Preparation method of double-protein condensation product for packaging and stabilizing spice essential oil, double-protein condensation product and application of double-protein condensation product

Abstract

The invention discloses a preparation method of a double-protein aggregate for packaging and stabilizing spice essential oil, the double-protein aggregate and application of the double-protein aggregate. According to the process, ovalbumin and lysozyme are used as raw materials, stock solutions of the ovalbumin and the lysozyme are mixed in proportion, then pH adjustment and NaCl concentration adjustment are conducted, pulsed electric field treatment is introduced, protein is promoted to be subjected to liquid-liquid phase separation under the synergistic effect to form a composite condensate, and the composite condensate is used for packaging and stabilizing spice essential oil. Through multi-dimensional synergistic regulation, the performance of the composite coagulator is remarkably improved, the embedding rate of the emulsion to the spice essential oil reaches 90% or above, the particle size increase is smaller than or equal to 10% after the emulsion is refrigerated for 6 months at 4 DEG C, the layering rate is smaller than or equal to 5%, the release rate of irritant peculiar smell components in the spice essential oil can be effectively reduced, the sensory characteristics of the spice essential oil can be greatly improved, and the flavor of the spice essential oil can be improved. The application range in the fields of food, health care products and the like is expanded.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of preparation of functional food ingredients and stabilization of natural active ingredients, in particular, a preparation method of double-protein coacervate for encapsulating and stabilizing spice essential oil, double-protein coacervate and application thereof. BACKGROUND

[0002] Spice essential oil is a mixture of volatile secondary metabolites extracted from spices, rich in active ingredients such as terpenes, phenols, aldehydes and ketones, and has unique flavor, aroma and physiological activity (such as antibacterial, antioxidant, etc.), and has wide application prospects in food, medicine, cosmetics and other fields. However, its poor chemical stability, easy volatility and irritating odor limit its application in food and other fields; in addition, the hydrophobicity of spice essential oil is strong, which makes it difficult to disperse uniformly in aqueous systems, further affecting its bioavailability and functional exertion.

[0003] To solve the above problems, the existing technology usually adopts microcapsule embedding (such as the preparation method of high-embedding-rate microcapsules for powderization of spice essential oil disclosed in the patent with publication number CN114568674A, the preparation method of a Chinese waxtree essential oil embedding material with antioxidant activity disclosed in the patent with publication number CN108913360A, etc.), emulsion stabilization (such as a complex bacteriostatic agent, its preparation method and application disclosed in the patent with publication number CN118383470A, and the preparation method and application of a Zanthoxylum bungeanum and Artemisia vulgaris essential oil microemulsion disclosed in the patent with publication number CN117323372A, etc.) and other methods to process spice essential oil. Among them, the complex coacervation technology based on protein has become a research hotspot due to its advantages such as wide source of raw materials and good biocompatibility. The core principle of complex coacervation is that two or more than two oppositely charged polymers (such as proteins) interact and aggregate to form insoluble coacervate under specific pH and ionic strength conditions by overcoming intermolecular repulsion through electrostatic attraction, and the three-dimensional network structure inside the coacervate can physically trap or chemically bind active ingredients, achieving embedding and protection.

[0004] In recent years, liquid-liquid phase separation (LLPS) as a special self-assembly phenomenon of biomolecules has attracted high attention in the field of protein coacervation. Its principle is that protein solution will spontaneously separate into a dense phase rich in protein and a dilute phase poor in protein under certain conditions (such as pH close to the isoelectric point and appropriate ionic strength), and the dense phase can form a complex coacervate with unique rheological properties by further aggregation. This coacervate is between liquid and solid, has high interfacial activity and embedding capacity, and provides a new carrier selection for the stabilization of active ingredients.

[0005] Currently, the protein system for spice essential oil embedding is mostly single protein or protein-polysaccharide complex system. The single protein system has weak interfacial activity and embedding capacity due to single charge distribution and limited intermolecular interaction form, and is difficult to form a stable three-dimensional network structure, resulting in easy leakage of spice essential oil during storage. Although the protein-polysaccharide complex system can improve stability through charge complementation, the interaction between polysaccharide and protein is mainly non-specific adsorption, and is significantly affected by pH (for example, polysaccharide is easily protonated under acidic conditions, thereby weakening the binding with protein), and it is difficult to precisely control the structure of the coagulum. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application provides, on the one hand, a preparation method of a double-protein coagulum for packaging and stabilizing spice essential oil, and on the other hand, a double-protein coagulum and application of the double-protein coagulum in preparing a spice essential oil emulsion product.

[0007] The technical solution adopted by the present application to solve the above technical problems is as follows: A preparation method of a double-protein coagulum for packaging and stabilizing spice essential oil, and a method for preparing a spice essential oil emulsion product by using the double-protein coagulum, specifically comprising the following steps: (1) Respectively weigh egg white protein powder and lysozyme powder, and dissolve them in deionized water to prepare egg white protein solution and lysozyme solution with a certain concentration; place the two solutions in a room temperature environment, and stir them at a certain speed for a certain time to ensure that the proteins are completely dissolved, thereby obtaining uniform egg white protein stock solution and lysozyme stock solution; (2) Select a spice essential oil with a certain purity, and store it under suitable conditions for later use. Before use, restore it to room temperature; (3) Mix the egg white protein stock solution and the lysozyme stock solution according to a certain proportion, and stir them at a certain speed for a certain time at room temperature to allow the two proteins to fully contact and initially interact, thereby obtaining a mixed protein solution; (4) Static electric field treatment: adjust the pH of the mixed protein solution to a set value, stir uniformly, and then stand for a certain period of time to stabilize the pH of the solution; add NaCl powder to the mixed protein solution with adjusted pH, stir to dissolve the NaCl completely, and prepare a system with a set concentration, and continue to stir at a certain speed for a certain time to form a protein solution in different static electric field environments; (5) Dynamic electric field treatment: introduce the protein solution with adjusted pH and salt concentration into a pulse electric field treatment device, and treat it under set parameters; through the action of the external electric field of the pulse electric field, induce the protein molecules to produce a dipole moment, promote the redistribution of electric charges and optimize the molecular conformation, and strengthen the electrostatic interaction between egg white protein and lysozyme and the liquid-liquid phase separation behavior; (6) Liquid-liquid phase separation treatment: after the pulse electric field treatment, the protein solution is continuously stirred at a certain speed for a certain time, so that the protein is fully subjected to liquid-liquid phase separation under the synergistic action of a certain pH, salt concentration and electric field, to form a dense phase (i.e. heteroprotein complex coagulum) rich in protein, and a bulk phase solution containing the heteroprotein complex coagulum is obtained; (7) The bulk phase solution containing the complex heteroprotein coagulum is mixed with the spice essential oil at a certain ratio. (8) A high-speed disperser is used to disperse for a certain time at a set speed, so that the spice essential oil is fully broken into small oil droplets, and at the same time, the complex coagulum is uniformly adsorbed to the essential oil-water interface to form a stable protective layer by electrostatic adsorption and interfacial spreading, realizing efficient encapsulation of the spice essential oil, and obtaining a stabilized emulsion system.

[0008] As a preferred, in step (1), the concentration of the egg white protein solution and the lysozyme solution is 3-7 mg / mL; the stirring speed is 150-250 r / min, and the stirring time is 20-40 min.

[0009] As a preferred, in step (2), the purity of the spice essential oil is more than 90%; the storage condition is 2-8 ℃ in the dark; and the spice essential oil is garlic essential oil, pepper essential oil, Chinese toon essential oil or prickly ash essential oil.

[0010] As a preferred, in step (3), the mixing ratio of the egg white protein stock solution and the lysozyme stock solution is 1:0.8-1:1.2 (v / v); the stirring speed is 100-300 r / min, and the stirring time is 10-50 min.

[0011] As a preferred, in step (4), the pH setting value of the static electric field treatment is 5.5-8.5; and the standing time is 3-8 min.

[0012] As a preferred, in step (4), the NaCl system concentration of the dynamic electric field treatment is 10-70 mmol / L; the stirring speed is 50-200 r / min, and the stirring time is 5-15 min.

[0013] As a preferred, in step (5), the pulse electric field treatment parameters are: electric field strength 10-35 kV / cm, pulse width 10-60 μs, pulse frequency 30-120 Hz, and treatment time 1-8 min; and the system temperature after treatment is controlled at ≤40 ℃.

[0014] As a preferred, in step (6), the stirring speed is 100-300 r / min, and the stirring time is 20-40 min.

[0015] Preferably, in step (7), the mixing ratio of the bulk solution containing the complex coacervate and the spice essential oil is 2:1-6:1 (v / v).

[0016] Preferably, in step (8), the rotation speed of the high-speed disperser is 6000-12000 r / min, and the dispersion time is 0.5-4 min.

[0017] The application also provides a double-protein coacervate prepared by the method.

[0018] The application of the double-protein coacervate in preparing a spice essential oil emulsion product.

[0019] The application of the double-protein coacervate in preparing a spice essential oil emulsion product is a stabilized emulsion system obtained by encapsulating and stabilizing the spice essential oil by the method.

[0020] The main component of ordinary vegetable oil (such as soybean oil, rapeseed oil) is triglyceride, which has high chemical stability, low volatility, no significant irritating odor, and does not need to rely on complex coacervate for active protection or odor masking. The emulsification of ordinary vegetable oil is more for improving dispersibility, and there is no need for active protection and odor regulation of coacervate. Therefore, the simple composition and high stability of ordinary vegetable oil can meet the needs of emulsification and stability by using ordinary surfactants, without the need for the synergistic regulation mechanism of the present application.

[0021] However, due to the relatively unstable chemical properties and high volatility of spice essential oil, a more dense encapsulation wall material that can hinder the escape of volatile small molecules is needed. The main purpose of embedding spice essential oil is to solve the problems of strong volatility, poor stability and obvious odor, and to rely on the three-dimensional network structure of complex coacervate for controlled release, stability and odor shielding.

[0022] Ovalbumin with an isoelectric point of about 4.5 and lysozyme with an isoelectric point of about 11.0 are two common food-grade proteins that can carry negative and positive charges, respectively, under natural conditions. In theory, they can be complex coacervated by electrostatic attraction, and have the potential to form stable coacervates through liquid-liquid phase separation. Since the salt concentration can adjust the strength of electrostatic interaction through ion shielding effect, and the pH can affect the interaction direction by changing the surface charge density of the protein, the existing technology focuses on the influence of single pH or salt concentration on the coacervation behavior, without forming a multi-dimensional synergistic regulation strategy. As a new type of non-thermal processing physical field technology, pulsed electric field can induce protein molecules to produce dipole moment, promote charge redistribution and change molecular conformation (when the electric field strength is sufficient, it can open part of the hydrophobic region and enhance the interaction between molecules).

[0023] Compared with the prior art, the application has the beneficial effects of: The application constructs a double-protein system based on ovalbumin-lysozyme, and through the synergistic regulation of liquid-liquid phase separation principle, pulse electric field, pH and salt concentration, self-assembly behavior between heteroproteins is realized to construct a colloidal carrier, form a dense encapsulation wall material that can hinder the escape of volatile small molecules, prepare a complex coacervate with high interfacial activity and stable structure, solve the problems of low embedding rate, poor stability and strong irritating odor of spice essential oil, realize controllable slow release of spice essential oil, and expand the application range of the spice essential oil in food and related fields, which has important theoretical significance and application value. Specifically: 1. The application first combines the liquid-liquid phase separation principle with the pulse electric field technology, pH and salt concentration adjustment to form an innovative regulation mechanism of "electrostatic-electric field synergistic driving". Through the precise modification of protein conformation by the pulse electric field, and the regulation of electrostatic interaction by pH and salt concentration, the complex coacervation efficiency of ovalbumin and lysozyme is increased by more than 30%, and the elastic modulus (G') of the coacervate body phase can reach 150-300 Pa, which is significantly higher than that of the single pH / salt regulation system (80-120 Pa). This multi-dimensional regulation strategy not only realizes the controllable construction of the microstructure of the coacervate, but also breaks through the limitations of traditional single regulation means, providing new theoretical and methodological support for double-protein complex coacervation technology.

[0024] 2. The embedding rate of the stable emulsion prepared by the application to garlic essential oil can reach more than 90%, which is significantly higher than that of the existing single protein carrier; after being stored at 4 DEG C for 6 months, the emulsion particle size increases by ≤10%, and the separation rate is ≤5%. At the same time, through the stabilizing effect of the complex coacervate, the release rate of the irritating odor components of garlic essential oil is reduced to less than 20%, which significantly expands the application scenarios of the garlic essential oil in the fields of food, health care products and the like. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a laser confocal image of the complex coacervate of Example 1 and a co-localization analysis diagram of ovalbumin and lysozyme; Among them: FITC labeled ovalbumin, RBITC labeled lysozyme; Figure 2 It is an encapsulation situation diagram of the complex coacervate in Example 1 to garlic essential oil; Figure 3 It is an encapsulation situation diagram of the complex coacervate in Example 4 to garlic essential oil; Figure 4 It is an encapsulation situation diagram of single ovalbumin in Comparative Example 1 to garlic essential oil; Figure 5 It is an encapsulation situation diagram of single lysozyme in Comparative Example 1 to garlic essential oil. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and effects of the present application more clear and explicit, the present application will be further described in detail below in conjunction with the drawings and specific examples.

[0027] Example 1 A double-protein coacervate for encapsulating and stabilizing spice essential oil, and a method for preparing a spice essential oil emulsion product using the double-protein coacervate, comprising the following steps: (1) Respectively weigh the egg white protein powder and lysozyme powder, and dissolve them in deionized water to prepare an egg white protein solution and a lysozyme solution with a concentration of 5 mg / mL; place the two solutions in a room temperature environment and stir at a speed of 200 r / min for 30 min to obtain a uniform egg white protein stock solution and a lysozyme stock solution; (2) Select garlic essential oil with a purity of 95%, and store it in a 4 ℃ light-proof environment for standby, and restore it to room temperature before use; (3) Mix the egg white protein stock solution and the lysozyme stock solution in a ratio of 1:1 (v / v), and stir at a speed of 200 r / min for 30 min at room temperature to obtain a mixed protein solution; (4) Adjust the pH of the mixed protein solution to 6.0, and stir uniformly and then stand for 5 min to stabilize the pH of the solution; (5) Add NaCl powder to the mixed protein solution after adjusting the pH, stir to completely dissolve the NaCl, and prepare a system with a concentration of 40 mmol / L, and continue to stir at a speed of 100 r / min for 10 min to form a protein solution in different electrostatic field environments; (6) Introduce the protein solution after adjusting the pH and salt concentration into a pulsed electric field treatment device, and treat it under the conditions of an electric field intensity of 20 kV / cm, a pulse width of 30 μs, a pulse frequency of 80 Hz, and a treatment time of 3 min, and the temperature of the system after treatment is 32 ℃; (7) After pulsed electric field treatment, continue to stir the protein solution at a speed of 200 r / min for 30 min to form a bulk solution containing heteroprotein complex coacervates; (8) Mix the bulk solution containing complex coacervates with garlic essential oil in a ratio of 3:1 (v / v); (9) Use a high-speed disperser to disperse at a speed of 10,000 r / min for 1 min to obtain a stabilized emulsion.

[0028] Example 2 (1) Take egg white protein powder and lysozyme powder respectively, dissolve them in deionized water to prepare egg white protein solution and lysozyme solution with a concentration of 3 mg / mL; place the two solutions at room temperature and stir at a speed of 150 r / min for 40 min to obtain uniform egg white protein stock solution and lysozyme stock solution; (2) Select garlic essential oil with a purity of 90%, store it in a 2°C light-proof environment for standby, and restore it to room temperature before use; (3) Mix the egg white protein stock solution and the lysozyme stock solution at a ratio of 1:0.8 (v / v), stir at a speed of 100 r / min for 50 min at room temperature to obtain a mixed protein solution; (4) Adjust the pH of the mixed protein solution to 5.5, stir uniformly, and then stand for 3 min to stabilize the pH of the solution; (5) Add NaCl powder to the mixed protein solution with adjusted pH, stir to dissolve the NaCl completely, prepare a system with a concentration of 10 mmol / L, and continue to stir at a speed of 50 r / min for 15 min to form a protein solution in different electrostatic field environments; (6) Introduce the protein solution with adjusted pH and salt concentration into a pulsed electric field treatment device, and treat it under the conditions of an electric field intensity of 10 kV / cm, a pulse width of 10 μs, a pulse frequency of 30 Hz, and a treatment time of 8 min. The temperature of the system after treatment is 30°C; (7) After pulsed electric field treatment, continue to stir the protein solution at a speed of 100 r / min for 40 min to form a bulk solution containing heteroprotein complex coaggregates; (8) Mix the bulk solution containing complex coaggregates with garlic essential oil at a ratio of 2:1 (v / v); (9) Use a high-speed disperser to disperse at a speed of 6000 r / min for 4 min to obtain a stabilized emulsion.

[0029] Example 3 (1) Take egg white protein powder and lysozyme powder respectively, dissolve them in deionized water to prepare egg white protein solution and lysozyme solution with a concentration of 7 mg / mL; place the two solutions at room temperature and stir at a speed of 250 r / min for 20 min to obtain uniform egg white protein stock solution and lysozyme stock solution; (2) Select garlic essential oil with a purity of 98%, store it in an 8°C light-proof environment for standby, and restore it to room temperature before use; (3) Mix the egg white protein stock solution and the lysozyme stock solution at a ratio of 1:1.2 (v / v), stir at a speed of 300 r / min for 10 min at room temperature to obtain a mixed protein solution; (4) Adjust the pH of the mixed protein solution to 8.5, stir uniformly and stand for 8 min to stabilize the pH of the solution; (5) Add NaCl powder to the mixed protein solution after adjusting the pH, stir to dissolve the NaCl completely, configure the system with a concentration of 70 mmol / L, continue to stir at a speed of 200 r / min for 5 min to form a protein solution in different electrostatic field environments; (6) Introduce the protein solution after adjusting the pH and salt concentration into a pulsed electric field treatment device, and treat it under the conditions of an electric field intensity of 35 kV / cm, a pulse width of 60 μs, a pulse frequency of 120 Hz, and a treatment time of 1 min. The temperature of the system after treatment is 38 ℃; (7) After pulsed electric field treatment, continue to stir the protein solution at a speed of 300 r / min for 20 min to form a bulk solution containing heteroprotein complex coaggregates; (8) Mix the bulk solution containing complex coaggregates with garlic essential oil according to a ratio of 6:1 (v / v); (9) Use a high-speed disperser to disperse at a speed of 12000 r / min for 0.5 min to obtain a stabilized emulsion.

[0030] Example 4 (1) Respectively weigh the egg white protein powder and lysozyme powder, dissolve them in deionized water to configure an egg white protein solution and a lysozyme solution with a concentration of 4 mg / mL; place the two solutions at room temperature, stir at a speed of 180 r / min for 35 min to obtain uniform egg white protein stock solution and lysozyme stock solution; (2) Select garlic essential oil with a purity of 92%, store it in a 5 ℃ light-proof environment for standby, and restore it to room temperature before use; (3) Mix the egg white protein stock solution and the lysozyme stock solution according to a ratio of 1:0.9 (v / v), stir at a speed of 150 r / min for 40 min at room temperature to obtain a mixed protein solution; (4) Adjust the pH of the mixed protein solution to 7.0, stir uniformly and stand for 6 min to stabilize the pH of the solution; (5) Add NaCl powder to the mixed protein solution after adjusting the pH, stir to dissolve the NaCl completely, configure the system with a concentration of 20 mmol / L, continue to stir at a speed of 80 r / min for 12 min to form a protein solution in different electrostatic field environments; (6) The protein solution adjusted in pH and salt concentration was introduced into the pulse electric field treatment device, and was treated under the conditions of electric field intensity 15 kV / cm, pulse width 20 μs, pulse frequency 50 Hz, and treatment time 5 min, and the system temperature after treatment was 34 ℃; (7) After the pulse electric field treatment, the protein solution was continuously stirred at a speed of 150 r / min for 35 min to form a bulk solution containing heteroprotein complex coaggregates; (8) The bulk solution containing the complex coaggregates was mixed with garlic essential oil at a ratio of 4:1 (v / v); (9) A high-speed disperser was used to disperse at a speed of 8000 r / min for 2 min to obtain a stable emulsion.

[0031] Example 5 (1) Egg white protein powder and lysozyme powder were weighed respectively, and were dissolved in deionized water to prepare egg white protein solution and lysozyme solution with a concentration of 6 mg / mL; the two solutions were stirred at a speed of 220 r / min for 25 min at room temperature to obtain uniform egg white protein stock solution and lysozyme stock solution; (2) Garlic essential oil with a purity of 96% was selected and stored in a 6 ℃ dark environment for standby, and was restored to room temperature before use; (3) The egg white protein stock solution and the lysozyme stock solution were mixed at a ratio of 1:1.1 (v / v), and were stirred at a speed of 250 r / min for 20 min at room temperature to obtain a mixed protein solution; (4) The pH of the mixed protein solution was adjusted to 8.0, and after stirring uniformly, it was left to stand for 4 min to stabilize the solution pH; (5) NaCl powder was added to the mixed protein solution adjusted in pH, and was stirred to completely dissolve the NaCl to prepare a system with a concentration of 60 mmol / L, and was continuously stirred at a speed of 150 r / min for 8 min to form a protein solution in different electrostatic field environments; (6) The protein solution adjusted in pH and salt concentration was introduced into the pulse electric field treatment device, and was treated under the conditions of electric field intensity 30 kV / cm, pulse width 50 μs, pulse frequency 100 Hz, and treatment time 2 min, and the system temperature after treatment was 36 ℃; (7) After the pulse electric field treatment, the protein solution was continuously stirred at a speed of 250 r / min for 25 min to form a bulk solution containing heteroprotein complex coaggregates; (8) The bulk solution containing the complex coaggregates was mixed with garlic essential oil at a ratio of 5:1 (v / v); (9) The high-speed disperser was used to disperse at a speed of 11000 r / min for 1.5 min to obtain a stable emulsion. Comparative Example 1 (1) Egg white protein powder was weighed and dissolved in deionized water to prepare an egg white protein solution with a concentration of 5 mg / mL; the solution was placed at room temperature and stirred at a speed of 200 r / min for 30 min to obtain a uniform egg white protein stock solution; (2) Garlic essential oil with a purity of 95% was selected and stored in a dark environment at 4 ℃ for standby, and was restored to room temperature before use; (3) The same volume of egg white protein stock solution as the mixed protein solution in Example 1 was taken and stirred at a speed of 200 r / min for 30 min at room temperature as a single protein solution; (4) The pH of the single protein solution was adjusted to 6.0, and after stirring uniformly, it was left to stand for 5 min to stabilize the pH of the solution; (5) NaCl powder was added to the single protein solution with adjusted pH, stirred to dissolve the NaCl completely, and prepared into a system with a concentration of 40 mmol / L, and continued to be stirred at a speed of 100 r / min for 10 min to form a protein solution in a specific electrostatic field environment; (6) The protein solution with adjusted pH and salt concentration was introduced into the pulse electric field treatment device, and treated under the conditions of electric field intensity 20 kV / cm, pulse width 30 μs, pulse frequency 80 Hz, and treatment time 3 min, and the temperature of the system after treatment was 32 ℃; (7) After pulse electric field treatment, the protein solution was continuously stirred at a speed of 200 r / min for 30 min to obtain a bulk solution containing egg white protein aggregates; (8) The bulk solution containing egg white protein aggregates was mixed with garlic essential oil at a ratio of 3:1 (v / v); (9) The high-speed disperser was used to disperse at a speed of 10000 r / min for 1 min to obtain an emulsion. Comparative Example 2 (1) Lysozyme powder was weighed and dissolved in deionized water to prepare a lysozyme solution with a concentration of 5 mg / mL; the solution was placed at room temperature and stirred at a speed of 200 r / min for 30 min to obtain a uniform lysozyme stock solution; (2) Garlic essential oil with a purity of 95% was selected and stored in a dark environment at 4 ℃ for standby, and was restored to room temperature before use; (3) The same volume of lysozyme stock solution as the mixed protein solution in Example 1 was taken and stirred at a speed of 200 r / min for 30 min at room temperature as a single protein solution; (4) Adjust the pH of the single protein solution to 6.0, stir evenly and stand for 5 min to stabilize the pH of the solution; (5) Add NaCl powder to the single protein solution after adjusting the pH, stir to dissolve the NaCl completely, configure the system with a concentration of 40 mmol / L, continue to stir at a speed of 100 r / min for 10 min to form a protein solution in a specific electrostatic field environment; (6) Introduce the protein solution after adjusting the pH and salt concentration into a pulsed electric field treatment device, and treat it under the conditions of an electric field intensity of 20 kV / cm, a pulse width of 30 μs, a pulse frequency of 80 Hz, and a treatment time of 3 min. The temperature of the system after treatment is 32°C; (7) After pulsed electric field treatment, continue to stir the protein solution at a speed of 200 r / min for 30 min to obtain a bulk solution containing lysozyme aggregates; (8) Mix the bulk solution containing lysozyme aggregates with garlic essential oil according to a ratio of 3:1 (v / v); (9) Use a high-speed disperser to disperse at a speed of 10,000 r / min for 1 min to obtain an emulsion.

[0032] Comparative Example 3 (1) Respectively weigh the ovalbumin powder and lysozyme powder, dissolve them in deionized water to configure ovalbumin solution and lysozyme solution with a concentration of 5 mg / mL; place the two solutions at room temperature, stir at a speed of 200 r / min for 30 min to obtain uniform ovalbumin stock solution and lysozyme stock solution; (2) Select garlic essential oil with a purity of 95%, store it in a 4°C light-proof environment for standby, and restore it to room temperature before use; (3) Mix the ovalbumin stock solution and the lysozyme stock solution according to a ratio of 1:1 (v / v), stir at a speed of 200 r / min for 30 min at room temperature to obtain a mixed protein solution; (4) Adjust the pH of the mixed protein solution to 6.0, stir evenly and stand for 5 min to stabilize the pH of the solution; (5) Add NaCl powder to the mixed protein solution after adjusting the pH, stir to dissolve the NaCl completely, configure the system with a concentration of 40 mmol / L, continue to stir at a speed of 100 r / min for 10 min to form a protein solution in a different electrostatic field environment; (6) Omit the pulsed electric field treatment step; (7) The protein solution adjusted in pH and salt concentration is directly stirred at a speed of 200 r / min for 30 min, so that the protein is subjected to liquid-liquid phase separation under the action of specific pH and salt concentration to form a bulk phase solution containing heteroprotein complex coaggregates; (8) The bulk phase solution containing complex coaggregates is mixed with garlic essential oil at a ratio of 3:1 (v / v); (9) An emulsion is obtained by using a high-speed disperser at a speed of 10,000 r / min for 1 min.

[0033] Comparative Example 4 (1) Ovalbumin powder and lysozyme powder are weighed respectively, and dissolved in deionized water to prepare ovalbumin solution and lysozyme solution with a concentration of 5 mg / mL; the two solutions are placed at room temperature and stirred at a speed of 200 r / min for 30 min to obtain uniform ovalbumin stock solution and lysozyme stock solution; (2) Garlic essential oil with a purity of 95% is selected and stored in a 4 ℃ light-proof environment for standby, and restored to room temperature before use; (3) The ovalbumin stock solution and the lysozyme stock solution are mixed at a ratio of 1:1 (v / v) and stirred at a speed of 200 r / min for 30 min at room temperature to obtain a mixed protein solution, maintaining the natural pH value and the basic ionic environment of deionized water; (4) The mixed protein solution without pH and ionic strength adjustment is introduced into a pulsed electric field treatment device, and treated under the conditions of electric field intensity 20 kV / cm, pulse width 30 μs, pulse frequency 80 Hz, and treatment time 3 min, and the temperature of the system after treatment is 32 ℃; (5) After pulsed electric field treatment, the protein solution is continuously stirred at a speed of 200 r / min for 30 min to obtain a bulk phase solution containing protein coaggregates; (6) The bulk phase solution containing coaggregates is mixed with garlic essential oil at a ratio of 3:1 (v / v); (7) An emulsion is obtained by using a high-speed disperser at a speed of 10,000 r / min for 1 min.

[0034] Comparative Example 5 (1) Ovalbumin powder and lysozyme powder are weighed respectively, and dissolved in deionized water to prepare ovalbumin solution and lysozyme solution with a concentration of 5 mg / mL; the two solutions are placed at room temperature and stirred at a speed of 200 r / min for 30 min to obtain uniform ovalbumin stock solution and lysozyme stock solution; (2) Japanese toona essential oil with a purity of 95% is selected and stored in a 4 ℃ light-proof environment for standby, and restored to room temperature before use; (3) The egg albumin stock solution was mixed with the lysozyme stock solution at a ratio of 1:1 (v / v), stirred at 200 r / min for 30 min at room temperature, to obtain a mixed protein solution; (4) The pH of the mixed protein solution was adjusted to 6.0, and after stirring evenly, it was left to stand for 5 min to stabilize the solution pH; (5) NaCl powder was added to the mixed protein solution after pH adjustment, stirred to dissolve the NaCl completely, and a system with a concentration of 40 mmol / L was configured, and the stirring was continued at a speed of 100 r / min for 10 min to form a protein solution in different electrostatic field environments; (6) The protein solution after pH and salt concentration adjustment was introduced into a pulsed electric field treatment device, and the treatment was carried out under the conditions of electric field intensity 20 kV / cm, pulse width 30 μs, pulse frequency 80 Hz, and treatment time 3 min, and the system temperature after treatment was 32 ℃; (7) After pulsed electric field treatment, the protein solution was continuously stirred at a speed of 200 r / min for 30 min to form a bulk solution containing heteroprotein complex coaggregates; (8) The bulk solution containing complex coaggregates was mixed with the Japanese toona oil at a ratio of 3:1 (v / v); (9) A high-speed disperser was used to disperse at a speed of 10,000 r / min for 1 min to obtain a stabilized emulsion.

[0035] Performance determination The properties of the emulsions prepared in each example and comparative example were determined, including: (1) Coagulant elastic modulus determination method: The bulk solution containing complex coaggregates in the examples and each comparative example was centrifuged to separate the dense phase rich in coagulants, and the supernatant (dilute phase) was discarded, and the bottom coagulants were retained as the sample to be tested. A rotational rheometer was used, equipped with a 40 mm parallel plate rotor, and the excess sample on the edge was scraped off. First, strain scanning was performed to determine the linear viscoelastic region of the sample, and the G' value at 1 Hz frequency under the determined strain condition was taken as the elastic modulus of the coagulants.

[0036] (2) Essential oil embedding rate determination method: The emulsion sample in the example and each comparative example was carefully extracted, and the mass of the unemulsified essential oil was determined. The embedding rate was calculated according to "(total essential oil amount-unembedded essential oil amount) / total essential oil amount x 100%".

[0037] (3) Particle size increase determination method: Freshly prepared emulsion and the same batch of emulsion after storage were diluted 10 times, and the average particle size was determined using a laser particle size analyzer. The particle size increase was calculated according to "(particle size after storage-initial particle size) / initial particle size x 100%".

[0038] (4) Layering rate determination method: take a certain volume of emulsion sample in a stoppered test tube, record the volume of the demulsified layer after storage and the initial total volume of the emulsion, and calculate the layering rate according to "demulsified layer volume / initial total volume of emulsion x 100%".

[0039] (5) Diallyl disulfide release rate determination method: take the emulsion sample and place it in a headspace bottle, equilibrate at 25°C for 10 min, then use solid-phase microextraction fiber to adsorb the diallyl disulfide in the headspace, separate and detect by gas chromatography, quantify the release amount by standard curve, and calculate the release rate according to "actual release amount / theoretical total content x 100%".

[0040] The results are shown in the following table: Note: The particle size increase after storage, the layering rate after storage and the release rate of diallyl disulfide refer to the results measured after 6 months of storage at 4°C.

[0041] Figure 1 Specifically, the appearance of the coagulum formed by ovalbumin and lysozyme under the synergistic regulation of the electrostatic field and the pulsed electric field, and the co-localization analysis results of the two proteins; the results show that the coagulum appears as a sphere and the fusion degree of the two proteins inside the coagulum is high, indicating that the synergistic regulation of the electrostatic field and the pulsed electric field can effectively promote the coagulation process, thereby forming a coagulum with uniform interior and regular appearance.

[0042] The above examples have made a detailed description of the present application, but it is only a part of the embodiments of the present application, not all the embodiments, and people can also obtain other embodiments according to the present embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A method for preparing a dual-protein coagulant for encapsulating and stabilizing spice essential oils, comprising the following steps: (1) Weigh out ovalbumin powder and lysozyme powder respectively and dissolve them in deionized water to prepare ovalbumin stock solution and lysozyme stock solution of a certain concentration; (2) Mix the ovalbumin stock solution and the lysozyme stock solution in a certain proportion, and stir at a certain speed for a certain time at room temperature to obtain a mixed protein solution; (3) The mixed protein solution was subjected to static electric field treatment and dynamic electric field treatment in sequence to obtain a pretreated mixed protein solution; (4) The pretreated mixed protein solution is subjected to liquid-liquid phase separation to obtain a bulk solution containing heteroprotein complex aggregates, which is the dual protein complex aggregate.

2. The method according to claim 1, characterized in that: The static electric field treatment includes pH adjustment and ion concentration adjustment: the pH of the mixed protein solution obtained in step (2) is adjusted to a set value; NaCl is added to the mixed protein solution after pH adjustment to prepare a set concentration system, and the mixture is stirred at a certain speed for a certain time to form protein solutions with different electrostatic field environments; The dynamic electric field treatment involves introducing a protein solution, whose pH and salt concentration have been adjusted, into a pulsed electric field treatment device for pulsed electric field treatment to obtain a pretreated mixed protein solution. The liquid-liquid phase separation process is as follows: after pulsed electric field treatment, the pretreated mixed protein solution is stirred at a certain speed for a certain time, so that the protein can fully undergo liquid-liquid phase separation under the synergistic effect of specific pH, salt concentration and electric field, forming a dense phase rich in protein, and obtaining a bulk solution containing heteroprotein complex aggregates.

3. The method according to claim 1, characterized in that: In step (1), the concentrations of the ovalbumin stock solution and the lysozyme stock solution are both 3-7 mg / mL.

4. The method according to claim 1 or 3, characterized in that: In step (2), the mixing ratio of the ovalbumin stock solution and the lysozyme stock solution is 1:0.8-1:1.2 (v / v); the stirring speed is 100-300 r / min and the stirring time is 10-50 min.

5. The method according to claim 2, characterized in that: In the static electric field treatment, the pH setting is 5.5-8.5; the system concentration after adding NaCl is 10-70 mmol / L; the stirring speed is 50-200 r / min; and the stirring time is 5-15 min. In the dynamic electric field treatment, the pulsed electric field treatment parameters are: electric field strength 10-35 kV / cm, pulse width 10-60 μs, pulse frequency 30-120 Hz, and treatment time 1-8 min; the system temperature after treatment is ≤40 ℃. In the liquid-liquid phase separation process, the stirring speed is 100-300 r / min and the stirring time is 20-40 min.

6. A dual-protein aggregate, characterized in that, Prepared using the method described in any one of claims 1-5.

7. The use of the dual protein aggregate of claim 6 in the preparation of spice essential oil emulsion products.

8. The application according to claim 7, characterized in that, The method for preparing spice essential oil emulsion products using the aforementioned dual protein coagulant comprises the following steps: (1) Mix the above bulk solution containing heteroprotein complex aggregates with spice essential oils at room temperature in a certain proportion; (2) A stable emulsion system is obtained by dispersing the emulsion at a set speed for a certain period of time using a high-speed disperser.

9. The application according to claim 8, characterized in that: The bulk solution is mixed with spice essential oil in a ratio of 2:1 to 6:1 (v / v).

10. The application according to any one of claims 7-9, characterized in that: The spice essential oil is garlic essential oil, chili essential oil, toon essential oil or Sichuan pepper essential oil, and the purity of the spice essential oil is above 90%.

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