A process for extracting wheat protein by ultrasonic method and wheat protein and its application

Abstract

The invention relates to the technical field of protein extraction, in particular to an ultrasonic wheat protein extraction process and wheat protein and application, which solves the problems of active oxygen and hydroxyl free radicals causing oxidation of protein, reducing protein active sites, deteriorating stability and poor protein dispersibility in the process of ultrasonic wheat protein extraction. The scheme specifically comprises: selecting high-quality wheat, defatting, crushing and screening, mixing the obtained defatted wheat flour with polyphenol compounds, and then embedding, adding the embedded particles into a phosphate buffer, mixing trehalose with water, ultrasonically crushing the two after blending, slowly adding trehalose, placing in ice water after the reaction is completed to terminate the reaction, obtaining a glycosylated wheat protein crude extract, and centrifugally drying to obtain wheat protein; the invention can realize high-efficiency extraction under an ultrasonic environment, while making the protein have better antioxidant capacity and higher group activity, and better dispersibility in water.

Description

Technical Field

[0001] The invention relates to the technical field of protein extraction, and in particular to an ultrasonic method for extracting wheat protein, wheat protein and application thereof. Background Art

[0002] Wheat contains about 13% protein, of which there are four main types of proteins, namely albumin, globulin, prolamin and glutenin; albumin accounts for 3% to 5% of the total wheat protein, mainly exists in the germ, is soluble in water and dilute salt solution, has poor thermal stability, and is denatured at 60°C; globulin accounts for 6% to 10% of wheat protein, is soluble in 10% NaCl, and is insoluble in water; prolamin accounts for 40% to 50% of the total wheat protein, is the main protein of wheat, is insoluble in water and neutral salt solution, and is soluble in 70% to 90% ethanol; glutenin accounts for 30% to 40% of the total wheat protein, is insoluble in water or ethanol, and is soluble in acid or alkali solutions.

[0003] In the prior art, alkali dissolution and acid precipitation are mostly used to extract wheat protein, and the extraction rate is about 70%. In order to improve the extraction efficiency, the wheat germ is physically crushed before treatment by ultrasonic crushing, which can greatly improve the extraction efficiency of wheat protein without destroying the primary structure of the protein. However, due to the cavitation effect generated by ultrasound, active oxygen and hydroxyl free radicals will be generated in the solution to react with the protein after ultrasound treatment. After the ultrasonic treatment, the disulfide bonds of the protein are broken, resulting in the exposure of the hydrophobic groups. The dispersibility in the aqueous solution is poor, the spatial stability of the amino acid residues and the protein is poor, and the active groups are easily inactivated. As a result, compared with the protein obtained by other routes, the functional characteristics of the wheat protein extracted by ultrasound cannot be fully exerted, and the ability to form hydrogen bonds is not as strong as other proteins. Therefore, the protein after ultrasonic cavitation treatment cannot meet the requirements for protein in the cosmetics field, and the protein obtained by alkali dissolution and acid precipitation has low purity and the extraction rate is not high enough. Summary of the invention

[0004] In order to solve the problems that during the ultrasonic extraction of wheat protein, the active oxygen and hydroxyl free radicals generated by the cavitation effect react with the active groups of the protein, resulting in a reduction in active sites and poor protein stability; and the hydrophobic groups are exposed due to the looseness of the protein, resulting in poor protein dispersibility, etc., the present invention provides a process for extracting wheat protein by ultrasonic method to solve the above problems.

[0005] First, this application includes the following steps:

[0006] S1. Select high-quality wheat, defatted the wheat, crushed and sieved it to obtain defatted wheat flour;

[0007] S2, mixing defatted wheat flour and polyphenol compounds in a mass ratio of 1:0.4-1.2, and then using carboxymethyl cellulose for embedding treatment to form embedded particles, adding the embedded particles into phosphate buffer to obtain a base solution, mixing trehalose with water to obtain a glycosylation base solution, and mixing the base solution with the glycosylation base solution to obtain a pre-ultrasonication solution;

[0008] S3, subjecting the pre-ultrasonic treatment solution to ultrasonic crushing, and slowly adding trehalose, the ultrasonic treatment power is 150-300W, the temperature is 35-60°C, and the time is 20-70min. After the reaction is completed, the solution is placed in ice water to terminate the reaction, and a glycosylated wheat protein crude extract is obtained, which is centrifugally dried to obtain wheat protein;

[0009] The mass ratio of defatted wheat flour to trehalose in the liquid before ultrasonic treatment is 8:1-6.

[0010] In the method steps of the present application, wheat is first pretreated to remove lipid substances in the wheat that affect the dissolution of protein, so as to avoid covalent bonding between lipids and proteins in the subsequent extraction process. Then, polyphenol compounds are added to the defatted wheat flour containing wheat protein. The hydroxyl groups of the polyphenol compounds have a strong electron donating effect, can undergo nucleophilic addition, and have good antioxidant capacity. The antioxidant capacity brought about by the simple addition of polyphenol compounds not only relies on the formation of hydrogen bonds between the hydroxyl groups of the polyphenols and the hydroxyl groups or amino groups of the proteins, but also the non-polar aromatic ring groups in the polyphenols can interact with the hydrophobic amino acids of the proteins to form non-covalent forces, thereby protecting the effective active groups of the proteins.

[0011] Secondly, because the polyphenols themselves are affected by the ultrasonic cavitation effect, and the proteins are also attacked by active oxygen and hydroxyl radicals in the solution, the present invention uses carboxymethyl cellulose to embed the defatted wheat flour and the polyphenol compounds to avoid excessive consumption of polyphenols. During the ultrasonic treatment, the cavitation effect destroys the embedding layer, which causes the wheat protein therein to be slowly released and the active sites to be exposed. Then, the glycosylation reaction is carried out with the trehalose in the glycosylation base liquid to form a sugar chain, thereby stabilizing the protein whose disulfide bond is broken due to the ultrasound. While stabilizing the protein, the hydroxyl group of the trehalose is combined with the amide group of the protein in the form of hydrogen bonds, thereby improving the denaturation resistance of the protein. The trehalose on the sugar chain and the free trehalose are wrapped around the wheat protein molecules and filled in the spatial structure of the wheat protein molecules, thereby protecting the active sites and fixing the protein spatial structure loosened due to the ultrasonic treatment.

[0012] Furthermore, the step of defatting the wheat is: first, the wheat is placed in a drying oven at a temperature of 110-130°C for dehydration for 50-60 minutes, and then the wheat is treated by supercritical carbon dioxide extraction, with an extraction pressure of 32MPa, an extraction temperature of 40-50°C, a separation pressure of 5MPa, a separation temperature of 35°C, and a sieved particle size of 100-300 meshes.

[0013] The proportion of wheat protein in wheat flour increases after defatted, and the dehydration treatment makes it easy to remove impurities such as non-protein starch, making wheat protein easier to separate.

[0014] Furthermore, the polyphenol compound is one of chlorogenic acid, resveratrol, anthocyanidin, isoflavone, catechin, cocoa polyphenol, tartrate, quercetin, rutin or curcumin.

[0015] Furthermore, the pH of the phosphate buffer is 7.0-7.8.

[0016] Further, the steps of the embedding treatment are as follows:

[0017] A mixture of defatted wheat flour and polyphenol compounds is added to an ethanol aqueous solution for mixing to form a wheat protein-polyphenol mixed solution; carboxymethyl cellulose is then dissolved in clean water to obtain a carboxymethyl cellulose solution, the wheat protein-polyphenol mixed solution is added dropwise to the carboxymethyl cellulose solution, and carboxymethyl cellulose embedded particles are obtained after homogenization and stirring, the carboxymethyl cellulose embedded particles are subjected to rotary evaporation, the carboxymethyl cellulose embedded particles are washed with deionized water, the pH value of the embedded particles is adjusted to 4-5, and impurities are removed by strong centrifugation to obtain the embedded particles.

[0018] In the present application, defatted wheat flour and polyphenol compounds are dissolved in an ethanol solution, and then dispersed in an aqueous solution of carboxymethyl cellulose by dripping. By continuous stirring, the wheat protein-polyphenol mixed solution can be well dispersed, which is convenient for carboxymethyl cellulose to form a thin film of uniform size on the outside of the wheat protein-polyphenol composite molecules. The thickness of the carboxymethyl cellulose film is gradually increased by flow, thereby delaying the direct reaction of defatted wheat flour with active oxygen and hydroxyl free radicals in the subsequent ultrasonic treatment step, forming an effect of slowly releasing wheat protein in the carboxymethyl cellulose embedding layer, and increasing the glycosylation reaction ratio. In order to reduce the influence of ethanol on wheat protein in the embedded particles, ethanol is removed by rotary evaporation to isolate the influence of hydroxyl groups, and the pH is adjusted to place the protein in a slightly acidic environment to reduce the influence of hydroxyl free radicals.

[0019] Furthermore, the liquid volume ratio of the wheat protein-polyphenol mixed solution to the carboxymethyl cellulose solution is 1:2-5, the rotation speed of the homogenizing stirring is 900-1200 r / min, and the stirring time is 0.5-2 h.

[0020] Furthermore, the volume fraction of the ethanol aqueous solution is 75%, and the solid-liquid ratio of defatted wheat flour to the ethanol aqueous solution is 8-10g:100ml.

[0021] Furthermore, the solid-liquid ratio of the carboxymethyl cellulose to the clean water is 1g:500ml.

[0022] On the other hand, the present invention also obtains a wheat protein prepared by any of the above-mentioned ultrasonic wheat protein extraction processes.

[0023] The wheat protein of the present application can be applied to the field of cosmetics.

[0024] The wheat protein prepared in the present application retains more active groups and connection sites, and can be used in toiletries. It increases the ability of wheat protein to combine with keratin through hydrogen bonds, and the protein molecules also have sugar chains that can attach to the attachment sites, thereby enhancing the connection with the skin surface or keratin such as hair, and has a strong repair ability. The protein is highly stable and is not easily denatured when adapted with other chemical substances.

[0025] The beneficial effects of the present invention are:

[0026] The wheat protein obtained in the present application has a high extraction rate and a stable structure. After ultrasonic treatment, it also has a strong antioxidant ability and is not easily affected by hydroxyl free radicals and reactive oxygen. Sugar chains are formed on the molecular chains of wheat protein through glycosylation reactions. The protein has more hydroxyl groups, which increases the solubility of wheat protein in water and solves the problem of decreased solubility of protein in water caused by exposed hydrophobic groups due to ultrasonic treatment. The trehalose used on the sugar chains can be connected to the amide groups through hydrogen bonds, which can further stabilize the protein molecules whose secondary structures are destroyed due to ultrasonic treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the fluorescence intensity diagram of wheat in the embodiment of the present invention and the comparative example. DETAILED DESCRIPTION

[0028] The following will refer to the attached Figure 1 The embodiments of the present invention are described in detail. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the protection scope of the present invention.

[0029] The wheat used in this application is selected from selected varieties with a protein content of up to 13%, specifically the following:

[0030] The Xi'nong 20 wheat variety in Shaanxi Province was bred by the College of Agriculture of Northwest A&F University. The protein content of Xi'nong 20 was measured to be as high as 16.7%, with good stability and high extraction and application value.

[0031] Chengmai 791 is a variety of wheat grown in the southern part of the Huanghuai winter wheat area. This variety of wheat was selected and bred by Henan Dacheng Seed Co., Ltd. Chengmai 791 was found to have a protein content of up to 14.2%, high water absorption rate, and strong stability in water, which is more in line with the application requirements in the field of water-soluble cosmetics.

[0032] The wheat used in this application is not limited to the above wheat varieties. High-quality wheat with a protein content higher than 13% can be used for the ultrasonic wheat protein extraction process of this application.

[0033] Example 1: Preparation of embedded particles

[0034] Wheat defatting treatment: The wheat was placed in a drying oven at a temperature of 120°C for dehydration for 50 minutes, and then the wheat was treated by supercritical carbon dioxide extraction, with an extraction pressure of 32MPa, an extraction temperature of 45°C, a separation pressure of 5MPa, a separation temperature of 35°C, and a sieved particle size of 180 meshes to obtain defatted wheat flour;

[0035] Particle embedding treatment: weigh a certain amount of defatted wheat flour, mix catechins and defatted wheat flour at a weight ratio of 0.8:1, add the mixed catechins and defatted wheat flour into a 75% volume fraction ethanol aqueous solution, use 100 ml of ethanol aqueous solution for every 10 g of defatted wheat flour, mix at 700 r / min for 2 h, and form a wheat protein-polyphenol mixed solution;

[0036] Weigh carboxymethyl cellulose and dissolve it in clean water to obtain a carboxymethyl cellulose solution, dissolve 1 g of carboxymethyl cellulose in 500 ml of clean water, add 1 unit volume of wheat protein-polyphenol mixed solution dropwise into 3.5 units of carboxymethyl cellulose solution according to the volume ratio, stir at a speed of 1000 r / min for 1 hour to obtain embedded particles wrapped with carboxymethyl cellulose, use a rotary evaporator to treat the embedded particles to remove ethanol, then use deionized water to soak the embedded particles, use 1 mol / L HCl solution to adjust the pH value of the deionized water to 5.0, finally centrifuge at a centrifugal speed of 5000 r / min for 5 minutes to remove large particle impurities, collect the residue and dry it to obtain embedded particles.

[0037] Example 2: Preparation of embedded particles

[0038] Wheat defatting treatment: The wheat was placed in a drying oven at a temperature of 110°C for dehydration for 60 minutes, and then the wheat was treated by supercritical carbon dioxide extraction, with an extraction pressure of 32MPa, an extraction temperature of 40°C, a separation pressure of 5MPa, a separation temperature of 35°C, and a sieved particle size of 120 meshes to obtain defatted wheat flour;

[0039] Particle embedding treatment: weigh a certain amount of defatted wheat flour, mix cocoa polyphenols and defatted wheat flour at a weight ratio of 0.4:1, add the mixed cocoa polyphenols and defatted wheat flour into a 75% volume fraction ethanol aqueous solution, use 100 ml of ethanol aqueous solution for every 8 g of defatted wheat flour, mix at 700 r / min for 2 h, and form a wheat protein-polyphenol mixed solution;

[0040] Weigh carboxymethyl cellulose and dissolve it in clean water to obtain a carboxymethyl cellulose solution, dissolve 1 g of carboxymethyl cellulose in 500 ml of clean water, add 1 unit volume of wheat protein-polyphenol mixed solution dropwise into 2 units of carboxymethyl cellulose solution according to the volume ratio, stir at a speed of 900 r / min for 2 hours to obtain embedded particles wrapped with carboxymethyl cellulose, use a rotary evaporator to treat the embedded particles to remove ethanol, then use deionized water to soak the embedded particles, use 1 mol / L HCl solution to adjust the pH value of the deionized water to 4.5, finally centrifuge at a centrifugal speed of 4000 r / min for 10 minutes to remove large particle impurities, collect the residue and dry it to obtain embedded particles.

[0041] Example 3: Preparation of embedded particles

[0042] Wheat defatting treatment: The wheat was placed in a drying oven at a temperature of 130°C for dehydration for 55 minutes, and then the wheat was treated by supercritical carbon dioxide extraction, with an extraction pressure of 32MPa, an extraction temperature of 50°C, a separation pressure of 5MPa, a separation temperature of 35°C, and a sieved particle size of 240 meshes to obtain defatted wheat flour;

[0043] Particle embedding treatment: weigh a certain amount of defatted wheat flour, mix isoflavones and defatted wheat flour in a weight ratio of 1.2:1, add the mixed isoflavones and defatted wheat flour into a 75% volume fraction ethanol aqueous solution, use 100 ml of ethanol aqueous solution for every 9 g of defatted wheat flour, mix at 700 r / min for 2 h, and form a wheat protein-polyphenol mixture;

[0044] Weigh carboxymethyl cellulose and dissolve it in clean water to obtain a carboxymethyl cellulose solution, dissolve 1 g of carboxymethyl cellulose in 500 ml of clean water, add 1 unit volume of wheat protein-polyphenol mixed solution dropwise into 5 units of carboxymethyl cellulose solution according to the volume ratio, stir at a speed of 1200 r / min for 0.5 h to obtain embedded particles wrapped with carboxymethyl cellulose, use a rotary evaporator to treat the embedded particles to remove ethanol, then use deionized water to soak the embedded particles, use 1 mol / L HCl solution to adjust the pH value of the deionized water to 4.0, finally centrifuge at a centrifugal speed of 5000 r / min for 5 min to remove large particle impurities, collect the residue and dry it to obtain embedded particles.

[0045] The polyphenol compounds in the above embodiments may also be replaced by chlorogenic acid, resveratrol, anthocyanidin, tartrate, quercetin, rutin or curcumin.

[0046] Comparative preparation example 1 of embedded particles

[0047] The difference between this comparative example and the embedded particle preparation example 1 is that the polyphenol compound and defatted wheat flour are not compounded and mixed. The specific differences are as follows:

[0048] Wheat defatting treatment: The wheat was placed in a drying oven at a temperature of 120°C for dehydration for 50 minutes, and then the wheat was treated by supercritical carbon dioxide extraction, with an extraction pressure of 32MPa, an extraction temperature of 45°C, a separation pressure of 5MPa, a separation temperature of 35°C, and a sieved particle size of 180 meshes to obtain defatted wheat flour;

[0049] Particle embedding treatment: weigh a certain amount of defatted wheat flour, add the defatted wheat flour into 75% volume fraction ethanol aqueous solution, use 100 ml ethanol aqueous solution for every 10 g defatted wheat flour, mix at 700 r / min for 2 h, to form wheat protein solution;

[0050] Weigh carboxymethyl cellulose and dissolve it in clean water to obtain a carboxymethyl cellulose solution. Dissolve 1 g of carboxymethyl cellulose in 500 ml of clean water. Add 1 unit volume of wheat protein solution dropwise to 3.5 units of carboxymethyl cellulose solution according to the volume ratio. Stir at a speed of 1000 r / min for 1 hour to obtain embedded particles wrapped with carboxymethyl cellulose. Use a rotary evaporator to treat the embedded particles to remove ethanol. Then use deionized water to soak the embedded particles. Use 1 mol / L HCl solution to adjust the pH value of the deionized water to 5.0. Finally, centrifuge at a centrifugal speed of 5000 r / min for 5 minutes to remove large particle impurities. Collect the residue and dry it to obtain embedded particles.

[0051] Comparative Preparation Example 2 of Embedded Particles

[0052] The difference between this comparative example and the embedded particle preparation example 1 is that carboxymethyl cellulose and defatted wheat flour are not compounded and mixed. The specific differences are as follows:

[0053] Wheat defatting treatment: The wheat was placed in a drying oven at a temperature of 120°C for dehydration for 50 minutes, and then the wheat was treated by supercritical carbon dioxide extraction, with an extraction pressure of 32MPa, an extraction temperature of 45°C, a separation pressure of 5MPa, a separation temperature of 35°C, and a sieved particle size of 180 meshes to obtain defatted wheat flour;

[0054] Particle embedding treatment: weigh a certain amount of defatted wheat flour, mix catechins and defatted wheat flour at a weight ratio of 0.8:1, add the mixed catechins and defatted wheat flour into a 75% volume fraction ethanol aqueous solution, use 100 ml of ethanol aqueous solution for every 10 g of defatted wheat flour, mix at 700 r / min for 2 h, and form a wheat protein-polyphenol mixed solution;

[0055] Weigh gelatin and dissolve it in clean water to obtain a gelatin solution. Dissolve 2 g of gelatin in 500 ml of clean water at 50° C. Add 1 unit volume of wheat protein-polyphenol mixed solution dropwise into 3.5 units of gelatin solution according to the volume ratio. Stir at a speed of 1000 r / min for 1 hour to obtain gelatin-wrapped embedded particles. Use a rotary evaporator to treat the embedded particles to remove ethanol. Then use deionized water to soak the embedded particles. Use 1 mol / L HCl solution to adjust the pH value of the deionized water to 5.0. Finally, centrifuge at a centrifugal speed of 5000 r / min for 5 minutes to remove large particle impurities. Collect the residue and dry it to obtain embedded particles.

[0056] Comparative preparation example 3 of embedded particles

[0057] The difference between this comparative preparation example and the embedded particle preparation example 1 is that the wheat is not defatted. The specific differences are as follows:

[0058] Wheat pretreatment: The wheat was placed in a drying oven at 120°C for dehydration for 50 minutes, and air-milled at 35°C to obtain wheat flour with a sieved particle size of 200 mesh;

[0059] Particle embedding treatment: weigh a certain amount of wheat flour, mix catechins and wheat flour at a weight ratio of 0.8:1, add the mixed catechins and wheat flour into a 75% volume fraction ethanol aqueous solution, use 100 ml ethanol aqueous solution for every 10 g wheat flour, mix at 700 r / min for 2 h, and form a wheat protein-polyphenol mixed solution;

[0060] Weigh carboxymethyl cellulose and dissolve it in clean water to obtain a carboxymethyl cellulose solution, dissolve 1 g of carboxymethyl cellulose in 500 ml of clean water, add 1 unit volume of wheat protein-polyphenol mixed solution dropwise into 3.5 units of carboxymethyl cellulose solution according to the volume ratio, stir at a speed of 1000 r / min for 1 hour to obtain embedded particles wrapped with carboxymethyl cellulose, use a rotary evaporator to treat the embedded particles to remove ethanol, then use deionized water to soak the embedded particles, use 1 mol / L HCl solution to adjust the pH value of the deionized water to 5.0, finally centrifuge at a centrifugal speed of 5000 r / min for 5 minutes to remove large particle impurities, collect the residue and dry it to obtain embedded particles. Example 1

[0061] This embodiment provides a wheat protein, which is as follows:

[0062] The embedded particles prepared in the embedded particles preparation example 1 are added into a phosphate buffer having a total phosphate concentration of 0.05 mol / L and a pH of 7.4 at a material ratio of 1 g:100 mL to obtain a base solution;

[0063] Mixing trehalose and water to prepare a 0.10 mol / L glycosylation base solution, mixing the base solution with the glycosylation base solution, wherein the mass ratio of defatted wheat flour to trehalose is 8:3, to obtain a solution before ultrasonic treatment;

[0064] The liquid before ultrasonic treatment was ultrasonically crushed and trehalose was slowly added to maintain the trehalose concentration in the reaction solution at 0.10 mol / L. The ultrasonic treatment power was 180 W, the temperature was 50°C, and the time was 50 min. After the reaction was completed, it was placed in ice water to terminate the reaction to obtain a crude extract of glycosylated wheat protein, which was then centrifuged and dried to obtain wheat protein. Example 2

[0065] This embodiment provides a wheat protein, which is as follows:

[0066] The embedded particles prepared in the embedded particles preparation example 2 are added into a phosphate buffer having a total phosphate concentration of 0.05 mol / L and a pH of 7.0 at a material ratio of 1 g:100 mL to obtain a base solution;

[0067] Mixing trehalose and water to prepare a 0.5 mol / L glycosylation base solution, mixing the base solution with the glycosylation base solution, wherein the mass ratio of defatted wheat flour to trehalose is 8:1, to obtain a solution before ultrasonic treatment;

[0068] The liquid before ultrasonic treatment was ultrasonically crushed and trehalose was slowly added to maintain the trehalose concentration in the reaction solution at 0.5 mol / L. The ultrasonic treatment power was 150 W, the temperature was 55° C., and the time was 70 min. After the reaction was completed, it was placed in ice water to terminate the reaction to obtain a crude extract of glycosylated wheat protein, which was then centrifuged and dried to obtain wheat protein. Example 3

[0069] This embodiment provides a wheat protein, which is as follows:

[0070] The embedded particles prepared in the embedded particles preparation example 3 are added into a phosphate buffer having a total phosphate concentration of 0.05 mol / L and a pH of 7.8 at a material ratio of 1 g:100 mL to obtain a base solution;

[0071] Mixing trehalose and water to prepare a 0.15 mol / L glycosylation base solution, mixing the base solution with the glycosylation base solution, wherein the mass ratio of defatted wheat flour to trehalose is 8:6, to obtain a solution before ultrasonic treatment;

[0072] The liquid before ultrasonic treatment was ultrasonically crushed and trehalose was slowly added to maintain the trehalose concentration in the reaction solution at 0.15 mol / L. The ultrasonic treatment power was 300 W, the temperature was 40°C, and the time was 30 min. After the reaction was completed, it was placed in ice water to terminate the reaction to obtain a crude extract of glycosylated wheat protein, which was then centrifuged and dried to obtain wheat protein.

[0073] Comparative Example 1

[0074] The difference between this comparative example and Example 1 is that the embedded particles in Comparative Preparation Example 1 are used to extract wheat protein, specifically as follows:

[0075] The embedded particles prepared in the embedded particles comparative preparation example 1 were added into a phosphate buffer having a total phosphate concentration of 0.05 mol / L and a pH of 7.4 at a material ratio of 1 g:100 mL to obtain a base solution;

[0076] Mixing trehalose and water to prepare a 0.10 mol / L glycosylation base solution, mixing the base solution with the glycosylation base solution, wherein the mass ratio of defatted wheat flour to trehalose is 8:3, to obtain a solution before ultrasonic treatment;

[0077] The liquid before ultrasonic treatment was ultrasonically crushed and trehalose was slowly added to maintain the trehalose concentration in the reaction solution at 0.10 mol / L. The ultrasonic treatment power was 180 W, the temperature was 50°C, and the time was 50 min. After the reaction was completed, it was placed in ice water to terminate the reaction to obtain a crude extract of glycosylated wheat protein, which was then centrifuged and dried to obtain wheat protein.

[0078] Comparative Example 2

[0079] The difference between this comparative example and Example 1 is that the embedded particles in Comparative Preparation Example 2 are used to extract wheat protein, specifically as follows:

[0080] The embedded particles prepared in the embedded particles comparative preparation example 2 were added into a phosphate buffer having a total phosphate concentration of 0.05 mol / L and a pH of 7.4 at a material ratio of 1 g:100 mL to obtain a base solution;

[0081] Mixing trehalose and water to prepare a 0.10 mol / L glycosylation base solution, mixing the base solution with the glycosylation base solution, wherein the mass ratio of defatted wheat flour to trehalose is 8:3, to obtain a solution before ultrasonic treatment;

[0082] The liquid before ultrasonic treatment was ultrasonically crushed and trehalose was slowly added to maintain the trehalose concentration in the reaction solution at 0.10 mol / L. The ultrasonic treatment power was 180 W, the temperature was 50°C, and the time was 50 min. After the reaction was completed, it was placed in ice water to terminate the reaction to obtain a crude extract of glycosylated wheat protein, which was then centrifuged and dried to obtain wheat protein.

[0083] Comparative Example 3

[0084] The difference between this comparative example and Example 1 is that the embedded particles in Comparative Preparation Example 3 are used to extract wheat protein, as follows:

[0085] The embedded particles prepared in the embedded particles comparative preparation example 3 were added into a phosphate buffer having a total phosphate concentration of 0.05 mol / L and a pH of 7.4 at a material ratio of 1 g:100 mL to obtain a base solution;

[0086] Mixing trehalose and water to prepare a 0.10 mol / L glycosylation base solution, mixing the base solution with the glycosylation base solution, wherein the mass ratio of defatted wheat flour to trehalose is 8:3, to obtain a solution before ultrasonic treatment;

[0087] The liquid before ultrasonic treatment was ultrasonically crushed and trehalose was slowly added to maintain the trehalose concentration in the reaction solution at 0.10 mol / L. The ultrasonic treatment power was 180 W, the temperature was 50°C, and the time was 50 min. After the reaction was completed, it was placed in ice water to terminate the reaction to obtain a crude extract of glycosylated wheat protein, which was then centrifuged and dried to obtain wheat protein.

[0088] Comparative Example 4

[0089] The difference between this comparative example and Example 1 is that no embedding treatment is performed, but direct glycosylation is performed. The specific differences are as follows:

[0090] Wheat defatting treatment: The wheat was placed in a drying oven at a temperature of 120°C for dehydration for 50 minutes, and then the wheat was treated by supercritical carbon dioxide extraction, with an extraction pressure of 32MPa, an extraction temperature of 45°C, a separation pressure of 5MPa, a separation temperature of 35°C, and a sieved particle size of 180 meshes to obtain defatted wheat flour;

[0091] Weigh a certain amount of defatted wheat flour, mix catechins and defatted wheat flour in a weight ratio of 0.8:1, add distilled water, stir at a speed of 600 r / min, stir for 2 h, and vacuum dry to obtain wheat-polyphenol powder;

[0092] The wheat-polyphenol powder is added into a phosphate buffer having a total phosphate concentration of 0.05 mol / L and a pH of 7.4 at a material ratio of 1 g:100 mL to obtain a base solution;

[0093] Mixing trehalose and water to prepare a 0.10 mol / L glycosylation base solution, mixing the base solution with the glycosylation base solution, wherein the mass ratio of defatted wheat flour to trehalose is 8:3, to obtain a solution before ultrasonic treatment;

[0094] The liquid before ultrasonic treatment was ultrasonically crushed and trehalose was slowly added to maintain the trehalose concentration in the reaction solution at 0.10 mol / L. The ultrasonic treatment power was 180 W, the temperature was 50°C, and the time was 50 min. After the reaction was completed, it was placed in ice water to terminate the reaction to obtain a crude extract of glycosylated wheat protein, which was then centrifuged and dried to obtain wheat protein.

[0095] Comparative Example 5

[0096] The difference between this comparative example and Example 1 is that trehalose is not used for glycosylation, but glucose is used for glycosylation. The specific differences are as follows:

[0097] The embedded particles prepared in the embedded particles comparative preparation example 1 were added into a phosphate buffer having a total phosphate concentration of 0.05 mol / L and a pH of 7.4 at a material ratio of 1 g:100 mL to obtain a base solution;

[0098] Mixing glucose and water to prepare a 0.10 mol / L glycosylation base solution, mixing the base solution with the glycosylation base solution, wherein the mass ratio of defatted wheat flour to glucose is 8:6, to obtain a solution before ultrasonic treatment;

[0099] The liquid before ultrasonic treatment was ultrasonically crushed and glucose was slowly added to maintain the glucose concentration in the reaction solution at 0.10 mol / L. The ultrasonic treatment power was 180 W, the temperature was 50°C, and the time was 50 min. After the reaction was completed, it was placed in ice water to terminate the reaction to obtain a crude extract of glycosylated wheat protein, which was then centrifuged and dried to obtain wheat protein.

[0100] Experiments and data

[0101] The wheat proteins prepared in Examples 1-3 and Comparative Examples 1-5 were tested using the following methods:

[0102] Stability test method: The crystallization ability of the protein is compared to determine the stability of the protein's spatial structure. The protein is dissolved in the organic solvent MPD and PEG is added dropwise to cause the wheat protein to crystallize.

[0103] Test standard: weigh 500 mg of protein and dissolve it in 10 ml of MPD, then add a certain amount of PEG and measure the amount of precipitation.

[0104] Activity test method: Since the proteins in this application are mostly applicable to the cosmetics field, the interaction activity effect of anthocyanin, the core product of cosmetics research and development, is now determined. The test method is: use a phosphate buffer with a pH of 7.4 to prepare a 50 mg / L anthocyanin solution, weigh the wheat protein for the experiment, dissolve it in a 75% ethanol solution, and prepare it to 100 mg / L; add the anthocyanin solution to the wheat protein solution, and bathe it in a constant temperature water bath for 1 hour, and perform fluorescence spectrum measurement, with an excitation wavelength of 280nm, a wavelength of 300-450nm, and a slit width of 5nm between the excitation wavelength and the emission wavelength; the synchronous fluorescence spectrum measurement conditions are: 25℃. The fixed wavelength difference between the excitation wavelength and the emission wavelength is 15nm and 60nm, and the spectrum is scanned.

[0105] Test standard: The fluorescence quenching phenomenon is mainly used to compare the fluorescence intensity emitted by tryptophan in wheat protein. The fluorescence intensity of tryptophan affected by active oxygen and hydroxyl free radicals is low, while the fluorescence intensity of unaffected tryptophan is high.

[0106] Dispersibility test method: Mix the protein with water, stir and centrifuge, and measure the PDI value of the protein mass of the solution and the total mass of added protein.

[0107] The measurement results are shown in Table 1:

[0108]

[0109] The fluorescence intensity of each embodiment and comparative example is plotted as a line graph. Figure 1 shown.

[0110] analyze

[0111] As can be seen from Table 1, the wheat protein prepared in the examples of the present application has high crystallinity, fluorescence intensity and protein dispersion index, which proves that the spatial conformation of its protein structure is stable, its activity is high enough, and it can provide enough active sites, which is suitable for use in cosmetics. The protein has good solubility and is easy to be added to an aqueous solution for use.

[0112] No polyphenol compounds were added during the preparation of the wheat protein in Comparative Example 1, and the remaining steps were the same as those in the examples of the present application. The data of Comparative Example 1 showed that its fluorescence intensity decreased significantly, indicating that it had fewer active sites that could bind to anthocyanins, proving that the mixed compound of polyphenol compounds can help wheat protein resist oxidation during the ultrasonic process, thereby improving the activity of wheat protein.

[0113] In the preparation of the wheat protein of Comparative Example 2, carboxymethyl cellulose was not used, but gelatin was used to embed defatted wheat flour. The remaining steps were the same as those in the embodiment. The data of Comparative Example 1 showed that its crystallinity was low, proving that the wheat protein of Comparative Example 1 had poor structural stability, was not easy to refold, had a bad spatial conformation, and had the lowest fluorescence intensity, proving that the wheat in Comparative Example 1 had few active sites. The reason was that gelatin and trehalose reacted under an ultrasonic environment, reducing the degree of protein glycosylation. Therefore, the protein had fewer sugar chains and hydroxyl groups on the surface, and its solubility and dispersibility in water were lower. It can be proved that the use of carboxymethyl cellulose can ensure the glycosylation of protein to the greatest extent.

[0114] The wheat protein in Comparative Example 3 was not defatted during preparation, and there was still a certain amount of lipid on the protein surface. The data showed that the crystallinity was poor, the fluorescence intensity was medium, and the water solubility was poor, proving that defatting treatment could enhance the glycosylation process and slightly reduce the activity. The lipids also had a certain antioxidant capacity. Because lipids were insoluble in water, the water solubility of Comparative Example 3 was poor, proving that defatting treatment could affect the spatial conformation of wheat protein and its dispersibility in water.

[0115] The wheat protein in Comparative Example 4 was not encapsulated during preparation, but was directly glycosylated. The data in Table 1 show that its crystallinity and solubility and dispersibility are good, but the activity of the wheat protein in Comparative Example 4 is poor, which proves that the use of encapsulation can prevent the wheat protein from being affected by external factors during the ultrasound process. The spatial structure and dispersibility in water prove that encapsulation does not affect the glycosylation process, but reduces the reaction of wheat protein with active oxygen and hydroxyl radicals during the glycosylation process.

[0116] The wheat protein in Comparative Example 5 was not glycosylated with trehalose, but was glycosylated with glucose. The data showed that the crystallinity and solubility and dispersibility of Comparative Example 5 were better, but the fluorescence intensity was moderate, and the activity was lower than that of the example, proving that the activity protection of wheat protein after glycosylation with glucose was not as high as that of trehalose, but the stability of the spatial conformation and water solubility and dispersibility were better.

[0117] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

Claims

1. A process for extracting wheat protein by ultrasonic method, characterized in that: The following steps are involved: S1. Select high-quality wheat, defatted the wheat, crushed and sieved it to obtain defatted wheat flour; S2, mixing defatted wheat flour and polyphenol compounds in a mass ratio of 1:0.4-1.2, and then using carboxymethyl cellulose for embedding treatment to form embedded particles, adding the embedded particles into phosphate buffer to obtain a base solution, mixing trehalose with water to obtain a glycosylation base solution, and mixing the base solution with the glycosylation base solution to obtain a pre-ultrasonication solution; S3, subjecting the pre-ultrasonic treatment solution to ultrasonic crushing, and slowly adding trehalose, the ultrasonic treatment power is 150-300W, the temperature is 35-60°C, and the time is 20-70min. After the reaction is completed, the solution is placed in ice water to terminate the reaction, and a glycosylated wheat protein crude extract is obtained, which is centrifugally dried to obtain wheat protein; The steps of the embedding process are as follows: The mixture of defatted wheat flour and polyphenol compounds is added to an ethanol aqueous solution for mixing to form a wheat protein-polyphenol mixed solution; carboxymethyl cellulose is then dissolved in clean water to obtain a carboxymethyl cellulose solution, the wheat protein-polyphenol mixed solution is added dropwise to the carboxymethyl cellulose solution, and the carboxymethyl cellulose embedded particles are obtained after homogenization and stirring, the carboxymethyl cellulose embedded particles are subjected to rotary evaporation, the carboxymethyl cellulose embedded particles are washed with deionized water, the pH value of the embedded particles is adjusted to 4-5, and impurities are removed by strong centrifugation to obtain embedded particles; The mass ratio of defatted wheat flour to trehalose in the liquid before ultrasonic treatment is 8:1-6; The polyphenol compound is isoflavone, catechin or cocoa polyphenol; Catechin and defatted wheat flour were mixed in a weight ratio of 0.8:1, and then added to an ethanol-water solution, using 100 ml of ethanol-water solution for every 10 g of defatted wheat flour; Cocoa polyphenols and defatted wheat flour are mixed in a weight ratio of 0.4:1, and then added to an ethanol-water solution, using 100 ml of ethanol-water solution for every 8 g of defatted wheat flour; Isoflavones and defatted wheat flour were mixed in a weight ratio of 1.2:1 and then added to an ethanol-water solution, using 100 ml of the ethanol-water solution for every 9 g of defatted wheat flour.

2. The process for extracting wheat protein by ultrasonic method according to claim 1, characterized in that: The steps of defatting the wheat are as follows: firstly, the wheat is placed in a drying oven at a temperature of 110-130° C. for dehydration for 50-60 minutes, and then the wheat is treated by supercritical carbon dioxide extraction, with an extraction pressure of 32 MPa, an extraction temperature of 40-50° C., a separation pressure of 5 MPa, a separation temperature of 35° C., and a sieved particle size of 100-300 meshes.

3. The process for extracting wheat protein by ultrasonic method according to claim 1, characterized in that: The pH of the phosphate buffer is 7.0-7.

8.

4. The process for extracting wheat protein by ultrasonic method according to claim 1, characterized in that: The liquid volume ratio of the wheat protein-polyphenol mixed solution to the carboxymethyl cellulose solution is 1:2-5, the rotation speed of the homogenizing stirring is 900-1200 r / min, and the stirring time is 0.5-2h.

5. The process for extracting wheat protein by ultrasonic method according to claim 1, characterized in that: The volume fraction of the ethanol aqueous solution is 75%, and the solid-liquid ratio of defatted wheat flour to the ethanol aqueous solution is 8-10g:100ml.

6. The process for extracting wheat protein by ultrasonic method according to claim 1, characterized in that: The solid-liquid ratio of the carboxymethyl cellulose to the clean water is 1g:500ml.

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