. Bulk phase nanobubbles in aqueous solution have a higher zeta potential (-15~-60mV) that is independent of particle size, a longer retention time, excellent anti-agglomeration stability, a larger specific surface area, higher gas solubility and dissolution rate; a large amount of energy can be released through bubble rupture or collapse, and free radicals are generated at the same time, changing the physical properties and environmental conditions of the aqueous medium. In the embodiments of the present invention, the hydrophobic properties of hydrogen gas phase nanobubbles as gas and the hydrophilic properties of the bubbles due to the charge on their surface are utilized, as well as the natural amphiphilicity and easy adsorption at the gas-liquid interface of lysozyme (such as egg white lysozyme). By adjusting the magnitude of electrostatic interaction forces, hydrophobic interaction forces and hydrogen bond interaction forces, lysozyme is promoted to unfold and adsorb on the surface of hydrogen gas phase nanobubbles to form core-shell structure nanoparticles with different nano-sizes and improved enzyme activity compared to natural lysozyme. Furthermore, in the embodiments of the present invention, by adjusting the type of buffer solution, lysozyme concentration, hydrogen gas phase nanobubble concentration and pH value in the reaction system, the hydrophobic interaction, electrostatic interaction and hydrogen bond interaction strength between hydrogen gas phase nanobubbles and lysozyme are adjusted, thereby changing the adsorption amount, adsorption strength and adsorption stability of lysozyme at the interface of hydrogen gas phase nanobubbles.

[0045] The method for preparing the core-shell structured lysozyme nanoparticles in the embodiment of the present invention is simple to operate, green, and efficient, has little damage to the nutritional value of lysozyme, and is non-toxic. The obtained core-shell structured lysozyme nanoparticles have stronger enzyme activity, higher stability, and good biocompatibility.

[0046] In an embodiment of the present invention, taking lysozyme as hen egg white lysozyme as an example, the hydrated particle size of the prepared gas-core core-shell structured lysozyme nanoparticles is specifically 499.2~1410.8nm, the ζ potential value is specifically -1.5~17.6mV, and the relative enzyme activity is increased by 12.6~15.4% compared with natural hen egg white lysozyme; on this basis, the liquid-core core-shell structured lysozyme nanoparticles prepared have a relative enzyme activity increased by 24.1~34.6% compared with natural hen egg white lysozyme.

[0047] The present invention provides the use of the core-shell structure lysozyme nanoparticles described in the above technical solution or the core-shell structure lysozyme nanoparticles prepared by the preparation method described in the above technical solution in preparing enzyme preparations with improved enzymatic activity or preparing nanocarrier delivery systems.

[0048] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Example 1

[0050] 14 mL of a 10 mM Tris-HCl buffer solution was placed in a sealed container, and 20 mL of hydrogen at 25° C. was filled into the sealed container; the pressure in the sealed container was increased from atmospheric pressure to 455 kPa within 2 seconds, and then the pressure in the sealed container was reduced to atmospheric pressure within 2 seconds, and the pressurization and decompression operations were repeated, with one pressurization and one decompression being recorded as one cycle, and a total of 200 cycles were performed to generate hydrogen gas phase nanobubbles in the Tris-HCl buffer solution to obtain a hydrogen gas phase nanobubble solution, wherein the particle size of the hydrogen nanobubbles in the hydrogen gas phase nanobubble solution was 200-750 nm, and the concentration was (1.54±0.12)×10 10 Pieces / mL;

[0051] The hen egg white lysozyme was mixed with a 10 mM Tris-HCl buffer solution, and stirred at 400 rpm for 2 h at 25 °C to obtain a solution with a 1.1 mg / mL hen egg white lysozyme concentration. The solution was then allowed to stand at 4 °C for 24 h to allow the hen egg white lysozyme to be fully hydrated. The insoluble aggregates were then removed by filtering through a 0.22 μm water filter membrane, and the filtrate was collected as a hen egg white lysozyme solution (the hen egg white lysozyme concentration was 1.0 mg / mL).

[0052] According to the volume ratio of the hen egg white lysozyme solution to the hydrogen gas phase nano bubble solution being 1:1, the hydrogen gas phase nano bubble solution is added dropwise at a speed of 2.0 mL / min to the continuously stirred hen egg white lysozyme solution, after the dropwise addition is completed, the hen egg white lysozyme concentration is 0.5 mg / mL, and the pH value of the obtained system is adjusted to 2 with hydrochloric acid having a concentration of 0.1 mol / L, and then stirred at a speed of 400 rpm for 5 min at 25° C. to allow the hen egg white lysozyme to self-assemble, to obtain a dispersion of nanoparticles with a gas-containing core-shell structure (denoted as A1 nanoparticles), wherein the shell of the nanoparticles with a gas-containing core-shell structure is formed by self-assembly of hen egg white lysozyme, and the cavity of the shell is filled with hydrogen (i.e., the hydrogen gas phase nanobubbles are the gas core);

[0053] The dispersion of the gas-containing core-shell structure nanoparticles was pre-frozen at -80°C for 12 hours under atmospheric pressure, and then vacuum freeze-dried at -70°C for 36 hours under a vacuum degree of 0.05 Pa to remove all moisture and hydrogen, and the residue was redissolved with ultrapure water with a conductivity of 18.2 MΩ·cm. The total volume of the dispersion was consistent before and after freeze-drying and redissolution to obtain a dispersion of liquid-containing core-shell structure nanoparticles (denoted as B1 nanoparticles), wherein the shell of the liquid-containing core-shell structure nanoparticles was formed by self-assembly of hen egg white lysozyme, and the cavity of the shell was filled with ultrapure water (i.e., ultrapure water was the liquid core).

[0054] Embodiments 2 to 8

[0055] The method of Example 1 was followed, except that the pH values ​​of the systems during self-assembly in Examples 2 to 8 were 3, 4, 5, 6, 7, 8, and 9, respectively, and the corresponding gas-containing core-shell structure nanoparticles were denoted as A2 to A8 nanoparticles, and the liquid-containing core-shell structure nanoparticles were denoted as B2 to B8 nanoparticles.

[0056] Comparative Examples 1 to 8

[0057] Comparative Examples 1 to 8 were respectively prepared according to the methods of Examples 1 to 8, except that the "hydrogen gas phase nanobubble solution" was replaced by "Tris-HCl buffer solution", and the nanoparticles (not having a core-shell structure) obtained by self-assembly were respectively recorded as C1 to C8 nanoparticles, and the nanoparticles obtained by freeze-drying and re-dissolving were respectively recorded as D1 to D8 nanoparticles.

[0058] Table 1 shows the average hydrated particle size and ζ potential of the nanoparticles prepared in Examples 1 to 8 and Comparative Examples 1 to 8. The results show that with the increase of the pH value of the system during self-assembly, the average hydrated particle size of the obtained nanoparticles gradually increases, which is significantly higher than the average hydrated particle size of the nanoparticles prepared under the same pH conditions in the comparative example; and with the increase of the pH value of the system during self-assembly, the average ζ potential of the obtained nanoparticles is between hydrogen gas phase nanobubbles (12.31±0.82mV, -2.54±0.57mV and -14.24±0.49mV at pH 3 to 5, respectively) and hen egg white lysozyme (23.10±0.97mV, 16.50±0.31mV and 6.60±0.40mV at pH 3 to 5, respectively) when the system pH is 3 to 5, and is basically consistent with the average ζ potential of hen egg white lysozyme when the system pH is 6 or above.

[0059] Table 1 Average hydrated particle size and average zeta potential of nanoparticles prepared in Examples 1 to 8 and Comparative Examples 1 to 8

[0060]

[0061] Embodiments 9 to 12

[0062] The method of Example 2 was followed (i.e., the pH value of the system during self-assembly was 3), except that the concentrations of the hen egg white lysozyme in the hen egg white lysozyme solutions in Examples 9 to 12 were 0.25 mg / mL, 1.00 mg / mL, 1.50 mg / mL, and 2.00 mg / mL, respectively, and the corresponding gas-containing core-shell structure nanoparticles were denoted as A9 to A12 nanoparticles, respectively, and the liquid-containing core-shell structure nanoparticles were denoted as B9 to B12 nanoparticles, respectively.

[0063] Embodiments 13 to 16

[0064] The method of Example 5 was followed (i.e., the pH value of the system during self-assembly was 6), except that the concentrations of the hen egg white lysozyme in the hen egg white lysozyme solutions in Examples 13 to 16 were 0.25 mg / mL, 1.00 mg / mL, 1.50 mg / mL, and 2.00 mg / mL, respectively, and the corresponding gas-containing core-shell structure nanoparticles were denoted as A13 to A16 nanoparticles, respectively, and the liquid-containing core-shell structure nanoparticles were denoted as B13 to B16 nanoparticles, respectively.

[0065] Embodiments 17 to 20

[0066] The method of Example 8 was followed (i.e., the pH value of the system during self-assembly was 9), except that the concentrations of the hen egg white lysozyme in the hen egg white lysozyme solutions in Examples 17 to 20 were 0.25 mg / mL, 1.00 mg / mL, 1.50 mg / mL, and 2.00 mg / mL, respectively, and the corresponding gas-containing core-shell structure nanoparticles were denoted as A17 to A20 nanoparticles, respectively, and the liquid-containing core-shell structure nanoparticles were denoted as B17 to B20 nanoparticles, respectively.

[0067] Comparative Examples 9-20

[0068] Comparative Examples 9 to 20 were respectively prepared according to the methods of Examples 9 to 20, except that the "hydrogen gas phase nanobubble solution" was replaced by "Tris-HCl buffer solution", and the nanoparticles (not having a core-shell structure) obtained by self-assembly were respectively recorded as C9 to C20 nanoparticles, and the nanoparticles obtained by freeze-drying and re-dissolving were respectively recorded as D9 to D20 nanoparticles.

[0069] Tables 2 to 4 show the average hydrated particle sizes of the nanoparticles prepared in Examples 9 to 20 and Comparative Examples 9 to 20. The results show that with the increase of the egg white lysozyme concentration, when the pH value of the system is 6 and 9 during self-assembly, the average hydrated particle size of the obtained nanoparticles first increases and then decreases. The egg white lysozyme concentration corresponding to the maximum average hydrated particle size is 1.50 mg / mL, and the average hydrated particle size increases from 922.1±65.8 nm to 1406. The average hydrated particle size of the nanoparticles was 0±230.8nm and then decreased to 1232.0±113.4nm, and increased from 1008.0±102.4nm to 1689.0±66.7nm and then decreased to 1216.0±24.7nm, which is much higher than the particle size of egg white lysozyme (average hydrated particle size 4.81±0.70nm and 5.08±0.65nm) and hydrogen gas phase nanobubbles (average hydrated particle size 241.0±8.5nm and 219.7±0.6nm). When the pH value of the system is 3 during self-assembly, the average hydrated particle size of the nanoparticles obtained has a poor change in significance, and its size is close to that of hydrogen gas phase nanobubbles.

[0070] Table 2 Average hydrated particle size of nanoparticles prepared in Examples 9 to 12 and Comparative Examples 9 to 12

[0071]

[0072] Table 3 Average hydrated particle size of nanoparticles prepared in Examples 13 to 16 and Comparative Examples 13 to 16

[0073]

[0074] Table 4 Average hydrated particle size of nanoparticles prepared in Examples 17 to 20 and Comparative Examples 17 to 20

[0075]

[0076] Embodiments 21 to 24

[0077] The method of Example 2 was followed (i.e., the pH value of the system during self-assembly was 3), except that the volume ratios of the hen's egg white lysozyme solution to the hydrogen gas phase nanobubble solution in Examples 21 to 24 were 3:1, 2:1, 1:2, and 1:3, respectively, and the corresponding gas-containing core-shell structure nanoparticles were respectively recorded as A21 to A24 nanoparticles, and the liquid-containing core-shell structure nanoparticles were respectively recorded as B21 to B24 nanoparticles.

[0078] Embodiments 25 to 28

[0079] The method of Example 5 was followed (i.e., the pH value of the system during self-assembly was 6), except that the volume ratios of the hen's egg white lysozyme solution to the hydrogen gas phase nanobubble solution in Examples 25 to 28 were 3:1, 2:1, 1:2, and 1:3, respectively, and the corresponding gas-containing core-shell structure nanoparticles were respectively recorded as A25 to A28 nanoparticles, and the liquid-containing core-shell structure nanoparticles were respectively recorded as B25 to B28 nanoparticles.

[0080] Embodiments 29 to 32

[0081] The method of Example 8 was followed (i.e., the pH value of the system during self-assembly was 9), except that the volume ratios of the hen's egg white lysozyme solution to the hydrogen gas phase nanobubble solution in Examples 29 to 32 were 3:1, 2:1, 1:2, and 1:3, respectively, and the corresponding gas-containing core-shell structure nanoparticles were denoted as A29 to A32 nanoparticles, respectively, and the liquid-containing core-shell structure nanoparticles were denoted as B29 to B32 nanoparticles, respectively.

[0082] Comparative Examples 21-32

[0083] Comparative Examples 21 to 32 were respectively prepared according to the methods of Examples 21 to 32, except that the "hydrogen gas phase nanobubble solution" was replaced by "Tris-HCl buffer solution", and the nanoparticles (not having a core-shell structure) obtained by self-assembly were respectively recorded as C21 to C32 nanoparticles, and the nanoparticles obtained by freeze-drying and re-dissolving were respectively recorded as D21 to D32 nanoparticles.

[0084] Tables 5 to 7 show the average hydrated particle size of the nanoparticles prepared in Examples 21 to 32 and Comparative Examples 21 to 32. The results show that with the increase of the concentration of hydrogen gas phase nanobubbles in the system, when the pH value of the system is 3 during self-assembly, the average hydrated particle size of the obtained nanoparticles continues to increase, from 543.5±33.6nm to 1250.0±84.8nm; when the pH value of the system is 6 during self-assembly, the average hydrated particle size of the obtained nanoparticles first decreases and then increases, from 1147.0±19.0nm to 918.6±62.3nm and then increases to 1200.0±68.1nm; when the pH value of the system is 9 during self-assembly, the average hydrated particle size of the obtained nanoparticles does not show obvious difference.

[0085] Table 5 Average hydrated particle size of nanoparticles prepared in Examples 21 to 24 and Comparative Examples 21 to 24

[0086]

[0087] Table 6 Average hydrated particle size of nanoparticles prepared in Examples 25 to 28 and Comparative Examples 25 to 28

[0088]

[0089] Table 7 Average hydrated particle size of nanoparticles prepared in Examples 29 to 32 and Comparative Examples 29 to 32

[0090]

[0091] Figure 1 This is a transmission electron microscope (TEM) image of the nanoparticles (numbered A5) prepared in Example 5, where the right side is an enlarged view of the left side; the results show that the particle sizes of the nanoparticles are mainly distributed in the range of 200-350 nm and 450-550 nm.

[0092] Figure 2 This is a transmission electron microscopy image of the nanoparticles (numbered C5) prepared in Comparative Example 5. The results show that the nanoparticles are in a monomer dispersion state, and the particle size is mainly distributed in the range of 4 to 6 nm.

[0093] Table 8 shows the enzyme activity test results of the nanoparticles prepared in Examples 5 and 8 and Comparative Examples 5 and 8. Specifically, the enzyme activities of the nanoparticles (numbered C5 and D5) in Comparative Example 5 are defined as 100% respectively, and the relative enzyme activities of the nanoparticles (numbered A5 and B5) in Example 5 are calculated; the enzyme activities of the nanoparticles (numbered C8 and D8) in Comparative Example 8 are defined as 100% respectively, and the relative enzyme activities of the nanoparticles (numbered A8 and B8) in Example 8 are calculated. It can be seen from Table 8 that the nanoparticles prepared by the method of the embodiment of the present invention have higher enzyme activity.

[0094] Table 8 Enzyme activity of nanoparticles prepared in Examples 5 and 8 and Comparative Examples 5 and 8

[0095]

[0096] It can be seen from the above embodiments that the present invention has at least the following beneficial effects:

[0097] (1) In the embodiment of the present invention, hydrogen gas phase nanobubbles are used to induce lysozyme to unfold and self-assemble at the gas-liquid interface to form a core-shell structure. The relevant structural changes are beneficial to the improvement of its enzyme activity. The hydrogen gas phase nanobubbles are formed by the bubble nucleation process when the pressure of hydrogen introduced into the liquid (i.e., the first buffer solution) changes. The hydrogen gas phase nanobubbles are green and non-toxic, and there is no safety problem.

[0098] (2) The hen egg white lysozyme used in the embodiments of the present invention is widely available and low in cost. The hydrogen gas phase nanobubbles have both hydrophobic properties as a gas and hydrophilic properties due to the charge on the bubble surface. Combined with the inherent amphiphilicity of hen egg white lysozyme and its easy adsorption and self-assembly at the gas-liquid interface, the hen egg white lysozyme spontaneously adsorbs on the surface of the hydrogen gas phase nanobubbles to form core-shell structured nanoparticles with different nanometer sizes. During the formation process, the hydrogen gas phase nanobubbles affect the surface hydrogen bond network of the hen egg white lysozyme. The changes in the secondary and tertiary structures of the hen egg white lysozyme itself increase its enzyme activity. The size is controllable, and the stability and safety are good.

[0099] (3) In the embodiments of the present invention, by adjusting the concentration of hydrogen gas phase nanobubbles, lysozyme concentration and pH value in the reaction system, the adsorption amount, adsorption strength and adsorption stability of hen egg white lysozyme at the gas-liquid interface of the hydrogen gas phase nanobubbles in the system are changed, thereby controlling the size, stability and enzyme activity of the formed core-shell structure nanoparticles.

[0100] (4) The preparation process of the core-shell structured lysozyme nanoparticles in the embodiment of the present invention is simple and easy to operate, does not introduce additional reaction substrates and does not use chemical additives, does not require complex equipment, is low-cost, green and non-toxic, safe and reliable, and easy to promote.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A core-shell structured lysozyme nanoparticle, comprising an outer shell and a core wrapped in the outer shell, wherein the outer shell is formed by self-assembly of lysozyme, and the core is a gas core formed by hydrogen or a liquid core formed by an aqueous solution; The method for preparing the core-shell structured lysozyme nanoparticles comprises the following steps: The hydrogen gas is mixed with the first buffer solution, and the mixture is pressurized and depressurized in sequence under a closed condition to obtain a hydrogen gas phase nanobubble solution; the concentration of hydrogen nanobubbles in the hydrogen gas phase nanobubble solution is (0.1-5)×10 10 Pieces / mL; The lysozyme is mixed with the second buffer solution and hydrated to obtain a lysozyme solution; the concentration of the lysozyme solution is 0.25-2.00 mg / mL; The lysozyme solution is mixed with a hydrogen gas phase nanobubble solution, and self-assembled at a pH value of 2 to 9 to obtain a dispersion of lysozyme nanoparticles with a core-shell structure containing a gas core; the volume ratio of the lysozyme solution to the hydrogen gas phase nanobubble solution is 3:1 to 1:3; The dispersion of the core-shell structured lysozyme nanoparticles containing a gas core is freeze-dried, and the obtained residue is mixed with water to obtain the core-shell structured lysozyme nanoparticles containing a liquid core.

2. The core-shell structured lysozyme nanoparticles according to claim 1, characterized in that: The particle size of the core-shell structured lysozyme nanoparticles is 200-550 nm, and the particle size of the core-shell structured lysozyme nanoparticles refers to the particle size displayed in a transmission electron microscope image.

3. The core-shell structured lysozyme nanoparticles according to claim 1 or 2, characterized in that: The lysozyme includes hen egg white lysozyme.

4. The method for preparing the core-shell structured lysozyme nanoparticles according to any one of claims 1 to 3, comprising the following steps: The hydrogen gas is mixed with the first buffer solution, and the mixture is pressurized and depressurized in sequence under a closed condition to obtain a hydrogen gas phase nanobubble solution; the concentration of hydrogen nanobubbles in the hydrogen gas phase nanobubble solution is (0.1-5)×10 10 Pieces / mL; The lysozyme is mixed with the second buffer solution and hydrated to obtain a lysozyme solution; the concentration of the lysozyme solution is 0.25-2.00 mg / mL; The lysozyme solution is mixed with a hydrogen gas phase nanobubble solution, and self-assembled at a pH value of 2 to 9 to obtain a dispersion of lysozyme nanoparticles with a core-shell structure containing a gas core; the volume ratio of the lysozyme solution to the hydrogen gas phase nanobubble solution is 3:1 to 1:3; The dispersion of the core-shell structured lysozyme nanoparticles containing a gas core is freeze-dried, and the obtained residue is mixed with water to obtain the core-shell structured lysozyme nanoparticles containing a liquid core.

5. The preparation method according to claim 4, characterized in that: The first buffer solution and the second buffer solution independently include Tris-HCl buffer solution, Na2HPO4-NaH2PO4 buffer solution, Na2HPO4-C6H8O7 buffer solution or C6H8O7-NaOH-HCl buffer solution.

6. The preparation method according to claim 4, characterized in that: The hydrated particle size of the hydrogen nanobubbles in the hydrogen gas phase nanobubble solution is 200-750 nm.

7. The preparation method according to claim 4, characterized in that: The pressurization includes: increasing the pressure from atmospheric pressure to 400-500 kPa within 2 seconds; the decompression includes: restoring the pressure to atmospheric pressure within 2 seconds; one pressurization and one decompression is counted as one cycle, and the number of cycles is 20-500 times.

8. The preparation method according to claim 4, characterized in that: The temperature of the hydration treatment is 2-8°C and the time is 12-36h; the hydration treatment is carried out under static conditions.

9. The preparation method according to claim 4, characterized in that: The self-assembly conditions include: temperature of 4-30° C. and time of 3-10 min; the self-assembly is performed under stirring conditions.

10. Use of the core-shell structure lysozyme nanoparticles according to any one of claims 1 to 3 or the core-shell structure lysozyme nanoparticles prepared by the preparation method according to any one of claims 4 to 9 in preparing enzyme preparations with improved enzymatic activity or preparing nanocarrier delivery systems.

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