Method for extracting insoluble protein powder from wheat bran

Abstract

The invention relates to the technical field of biochemical engineering, in particular to a method for extracting insoluble protein powder from wheat bran. Comprising the following steps: S1, adding lecithin and octyl glycoside into water, stirring for 10-15 minutes, adding wheat bran powder, heating and stirring for 1-3 hours, filtering, washing, drying, adding into a eutectic solvent, heating and stirring for 2-4 hours, centrifuging, washing and drying to obtain pretreated wheat bran powder; s2, adding a complex microbial inoculant into the suspension, and treating for 6-8 hours; centrifuging to obtain supernate and precipitate; wherein the mass ratio of the suspension to the complex microbial inoculant is (60-70): (0.3-0.6); and S3, washing the precipitate, and freeze-drying to obtain the insoluble protein powder. The method is low in cost, green and safe, and the prepared insoluble protein powder is high in purity and suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and particularly relates to a method for extracting insoluble protein powder from wheat bran. Background Art

[0002] Wheat bran is the main by-product in the wheat processing process, and its output accounts for about 10%-15% of the total wheat output. Wheat bran is rich in various nutrients such as protein, dietary fiber, vitamins, and minerals. However, the current utilization of wheat bran mainly focuses on the feed field, with relatively low added value.

[0003] The protein in wheat bran mainly exists in the form of insoluble protein, and its content accounts for about 15%-20% of the dry weight of bran. Insoluble protein has good functional properties such as water-holding capacity, oil-holding capacity, and gelation, and has broad application prospects in the fields of food, medicine, cosmetics, etc. However, traditional wheat bran protein extraction methods have problems such as low extraction rate, low product purity, complex process, and high cost, which limit the large-scale production and application of wheat bran insoluble protein powder. Therefore, it is of great practical significance to develop an efficient, low-cost, and environmentally friendly method for extracting wheat bran insoluble protein powder. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for extracting insoluble protein powder from wheat bran, which can improve the extraction rate and purity of insoluble protein powder, reduce environmental pollution at the same time, and realize the high-value utilization of wheat bran.

[0005] To solve the above technical problems, the present invention adopts the following technical solutions:

[0006] A method for extracting insoluble protein powder from wheat bran, comprising the following steps:

[0007] S1. Add lecithin and octyl glucoside to deionized water, stir for 10-15 min, add wheat bran powder, heat to 40-50 °C, stir for 1-3 h, filter, wash, dry, add to a deep eutectic solvent, heat to 50-60 °C, stir for 2-4 h, centrifuge, wash, and dry to obtain pretreated wheat bran powder;

[0008] S2. Add the pretreated wheat bran powder to water to prepare a bran suspension with a mass fraction of 10%-30%; add a compound bacterial agent to the suspension and treat for 6-8 h; centrifuge to obtain a supernatant and a precipitate; wherein, the mass ratio of the suspension to the compound bacterial agent is 60-70:0.3-0.6;

[0009] S3. Wash the precipitate, freeze-dry to obtain insoluble protein powder.

[0010] Preferably, the deep eutectic solvent in step S1 is prepared by mixing sulfonic acid group-modified choline chloride, citric acid, and glycerol in a mass ratio of 1-2:0.8-1.2:2-3.

[0011] Preferably, the sulfonic acid group-modified choline chloride is prepared by the following method:

[0012] Disperse choline chloride in dimethyl sulfoxide, stir for 5-8 min, add sulfonated-β-cyclodextrin, heat and stir for 45-65 min, then add gallic acid, heat to 60-70 °C, stir for 2-3 h, carry out vacuum distillation, and freeze-dry to obtain sulfonic acid group-modified choline chloride.

[0013] Preferably, the sulfonic acid group-modified choline chloride comprises the following components in parts by mass: 12-16 parts of choline chloride, 1.5-2 parts of sulfonated-β-cyclodextrin, 0.01-0.05 parts of gallic acid, and 70-80 parts of dimethyl sulfoxide.

[0014] Preferably, the chitosan-modified xylanase in step S2 is prepared by the following method:

[0015] A1. Add chitosan to an acetic acid-sodium acetate buffer solution, stir for 15-25 min, and adjust the pH to 5-6 to obtain a chitosan solution; wherein, 3-5 parts of chitosan and 60-70 parts of acetic acid-sodium acetate buffer solution;

[0016] A2. Add xylanase to an acetic acid-sodium acetate buffer solution, stir for 10-15 min to obtain a xylanase solution; wherein, 1-2 parts of xylanase and 60-70 parts of acetic acid-sodium acetate buffer solution;

[0017] A3. Mix the chitosan solution and the xylanase solution, stir for 20-25 min, add a catalytic aid, stir and react for 3-5 h, heat to 60-70 °C, keep warm for 5-8 min, terminate the reaction, centrifuge, collect the supernatant, dialyze with deionized water for 24-36 h, and freeze-dry to prepare chitosan-modified xylanase; wherein, 5-8 parts of chitosan solution, 40-50 parts of xylanase solution, and 0.5-1 part of catalytic aid.

[0018] Preferably, the catalytic aid is prepared by mixing ellagic acid and horseradish peroxidase in a mass ratio of 5-8:0.1-0.3.

[0019] Preferably, step S1 comprises the following components in parts by mass: 0.3-0.6 part of lecithin, 0.1-0.3 part of octyl glucoside, 2-4 parts of deep eutectic solvent, 70-80 parts of deionized water, and 10-15 parts of wheat bran powder.

[0020] Preferably, the compound microbial agent comprises chitosan-modified xylanase, Phanerochaete chrysosporium, and Trichoderma harzianum with a mass ratio of 0.2-0.3:0.8-1.2:1-1.5.

[0021] Preferably, the number of active enzyme units of the chitosan-modified xylanase is 1100-1500 IU / g, the number of viable cells of Phanerochaete chrysosporium is 1×10 8 -2×10 8 CFU / g, and the number of viable cells of Trichoderma harzianum is 1×10 7 -3×10 7 CFU / g.

[0022] Preferably, in the step S2, the treatment steps specifically include: heating to 40-50 °C and standing for 6-8 h at a pH of 4.5-5.5.

[0023] In summary, the present invention has the following beneficial effects:

[0024] In the present invention, sulfonated-β-cyclodextrin and gallic acid are used to treat choline chloride, and sulfonic acid group-modified choline chloride is successfully prepared. The sulfonic acid group is introduced into the choline chloride molecule, significantly enhancing the reaction activity of choline chloride, enabling it to widely participate in more types of chemical reactions; then, the sulfonic acid group-modified choline chloride is mixed with citric acid and glycerol to prepare a deep eutectic solvent. Among them, the presence of the sulfonic acid group can interact with the groups in the cellulose-hemicellulose-lignin composite structure in wheat bran, effectively helping to destroy the composite structure and making the encapsulated insoluble gluten more easily exposed. The addition of citric acid and glycerol further optimizes the solubility and extraction ability of the deep eutectic solvent for proteins, laying a foundation for improving the extraction efficiency of protein powder.

[0025] In the present invention, lecithin and octyl glucoside are used to initially treat wheat bran. This step can destroy part of the cell wall structure of wheat bran and remove impurities at the same time, creating favorable conditions for subsequent treatment. Subsequently, the deep eutectic solvent is used to perform a secondary treatment on wheat bran. By using the method of first treating with lecithin and octyl glucoside, the structure of wheat bran can be preliminarily destroyed, laying a solid foundation for the subsequent in-depth action of the deep eutectic solvent. Such an arrangement of the treatment sequence has multiple advantages. On the one hand, it can make the deep eutectic solvent more efficiently perform its function, reduce the usage amount and treatment time of the deep eutectic solvent, and thus reduce the production cost; on the other hand, first performing a relatively mild treatment with lecithin and octyl glucoside and then using the deep eutectic solvent can effectively avoid the excessive damage to proteins that may occur when the deep eutectic solvent is directly used, which is beneficial to better protecting the activity and structure of proteins, and ultimately realizing the extraction of high-quality wheat bran protein powder.

[0026] With the synergistic catalytic effect of ellagic acid and horseradish peroxidase, the present invention successfully realizes the graft modification of xylanase by chitosan. Horseradish peroxidase initiates an oxidative cross-linking reaction in the system, promoting the formation of a chemical bond connection between chitosan and xylanase. Ellagic acid plays a key role in precisely regulating the reactive sites, optimizing the reaction pathway through its interaction with reaction intermediates or enzyme molecules. The two cooperate with each other, greatly improving the efficiency of the graft reaction, ensuring that chitosan can be evenly and stably grafted onto xylanase, so that the modified xylanase can fully exert its best catalytic function.

[0027] In the enzymatic hydrolysis process of wheat bran, the present invention uses a combined action system of chitosan-modified xylanase, Phanerochaete chrysosporium, and Trichoderma harzianum. Chitosan-modified xylanase can specifically recognize and efficiently degrade non-starch polysaccharide components such as xylan in wheat bran. This process effectively destroys part of the structure of the wheat bran cell wall, creating favorable conditions for the release of the protein encapsulated therein. Phanerochaete chrysosporium can secrete a series of lignin-degrading enzyme systems, such as lignin peroxidase, etc. These enzymes can target and degrade the lignin in wheat bran, further disintegrating the firm structure of the cell wall and promoting the release of protein from the cell wall binding. Trichoderma harzianum can produce a variety of extracellular enzymes, and its role is highly synergistic with that of chitosan-modified xylanase and Phanerochaete chrysosporium. On the one hand, the enzymes it produces can act together with other enzymes to further destroy the cell wall structure and fully expose the protein; on the other hand, the protease it produces can directly act on the protein, hydrolyzing the protein into small peptides and amino acids, significantly improving the protein extraction rate and solubility. This combined action system has significant advantages. Chitosan-modified xylanase, Phanerochaete chrysosporium, and Trichoderma harzianum synergistically destroy the cell wall structure of wheat bran from multiple dimensions, making the protein fully exposed and greatly improving the accessibility of the protein. This effect is far better than the individual action of a single enzyme or microorganism. In the synergistic action mechanism, the destruction of the cell wall polysaccharide structure by chitosan-modified xylanase provides more action targets for the lignin-degrading enzymes of Phanerochaete chrysosporium, making lignin easier to degrade. And the degradation of lignin in turn creates more favorable space and substrate conditions for chitosan-modified xylanase to further act on the internal polysaccharides. At the same time, the moderate hydrolysis of the protein by the protease of Trichoderma harzianum not only increases the solubility of the protein but also alleviates the hindrance of the cell wall structure to the enzyme action to a certain extent, helping other enzymes to more efficiently destroy the cell wall structure, thus comprehensively improving the efficiency and effect of the entire fermentation process. Specific embodiments

[0028] The following further illustrates the present invention in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form.

[0029] Sulfonated-β-cyclodextrin was purchased from Beijing Bailingwei Technology Co., Ltd.; lecithin was purchased from Shaanxi Jinrun Biotechnology Co., Ltd., item number: 2365; octyl glycoside was purchased from Linyi Lvsen Chemical Co., Ltd.; xylanase was purchased from Zhejiang Fuxuan Biotechnology Co., Ltd., production license number: SC20137148200057; Phanerochaete chrysosporium was purchased from Shanghai Jiachu Bioengineering Co., Ltd.; Trichoderma harzianum was purchased from Jinan Binhai Trading Co., Ltd., item number: xls-109; choline chloride was purchased from Anhui Weimao Biotechnology Co., Ltd.; ellagic acid was purchased from Xi'an Xihai Biotechnology Co., Ltd.; horseradish peroxidase was purchased from Xiahe (Shenzhen) Biotechnology Co., Ltd., item number: PE033; acetic acid-sodium acetate buffer solution was purchased from Henan Standard Material Research and Development Center, number: SH-10494-2, pH 4.5.

[0030] Example 1

[0031] A method for extracting insoluble protein powder from wheat bran comprises the following steps:

[0032] S1. Screen the wheat bran to remove impurities, then rinse it with clean water and dry it at 40°C until the moisture content is less than 10%. Crush the dried wheat bran and pass it through a 40-mesh sieve to obtain wheat bran powder. Add lecithin and octyl glucoside to deionized water, stir for 10 minutes, add wheat bran powder, heat to 40°C, stir at 100rpm for 1h, filter, wash 3 times with deionized water, dry at 60°C for 6h, add to a low eutectic solvent, heat to 50°C, stir at 100rpm for 2h, centrifuge at 5000rpm for 3min, wash 3 times with deionized water, dry at 60°C for 6h to obtain pretreated wheat bran powder; wherein, lecithin 0.3 parts, octyl glucoside 0.1 parts, low eutectic solvent 2 parts, deionized water 70 10 parts of wheat bran powder; the low eutectic solvent is prepared by mixing sulfonic acid modified choline chloride, citric acid and glycerol in a mass ratio of 1:0.8:2; the sulfonic acid modified choline chloride is prepared by the following method: dispersing 12 parts of choline chloride in 70 parts of dimethyl sulfoxide, stirring for 5 minutes, adding 1.5 parts of sulfonated-β-cyclodextrin, heating to 50°C, stirring at 100rpm for 45 minutes, then adding 0.01 parts of gallic acid, heating to 60°C, stirring for 2 hours, distilling under reduced pressure, and freeze-drying at -30°C for 24 hours to obtain sulfonic acid modified choline chloride;

[0033] S2. Add the pretreated wheat bran powder to water to prepare a wheat bran suspension with a mass fraction of 10%. Add a compound bacterial agent to the suspension, and let it stand for 6 h at 40 °C and a pH of 4.5. Centrifuge at 3000 rpm for 8 min to obtain a supernatant and a precipitate. Among them, the mass ratio of the suspension to the compound bacterial agent is 60:0.3. The compound bacterial agent includes chitosan-modified xylanase, Phanerochaete chrysosporium, and Trichoderma harzianum with a mass ratio of 0.2:0.8:1. The number of active enzyme units of the chitosan-modified xylanase is 1100 IU / g, the number of viable bacteria of Phanerochaete chrysosporium is 1×10 8 CFU / g, and the number of viable bacteria of Trichoderma harzianum is 1×10 7 CFU / g;

[0034] S3. Wash the precipitate 3 times with deionized water until no reducing sugar is detected in the washing liquid, and freeze-dry at -40 °C for 24 h to obtain an insoluble protein powder.

[0035] Among them, the chitosan-modified xylanase is prepared by the following method:

[0036] A1. Add chitosan to an acetic acid-sodium acetate buffer solution, stir at 80 rpm for 15 min, and adjust the pH to 5 to obtain a chitosan solution. Among them, there are 3 parts of chitosan and 60 parts of the acetic acid-sodium acetate buffer solution;

[0037] A2. Add xylanase to an acetic acid-sodium acetate buffer solution, stir at 80 rpm for 10 min to obtain a xylanase solution. Among them, there is 1 part of xylanase and 60 parts of the acetic acid-sodium acetate buffer solution;

[0038] A3. Mix the chitosan solution and the xylanase solution, stir at 100 rpm for 20 min, add a catalytic assistant, stir and react for 3 h, heat to 60 °C, keep warm for 5 min, terminate the reaction, centrifuge at 5000 rpm for 5 min, collect the supernatant, dialyze with deionized water, select a dialysis bag with a cut-off molecular weight of 20 kDa, dialyze at 4 °C for 24 h, change the deionized water every 12 h, and the volume of the deionized water each time is 20 times the volume of the supernatant. Freeze-dry at -30 °C for 12 h to obtain the chitosan-modified xylanase. Among them, there are 5 parts of the chitosan solution, 40 parts of the xylanase solution, and 0.5 part of the catalytic assistant. The catalytic assistant is prepared by mixing ellagic acid and horseradish peroxidase with a mass ratio of 5:0.1.

[0039] Example 2

[0040] A method for extracting insoluble protein powder from wheat bran includes the following steps:

[0041] S1. Screen wheat bran to remove impurities, then rinse it with clean water and dry it at 60 °C until the moisture content is lower than 10%. Crush the dried wheat bran and pass it through an 80-mesh sieve to obtain wheat bran powder. Add lecithin and octyl glucoside to deionized water, stir for 15 min, add the wheat bran powder, heat to 50 °C, stir at 150 rpm for 3 h, filter, wash with deionized water 3 times, dry at 70 °C for 9 h, add it to the deep eutectic solvent, heat to 60 °C, stir at 150 rpm for 4 h, centrifuge at 5800 rpm for 6 min, wash with deionized water 3 times, and dry at 70 °C for 8 h to obtain pretreated wheat bran powder; among them, 0.6 parts of lecithin, 0.3 parts of octyl glucoside, 4 parts of deep eutectic solvent, 80 parts of deionized water, and 15 parts of wheat bran powder; the deep eutectic solvent is prepared by mixing sulfonic acid group-modified choline chloride, citric acid, and glycerol in a mass ratio of 2:1.2:3; the sulfonic acid group-modified choline chloride is prepared by the following method: Disperse 16 parts of choline chloride in 80 parts of dimethyl sulfoxide, stir for 8 min, add 2 parts of sulfonated-β-cyclodextrin, heat to 60 °C, stir at 200 rpm for 65 min, then add 0.05 parts of gallic acid, heat to 70 °C, stir for 3 h, carry out vacuum distillation, and freeze-dry at -30 °C for 36 h to obtain sulfonic acid group-modified choline chloride;

[0042] S2. Add the pretreated wheat bran powder to water to prepare a wheat bran suspension with a mass fraction of 30%; add a compound microbial agent to the suspension and let it stand at 50 °C and pH 5.5 for 8 h; centrifuge at 5000 rpm for 12 min to obtain a supernatant and a precipitate; among them, the mass ratio of the suspension to the compound microbial agent is 70:0.6; the compound microbial agent includes chitosan-modified xylanase, Phanerochaete chrysosporium, and Trichoderma harzianum with a mass ratio of 0.3:1.2:1.5; the number of active enzyme units of the chitosan-modified xylanase is 1500 IU / g, the number of viable cells of Phanerochaete chrysosporium is 2×10 8 CFU / g, and the number of viable cells of Trichoderma harzianum is 3×10 7 CFU / g;

[0043] S3. Wash the precipitate with deionized water 3 times until no reducing sugar is detected in the washing solution, and freeze-dry at -40 °C for 36 h to obtain insoluble protein powder.

[0044] Among them, the chitosan-modified xylanase is prepared by the following method:

[0045] A1. Add chitosan to an acetic acid-sodium acetate buffer solution, stir at 100 rpm for 25 min, and adjust the pH to 6 to obtain a chitosan solution; among them, 5 parts of chitosan and 70 parts of acetic acid-sodium acetate buffer solution;

[0046] A2. Add xylanase to acetic acid - sodium acetate buffer solution and stir at 100 rpm for 15 min to obtain xylanase solution. Among them, there are 2 parts of xylanase and 70 parts of acetic acid - sodium acetate buffer solution.

[0047] A3. Mix the chitosan solution and xylanase solution, stir at 150 rpm for 25 min, add a catalytic auxiliary agent, stir and react for 5 h, heat to 70 °C, keep warm for 8 min, terminate the reaction, centrifuge at 7000 rpm for 7 min, collect the supernatant, dialyze with deionized water. Select a dialysis bag with a molecular weight cut - off of 20 kDa, dialyze at 4 °C for 24 h, change deionized water every 12 h, and the volume of deionized water each time is 20 times the volume of the supernatant. Freeze - dry at - 30 °C for 12 h to obtain chitosan - modified xylanase. Among them, there are 8 parts of chitosan solution, 50 parts of xylanase solution, and 1 part of catalytic auxiliary agent. The catalytic auxiliary agent is prepared by mixing ellagic acid and horseradish peroxidase in a mass ratio of 8:0.3.

[0048] Example 3

[0049] A method for extracting insoluble protein powder from wheat bran, comprising the following steps:

[0050] S1. Screen wheat bran to remove impurities, then rinse it with clean water and dry it at 50 °C until the moisture content is less than 10%. Crush the dried wheat bran, pass it through an 80 - mesh sieve to obtain wheat bran powder. Add lecithin and octyl glucoside to deionized water, stir for 12 min, add wheat bran powder, heat to 45 °C, stir at 130 rpm for 2 h, filter, wash with deionized water 3 times, dry at 65 °C for 8 h, add it to a deep eutectic solvent, heat to 55 °C, stir at 130 rpm for 3 h, centrifuge at 7000 rpm for 5 min, wash with deionized water 3 times, dry at 65 °C for 7 h to obtain pretreated wheat bran powder. Among them, there are 0.5 parts of lecithin, 0.2 parts of octyl glucoside, 3 parts of deep eutectic solvent, 75 parts of deionized water, and 12 parts of wheat bran powder; the deep eutectic solvent is prepared by mixing sulfonic acid - modified choline chloride, citric acid, and glycerol in a mass ratio of 1.5:1:2.5; sulfonic acid - modified choline chloride is prepared by the following method: Disperse 14 parts of choline chloride in 75 parts of dimethyl sulfoxide, stir for 7 min, add 1.8 parts of sulfonated - β - cyclodextrin, heat to 55 °C, stir at 150 rpm for 55 min, then add 0.03 parts of gallic acid, heat to 65 °C, stir for 2.5 h, carry out vacuum distillation, and freeze - dry at - 30 °C for 30 h to obtain sulfonic acid - modified choline chloride.

[0051] S2. Add the pretreated wheat bran powder to water to prepare a wheat bran suspension with a mass fraction of 20%. Add a compound microbial agent to the suspension, and let it stand for 7 h at 45 °C and a pH of 5. Centrifuge at 4000 rpm for 10 min to obtain a supernatant and a precipitate. Among them, the mass ratio of the suspension to the compound microbial agent is 65:0.5. The compound microbial agent includes chitosan-modified xylanase, Phanerochaete chrysosporium, and Trichoderma harzianum with a mass ratio of 0.25:1:1. The number of active enzyme units of the chitosan-modified xylanase is 1300 IU / g, the number of viable cells of Phanerochaete chrysosporium is 1.5×10 8 CFU / g, and the number of viable cells of Trichoderma harzianum is 2×10 7 CFU / g;

[0052] S3. Wash the precipitate 3 times with deionized water until no reducing sugar is detected in the washing solution, and freeze-dry at -40 °C for 30 h to obtain an insoluble protein powder.

[0053] Among them, the chitosan-modified xylanase is prepared by the following method:

[0054] A1. Add chitosan to an acetic acid-sodium acetate buffer solution, stir at 90 rpm for 20 min, and adjust the pH to 6 to obtain a chitosan solution. Among them, there are 4 parts of chitosan and 65 parts of the acetic acid-sodium acetate buffer solution;

[0055] A2. Add xylanase to an acetic acid-sodium acetate buffer solution, stir at 90 rpm for 13 min to obtain a xylanase solution. Among them, there are 1.5 parts of xylanase and 65 parts of the acetic acid-sodium acetate buffer solution;

[0056] A3. Mix the chitosan solution and the xylanase solution, stir at 130 rpm for 23 min, add a catalytic assistant, stir and react for 4 h, heat to 65 °C, keep warm for 7 min, terminate the reaction, centrifuge at 6000 rpm for 6 min, collect the supernatant, dialyze with deionized water, select a dialysis bag with a cut-off molecular weight of 20 kDa, dialyze at 4 °C for 24 h, change the deionized water every 12 h, and the volume of deionized water each time is 20 times the volume of the supernatant. Freeze-dry at -30 °C for 12 h to obtain the chitosan-modified xylanase. Among them, there are 7 parts of the chitosan solution, 45 parts of the xylanase solution, and 0.8 part of the catalytic assistant. The catalytic assistant is prepared by mixing ellagic acid and horseradish peroxidase with a mass ratio of 7:0.2.

[0057] Comparative Example 1

[0058] Comparative Example 1 is the same as Example 1, except that the procedure in step S1 is different, which is specifically as follows:

[0059] S1. Screen the wheat bran, remove impurities, then rinse it with clean water and dry it at 40 °C until the moisture content is less than 10%. Crush the dried wheat bran and pass it through a 40-mesh sieve to obtain wheat bran powder. Add the wheat bran powder to the deep eutectic solvent, heat it to 50 °C, stir at 100 rpm for 2 h, centrifuge at 5000 rpm for 3 min, wash it 3 times with deionized water, and dry it at 60 °C for 6 h; add lecithin and octyl glucoside to deionized water, stir for 10 min, add the dried wheat bran powder, heat it to 40 °C, stir at 100 rpm for 1 h, filter, wash it 3 times with deionized water, and dry it at 60 °C for 6 h to obtain the pretreated wheat bran powder; among them, 0.3 parts of lecithin, 0.1 part of octyl glucoside, 2 parts of deep eutectic solvent, 70 parts of deionized water, and 10 parts of wheat bran powder; the deep eutectic solvent is prepared by mixing sulfonic acid group-modified choline chloride, citric acid, and glycerol in a mass ratio of 1:0.8:2; the sulfonic acid group-modified choline chloride is prepared by the following method: disperse 12 parts of choline chloride in 70 parts of dimethyl sulfoxide, stir for 5 min, add 1.5 parts of sulfonated-β-cyclodextrin, heat it to 50 °C, stir at 100 rpm for 45 min, then add 0.01 part of gallic acid, heat it to 60 °C, stir for 2 h, perform vacuum distillation, and freeze-dry at -30 °C for 24 h to obtain sulfonic acid group-modified choline chloride.

[0060] Comparative Example 2

[0061] Comparative Example 2 is the same as Example 1, the only difference is that the preparation method of the deep eutectic solvent is different, specifically as follows:

[0062] The deep eutectic solvent is prepared by mixing choline chloride, citric acid, and glycerol in a mass ratio of 1:0.8:2.

[0063] Comparative Example 3

[0064] Comparative Example 3 is the same as Example 1, the only difference is that the catalytic assistant is ellagic acid.

[0065] Comparative Example 4

[0066] Comparative Example 4 is the same as Example 1, the only difference is that the catalytic assistant is horseradish peroxidase.

[0067] Comparative Example 5

[0068] Comparative Example 5 is the same as Example 1, the only difference is that the composite bacterium agent includes chitosan-modified xylanase and Phanerochaete chrysosporium with a mass ratio of 0.2:1.8, the number of active enzyme units of chitosan-modified xylanase is 1100 IU / g, and the number of viable bacteria of Phanerochaete chrysosporium is 1×10 8 CFU / g.

[0069] Comparative Example 6

[0070] Comparative Example 6 is the same as Example 1, except that the composite microbial agent comprises chitosan-modified xylanase and Trichoderma harzianum with a mass ratio of 0.2:1.8. The number of active enzyme units of chitosan-modified xylanase is 1100 IU / g, and the number of viable bacteria of Trichoderma harzianum is 1×10 7 CFU / g.

[0071] Comparative Example 7

[0072] Comparative Example 7 is the same as Example 1, except that the composite microbial agent comprises Phanerochaete chrysosporium and Trichoderma harzianum with a mass ratio of 0.9:1.1. The number of viable bacteria of Phanerochaete chrysosporium is 1×10 8 CFU / g, and the number of viable bacteria of Trichoderma harzianum is 1×10 7 CFU / g.

[0073] Performance Test

[0074] The undissolved protein powders prepared in Examples 1-3 and Comparative Examples 1-7 were tested as follows. Extraction rate = (mass of undissolved protein powder obtained) / (mass of wheat bran raw material) × 100%; The test results are as follows:

[0075] Table 1 Performance Test Results of Protein Powder

[0076] Specimen Number Extraction Rate (%) Purity (%) Example 1 61.3 85.9 Example 2 62.5 88.0 Example 3 64.0 91.2 Comparative Example 1 53.7 80.4 Comparative Example 2 54.6 81.5 Comparative Example 3 54.2 82.0 Comparative Example 4 54.3 82.2 Comparative Example 5 52.7 79.6 Comparative Example 6 52.1 79.1 Comparative Example 7 50.4 78.2

[0077] As can be seen from the results in Table 1, by comparing Example 1 and Comparative Example 1, it can be known that if the deep eutectic solvent is used first, since its main function is to destroy the cell wall structure and dissolve proteins, and without the removal of surface impurities and preliminary structure destruction of bran by lecithin and octyl glucoside, the deep eutectic solvent is difficult to fully penetrate into the interior of bran, and longer time and higher dosage are required to achieve the same extraction effect, which will increase the cost and the risk of protein denaturation. Moreover, directly using the deep eutectic solvent without pretreatment may cause impurities to be dissolved together with proteins, affecting the subsequent separation and purification of proteins. Therefore, changing the order may reduce the efficiency of the entire extraction process and the product quality.

[0078] By comparing Example 1 and Comparative Example 2, it can be known that the introduction of sulfonic acid groups in the deep eutectic solvent can interact with the groups of the composite structure in bran, making the insoluble gluten exposed, creating favorable conditions for subsequent extraction; compared with the traditional extraction method, this deep eutectic solvent shows higher extraction efficiency and selectivity when extracting undissolved proteins from wheat bran. Components such as sulfonic acid groups and citric acid in the deep eutectic solvent can endow the protein powder with a special functional property during the extraction process, broadening the application fields of the protein powder.

[0079] By comparing Example 1 with Comparative Examples 3 and 4, it can be seen that ellagic acid and horseradish peroxidase show a significant synergistic effect in the catalytic process. Horseradish peroxidase first triggers the oxidative cross-linking reaction, building a bridge for the combination of chitosan and xylanase; ellagic acid accurately adjusts the reactive sites and optimizes the reaction path. The two complement each other, greatly improving the efficiency of the grafting reaction, and ensuring that chitosan is successfully grafted onto xylanase in a uniform and stable manner. The structure of the modified xylanase is optimized, and its optimal catalytic function can be fully exerted.

[0080] The experimental results of Example 1 and Comparative Examples 5, 6, and 7 clearly show the unique advantages of chitosan-modified xylanase, Phanerochaete chrysosporium, and Trichoderma harzianum in processing wheat bran. Chitosan-modified xylanase has a significant degradation effect on non-starch polysaccharides such as xylan in wheat bran. Under its action, the cell wall structure is initially destroyed, and the protein originally tightly wrapped therein is more easily released. This lays a good foundation for subsequent further treatment. Phanerochaete chrysosporium degrades lignin in wheat bran by secreting a lignin-degrading enzyme system, especially lignin peroxidase. With the gradual decomposition of lignin, the cell wall structure is further destroyed, and more proteins are freed from the complex structural constraints. The various extracellular enzymes produced by Trichoderma harzianum exert a significant synergistic effect with chitosan-modified xylanase and Phanerochaete chrysosporium. They act together on the cell wall of wheat bran, destroying its structure from multiple dimensions, so that the protein can be fully exposed. Compared with using only a single enzyme or microorganism, this combined treatment method has outstanding advantages and greatly improves the release degree and extraction efficiency of protein.

[0081] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for extracting insoluble protein powder from wheat bran, characterized in that, It includes the following steps: S1. Add lecithin and octyl glucoside to deionized water, stir for 10 - 15 min, add wheat bran powder, heat to 40 - 50 °C, stir for 1 - 3 h, filter, wash, dry, add to the eutectic solvent, heat to 50 - 60 °C, stir for 2 - 4 h, centrifuge, wash, dry to obtain pretreated wheat bran powder; S2. Add the pretreated wheat bran powder to water to prepare a bran suspension with a mass fraction of 10 - 30%; add a compound bacterium agent to the suspension and treat for 6 - 8 h; Centrifuge to obtain supernatant and precipitate; wherein, the mass ratio of the suspension to the compound bacterium agent is 60 - 70:0.3 - 0.6; S3. Wash the precipitate, freeze-dry to obtain insoluble protein powder.

2. The method for extracting insoluble protein powder from wheat bran according to claim 1, characterized in that, In step S1, the eutectic solvent is prepared by mixing sulfonic acid group-modified choline chloride, citric acid, and glycerol with a mass ratio of 1 - 2:0.8 - 1.2:2 - 3.

3. The method for extracting insoluble protein powder using wheat bran according to claim 2, wherein, The sulfonic acid group-modified choline chloride is prepared by the following method: Disperse choline chloride in dimethyl sulfoxide, stir for 5 - 8 min, add sulfonated-β-cyclodextrin, heat and stir for 45 - 65 min, then add gallic acid, heat to 60 - 70 °C, stir for 2 - 3 h, carry out vacuum distillation, freeze-dry to obtain sulfonic acid group-modified choline chloride.

4. The method for extracting insoluble protein powder from wheat bran according to claim 3, characterized in that, The sulfonic acid group-modified choline chloride includes the following components in parts by mass: 12 - 16 parts of choline chloride, 1.5 - 2 parts of sulfonated-β-cyclodextrin, 0.01 - 0.05 parts of gallic acid, and 70 - 80 parts of dimethyl sulfoxide.

5. The method for extracting insoluble protein powder using wheat bran according to claim 1, characterized in that, The chitosan-modified xylanase in step S2 is prepared by the following method: A1. Add chitosan to an acetic acid-sodium acetate buffer solution, stir for 15 - 25 min, adjust the pH to 5 - 6 to obtain a chitosan solution; wherein, 3 - 5 parts of chitosan and 60 - 70 parts of acetic acid-sodium acetate buffer solution; A2. Add xylanase to an acetic acid-sodium acetate buffer solution, stir for 10 - 15 min to obtain a xylanase solution; wherein, 1 - 2 parts of xylanase and 60 - 70 parts of acetic acid-sodium acetate buffer solution; A3. Mix the chitosan solution and the xylanase solution, stir for 20 - 25 min, add a catalytic assistant, stir and react for 3 - 5 h, heat to 60 - 70 °C, keep warm for 5 - 8 min, terminate the reaction, centrifuge, collect the supernatant, dialyze with deionized water for 24 - 36 h, freeze-dry to prepare chitosan-modified xylanase; wherein, 5 - 8 parts of chitosan solution, 40 - 50 parts of xylanase solution, and 0.5 - 1 part of catalytic assistant.

6. The method for extracting insoluble protein powder from wheat bran according to claim 5, characterized in that, The catalytic assistant is prepared by mixing ellagic acid and horseradish peroxidase with a mass ratio of 5 - 8:0.1 - 0.

3.

7. The method for extracting insoluble protein powder from wheat bran according to claim 1, wherein, Step S1 includes the following components in parts by mass: 0.3 - 0.6 parts of lecithin, 0.1 - 0.3 parts of octyl glucoside, 2 - 4 parts of eutectic solvent, 70 - 80 parts of deionized water, and 10 - 15 parts of wheat bran powder.

8. The method for extracting insoluble protein powder from wheat bran according to claim 1, characterized in that, In step S2, the compound bacterium agent includes chitosan-modified xylanase, Phanerochaete chrysosporium, and Trichoderma harzianum with a mass ratio of 0.2 - 0.3:0.8 - 1.2:1 - 1.

5.

9. The method for extracting insoluble protein powder from wheat bran according to claim 8, characterized in that, The number of active enzyme units of the chitosan-modified xylanase is 1,100 - 1,500 IU / g, and the number of viable cells of *Phanerochaete chrysosporium* is 1×10 8 - 2×10 8 CFU / g, and the number of viable cells of *Trichoderma harzianum* is 1×10 7 - 3×10 7 CFU / g.

10. The method for extracting insoluble protein powder using wheat bran according to claim 1, characterized in that, In the step S2, the processing steps specifically include: heating to 40-50°C and standing for 6-8 h at a pH of 4.5-5.5.