Organic-water double-solvent evaporation induced protein gel toughening method

Abstract

The invention discloses an organic-water double-solvent evaporation induced protein gel toughening method, and belongs to the technical field of protein gel preparation. The method comprises the following steps: mixing an organic solvent with water to prepare a double-solvent mixed solution; after protein is dissolved in the double-solvent mixed solution, gelatinization treatment is carried out, and protein gel is obtained; and carrying out airflow-assisted moisture volatilization on the protein gel to obtain the strengthened and toughened protein gel. According to the organic-water double-solvent protein gel system, the environmental adaptability of a protein gel material can be improved, the functional attribute of the protein gel material is increased, the mechanical property and the storage stability of the gel are improved, and the organic-water double-solvent protein gel system has the advantages of mildness, simplicity, convenience and universality and is beneficial to large-scale production of the protein gel in actual industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of protein gel preparation, and particularly to a method for toughening protein gels induced by organic-water dual-solvent evaporation. Background Art

[0002] Protein gels refer to substances in which proteins denature under certain conditions (such as temperature, pH, ionic strength, etc.) and aggregate from a solution to form a three-dimensional network structure. This structure can fix a large amount of water to form a gel system with a certain strength and elasticity. In the food industry, protein gels are used as food additives to improve the texture, taste and stability of foods, such as meat products, dairy products, candies, etc. In the field of biomaterials, protein gels can be used to prepare scaffold materials, drug carriers, etc. In the medical field, protein gels can be used in drug delivery systems and biomedical materials.

[0003] Protein gels have certain limitations in practical applications, such as insufficient strength, easy cracking, poor elasticity, etc. In order to broaden the application scope of protein gels and improve their performance, it is necessary to toughen and modify them. Existing methods include: 1) Physical methods: improving gel properties by changing processing conditions (such as temperature, pressure, time, etc.); 2) Chemical methods: introducing chemical substances such as cross-linking agents and modifiers to improve the network structure and strength of gels; 3) Biotechnology methods: using biotechnology means such as enzymes and microorganisms to modify proteins and improve gel properties. However, during the toughening process, other functional properties of the protein gel need to be maintained, such as transparency, taste, biocompatibility, etc. The toughening agents and modification methods used must also meet the requirements of food safety and biocompatibility. In industrial production, it is necessary to ensure the controllability, stability and simplicity of the toughening process.

[0004] However, protein gel toughening technology still faces some bottleneck problems in the research and application process, such as: 1) When toughening protein gels, improving their mechanical strength often leads to a loss of gel toughness; 2) Protein gels may have stability problems during storage, such as water loss, structural collapse or microbial contamination; improving the long-term stability of gels is also a technical bottleneck; 3) The formation and toughening process of protein gels are very sensitive to processing conditions, such as temperature, pH value, ionic strength and stirring speed, etc. Precise control of these conditions for industrial production is also a challenge; 4) Efficient toughening methods often involve expensive raw materials or complex processing steps, which will increase production costs and limit their commercial feasibility; 5) Laboratory-scale toughening technology may be difficult to directly apply to industrial production; maintaining the consistency of gel quality and performance in large-scale production is a difficult problem; 6) There is a lack of in-depth understanding of the relationship between the structure and properties of protein gels, which limits the development of more effective toughening strategies.

[0005] To overcome these bottlenecks, researchers are exploring new toughening strategies, such as using natural polymers, nanomaterials, and bioengineering techniques, as well as developing more advanced processing technologies and equipment. Through these efforts, it is expected to further improve the performance of protein gels and broaden their application scope. Summary of the Invention

[0006] The object of the present invention is to provide a method for toughening protein gels induced by organic-water dual-solvent evaporation to solve the problems existing in the above-mentioned prior art. The organic-water dual-solvent protein gel system of the present invention can enhance the environmental adaptability of protein gel materials, increase their functional properties, improve the mechanical properties and storage stability of the gels, and has the advantages of mildness, simplicity, and universality, which is conducive to the large-scale production of protein gels in actual industrial production.

[0007] It helps to better understand the formation mechanism of protein gels and also provides new ideas and methods for the development of multiple fields such as materials science, food science, and biomedicine.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a method for toughening protein gels induced by organic-water dual-solvent evaporation, comprising the following steps:

[0010] Mix an organic solvent with water to prepare a dual-solvent mixed solution;

[0011] Dissolve a protein in the dual-solvent mixed solution and then perform gelation treatment to obtain a protein gel;

[0012] Perform air-flow assisted water evaporation on the protein gel to obtain a toughened protein gel.

[0013] Optionally, the organic solvent is a solvent that can be dissolved in water.

[0014] Optionally, the organic solvent includes one or more of tetrahydrofuran, ethanol, methanol, ethylene glycol, triethylene glycol, tetraethylene glycol, glycerol, dimethyl sulfoxide, N,N-dimethylformamide, and dioxane.

[0015] Optionally, the protein includes a natural protein or a protein obtained by chemically modifying the natural protein.

[0016] Optionally, the protein includes one or more of gelatin protein, methacrylated gelatin protein, silk protein, royal jelly protein, yeast protein, and microbial protein.

[0017] Optionally, the organic solvent and water can be mixed in any ratio.

[0018] Optionally, the mass ratio of the organic solvent to water is 1:(1 - 10).

[0019] Optionally, the mass ratio of the organic solvent to the protein is (1 - 3):1.

[0020] Optionally, when the airflow assists in water volatilization, the temperature is 15 - 40°C, and the water evaporation rate is 0.1 - 0.2 g / h -1 .

[0021] Optionally, when the protein is gelatin protein, the gelation treatment includes a step of cooling and solidifying;

[0022] When the protein is methacrylated gelatin protein, the gelation treatment includes a step of irradiating with ultraviolet light after adding a photoinitiator.

[0023] In the present invention, the gelation treatment process includes a step of adding other coagulation-promoting substances and performing coagulation-promoting treatment according to the gel properties of the protein.

[0024] The present invention discloses the following technical effects:

[0025] (1) In the organic-water dual-solvent protein gel system of the present invention, the selective interaction between the solvent and protein molecules is induced and regulated through the controllable evaporation of the solvent (evaporation rate, evaporation method, etc.), and the precise regulation of the molecular chain aggregation structure in the protein gel is achieved through key influencing factors such as protein concentration and solubility. This regulation strategy avoids the requirements for equipment and complex process flows of traditional external force assistance methods, and can continuously track and characterize the entire regulation process. This method has the advantages of mildness, simplicity, and universality, which is conducive to large-scale production of protein gels in actual industrial production.

[0026] (2) The advantage of the dual-solvent is that its adjustment range is wider and the regulation effect is more obvious. The organic-water dual-solvent gel system effectively improves or solves the pain points faced by single-solvent protein gels, such as water loss and freezing, weak ion conduction ability in organic solvents, etc., enhances the environmental adaptability of protein gel materials while increasing their functional properties, and improves the mechanical properties and storage stability of the gels. On the other hand, the mixed solvent systems with different properties are directly related to the diversity of protein gel properties, which can help develop new protein gel materials and broaden the application range of gels.

[0027] (3) A simple and gentle toughening strategy not only brings great convenience to experimental operations but also effectively improves the toughness of protein gels without damaging the proteins themselves. The implementation of this strategy has greatly promoted the systematic study of the complex structure-property relationship between the internal structure of protein gels and their mechanical properties. Through this method, the microscopic structural changes during the formation of protein gels can be observed and analyzed more carefully, as well as how these changes affect the final mechanical properties. The application of this toughening strategy provides a powerful tool for scientific research, facilitating the accurate revelation of the evolution process of the protein aggregate structure induced during solvent evaporation. This process is crucial for understanding the mechanical properties of protein gels because it is directly related to the network structure and stability of the gels. By systematically studying this structure-property relationship, not only can the physical and chemical nature of protein gels be deeply understood, but also theoretical guidance and practical basis can be provided for the design and preparation of protein gel materials with specific mechanical properties.

[0028] In summary, this simple and gentle toughening strategy has opened up a new path for research in the field of protein gels, not only helping to better understand the formation mechanism of protein gels but also providing new ideas and methods for the development of multiple fields such as materials science, food science, and biomedicine. Brief Description of the Drawings

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0030] Figure 1 It is the preparation flow chart of the toughened protein gel in Example 1;

[0031] Figure 2 It is the change in crystallinity and mechanical properties during the water evaporation process of the toughened protein gel in Example 1; where a is the X-ray diffraction pattern; b is the mechanical property test result; 3:1, 9:10, 6:10, 3:10, 1:10 are the changes in the mass ratio of water to ethylene glycol (EG) during the water evaporation process of the toughened protein gel.

[0032] Figure 3Mechanical property test results of the toughened protein gel in Example 1; among them, a is the fatigue test of the protein gel before and after solvent evaporation; b is the cyclic tensile test of the protein gel; c is the mechanical properties of the protein gel at different storage times (1d, 30d, 50d, 100d) in an open environment at room temperature; d is the rheological characterization of the protein gel at 1% strain at different temperatures before and after solvent evaporation; e is the shear strain of the protein gel at 20 °C before and after solvent evaporation.

[0033] Figure 4 Gel mechanical properties of the toughened protein gel by the double-solvent evaporation method and the protein hydrogel by the single-solvent evaporation method before and after solvent evaporation in Example 2; among them, 3:1 represents before solvent evaporation of the toughened protein gel by the double-solvent evaporation method, 1:10 represents after solvent evaporation of the toughened protein gel by the double-solvent evaporation method; Hydrogel represents before solvent evaporation of the protein hydrogel by the single-solvent evaporation method, and Dried represents after solvent evaporation of the protein hydrogel by the single-solvent evaporation method.

[0034] Figure 5 SEM images of the toughened protein gel by the double-solvent evaporation method and the protein hydrogel by the single-solvent evaporation method before and after solvent evaporation in Example 2; among them, a is the SEM image of the toughened protein gel by the double-solvent evaporation method; b is the SEM image of the protein hydrogel by the single-solvent evaporation method; 3:1 represents before solvent evaporation of the toughened protein gel by the double-solvent evaporation method, 1:10 represents after solvent evaporation of the toughened protein gel by the double-solvent evaporation method; Hydrogel represents before solvent evaporation of the protein hydrogel by the single-solvent evaporation method, and Dried represents after solvent evaporation of the protein hydrogel by the single-solvent evaporation method.

[0035] Figure 6 Effects of protein concentration (a) and different moisture removal methods (b) on the crystallization performance of the protein gel. Detailed implementation manners

[0036] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0038] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0039] Without departing from the scope or spirit of this invention, various modifications and variations can be made to the specific embodiments of the specification of this invention, which will be obvious to those skilled in the art. Other embodiments obtained from the specification of this invention will be obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0040] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0041] Example 1

[0042] 1) Preparation of methacrylated gelatin (GelMA)

[0043] First, in a 250 mL flask, 10 g of gelatin powder was added to 100 mL of PBS buffer solution, and it was heated and stirred at 40 °C to form a homogeneous solution. Then, 2.3 mL of methacrylic anhydride was slowly added dropwise to the above gelatin solution, and stirring was continued. The reaction mixture was reacted at 30 °C for 12 h. After the reaction was completed, the reaction mixture was neutralized with sodium bicarbonate solution to neutralize the unreacted methacrylic anhydride. The reaction mixture was transferred to a dialysis bag and dialyzed using phosphate buffer solution (PBS) to remove the unreacted methacrylic anhydride and other small molecule impurities. The dialyzed GelMA solution was transferred to a freeze dryer and freeze-dried to remove water, obtaining dry GelMA powder. The final product was stored in a dry and light-proof environment to prevent moisture absorption and degradation.

[0044] 2) Preparation of toughened protein gel

[0045] The preparation flow chart of the toughened protein gel is as Figure 1 shown. 1 g of GelMA powder was added to a mixed solution of 10 mL of water and ethylene glycol (EG) (W H2O :W EGIn (3:1), it was stirred in a 40 °C water bath for 30 min. After it was completely dissolved, it was cooled to room temperature, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator was added and stirred in the dark for 10 min. Then, the stirred reaction solution was poured into a mold, and the air bubbles in the solution were removed by the vacuum method. After being irradiated with ultraviolet light (365 nm) for 5 min, the gelatin protein gel after double bond polymerization and crosslinking was obtained. Finally, the gel obtained by this preparation was placed under the condition of constant temperature of 25 °C and open air for air-assisted water evaporation, and the evaporation rate was controlled at 0.15 g h -1 , and the change in the gel weight was recorded until it reached equilibrium. At this time, the protein gel was the final methacrylated gelatin toughened gel.

[0046] The crystallization situation and mechanical properties during the water evaporation process of the above gel were monitored in real time, and the results were as Figure 2 shown. As can be seen from a, with the evaporation of water, the crystallinity of the gel gradually increased. When the gel weight reached equilibrium, the ratio of water to EG was 1:10, and the gel had the highest crystallinity. As can be seen from b, with the evaporation of water, the mechanical properties of the gel were gradually improved. When the gel weight reached equilibrium, the mechanical properties were the best, with a fracture stress of 1.6 MPa and a fracture energy of 3.2 kJ m -2 , and the fracture strain was 600%.

[0047] The properties of the methacrylated gelatin toughened gel after solvent evaporation were detected. The results were as Figure 3 shown. Among them, a was the fatigue test of the protein gel before and after solvent evaporation. The fatigue threshold before evaporation was 50 J m -2 , and the fatigue threshold after evaporation increased to 800 J m -2 . b was the cyclic tensile test of the protein gel. It can be seen that after 1000 consecutive loading-unloading cycles, the stress still remained stable. c was the mechanical properties of the protein gel at different storage times (1 d, 30 d, 50 d, 100 d) in an open environment at room temperature. It can be seen that the toughened gel prepared by this method had good long-term stability. d was the rheological characterization of the protein gel before and after solvent evaporation at 1% strain at different temperatures. e was the shear strain of the protein gel before and after solvent evaporation at 20 °C. It can be seen that by using the double-solvent evaporation method, after solvent evaporation, the protein gel had strong and tough mechanical properties and stable performance.

[0048] Example 2

[0049] To compare the properties of the protein gels prepared by the traditional single-solvent evaporation method and the double-solvent evaporation method of the present invention, in this example, the gelatin protein gels were prepared by the traditional single-solvent evaporation method and the double-solvent evaporation method. The preparation process was as follows:

[0050] Traditional single-solvent evaporation method: Add 1 g of gelatin protein (Gel) to 10 mL of water, stir in a 40 °C water bath for 30 min. After it is completely dissolved, pour it into a mold, use the vacuum method to remove the air bubbles in the solution, and then place the gelatin solution in the refrigerator (5 °C) to cool and solidify to form a physical gel. Then, place the gel under a constant temperature of 20 °C in an open environment for water evaporation, record the change in the gel weight until it reaches equilibrium, and the protein gel at this time is the final protein hydrogel.

[0051] Dual-solvent evaporation method: Add 1 g of gelatin protein (Gel) to a mixed solution of 10 mL of water and ethylene glycol (EG) (W H2O :W EG = 3:1), stir in a 40 °C water bath for 30 min. After it is completely dissolved, pour it into a mold, use the vacuum method to remove the air bubbles in the solution, and then place the gelatin solution in the refrigerator (5 °C) to cool and solidify to form a physical gel. Then, place the gel under a constant temperature of 20 °C in an open environment for air-assisted water evaporation, and control the evaporation rate at 0.15 g h -1 . Record the change in the gel weight until it reaches equilibrium, and the protein gel at this time is the final toughened protein gel.

[0052] Measure the gel mechanical properties of the toughened protein gel prepared by the dual-solvent evaporation method and the protein hydrogel prepared by the single-solvent evaporation method before and after solvent evaporation. The results are as Figure 4 shown. 3:1 represents before the solvent evaporation of the toughened protein gel, 1:10 represents after the solvent evaporation of the toughened protein gel; Hydrogel represents before the solvent evaporation of the protein hydrogel, and Dried represents after the solvent evaporation of the protein hydrogel. It can be seen that the protein hydrogel prepared by the single-solvent evaporation method is hard and brittle (stress 2.5 MPa, strain 47%), with low toughness (0.5 MJm -3 ); the toughened protein gel is strong and tough (stress 1.6 MPa, strain 600%), with relatively high toughness (4.5 MJ m -3 ).

[0053] Observe the surface structure characteristics of the toughened protein gel prepared by the dual-solvent evaporation method and the protein hydrogel prepared by the single-solvent evaporation method before and after solvent evaporation using a scanning electron microscope (SEM). The results are as Figure 5 shown, where a is the SEM image of the toughened protein gel prepared by the dual-solvent evaporation method before and after solvent evaporation, and b is the SEM image of the protein hydrogel prepared by the single-solvent evaporation method before and after solvent evaporation. It can be seen that after water evaporation in the single-solvent system, its morphology is very compact, and this structure is not conducive to improving the toughness of the gel. After water evaporation in the dual-solvent system, "gully" morphology appears inside, and this morphology can greatly improve the stretchability of the gel material, thereby endowing the protein gel with high strength and toughness mechanical properties.

[0054] Example 3

[0055] To verify the effect of protein concentration on the properties of protein gels in the double-solvent evaporation method, gelatin protein gels with different gelatin protein concentrations were prepared in this example. The preparation process is as follows:

[0056] Weigh the raw materials according to W EG :W 明胶 = 10:1, 4:1, 3:1, 2:1, 5:3 respectively, and prepare a double-solvent mixed solution according to W H2O :W EG = 3:1. Add gelatin to 10 mL of the double-solvent mixed solution, stir in a 40 °C water bath for 30 min. After it is completely dissolved, pour it into a mold, use the vacuum method to remove the bubbles in the solution, and then place the gelatin solution in a refrigerator (5 °C) to cool and solidify to form a physical gel. Then, place the gel under a constant temperature of 20 °C in an open environment for water evaporation, record the change in the gel weight until it reaches equilibrium. The protein gel at this time is the final toughened gel.

[0057] Perform X-ray powder diffraction analysis on the toughened gels prepared above. The results are as shown in Figure 6 a of. It can be seen that different protein concentrations have different effects on the properties of protein gels. When the ratio of organic solvent to protein is 5:3, the crystallization performance of the protein gel is the best.

[0058] Example 4

[0059] To verify the effect of the water removal method on the properties of protein gels, gelatin protein gels prepared by different water removal methods were prepared in this example. The preparation process is as follows:

[0060] Add 1 g of gelatin to 10 mL of the double-solvent mixed solution (W H2O :W EG = 3:1), stir in a 40 °C water bath for 30 min. After it is completely dissolved, cool it to room temperature, add lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator and stir in the dark for 10 min; then pour the stirred reaction solution into a mold, use the vacuum method to remove the bubbles in the solution, and irradiate it with ultraviolet light (365 nm) for 5 min to obtain the gelatin protein gel after double-bond polymerization crosslinking.

[0061] Place the gelatin protein gels prepared above under a constant temperature of 25 °C in an open environment for natural evaporation (Regular), or place them in an oven at 60 °C to dry the water (60 °C), or place them in an EG solution to remove the water by solvent conversion (In EG), or place them under a constant temperature of 25 °C in an open environment for air-assisted water evaporation, and control the evaporation rate at 0.15 g h -1(Blow-dry). In all cases, the change in the weight of the gel was recorded in real time until it reached equilibrium, which was taken as the node when evaporation was complete, to obtain the protein gel.

[0062] X-ray powder diffraction analysis was performed on the protein gelatin prepared by different moisture removal methods, and the results are as Figure 6 shown in b. It can be seen that compared with the other methods of removing the solvent (natural air drying, high-temperature drying, solvent replacement), room-temperature air flow-assisted evaporation can effectively improve the protein crystallinity.

[0063] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A protein gel toughening method induced by evaporation of an organic-water dual solvent, characterized in that: The steps include: Mixing an organic solvent with water to prepare a dual solvent mixed solution; After dissolving the protein in the dual-solvent mixed solution, performing gelation treatment to obtain a protein gel; The protein gel is subjected to airflow-assisted water volatilization to obtain a toughened protein gel.

2. The protein gel toughening method according to claim 1, characterized in that: The organic solvent is a solvent that is soluble in water.

3. The protein gel toughening method according to claim 2, characterized in that: The organic solvent includes one or more of tetrahydrofuran, ethanol, methanol, ethylene glycol, triethylene glycol, tetraethylene glycol, glycerol, dimethyl sulfoxide, N,N-dimethylformamide and dioxane.

4. The protein gel toughening method according to claim 1, characterized in that: The protein includes a natural protein or a protein obtained by chemically modifying the natural protein.

5. The protein gel toughening method according to claim 4, characterized in that: The protein includes one or more of gelatin protein, methacryloyl gelatin protein, silk protein, royal jelly protein, yeast protein and microbial protein.

6. The protein gel toughening method according to claim 1, characterized in that: The mass ratio of the organic solvent to the protein is (1-3):

1.

7. The protein gel toughening method according to claim 1, characterized in that: The temperature of the airflow-assisted water volatilization is 15-40°C, and the water evaporation rate is 0.1-0.2gh -1 .

8. The protein gel toughening method according to claim 1 or 4, characterized in that: When the protein is gelatin, the gelling process comprises the steps of cooling and solidifying; When the protein is methacryloyl gelatin, the gelation treatment comprises the steps of adding a photoinitiator and then irradiating under ultraviolet light.

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