Preparation method and application of protein adhesive

Abstract

The invention relates to the technical field of biological materials, in particular to a preparation method and application of a protein adhesive, and the method comprises the following steps: A, mixing protein with a protein denaturant under an acidic condition to obtain an unfolded protein solution; b, adding a reducing agent into the unfolded protein solution to obtain a mixed solution; and C, carrying out dehydration treatment on the mixed solution to obtain the cured adhesive. The adhesive produced by adopting the method is low in production cost, small in operation difficulty, non-toxic, harmless, environment-friendly and high in adhesion capacity, and particularly, the adhesion capacity reaches 1.73 MPa in the embodiment that the quinoa protein adhesive is used for bonding ebony sheets.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological materials, and particularly relates to a preparation method of a protein adhesive and application thereof. BACKGROUND

[0002] The adhesive is a material that uses the cohesion and interfacial adsorption capacity of the main material to tightly combine homogenous or heterogeneous materials. According to the annual meeting data of the China Adhesive and Adhesive Tape Industry Association, the development goal of the adhesive in China is that the annual growth rate of the output is 4.2%, and the annual growth rate of the sales is 4.3%. The adhesive industry in China will still develop rapidly. However, with the continuous advancement of urbanization, informatization and industrialization, the functional products of environmental protection, engineering glue and special application glue become the hot spot of the subdivided market, but the domestic products are faced with the situation of insufficient high-end products and excessive low-end products, and therefore it is urgent to explore new environmental protection materials as the main material of the adhesive.

[0003] According to whether the raw materials are biodegradable, adhesives can be divided into petroleum-based adhesives and biomass-based adhesives. Petroleum-based adhesives occupy the main share of the market due to their strong bonding performance and simple preparation process. However, some common petroleum-based adhesives, such as aldehyde-based resins and synthetic rubber-based adhesives, release toxic and harmful components such as formaldehyde, which endanger human health. In the field of biomass-based adhesives, renewable raw materials are used to obtain biopolymer materials or monomers through biological transformation. Traditional protein-based adhesives usually use physical (high temperature, high pressure, ultrasound, etc.) or chemical modification (strong acid, alkali, salt, etc.) to pretreat proteins to expose active groups, and then use covalent cross-linking, hybridization, etc. to enhance the cohesion of proteins. For example, the patent document with application number CN202011338276.2 discloses an adhesive made of oil tea cake protein using ultrasonic treatment to change the protein conformation. Pang et al. (Pang H, Wang Y, Chang Z, Xia C, Han C, Liu H, et al. Soy meal adhesive with high strength and water resistance via carboxymethylated wood fiber-induced crosslinking. Cellulose. 2021; 28(6): 3569-84.) used carboxymethylated wood fiber to cross-link with the side chains of soybean meal. Pradyawong et al. (Pradyawong S, Brown NH, Zhao J, Qi G, Zheng Y, Sun XS, et al. Improved soy protein adhesives by lignin and polyamide-epichlorohydrin: Adhesion performance and properties. Journal of Applied Polymer Science. 2022; 139(44): e53086.) used the interaction of protein carbonyl, hydroxyl, and amino groups to construct a stable cross-linked network with lignin. However, the preparation of the above materials usually involves multiple steps, takes a long time, and the developed adhesives usually perform poorly in high humidity conditions because they tend to interact with water rather than the material surface. Prolonged hydration gradually disintegrates the adhesive network.Waite et al. (Waite JH. Mussel adhesion-essential footwork. Journal of Experimental Biology. 2017; 220(4): 517-30.) found that some functional groups in the protein such as threonine, serine and lysine side chain rich in hydroxyl or amino effectively remove the hydration layer of the material surface, and the amino of mussel adhesion protein promotes the adhesion of the interface by replacing the cations adsorbed on the mineral surface, and finally deposits the mussel adhesion protein to the target surface. At the same time, the barnacle protein (Waite JH. Mussel adhesion-essential footwork. Journal of Experimental Biology. 2017; 220(4): 517-30.) also contains a large number of amino acid residues that promote the adhesion of the interface, and there are at least 20% of the disordered residues, and the flexibility of the adhesion-promoting functional groups and the disordered amino acids can help the barnacle protein self-assembled fiber to adapt to different microstructure substrates (materials), such as Mrcp20 protein can be strongly adsorbed on mild steel through thiol functional groups and high surface positive charge. Non-covalent forces such as electrostatic interactions seem to be the key to the adhesion of mussels and barnacle proteins to the substrate, but the adhesion effect of Mrcp20 protein to glass and other high polymers is poor.

[0004] The above-mentioned adhesive has the defects of difficult raw material extraction, difficult modification operation or poor bonding effect, and has certain difficulty in large-area industrial production, so it is urgent to explore a new method for preparing the gelling agent. SUMMARY

[0005] The present application aims to at least partially solve one of the technical problems in the related art. To this end, one object of the present application is to provide a method for preparing an adhesive. The method of the present application can quickly and conveniently produce the adhesive, which is low in price and non-toxic and environmentally friendly in the production process, and the produced adhesive has strong adhesion to wood.

[0006] In one aspect of the present application, a method for preparing an adhesive is provided, characterized in that the method comprises:

[0007] A. mixing a protein with a protein denaturant under acidic conditions to obtain an unfolded protein solution;

[0008] B. adding a reducing agent to the unfolded protein solution to obtain a mixed solution;

[0009] C. dehydrating the mixed solution to obtain a solidified adhesive.

[0010] The protein raw material used in the method for preparing the adhesive in the present application can be any protein known in the art, including commercial proteins or proteins extracted by those skilled in the art according to the technical means in the art, as long as the purity of the protein is not less than 80%. The method of the present application can quickly and conveniently produce the adhesive, which is low in price and non-toxic and environmentally friendly in the production process. The adhesive produced has a very strong adhesive capacity for wood, specifically, has a good bonding effect on glass samples, and the bonding strength for wood reaches 1.73 MPa.

[0011] According to a specific embodiment of the present application, the preparation of the adhesive can also have the following technical features:

[0012] According to a specific embodiment of the present application, in step A, the protein includes plant protein or animal protein.

[0013] According to a specific embodiment of the present application, the plant protein includes any one extracted from soybean, wheat, pea, peanut, and quinoa.

[0014] According to a specific embodiment of the present application, the protein denaturant includes guanidine hydrochloride or urea.

[0015] According to a specific embodiment of the present application, when mixed, the purity of the protein is not less than 80%, and when the purity of the protein is not less than 80%, the adhesive obtained has a stronger adhesive capacity.

[0016] According to a specific embodiment of the present application, when mixed, the concentration of the protein denaturant is 4-9 mol / L.

[0017] According to a specific embodiment of the present application, when mixed, the mass ratio of the protein to the protein denaturant is 1:(5-20), and when the mass ratio of the protein to the protein denaturant is 1:(5-20), the adhesive obtained has a stronger adhesive effect.

[0018] According to a specific embodiment of the present application, when mixed, the pH of the acidic condition is 1-4, and when mixed, the pH of the solution is in this range, the adhesive obtained has a better adhesive effect.

[0019] According to a specific embodiment of the present application, the reducing agent in step B can be any reagent known in the art that has a strong reducing effect on protein.

[0020] According to a specific embodiment of the present application, in step B, the reducing agent includes at least one of beta-mercaptoethanol and / or dithiothreitol.

[0021] According to a specific embodiment of the present application, in the mixed solution of step B, the concentration of the reducing agent is 10-40 mM.

[0022] According to a specific embodiment of the present application, in step C, the method of the dehydration treatment comprises: coating the mixed solution on a carrier, and evaporating water to obtain the cured adhesive.

[0023] According to a specific embodiment of the present application, the temperature used for evaporating water in the dehydration treatment is 20-35°C.

[0024] According to a specific embodiment of the present application, when the protein is derived from plant seeds, further comprising: drying and crushing the plant seeds to obtain plant seed powder, and then performing a defatting treatment, and then extracting the protein.

[0025] According to a specific embodiment of the present application, the defatting treatment comprises: mixing an organic solvent 1 with the plant seed powder to obtain defatted powder.

[0026] According to a specific embodiment of the present application, the water activity of the plant seed powder is not higher than 11%.

[0027] According to a specific embodiment of the present application, the organic solvent 1 comprises at least one of n-hexane, ethanol, and diethyl ether.

[0028] According to a specific embodiment of the present application, the mass ratio of the defatted powder to the organic solvent 1 is 1:(2.5-5).

[0029] According to a specific embodiment of the present application, the content of oil and fat in the defatted powder is not higher than 2%.

[0030] According to a specific embodiment of the present application, the method of extracting the protein comprises any one of alkaline solution and acid precipitation, enzyme method, and Osborne fractionation.

[0031] According to a specific embodiment of the present application, in the alkaline solution and acid precipitation, the pH of the alkaline solution is 8.5-9.5, and the pH of the acid solution is 4.4-4.6.

[0032] According to a more specific embodiment of the present application, the present application provides a production method of quinoa protein adhesive, comprising:

[0033] (1) defatting quinoa seeds, and then sequentially performing alkaline solution and acid precipitation, water washing, and freeze-drying, so as to obtain quinoa protein;

[0034] (2) mixing the quinoa protein with a urea solution, so as to obtain an unfolded protein solution;

[0035] (3) mixing the unfolded protein solution with mercaptoethanol, so as to break the disulfide bond of the protein, and thus increase the unfolding degree of the protein and enhance the adhesion;

[0036] (4) The above solution is coated between the plates at room temperature, and the moisture is volatilized to obtain the cured quinoa protein adhesive.

[0037] According to the method for improving the solubility of quinoa protein, the quinoa seeds are first defatted, and then subjected to alkali dissolution and acid precipitation and water washing to obtain quinoa protein. Then, the quinoa protein is mixed with a urea solution. The stronger dispersion interaction between urea (relative to water) and the hydrophobic side chain and the protein backbone drives the preferential binding of urea and allows it to invade the core of the globular protein to a certain extent. The preferential binding of urea promotes the dissolution of the hydrophobic region of the protein core, induces the protein to transform from a folded state to an unfolded state, and meanwhile, the addition of a strong acid solution causes the protein to gradually carry a positive charge. The intermolecular repulsion between the positively charged groups leads to the unfolded and relatively expanded conformation until the protein is maximally protonated. Then, mercaptoethanol is added to break the inter-chain or intra-chain disulfide bonds, and a stable and more unfolded protein chain is obtained. Finally, the obtained solution is coated between the substrates at room temperature. The stable hydration layer usually present on the surface of the substrate causes most of the adhesive to be strongly repelled, and the amino acid residues of the unfolded protein chain can cause the rupture of the hydration layer of the substrate. This is because the flexible hydrophobic region exposed by the unfolded protein chain can dry the wet surface, and then dynamically adjust the hydrophobic and hydrophilic residues to better interact with the solid surface, thereby providing strong interfacial adhesion. With the increase of time, the area of the hydration layer of the substrate is further expanded, and the protein drags the entire chain along the surface to pass through the hydration layer to contact the medium of the substrate surface. At the same time, the entire process is accompanied by the volatilization of water, and the exposure of the protein chain begins to aggregate, accompanied by the rotation of the amino acid residues, the content of the β-sheet structure is enhanced, and the protein chain is firmly adhered to the surface of the substrate, so that the substrates are bonded in the macroscopic view. The method of the present application can simply prepare a quinoa protein adhesive. Specifically, the bonding strength of the quinoa protein adhesive between black ebony wood slices reaches 1.73 MPa.

[0038] In addition, the preparation of the quinoa protein adhesive according to the above embodiments of the present application can also have the following technical features:

[0039] According to a more specific embodiment of the present application, in step (1), the defatting of the quinoa seeds comprises: drying the quinoa seeds, grinding and sieving them into quinoa powder, and then mixing them with an organic solvent for defatting.

[0040] According to a more specific embodiment of the present application, the drying temperature is 35-45℃.

[0041] According to a more specific embodiment of the present application, the water activity of the quinoa seeds after drying is 8.5%-10.5%.

[0042] According to a more specific embodiment of the present application, the mass ratio of the quinoa flour to the organic solvent is 1:(2.5-5).

[0043] According to a more specific embodiment of the present application, the oil content of the defatted quinoa flour is less than 2%.

[0044] According to a more specific embodiment of the present application, in step (1), the alkali dissolution and acid precipitation includes mixing the defatted quinoa flour with water, adding alkali to centrifugally collect supernatant, and mixing the supernatant with acid to centrifugally collect precipitate.

[0045] According to a more specific embodiment of the present application, the mass ratio of the defatted quinoa seed flour to the water is 1:(7-13).

[0046] According to a more specific embodiment of the present application, the process of adding alkali includes adjusting and maintaining pH between 8.5-9.5 for 60min-120min by adding 1mol / L-12mol / L NaOH solution.

[0047] According to a more specific embodiment of the present application, the concentration of the acid is 1mol / L-12mol / L, and the acid adjusts the pH of the supernatant to 4.4-4.6.

[0048] According to a more specific embodiment of the present application, in step (1), the water washing includes mixing the precipitate obtained by alkali dissolution and acid precipitation with water, and adding alkali to adjust pH so that the obtained quinoa protein has good resolubility.

[0049] According to a more specific embodiment of the present application, the mass ratio of the precipitate to the water is 1:(20-30).

[0050] According to a more specific embodiment of the present application, the process of adding alkali includes adjusting and maintaining pH between 6.5-7.5 for 30min-60min by adding 6mol / L-12mol / L NaOH solution.

[0051] According to a more specific embodiment of the present application, in step (2), the process includes mixing quinoa protein with urea solution, and adding acid solution to adjust pH so that protein non-covalent interaction is shielded and protein chain is unfolded.

[0052] According to a more specific embodiment of the present application, the concentration of the urea solution is 6mol / L-8mol / L.

[0053] According to a more specific embodiment of the present application, the mass ratio of the quinoa protein to the urea solution is 1:(5-20).

[0054] According to a more specific embodiment of the present application, the process of adjusting pH by adding acid solution comprises: adjusting pH to 1.0-3.0 by adding 6mol / L-12mol / L HCl solution.

[0055] According to a more specific embodiment of the present application, in step (3), the unfolded protein solution is mixed with mercaptoethanol to obtain stable and more relaxed unfolded protein chains.

[0056] According to a more specific embodiment of the present application, after the unfolded protein solution is mixed with mercaptoethanol, the concentration of mercaptoethanol in the mixture is 10mM-40mM.

[0057] According to a more specific embodiment of the present application, in step (4), the water evaporation time is 60-120min to make the protein exposed and aggregated to adhere to the surface of the substrate.

[0058] The second aspect of the present application provides an adhesive prepared by the method of the first aspect.

[0059] The third aspect of the present application provides the use of the adhesive of the second aspect in bonding glass. The adhesive prepared by the method of the present application has good bonding strength for glass materials.

[0060] The fourth aspect of the present application provides the use of the adhesive of the second aspect in bonding wood.

[0061] The method of the present application can be used to simply prepare an adhesive. Specifically, the bonding strength of the adhesive of the present application in black sandalwood slices reaches 1.73MPa.

[0062] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0063] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0064] Figure 1 is a preparation method of quinoa protein adhesive in a specific embodiment of the present application;

[0065] Figure 2 is an influence of urea concentration on the performance of protein adhesive in Example 1 of the present application;

[0066] Figure 3 is an influence of pH of urea solution on the performance of protein adhesive in Example 4 of the present application;

[0067] Figure 4Effect of protein concentration on the performance of protein adhesive of Example 6 of the present application

[0068] Figure 5 Effect of mercaptoethanol concentration on the performance of protein adhesive of Example 7 of the present application. DETAILED DESCRIPTION

[0069] Embodiments of the present application are described in detail below. The embodiments described below are examples for explaining the present application and should not be understood as limiting the present application.

[0070] It should be noted that the terms "first", "second" are used only for the purpose of description and should not be understood as indicating or implying relative importance or implying a specified number of technical features. Thus, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0071] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The ranges or values should be construed to be approximations that allow for significant variation. Within the ranges or values, endpoints are provided as a separate point for use in the claims.

[0072] In order that the present application can be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in this document concur in meaning with the general use of those terms by those skilled in the art to which the present application pertains.

[0073] In this document, the terms "comprising" or "including" are open-ended terms, i.e. the inclusion of the recited elements, but not the exclusion of other elements.

[0074] In this document, the terms "optionally", "optional" or "may" generally mean that the subsequently described event or circumstance can or can not occur, and the description includes instances where the event or circumstance occurs and instances where it does not.

[0075] In this document, the term "protein denaturant" refers to an agent that causes denaturation of a protein, which is the action of a protein being affected by physical or chemical factors, changing its internal molecular structure and properties. It is generally believed that the secondary structure and tertiary structure of the protein are changed or destroyed, which are the results of denaturation.

[0076] In the present text, the term "plant protein" is a kind of protein, which is derived from plants, is nutritionally complete, similar to animal protein, easy to be digested and absorbed by human body, and has multiple physiological and health care functions.

[0077] In the present text, the term "alkali-solvent acid precipitation method" is a method for achieving the purpose of purification or separation by using the property that a substance generates a soluble substance in an alkaline solution and generates a precipitate in an acidic solution, and extracting with alkali and precipitating with acid.

[0078] According to one embodiment of the present application, the present application provides a method for preparing an adhesive, characterized in that the method comprises:

[0079] A. mixing the protein with a protein denaturant under acidic conditions to obtain an unfolded protein solution;

[0080] B. obtaining a mixed solution after adding a reducing agent to the unfolded protein solution;

[0081] C. performing dehydration treatment on the mixed solution to obtain a solidified adhesive.

[0082] The protein raw material used in the method for preparing an adhesive in the present application can be any protein known in the art, including commercially available proteins or proteins extracted by a person skilled in the art according to technical means in the art. The method of the present application can be used to quickly and conveniently produce an adhesive, which is low in price and non-toxic and environmentally friendly in the production process. The produced adhesive has extremely strong adhesion to wood and glass, and specifically, the wood bonding strength reaches 1.73 MPa.

[0083] According to a more specific embodiment of the present application, the present application provides a method for preparing a quinoa protein adhesive, specifically as shown in Figure 1 The method comprises:

[0084] S100: after defatting quinoa seeds, sequentially performing alkali-solvent acid precipitation and water washing to obtain quinoa protein;

[0085] In this step, the quinoa seeds are washed and then dried at a temperature of 35-45°C until the water activity is 8.5%-10.5%, and then ground through a 60-80 mesh sieve. The sieved quinoa seed powder is mixed with an organic solvent at a mass ratio of 1:(2.5-5), and shaken in a shaker at 28-33°C and 180-250 r / min for 1-1.5 hours. The defatted quinoa seed powder is obtained by repeating the defatting 2-4 times and drying until the fat content is less than 2%. The defatted quinoa seed powder is mixed with water at a mass ratio of 1:(7-13), and then 1 mol / L-12 mol / L NaOH solution is added to adjust and maintain the pH at 8.5-9.5 for 60-120 min. The mixture is then centrifuged at 8000-10000 rpm for 13-17 min at room temperature, and the supernatant is collected by passing through a 150-250 mesh nylon cloth. The supernatant is repeatedly centrifuged and filtered 2-4 times, and the supernatant is collected. The pH of the obtained supernatant is adjusted to 4.4-4.6 with 1 mol / L-12 mol / L hydrochloric acid, and then centrifuged at 8000-10000 rpm for 13-18 min at room temperature. The protein precipitate is collected and washed with deionized water 2-4 times, with a mass ratio of 1:(2-5) of the protein precipitate to water each time. The protein precipitate is sheared with a shear machine at 8000-12000 rpm for 2-5 min. The precipitate is sheared again, the solution is adjusted to neutral, and then freeze-dried to obtain quinoa protein.

[0086] S200: mixing the quinoa protein with a urea solution and adding an acid solution to adjust the pH to obtain an unfolded protein solution;

[0087] In this step, the quinoa protein obtained in S100 is mixed with a 6 mol / L-8 mol / L urea solution at a mass ratio of 1:5-1:20 of the protein powder to the solvent, and 6 mol / L-12 mol / L HCl solution is added to adjust the pH to 1.0-4.0. The stronger dispersion interaction between urea (relative to water) and the hydrophobic side chain and the protein backbone drives the preferential binding of urea, and to some extent allows it to invade the core of the globular protein. Therefore, the preferential binding of urea promotes the dissolution of the hydrophobic region of the protein core, and induces the protein to transform from a folded state to an unfolded state. At the same time, the addition of a strong acid solution gradually charges the protein with positive charges, and the intermolecular repulsion between positively charged groups leads to an unfolded and relatively expanded conformation until the protein is maximally protonated.

[0088] According to an embodiment of the present application, the concentration of urea is 5-9 mol / L. The inventor has found that when the concentration of the protein denaturant is too low, most of the quinoa protein particles still exist in the solution in the form of >100 nm spherical aggregates, and according to the Stokes law sedimentation equation, large protein particles have a higher aggregation rate and are more likely to aggregate and precipitate, and the protein unfolding degree is limited; when the concentration of the protein denaturant is too high, the denaturant molecules always surround the protein chains, and the protein non-covalent interaction is partially shielded and cannot effectively aggregate and adhere to the substrate surface. Therefore, the urea solution of the present application has a concentration of 5-9 mol / L, which is beneficial to the enhancement of the protein adsorption capacity under the condition of stabilizing the unfolded protein chains.

[0089] According to an embodiment of the present application, the mass ratio of the protein powder to the solvent is 1:5-1:20. The inventor has found that when the mass ratio of the protein powder to the solvent is low, the cohesion generated by the adsorption of the protein chains cannot effectively resist the external pulling force or shear force, and cannot form a stable and effective bonding effect on the substrate surface; when the mass ratio of the protein powder to the solvent is high, the solution itself has high viscosity and low flowability, and the solution state changes from liquid to semi-solid, which makes sampling difficult in practical application. Therefore, the present application uses the mass ratio of the protein powder to the solvent of 1:5-1:20, which can save costs and obtain an unfolded protein solution with strong adsorption capacity.

[0090] According to an embodiment of the present application, after the protein and the protein denaturant are mixed, 6-12 mol / L of HCl solution is added to adjust the pH to 1.0-4.0. The inventor has found that the unfolding degree of the protein chain is positively correlated with the bonding strength of the protein, and when the pH of the solution is low, the addition of more strong acid causes protons and anions to be added to the solution, and when the pH is 1-2, the protein has been protonated to the maximum extent, so in principle, increasing more protons has no effect on its ionization state, but adding more anions will cause electrostatic interaction with the positively charged center of the protein, thereby shielding part of the electrostatic repulsion. The combination of anions eliminates the electrostatic repulsion, not only reduces the intramolecular repulsion and intermolecular repulsion, but also induces the unfolded protein conformation to change to a dense molten globule structure, that is, the acid-induced protein folding phenomenon occurs; when the pH of the solution is high, it is close to the isoelectric point of the protein, the electrostatic interaction of the protein decreases, the protein tends to aggregate, and subsequent interchain entanglement to form an adhesive network is difficult to occur, the adhesion strength decreases, and the solution changes obviously in color from a yellow transparent solution to a yellow-brown turbid solution. Therefore, the present application adjusts the pH of the protein and the protein denaturant to 1.0-3.0 after mixing, which can obtain an unfolded protein solution with strong adsorption capacity.

[0091] S300: The unfolded protein solution is mixed with mercaptoethanol to obtain a protein solution with broken disulfide bonds;

[0092] In this step, the unfolded protein solution obtained in S200 is mixed with a mercaptoethanol solution. After mixing, the concentration of mercaptoethanol in the mixture is 10 mM-40 mM. The degree of disulfide bond breakage of quinoa protein is related to the reducing ability and protein adsorption capacity of the reducing agent. Disulfide bonds are relatively stable covalent bonds that maintain the tertiary structure and biological function of proteins. The more disulfide bonds there are, the greater the stability of the protein molecule against external factors. Therefore, a reducing agent with strong reducing ability, such as mercaptoethanol or dithiothreitol, is selected. When the concentration of the reducing agent is low, the number of disulfide bonds broken is small, and the protein chain cannot be effectively unfolded. When the concentration of the reducing agent is too high, the cost increases. The concentration of the reducing agent in this range can produce a relatively stable bonding effect between the substrates. Thus, mercaptoethanol or dithiothreitol is used as the reducing agent, and the concentration of mercaptoethanol in the solution after mixing is 10 mM-40 mM, which can effectively break the disulfide bonds and unfold the protein chain.

[0093] S400: The unfolded protein solution is coated between the substrates under room temperature conditions, and the water is evaporated to obtain a gelling agent.

[0094] In this step, about 100 ul of the unfolded protein solution is coated between the substrates with an overlapping area of 1 cm 2 . The water is evaporated under room temperature conditions for 60-120 min. The amino acid residues of the unfolded protein chain can cause the hydration layer of the substrate to break. As time increases, the area of the broken hydration layer of the substrate expands further, and the protein drags the entire chain along the surface to pass through the hydration layer and contact the medium on the surface of the substrate. At the same time, the entire process is accompanied by water evaporation, and the exposed protein chain begins to aggregate. During this process, the content of beta-sheet structure is enhanced through the rotation of amino acid residues, and the protein chain is firmly adhered to the surface of the substrate, causing the substrates to bond together under a macroscopic view. If the time is too short, the bonding network is not ordered, and the bonding strength is low. If the time is too long, the protein chain has been basically adsorbed on the overlapping surface of the substrate, and the bonding strength will not change significantly. Thus, the coating type is used, and the water evaporation time is 60-120 min, which can allow the unfolded protein chain to have sufficient time to expose, aggregate, and adhere to the surface of the substrate.

[0095] The schemes of the present disclosure will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. If specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained by purchase.

[0096] Example 1

[0097] The quinoa seeds were washed to remove saponins, dried at 40°C to a water activity of 9.5%, then ground and passed through a 60-mesh sieve. The quinoa powder was mixed with n-hexane in a mass ratio of 1:3 in a fume hood, shaken at 30°C and 220 r / min for 1 h in a shaker, and filtered. The defatting process was repeated three times. The defatted quinoa powder was placed in a fume hood overnight to remove residual n-hexane. The defatted quinoa powder was added to water in a mass ratio of 1:10, adjusted to a pH of 9.0 with a 6 mol / L NaOH solution, and maintained for 90 min. The mixture was centrifuged at 10,000 r / min for 15 min at room temperature. The supernatant was passed through a 200-mesh nylon cloth, and the supernatant was collected. The supernatant was centrifuged and filtered twice, and the supernatant was collected. The supernatant was adjusted to a pH of 4.5 with a 6 mol / L HCl solution, and centrifuged at 10,000 r / min for 15 min at room temperature. The precipitate was washed three times with deionized water to remove soluble salts. The precipitate was sheared with a shear machine at 10,000 r / min for 3 min to disperse the precipitate evenly in water. The precipitate was sheared again at 10,000 r / min for 3 min, and the precipitate:water ratio was 1:1.5. The pH was adjusted to 7.0 with a 1 mol / L NaOH solution and maintained for 30 min. The quinoa protein was obtained by freeze-drying. The purity of the obtained quinoa protein was 85% as determined by the Kjeldahl method.

[0098] At room temperature, 50.0 g of urea was dissolved in 50.0 g of water to prepare an 8 mol / L urea solution. The pH of the urea solution was adjusted to 2.0 with a 6 mol / L HCl solution. 0.3 g of protein was dissolved in 2.7 g of 8 mol / L urea solution with a pH of 2.0, and mixed thoroughly to obtain an unfolded protein solution. At this time, the ratio of quinoa protein to urea solution was 1:9. 4 ul of 15 mol / L mercaptoethanol solution was added to the unfolded protein solution and mixed thoroughly. At this time, the concentration of mercaptoethanol in the mixture was 20 mmol / L.

[0099] 100 ul of the above solution was evenly coated on one end of a 3 cm*2 cm*0.6 cm black ebony wood strip, and the coating area was 1 cm 2After covering with another wood strip of the same size, stand for 120 min. Fix the two bonded wood pieces on the clamps of a universal material testing machine (Iinstron, 3400, USA) in the initial state, keeping the vertical height between the clamps at 4 cm. Select the tensile mode, set the initial shape of the sample (40 mm*20 mm*6 mm), the final shape of the sample (41 mm*20 mm*6 mm), and set the test procedure: a) before test: zero displacement and load; b) test: control displacement speed 1 mm / min, fixed slope 1, stretch at constant speed; c) end of test: 20% drop in peak load. The tensile strength is calculated from the breaking point, and the elastic modulus is determined from the average slope of the stress-elongation curve at 0-20% elongation. The effect of different urea concentrations on the performance of the protein adhesive is determined, and the results are shown in Figure 2 Figure 2, which shows that the adhesive bonding strength also increases with the increase of urea concentration. When the urea concentration is 8M, the measured bonding strength is the strongest, 1.73 MPa. 8M urea is the critical value, and subsequent increase of urea concentration has no significant effect on the bonding strength.

[0100] Example 2

[0101] The main difference between Example 2 and Example 1 is:

[0102] At room temperature, 37.5 g of urea is dissolved in 62.5 g of water to prepare a 6 mol / L urea solution. Use 6 mol / L HCl solution to adjust the pH of the urea solution to 2.0. Dissolve 0.3 g of protein in 2.7 g of 6 mol / L urea solution with pH 2.0, mix thoroughly to obtain an unfolded protein solution. At this time, the mass ratio of quinoa protein to urea solution is 1:9. Add 4 ul of 15 mol / L mercaptoethanol solution to the unfolded protein solution and mix thoroughly. At this time, the concentration of mercaptoethanol in the mixture is 20 mmol / L.

[0103] The test method of protein bonding strength is the same as that of Example 1. The measured protein bonding strength in Example 2 is 1.65 MPa.

[0104] Example 3

[0105] The main difference between Example 3 and Example 1 is:

[0106] At room temperature, 25.0 g urea was dissolved in 75.0 g water to prepare 4 mol / L urea solution, 6 mol / L HCl solution was used to adjust the pH of the urea solution to 2.0, 0.3 g protein was dissolved in 2.7 g 4 mol / L urea solution with pH of 2.0, and mixed thoroughly to obtain an unfolded protein solution, at this time, the mass ratio of quinoa protein to urea solution was 1:9, 4 ul 15 mol / L mercaptoethanol solution was added to the unfolded protein solution, and mixed thoroughly, at this time, the concentration of mercaptoethanol in the mixed solution was 20 mmol / L.

[0107] The test method of protein bonding strength was the same as that of Example 1, and the protein bonding strength measured in Example 3 was 1.11 MPa.

[0108] Example 4

[0109] The main difference between Example 4 and Example 1 is that:

[0110] At room temperature, 50.0 g urea was dissolved in 50.0 g water to prepare 8 mol / L urea solution, 6 mol / L HCl solution was used to adjust the pH of the urea solution to 1.0, 0.3 g protein was dissolved in 2.7 g 8 mol / L urea solution with pH of 1.0, and mixed thoroughly to obtain an unfolded protein solution, at this time, the mass ratio of quinoa protein to urea solution was 1:9, 4 ul 15 mol / L mercaptoethanol solution was added to the unfolded protein solution, and mixed thoroughly, at this time, the concentration of mercaptoethanol in the mixed solution was 20 mmol / L.

[0111] The test method of protein bonding strength was the same as that of Example 1, and the effect of urea solution on protein adhesive under different pH conditions was determined, and the results are shown in Figure 3 The protein bonding strength measured in Example 4 was 0.85 MPa when the pH was 1.

[0112] Example 5

[0113] The main difference between Example 5 and Example 1 is that:

[0114] At room temperature, 50.0 g urea was dissolved in 50.0 g water to prepare 8 mol / L urea solution, 6 mol / L HCl solution was used to adjust the pH of the urea solution to 3.0, 0.3 g protein was dissolved in 2.7 g 8 mol / L urea solution with pH of 3.0, and mixed thoroughly to obtain an unfolded protein solution, at this time, the mass ratio of quinoa protein to urea solution was 1:9, 4 ul 15 mol / L mercaptoethanol solution was added to the unfolded protein solution, and mixed thoroughly, at this time, the concentration of mercaptoethanol in the mixed solution was 20 mmol / L.

[0115] The method for testing the protein bonding strength is the same as that in Example 1. The protein bonding strength measured in Example 5 is 1.21 MPa.

[0116] Example 6

[0117] The main difference between Example 6 and Example 1 is that:

[0118] At room temperature, 50.0 g of urea was dissolved in 50.0 g of water to prepare an 8 mol / L urea solution. The pH of the urea solution was adjusted to 2.0 using a 6 mol / L HC1 solution. 0.15 g of protein was dissolved in 2.85 g of the 8 mol / L urea solution with a pH of 2.0, and mixed thoroughly to obtain an unfolded protein solution. At this time, the mass ratio of quinoa protein to urea solution was 1:19. 4 ul of 15 mol / L mercaptoethanol solution was added to the unfolded protein solution, and mixed thoroughly. At this time, the concentration of mercaptoethanol in the mixture was 20 mmol / L.

[0119] The method for testing the protein bonding strength is the same as that in Example 1. The effect of different protein concentrations on the protein adhesive was determined, and the results are shown in Table 1. Figure 4 As shown in Table 1, the bonding strength of the adhesive also increased with the increase of the protein concentration. The protein bonding strength measured in Example 6 was 0.53 MPa.

[0120] Example 7

[0121] The main difference between Example 7 and Example 1 is that:

[0122] At room temperature, 50.0 g of urea was dissolved in 50.0 g of water to prepare an 8 mol / L urea solution. The pH of the urea solution was adjusted to 2.0 using a 6 mol / L HC1 solution. 0.15 g of protein was dissolved in 2.85 g of the 8 mol / L urea solution with a pH of 2.0, and mixed thoroughly to obtain an unfolded protein solution. At this time, the mass ratio of quinoa protein to urea solution was 1:19. 4 ul of 15 mol / L mercaptoethanol solution was added to the unfolded protein solution, and mixed thoroughly. At this time, the concentration of mercaptoethanol in the mixture was 20 mmol / L.

[0123] The method for testing the protein bonding strength is the same as that in Example 1. The effect of different concentrations of reducing agent on the protein adhesive was determined, and the results are shown in Table 2. Figure 5 As shown in Table 2, the protein bonding strength was best when the concentration of mercaptoethanol was 20 mM. The protein bonding strength measured in Example 7 was 1.73 MPa.

[0124] Example 8

[0125] The main difference between Example 8 and Example 1 is that:

[0126] At room temperature, 50.0 g urea was dissolved in 50.0 g water to prepare 8 mol / L urea solution, 8 mol / L HCl solution was used to adjust the pH of the urea solution to 2.0, 0.3 g protein was dissolved in 2.7 g 8 mol / L urea solution with pH 2.0, and mixed well to obtain an unfolded protein solution, at this time the mass ratio of quinoa protein to urea solution was 1:9, 4 ul 15 mol / L mercaptoethanol solution was added to the unfolded protein solution, and mixed well, at this time the concentration of mercaptoethanol in the mixed solution was 20 mmol / L.

[0127] The test method of protein bonding strength was the same as that of Example 1, and the protein bonding strength measured in Example 8 was 1.23 MPa.

[0128] Example 9

[0129] The main difference between Example 9 and Example 1 is that:

[0130] At room temperature, 50.0 g urea was dissolved in 50.0 g water to prepare 8 mol / L urea solution, 8 mol / L HCl solution was used to adjust the pH of the urea solution to 2.0, 0.3 g protein was dissolved in 2.7 g 8 mol / L urea solution with pH 2.0, and mixed well to obtain an unfolded protein solution, at this time the mass ratio of quinoa protein to urea solution was 1:9, 4 ul 15 mol / L mercaptoethanol solution was added to the unfolded protein solution, and mixed well, at this time the concentration of mercaptoethanol in the mixed solution was 20 mmol / L.

[0131] The test method of protein bonding strength was the same as that of Example 1, and the protein bonding strength measured in Example 8 was 1.23 MPa.

[0132] Example 10

[0133] The main difference between Example 10 and Example 1 is that:

[0134] At room temperature, 50.0 g urea was dissolved in 50.0 g water to prepare 8 mol / L urea solution, 8 mol / L HCl solution was used to adjust the pH of the urea solution to 2.0, 0.3 g protein was dissolved in 2.7 g 8 mol / L urea solution with pH 2.0, and mixed well to obtain an unfolded protein solution, at this time the mass ratio of quinoa protein to urea solution was 1:9, 4 ul 15 mol / L mercaptoethanol solution was added to the unfolded protein solution, and mixed well, at this time the concentration of mercaptoethanol in the mixed solution was 20 mmol / L.

[0135] Two pieces of 7.5cm*2.5cm*0.1cm ordinary glass slides were taken, holes were drilled at the position 1cm away from the short edge of the two glass slides, 100ul of the above unfolded protein solution was smeared on the end without drilling holes, and the other glass plate was overlapped to form a 1cm 2 After the water was drained, the protein adhesive was solidified, a 100g weight was tied on one end with a rope, the upper part of the glass slide that was not overlapped was pinched, and the whole was lifted vertically in the air, and it was observed that the overlapped part of the glass slide did not move obviously.

[0136] Example 11

[0137] The main difference between Example 11 and Example 1 is that:

[0138] At room temperature, 50.0g of urea was dissolved in 50.0g of water to prepare 8mol / L urea, 6mol / L HCl solution was used to adjust the pH of the urea solution to 2.0, 0.3g of protein was dissolved in 2.7g of 8mol / L urea solution with pH 2.0, and was mixed thoroughly to obtain an unfolded protein solution, at this time the mass ratio of quinoa protein to urea solution was 1:9, 4ul of 15mol / L mercaptoethanol solution was added to the unfolded protein solution, and was mixed thoroughly, at this time the concentration of mercaptoethanol in the mixture was 20mmol / L.

[0139] The test method of the protein bonding strength was changed to bamboo skin, and the rest of the conditions were the same as in Example 1, the protein bonding strength measured in Example 11 was 0.63MPa, which showed that the protein adhesive prepared by the present application had different bonding strengths for different materials, and was suitable for gluing wood and glass.

[0140] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", "some embodiments" or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms is not necessarily directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and modified by those skilled in the art without contradiction.

[0141] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A method for preparing an adhesive, characterized in that: The method comprises: A. Mix the protein with a protein denaturant under acidic conditions to obtain an unfolded protein solution; B. adding a reducing agent to the unfolded protein solution to obtain a mixed solution; C. Dehydrating the mixed solution to obtain a solidified adhesive.

2. The method according to claim 1, characterized in that In step A, the protein includes plant protein or animal protein; Optionally, the plant protein comprises any one extracted from soybean, wheat, pea, peanut, and quinoa; Optionally, the protein denaturant comprises guanidine hydrochloride or urea; Optionally, when mixed, the purity of the protein is not less than 80%; Optionally, during mixing, the concentration of the protein denaturant is 4-9 mol / L; Optionally, during mixing, the mass ratio of the protein to the protein denaturant is 1:(5-20); Optionally, the acidic conditions have a pH of 1-4 during mixing.

3. The method according to claim 1, characterized in that In step B, the reducing agent comprises at least one selected from β-mercaptoethanol and / or dithiothreitol; Optionally, in the mixed solution, the concentration of the reducing agent is 10-40 mM.

4. The method according to claim 1, wherein In step C, the dehydration treatment method includes: applying the mixed solution on a carrier, and volatilizing the water to obtain a solidified adhesive; Optionally, the temperature used for volatilizing water during the dehydration treatment is 20-35°C.

5. The method according to claim 1, wherein When the protein is derived from plant seeds, the method further comprises drying and crushing the plant seeds to obtain plant seed powder, defatting the powder, and then extracting the protein; Optionally, the defatting treatment comprises: mixing an organic solvent 1 with the plant seed powder to obtain defatted powder; Optionally, the water activity of the plant seed powder is not higher than 11%; Optionally, the organic solvent 1 comprises at least one selected from n-hexane, diethyl ether, and ethanol; Optionally, the mass ratio of the defatted powder to the organic solvent 1 is 1:(2.5-5); Optionally, the fat content in the defatted powder is not higher than 2%; Optionally, the protein extraction method comprises any one of alkali dissolution and acid precipitation, enzymatic method, and Osborne fractional extraction; Optionally, in the alkali dissolution and acid precipitation, the pH of the alkaline solution is 8.5-9.5, and the pH of the acidic solution is 4.4-4.

6.

6. An adhesive, characterized in that: The adhesive is prepared by the method according to any one of claims 1 to 5.

7. Use of the adhesive according to claim 6 in bonding glass.

8. Use of the adhesive according to claim 6 in bonding wood.

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