Binary protein nanoparticle with multi-shell hollow structure and preparation method of binary protein nanoparticle

Abstract

The invention provides a binary protein nano-particle with a multi-shell hollow structure and a preparation method thereof, the binary protein nano-particle comprises a shell layer formed by crosslinking amphiphilic vegetable protein and a core layer formed by crosslinking hydrophobic vegetable protein, and the particle size of the binary protein nano-particle is less than 150nm. According to the binary protein nano-particle, the binary protein nano-particle with a multi-shell hollow structure is formed on the basis of a protein co-assembly principle, and the binary protein nano-particle has the advantages of smaller particle size, better dispersity, more cavities, higher cavitation rate and wide application prospect in the fields of chemical catalysis, medicine, food and health care products.

Description

Technical Field

[0001] The invention belongs to the field of protein processing, and in particular relates to a binary protein nanoparticle with a multi-shell hollow structure and a preparation method thereof. Background Art

[0002] Multi-compartmentalization is a basic structural feature of eukaryotic cells. This biomimetic structure can coherently regulate various functions in precisely specified intracellular subcompartments. The hollow multi-shell structure is composed of chambers or shells in multiple regions. It has a larger surface area, higher drug loading capacity and lower density than the solid, shell-core structure, which is conducive to more effective surface area for mass / energy transfer. Therefore, the hollow multi-shell structure has the characteristics of constructing independent reaction environments in each region in the same space, providing a structural basis for functions such as coordinated drug loading, precise control of targeted delivery sites, and flexible regulation of release rate.

[0003] Traditionally, multi-shell structures are used in the chemical industry, but their application in the food industry has not yet been explored. The common template method for preparing multi-shell hollow structures can be roughly divided into sacrificial template method and self-template method. The sacrificial template method grows multiple layers of shells on the basis of a prefabricated solid core. This method can easily lead to shell damage during the removal of the core. The self-template method forms a multi-chamber structure through the infiltration of solvents driven by surfactants or copolymerization of synthetic initiators pre-embedded in the template into the shell. However, the residual initiator leads to its low biocompatibility and safety.

[0004] Plant proteins themselves have complex high-level spatial structures, and proteins can easily self-assemble to form new structures through intermolecular interactions. However, the existing technology has not yet found a way to prepare multi-shell chambered nanoprotein particles.

[0005] Therefore, exploring efficient, controllable, green and safe methods to construct the precise spatial structure of proteins is of great significance for improving the functional properties of plant proteins and the commercialization of plant-based protein foods. Summary of the invention

[0006] In view of this, the present invention aims to provide a binary protein nanoparticle with a multi-shell hollow structure and a preparation method thereof, so as to achieve the compartmentalization or porous transformation of the protein structure at the molecular scale.

[0007] To achieve the above object, the technical solution of the present invention is achieved as follows: In a first aspect, the present invention provides a binary protein nanoparticle with a multi-shell hollow structure, wherein the binary protein nanoparticle comprises a shell layer formed by cross-linking amphiphilic plant proteins and a core layer formed by cross-linking hydrophobic plant proteins, and the particle size of the binary protein nanoparticle is less than 150 nm.

[0008] Furthermore, the PDI of the binary protein nanoparticles is less than 0.3.

[0009] Furthermore, the shell layer and the core layer have cavities and / or holes.

[0010] Furthermore, the amphiphilic plant protein is selected from one of soy protein, pea protein, mung bean protein and chickpea protein, and the hydrophobic plant protein is selected from one of corn protein, distiller's grains protein, walnut protein and alfalfa protein.

[0011] Furthermore, the preparation method of the binary protein nanoparticles includes the steps of adjusting the pH values ​​of the amphiphilic plant protein solution and the hydrophobic plant protein solution to alkaline to obtain two molten plant proteins, mixing the two molten plant proteins and adjusting the pH to neutral, then drying the supernatant to obtain the binary plant protein, mixing and dissolving the binary plant protein and a cross-linking agent to obtain a mixed solution, and centrifuging the mixed solution to obtain the supernatant and drying it.

[0012] Furthermore, the mass ratio of the amphiphilic plant protein to the hydrophobic plant protein is 0.2-4.

[0013] Furthermore, the cross-linking agent is selected from one of genipin, peppermint essential oil, cinnamon essential oil and eugenol.

[0014] In a second aspect, the present invention provides a method for preparing binary protein nanoparticles having a multi-shell hollow structure, the preparation method comprising the following steps: a. adjusting the pH values ​​of the amphiphilic plant protein solution and the hydrophobic plant protein solution to alkaline to obtain two molten plant protein solutions; b. Mixing the two molten vegetable protein solutions, adjusting the pH to neutral, centrifuging, taking the supernatant, and dialyzing and drying to obtain the binary vegetable protein; c. dissolving the binary plant protein and mixing it with a cross-linking agent to obtain a mixed solution; d. Centrifuge the mixed solution to obtain the supernatant, and dialyze and dry the supernatant to obtain the binary protein nanoparticles.

[0015] Furthermore, the preparation method of the binary protein nanoparticles of the present invention unfolds the amphiphilic plant protein and the hydrophobic plant protein respectively during the pH cycle to form a molten state, and then the molten plant proteins are mixed and self-assembled to form a binary protein complex consisting of a hydrophilic shell and a hydrophobic core, thereby forming a centripetal hydrophobic gradient difference. By utilizing this gradient-increasing hydrophobicity, a cross-linking agent is added to form an asymmetric diffusion inside and outside the protein, enter the interior of the molecule, and trigger the cross-linking between the protein molecules again, forming a multi-chamber or multi-shell hollow structure.

[0016] Furthermore, the operation method of step a is: after mixing the amphiphilic plant protein and the hydrophobic plant protein with water respectively, adjusting the pH value to 9-11, heating and stirring to obtain two molten plant protein solutions.

[0017] Furthermore, the mass (w / w) ratio of the amphiphilic plant protein to water is 0.1% to 5%; the mass (w / w) ratio of the hydrophobic plant protein to water is 0.1% to 5%.

[0018] Furthermore, the heating and stirring temperature in step a is 55-85°C.

[0019] Furthermore, the stirring rate of the heating and stirring in step a is 200-1500 rpm, and the stirring time is 30-120 min.

[0020] Furthermore, in step a, a 0.1M to 2M sodium hydroxide solution is used to adjust the pH value.

[0021] Furthermore, the mass ratio of the amphiphilic plant protein to the hydrophobic plant protein is 0.2-4.

[0022] Furthermore, in step b, the temperature at which the two molten vegetable protein solutions are mixed is 55-85°C.

[0023] Furthermore, in step b, the stirring rate of mixing the two molten vegetable protein solutions is 200-1500 rpm, and the stirring time is 30-120 min.

[0024] Furthermore, in step b, 0.1 M to 1 M HCl is used to adjust the pH value to neutral.

[0025] Furthermore, the centrifugal force of the centrifugation in step b is 5000-11000g, and the centrifugation time is 5-30min.

[0026] Furthermore, in step b, the dialysis molecular weight is 1000-5000 Da, and the dialysis time is 12-48 h.

[0027] Furthermore, in step c, the binary plant protein is dissolved and then stirred and mixed with the cross-linking agent; preferably, the stirring rate is 200-1500 rpm, and the stirring time is 30-120 min.

[0028] Furthermore, the cross-linking agent is selected from one of genipin, peppermint essential oil, cinnamon essential oil and eugenol.

[0029] Furthermore, the amount of the cross-linking agent added in step c is 0.08-0.25% of the mass of the solvent used to dissolve the binary plant protein.

[0030] Furthermore, the solvent used to dissolve the binary plant protein in step c is water.

[0031] Furthermore, the centrifugal force of the centrifugation in step d is 5000-11000 g, and the centrifugation time is 5-30 min.

[0032] Furthermore, in step d, the dialysis molecular weight is 1000-5000 Da, and the dialysis time is 12-48 h.

[0033] In a third aspect, the present invention provides the use of the binary protein nanoparticles as described in the first aspect or the binary protein nanoparticles prepared by the preparation method as described in the second aspect in the field of chemical catalysis, medicine, food or health products.

[0034] Compared with the prior art, the binary protein nanoparticles with a multi-shell hollow structure and the preparation method thereof of the present invention have the following advantages: (1) The binary protein nanoparticles of the present invention have smaller particle size and better dispersibility, more cavities, and higher cavitation rate, and have broad application prospects in the fields of chemical catalysis, medicine, food and health products.

[0035] (2) The binary protein nanoparticles described in the present invention are based on the principle of protein co-assembly. Alkaline solution is used to transform two plant proteins into a molten state. After pH cycling, a binary protein complex with a hydrophilic shell and a hydrophobic core is formed. On this basis, the cross-linking agent is used to diffuse into the protein molecules asymmetricly in the protein, which triggers the cross-linking of the internal molecules of the binary protein again. Finally, the cross-linking agent is removed by dialysis to form a binary protein nanoparticle with a multi-shell hollow structure. The protein structure can be compartmentalized or porous at the molecular scale without changing the primary structure of the plant protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings: Figure 1 Schematic diagram of the microstructure of binary protein nanoparticles with multi-shell hollow structures prepared in Examples 1-4 of the present invention; Figure 2 Schematic diagram of the microstructure of the plant protein products obtained in Comparative Examples 1-9 of the present invention. DETAILED DESCRIPTION

[0037] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0038] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0039] Example 1 (1) 1000 g of soy protein and 1000 g of distiller's grains protein were added to 10 L of water, adjusted to pH = 10 with NaOH solution, and stirred at 800 r / min for 30 min under heating at 65°C to obtain a molten protein solution; (2) The molten soy protein solution obtained in (1) and the distiller's grains protein solution were mixed in a mass ratio of 1:1, and the pH was adjusted to 7 with HCl to obtain a mixed solution. The mixed solution was then centrifuged at 10000g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and freeze-dried to obtain a binary plant protein; (3) The binary plant protein obtained in (2) was re-dissolved in water to form a 1% (w / v) binary plant protein solution, 8 mL of peppermint essential oil was added, and the mixture was stirred at 800 r / min for 60 min to obtain a cross-linked mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, and the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h. After freeze-drying, multi-shell hollow structure binary protein nanoparticles were obtained.

[0040] Example 2

[0041] (1) 1000 g of soy protein and 1000 g of distiller's grains protein were added to 10 L of water, adjusted to pH = 10 with NaOH, and stirred at 1000 r / min for 30 min under heating at 75°C to obtain a molten protein solution; (2) The molten soy protein solution obtained in (1) and the distiller's grains protein solution were mixed at a mass ratio of 2:1, and the pH was adjusted to 7 with HCl to obtain a mixed solution. The mixed solution was then centrifuged at 10000 g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and freeze-dried to obtain a binary plant protein; (3) The binary plant protein obtained in (2) was re-dissolved in water to form a 1% (w / v) binary plant protein solution, 8 mL of peppermint essential oil was added, and the mixture was stirred at 800 r / min for 60 min to obtain a cross-linked mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, and the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h. After freeze-drying, multi-shell hollow structure binary protein nanoparticles were obtained.

[0042] Example 3

[0043] (1) 1000 g of soy protein and 1000 g of distiller's grains protein were added to 10 L of water, adjusted to pH = 10 with NaOH, and stirred at 1000 r / min for 30 min under heating at 85°C to obtain a molten protein solution; (2) The molten soy protein solution obtained in (1) and the distiller's grains protein solution were mixed in a mass ratio of 1:1, and the pH was adjusted to 7 with HCl to obtain a mixed solution. The mixed solution was then centrifuged at 10000g for 15 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and freeze-dried to obtain a binary plant protein; (3) The binary plant protein obtained in (2) was re-dissolved in water to form a 1% (w / v) binary plant protein solution, 10 mL of eugenol was added, and the mixture was stirred at 1000 r / min for 60 min to obtain a cross-linked mixture; (4) the mixture obtained in (3) was centrifuged at 10000 g for 10 min, the supernatant was dialyzed with a 3000 Da dialysis bag for 16 h, and then freeze-dried to obtain binary protein nanoparticles with multi-shell hollow structures;

[0044] Example 4 (1) 1000 g of pea protein and 1000 g of corn protein were added to 10 L of water, adjusted to pH = 12 with NaOH, and stirred at 1000 r / min for 30 min under heating at 85°C to obtain a molten protein solution; (2) The molten soy protein solution obtained in (1) and the distiller's grains protein solution were mixed in a mass ratio of 1:1, and the pH was adjusted to 7 with HCl to obtain a mixed solution. The mixed solution was then centrifuged at 10000g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and freeze-dried to obtain a binary plant protein; (3) The binary plant protein obtained in (2) was re-dissolved in water to form a 1% (w / v) binary plant protein solution, 8 mL of peppermint essential oil was added, and the mixture was stirred at 800 r / min for 60 min to obtain a cross-linked mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, and the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h. After freeze-drying, multi-shell hollow structure binary protein nanoparticles were obtained.

[0045] Example 5

[0046] (1) 1000 g mung bean protein and 1000 g walnut protein were added to 10 L water, adjusted to pH = 12 with NaOH, and stirred at 1000 r / min for 30 min under heating at 85°C to obtain a molten protein solution; (2) The molten soy protein solution obtained in (1) and the distiller's grains protein solution were mixed at a mass ratio of 1:5, and the pH was adjusted to 7 with HCl to obtain a mixed solution. The mixed solution was then centrifuged at 10000 g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and freeze-dried to obtain a binary plant protein; (3) The binary plant protein obtained in (2) was re-dissolved in water to form a 1% (w / v) binary plant protein solution, 8 mL of genipin was added, and the mixture was stirred at 800 r / min for 60 min to obtain a cross-linked mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, and the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h. After freeze-drying, multi-shell hollow structure binary protein nanoparticles were obtained.

[0047] Example 6

[0048] (1) 1000 g of chickpea protein and 1000 g of alfalfa protein were added to 10 L of water, adjusted to pH = 12 with NaOH, and stirred at 1000 r / min for 30 min under heating at 85°C to obtain a molten protein solution; (2) The molten soy protein solution obtained in (1) and the distiller's grains protein solution were mixed at a mass ratio of 4:1, and the pH was adjusted to 7 with HCl to obtain a mixed solution. The mixed solution was then centrifuged at 10000 g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and freeze-dried to obtain a binary plant protein; (3) The binary plant protein obtained in (2) was re-dissolved in water to form a 1% (w / v) binary plant protein solution, 8 mL of cinnamon essential oil was added, and the mixture was stirred at 800 r / min for 60 min to obtain a cross-linked mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, and the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h. After freeze-drying, multi-shell hollow structure binary protein nanoparticles were obtained.

[0049] Example 7

[0050] (1) 1000 g of chickpea protein and 1000 g of alfalfa protein were added to 10 L of water, adjusted to pH = 12 with NaOH, and stirred at 1000 r / min for 30 min under heating at 85°C to obtain a molten protein solution; (2) The molten soy protein solution obtained in (1) and the distiller's grains protein solution were mixed in a mass ratio of 1:2, and the pH was adjusted to 7 with HCl to obtain a mixed solution. The mixed solution was then centrifuged at 10000g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and freeze-dried to obtain a binary plant protein; (3) The binary plant protein obtained in (2) was re-dissolved in water to form a 1% (w / v) binary plant protein solution, 8 mL of cinnamon essential oil was added, and the mixture was stirred at 800 r / min for 60 min to obtain a cross-linked mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, and the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h. After freeze-drying, multi-shell hollow structure binary protein nanoparticles were obtained.

[0051] Comparative Example 1 (1) 2000 g of soy protein was added to 10 L of water and adjusted to pH = 10 with NaOH to dissolve, and then heated at 65 ° C and stirred at 800 r / min for 30 min to obtain a molten protein solution; (2) The molten soybean protein solution obtained in (1) was adjusted to pH = 7 with HCl to obtain a mixed solution, and then the mixed solution was centrifuged at 10000g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and then freeze-dried to obtain plant protein; (3) re-dissolving the plant protein obtained in (2) with water to form a 1% (w / v) binary plant protein solution, adding 8 mL of peppermint essential oil, and stirring at 800 r / min for 60 min to obtain a mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h, and then freeze-dried to obtain a plant protein product.

[0052] Comparative Example 2 (1) 2000 g of vinasse protein was added to 10 L of water and adjusted to pH = 10 with NaOH to dissolve, and then heated at 65°C and stirred at 800 r / min for 30 min to obtain a molten protein solution; (2) The molten distiller's grains protein solution obtained in (1) was adjusted to pH = 7 with HCl to obtain a mixed solution, and then the mixed solution was centrifuged at 10000g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and then freeze-dried to obtain plant protein; (3) re-dissolving the plant protein obtained in (2) with water to form a 1% (w / v) binary plant protein solution, adding 8 mL of peppermint essential oil, and stirring at 800 r / min for 60 min to obtain a mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h, and then freeze-dried to obtain a plant protein product.

[0053] Comparative Example 3 (1) 1000 g of soy protein and 1000 g of distiller's grains protein were added to 10 L of water, adjusted to pH = 10 with NaOH, and stirred at 800 r / min for 30 min under heating at 65°C to obtain a molten protein solution; (2) The molten soy protein solution obtained in (1) and the distiller's grains protein solution were mixed at a mass ratio of 1:6, and the pH was adjusted to 7 with HCl to obtain a mixed solution. The mixed solution was then centrifuged at 10000 g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and freeze-dried to obtain a binary plant protein; (3) The binary plant protein obtained in (2) was re-dissolved in water to form a 1% (w / v) binary plant protein solution, 8 mL of peppermint essential oil was added, and the mixture was stirred at 800 r / min for 60 min to obtain a cross-linked mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h, and then freeze-dried to obtain a plant protein product.

[0054] Comparative Example 4 (1) 1000 g of soy protein and 1000 g of distiller's grains protein were added to 10 L of water, adjusted to pH = 10 with NaOH, and stirred at 800 r / min for 30 min under heating at 65°C to obtain a molten protein solution; (2) The molten soy protein solution obtained in (1) and the distiller's grains protein solution were mixed at a mass ratio of 5:1, and the pH was adjusted to 7 with HCl to obtain a mixed solution. The mixed solution was then centrifuged at 10000g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and freeze-dried to obtain a binary plant protein; (3) The binary plant protein obtained in (2) was re-dissolved in water to form a 1% (w / v) binary plant protein solution, 8 mL of peppermint essential oil was added, and the mixture was stirred at 800 r / min for 60 min to obtain a cross-linked mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h, and then freeze-dried to obtain a plant protein product.

[0055] Comparative Example 5 (1) 1000 g of soy protein and 1000 g of distiller's grains protein were added to 10 L of water, adjusted to pH = 10 with NaOH, and stirred at 800 r / min for 30 min under heating at 65°C to obtain a molten protein solution; (2) The molten soy protein solution obtained in (1) and the distiller's grains protein solution were mixed in a mass ratio of 1:1, and the pH was adjusted to 7 with HCl to obtain a mixed solution. The mixed solution was then centrifuged at 10000g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and freeze-dried to obtain a binary plant protein; (3) adding water to dissolve the binary vegetable protein obtained in (2) to form a 1% (w / v) binary vegetable protein solution, and then stirring at 800 r / min for 60 min to obtain a mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h, and then freeze-dried to obtain a plant protein product.

[0056] Comparative Example 6 (1) 1000 g of soy protein and 1000 g of distiller's grains protein were added to 10 L of water, adjusted to pH = 10 with NaOH, and stirred at 800 r / min for 30 min under heating at 65°C to obtain a molten protein solution; (2) The molten soy protein solution obtained in (1) and the distiller's grains protein solution were mixed in a mass ratio of 1:1, and the pH was adjusted to 7 with HCl to obtain a mixed solution. The mixed solution was then centrifuged at 10000g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and freeze-dried to obtain a binary plant protein; (3) The binary plant protein obtained in (2) was re-dissolved in water to form a 1% (w / v) binary plant protein solution, 8 mL of rosemary essential oil was added, and the mixture was stirred at 800 r / min for 60 min to obtain a cross-linked mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h, and then freeze-dried to obtain a plant protein product.

[0057] Comparative Example 7 (1) 1000 g of soy protein and 1000 g of distiller's grains protein were added to 10 L of water, adjusted to pH = 10 with NaOH, and stirred at 800 r / min for 30 min under heating at 65°C to obtain a molten protein solution; (2) The molten soy protein solution obtained in (1) and the distiller's grains protein solution were mixed in a mass ratio of 1:1, and the pH was adjusted to 7 with HCl to obtain a mixed solution. The mixed solution was then centrifuged at 10000g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and freeze-dried to obtain a binary plant protein; (3) The binary plant protein obtained in (2) was re-dissolved in water to form a 1% (w / v) binary plant protein solution, 8 mL of anhydrous ethanol was added, and the mixture was stirred at 800 r / min for 60 min to obtain a cross-linked mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h, and then freeze-dried to obtain a plant protein product.

[0058] Comparative Example 8 (1) 1000 g of soy protein and 1000 g of pea protein were added to 10 L of water, adjusted to pH = 10 with NaOH, and stirred at 800 r / min for 30 min under heating at 65°C to obtain a molten protein solution; (2) The molten soy protein solution obtained in (1) and the pea protein solution were mixed in a mass ratio of 1:1, and the pH was adjusted to 7 with HCl to obtain a mixed solution. The mixed solution was then centrifuged at 10000g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and freeze-dried to obtain a binary plant protein; (3) The binary plant protein obtained in (2) was re-dissolved in water to form a 1% (w / v) binary plant protein solution, 8 mL of peppermint essential oil was added, and the mixture was stirred at 800 r / min for 60 min to obtain a cross-linked mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h, and then freeze-dried to obtain a plant protein product.

[0059] Comparative Example 9 (1) 1000 g of wine lees protein and 1000 g of walnut protein were added to 10 L of water, adjusted to pH = 10 with NaOH, and stirred at 800 r / min for 30 min under heating at 65°C to obtain a molten protein solution; (2) The molten wine lees protein solution obtained in (1) and the walnut protein solution were mixed in a mass ratio of 1:1, and the pH was adjusted to 7 with HCl to obtain a mixed solution. The mixed solution was then centrifuged at 10000g for 10 min, and the supernatant was dialyzed with a 3500 Da dialysis bag for 8 h, and freeze-dried to obtain a binary plant protein; (3) The binary plant protein obtained in (2) was re-dissolved in water to form a 1% (w / v) binary plant protein solution, 8 mL of peppermint essential oil was added, and the mixture was stirred at 800 r / min for 60 min to obtain a cross-linked mixture; (4) The mixture obtained in (3) was centrifuged at 10000 g for 10 min, the supernatant was dialyzed using a 3000 Da dialysis bag for 12 h, and then freeze-dried to obtain a plant protein product.

[0060] Performance test example (1) Dynamic light scattering was used to measure the particle sizes of the raw plant protein and the plant proteins prepared in Examples 1-4 and Comparative Examples 1-9. The results are shown in Table 1.

[0061] (2) Using transmission electron microscopy to examine the microscopic morphology of plant proteins, the prepared protein solution was dropped onto a copper grid containing a carbon film and tested after the sample was dried. The results are as follows: Figure 1-2 shown.

[0062] (3) Image J software was used to analyze the number of cavities and cavitation rate of the plant proteins prepared in Examples 1-4 and Comparative Examples 1-9. The results are shown in Table 2.

[0063] Table 1 Particle size and dispersibility of multi-shell hollow structure binary protein nanoparticles

[0064] From the data in Table 1, it can be seen that the particle size of the multi-chamber binary nano-protein particles prepared in the above embodiments is less than 150nm, and the PDI values ​​are closer to 0 (less than 0.30). The particle size of the plant protein products prepared by Comparative Examples 1-2 (using a single type of plant protein), Comparative Examples 3-4 (excessive or insufficient amounts of two proteins), Comparative Examples 5-7 (no cross-linking agent or using a cross-linking agent different from the embodiment) and Comparative Examples 8-9 (using plant proteins different from the embodiment) is larger, and the polydispersity index is closer to 1 (greater than 0.58). It can be seen that, relative to the comparative examples, the particle size of the multi-chamber binary nano-protein particles prepared in the embodiments is significantly reduced, and the dispersion uniformity is significantly improved. In addition, the shell layer formed by cross-linking of the amphiphilic plant protein and the core layer formed by cross-linking of the hydrophobic plant protein cooperate with each other to form binary protein nanoparticles with a multi-shell hollow structure, which have a smaller particle size and better dispersibility.

[0065] Table 2 The number of chambers and the chamberization rate of multi-shell hollow structure binary protein nanoparticles

[0066] from Figure 1~2 It can be seen that the binary nano-protein particles prepared in the examples present an independent approximately spherical core-shell structure and are distributed with obvious cavities, proving that the preparation method of the present invention produces binary protein nano-particles with a multi-shell hollow structure. The plant protein products prepared in Comparative Examples 1-2 (using a single type of plant protein), Comparative Examples 3-4 (using too much or too little of the two proteins), Comparative Examples 5-7 (no cross-linking agent or using a cross-linking agent different from that in the examples) and Comparative Examples 8-9 (using a plant protein different from that in the examples) all present a flocculent structure and do not have a multi-shell hollow structure.

[0067] From the data in Table 2, it can be seen that cavities are formed in the multi-chamber binary nano-protein particles prepared in the embodiment, the number of cavities in a single particle is greater than 5, and the chamberization rate is greater than 60%. Compared with the comparative example, it has a larger number of cavities and a higher chamberization rate. However, the plant protein products prepared in Comparative Examples 1-2 (using a single type of plant protein), Comparative Examples 3-4 (excessive or insufficient amounts of two proteins), Comparative Examples 5-7 (no cross-linking agent is added or a cross-linking agent different from that in the embodiment) and Comparative Examples 8-9 (using plant proteins different from those in the embodiment) did not form cavities. Therefore, the binary nano-protein particles prepared in the embodiment of the present invention have high porosity and large specific surface area, realize the chamberization or porous transformation of the protein structure at the molecular scale, have better loading capacity, and have broad application prospects in the fields of chemical catalysis, medicine, food and health products.

[0068] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. A binary protein nanoparticle with a multi-shell hollow structure, characterized in that: The binary protein nanoparticles comprise a shell layer formed by cross-linking of amphiphilic plant proteins and a core layer formed by cross-linking of hydrophobic plant proteins. The particle size of the binary protein nanoparticles is less than 150 nm.

2. The binary protein nanoparticle according to claim 1, characterized in that: The amphiphilic plant protein is selected from one of soybean protein, pea protein, mung bean protein or chickpea protein, and the hydrophobic plant protein is selected from one of corn protein, distiller's grains protein, walnut protein or alfalfa protein.

3. A method for preparing binary protein nanoparticles having a multi-shell hollow structure according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: a. adjusting the pH values ​​of the amphiphilic plant protein solution and the hydrophobic plant protein solution to alkaline to obtain two molten plant protein solutions; b. Mixing the two molten vegetable protein solutions, adjusting the pH to neutral, centrifuging, taking the supernatant, and dialyzing and drying to obtain the binary vegetable protein; c. dissolving the binary plant protein and mixing it with a cross-linking agent to obtain a mixed solution; d. Centrifuge the mixed solution to obtain the supernatant, and dialyze and dry the supernatant to obtain the binary protein nanoparticles.

4. The preparation method according to claim 3, characterized in that: The operation method of step a is: after mixing the amphiphilic plant protein and the hydrophobic plant protein with water respectively, adjusting the pH value to 9-11, heating and stirring to obtain two molten plant protein solutions.

5. The preparation method according to claim 3, characterized in that: The mass ratio of the amphiphilic plant protein to the hydrophobic plant protein is 0.2-4.

6. The preparation method according to claim 3, characterized in that: The cross-linking agent is selected from one of genipin, peppermint essential oil, cinnamon essential oil and eugenol.

7. The preparation method according to claim 3, characterized in that: The amount of the cross-linking agent added in step c is 0.08-0.25% of the mass of the solvent used to dissolve the binary plant protein.

8. The preparation method according to claim 3, characterized in that: The centrifugal force of the centrifugation in steps b and d is 5000-11000 g, and the centrifugation time is 5-30 min.

9. The preparation method according to claim 3, characterized in that: In steps b and d, the dialysis molecular weight is 1000-5000 Da, and the dialysis time is 12-48 h.

10. Use of the binary protein nanoparticles according to claim 1 or 2 or the binary protein nanoparticles prepared by the preparation method according to any one of claims 3 to 9 in the fields of chemical catalysis, medicine, food or cosmetics.

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