Method for coupling pretreatment to prepare modified protein and use thereof

Abstract

The application belongs to the technical field of protein modification, and provides a method for preparing modified protein by coupling pretreatment and application thereof, the method comprising the following steps: mixing and stirring protein powder with water to prepare protein suspension slurry; under an inert atmosphere, simultaneously performing DBD plasma and alternating magnetic field coupling pretreatment on the protein suspension slurry; performing enzymolysis on the slurry after the pretreatment is completed; deactivating the enzyme, centrifuging, and collecting the enzymolysis liquid; drying the enzymolysis liquid to obtain modified protein. Under the inert gas, DBD low-temperature plasma and alternating magnetic field are integrally and synergistically designed, which greatly reduces the generation of oxidative free radicals, avoids protein oxidative deterioration and odor generation; the plasma and the magnetic field act synchronously, the alternating magnetic field continuously binds and stretches the protein molecules, inhibits the reoxidation and crosslinking of free sulfhydryl, prevents protein reaggregation, and effectively improves the raw material protein dissolution rate and the protein hydrolysis degree. The application can be widely applied in the fields of functional feed, food ingredients, bioactive peptide raw materials and the like.

Description

Technical Field

[0001] This invention belongs to the field of protein modification technology, specifically relating to a method for preparing modified proteins through coupled pretreatment and its application. Background Technology

[0002] Corn gluten meal is a core byproduct of corn starch processing, with a crude protein content of 60%-75%, rich in prolamins and glutenins, making it a high-quality plant protein resource. However, natural corn gluten meal suffers from problems such as dense protein structure, severe particle agglomeration, poor water solubility, and a high proportion of large-molecule proteins. When applied directly, it has low digestibility and poor functional properties, limiting its high-value utilization.

[0003] Existing modification technologies are mainly divided into two categories: physical pretreatment (extrusion, ultrasound, heat treatment) and single / combined enzymatic hydrolysis. For example, using twin-screw extrusion puffing coupled with alkaline protease hydrolysis can improve the degree of hydrolysis, but the high extrusion temperature can easily lead to protein denaturation and nutrient loss. Moreover, using only alkaline protease hydrolysis results in limited substrate specificity and an unbalanced amino acid composition in the hydrolysis products.

[0004] Current enzymatic hydrolysis processes mostly involve simultaneous hydrolysis with a single protease or mixed hydrolysis with multiple proteases. Due to the lack of efficient physical pretreatment, protein particles are not fully dissociated, resulting in a small contact area between the enzyme and substrate and low hydrolysis efficiency. Furthermore, the hydrolysis pH and temperature are often uniform, failing to match the optimal operating conditions for different proteases and hindering the maximization of enzyme activity. In addition, the hydrolysis products are mainly composed of common peptides with low levels of branched-chain amino acids (valine, leucine, and isoleucine), which cannot meet the requirements for functional protein raw materials. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects of existing protein processing technologies, such as nutrient loss, low enzymatic hydrolysis efficiency, unbalanced amino acid composition, and low content of functional components caused by physical pretreatment.

[0006] Therefore, the present invention provides a method for preparing modified proteins through coupling pretreatment, comprising the following steps:

[0007] Protein powder is mixed with water and stirred to form a protein suspension slurry;

[0008] Under an inert atmosphere, the protein suspension slurry was simultaneously pretreated by DBD plasma coupled with an alternating magnetic field;

[0009] The pretreated slurry is then enzymatically hydrolyzed.

[0010] Inactivate the enzyme, centrifuge, and collect the enzyme hydrolysate;

[0011] The enzymatic hydrolysate was dried to obtain the modified protein.

[0012] Specifically, the above-mentioned coupling pretreatment is as follows: the protein suspension slurry is fed into an integrated coupling reaction chamber; the integrated coupling reaction chamber is equipped with a DBD discharge electrode and an alternating magnetic field coil; the air in the integrated coupling reaction chamber is replaced with an inert gas; the DBD discharge electrode and the alternating magnetic field coil are simultaneously activated to perform coupling pretreatment on the slurry.

[0013] Specifically, the discharge power of the aforementioned DBD discharge electrode is 60~90W; the alternating magnetic field strength is 120~180kA / m and the frequency is 300~500Hz; the coupling pretreatment time is 30~50s.

[0014] Specifically, the aforementioned DBD discharge electrode adopts an intermittent pulse working mode, with a discharge time of 7 seconds and an interval of 4 seconds.

[0015] Specifically, the above enzymatic hydrolysis is as follows: after pretreatment, the slurry is heated to 48~52℃; acidic protease is added, and the enzymatic hydrolysis is carried out at a constant temperature for 1.5h~2.5h; the pH of the system is adjusted to 8.0~10, and the temperature is raised to 53~57℃; alkaline protease is added, and the enzymatic hydrolysis is carried out at a constant temperature for 2.5h~3.5h.

[0016] Specifically, based on the dry weight of the protein powder raw material, the amount of acidic protease added is 0.2%~0.5%, and the amount of alkaline protease added is 0.4%~0.8%.

[0017] Specifically, the enzymatic hydrolysis process also includes the addition of diatomaceous earth for debittering; the diatomaceous earth is added simultaneously with the alkaline protease.

[0018] Specifically, based on the total mass of the system, the amount of diatomaceous earth added is 2% to 4%.

[0019] The present invention also provides a modified protein, which is prepared by any of the above-described methods for preparing modified proteins using coupling pretreatment.

[0020] The present invention also provides a method for preparing modified proteins by the above-mentioned coupling pretreatment or the application of the above-mentioned modified proteins in the preparation of functional feeds, food ingredients, and bioactive peptide raw materials.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] This invention provides a method for preparing modified proteins through coupled pretreatment. It employs an integrated design of dielectric barrier discharge (DBD) low-temperature plasma and alternating magnetic field under inert gas conditions. This isolates oxygen, significantly reducing the generation of oxidative free radicals and preventing protein oxidation, deterioration, and off-odors. The plasma and magnetic field act synchronously within the same chamber, eliminating material transfer intervals. The alternating magnetic field continuously binds and relaxes protein molecules, inhibiting the re-oxidation and cross-linking of free thiol groups and preventing protein re-aggregation. Pulsed intermittent discharge allows for heat dissipation time, completely eliminating localized overheating and protein denaturation problems caused by continuous constant-power discharge. Compared to segmented air pretreatment, this method significantly improves protein dissolution rate, retains more free thiol groups, and reduces protein oxidation, effectively increasing the raw material protein dissolution rate and degree of hydrolysis. During enzymatic hydrolysis, the specificity of different proteases' cleavage sites is utilized to progressively degrade large protein molecules. Alkaline proteases can directionally enrich branched-chain amino acids, resulting in a product with high water solubility, high degree of hydrolysis, and high functionality. This product can be widely used in functional feeds, food ingredients, and bioactive peptide raw materials. In addition, the in-situ simultaneous debittering design during the alkaline enzymatic hydrolysis stage eliminates the need for an additional independent decolorization section, and the modified diatomaceous earth can be regenerated and recycled at high temperatures. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0024] This invention provides a method for preparing modified proteins through coupling pretreatment, comprising the following steps:

[0025] S1. Mix protein powder and water at a mass ratio of 1:5 to 1:8 and stir to prepare a protein suspension slurry;

[0026] S2. The protein suspension slurry is fed into an integrated coupling reaction chamber; the integrated coupling reaction chamber is equipped with a DBD discharge electrode and an alternating magnetic field coil.

[0027] Inert gas is continuously introduced into the reaction chamber to replace all the air in the chamber; DBD discharge electrodes and alternating magnetic field coils are simultaneously activated to perform DBD plasma and alternating magnetic field coupling pretreatment on the slurry;

[0028] The plasma operates in an intermittent pulse mode: 7 seconds of discharge followed by 4 seconds of intermittent discharge, with a discharge power controlled at 60-90W and a total processing time of 30-50 seconds. The material inside the chamber is kept at a constant temperature of 25-30℃ throughout the process. An alternating magnetic field operates synchronously with the plasma throughout the process, with a magnetic field strength of 120-180kA / m and a frequency of 300-500Hz. After the dual-field synchronous processing is completed, the slurry is directly transported to the enzymatic hydrolysis tank through pipelines.

[0029] S3. Heat to 48~52℃; add acidic protease at 0.2%~0.5% of the dry weight of protein powder, and hydrolyze at a constant temperature for 1.5h~2.5h.

[0030] Adjust the pH of the system to 8.0-10 and raise the temperature to 53-57℃; add alkaline protease at 0.4%-0.8% of the dry weight of the protein powder and diatomaceous earth at 2%-4% of the total mass of the system, and enzymatically hydrolyze at a constant temperature for 2.5-3.5 hours.

[0031] S4. After enzymatic hydrolysis, heat the solution to completely inactivate the protease and terminate the enzymatic hydrolysis reaction. After inactivation, cool the solution to room temperature and centrifuge to collect the supernatant (enzymatic hydrolysate). Place the enzymatic hydrolysate in a vacuum drying oven and vacuum dry it until the moisture content is ≤8%. After pulverizing, sieve the solution to obtain the modified protein powder product.

[0032] The following specific embodiments illustrate the method for preparing modified proteins through coupling pretreatment according to the present invention and the effects of its application.

[0033] Example 1:

[0034] This embodiment provides a method for preparing modified proteins through coupling pretreatment, including the following steps:

[0035] S1. Add corn gluten powder and deionized protein powder to the mixing tank at a mass ratio of 1:5 (g / mL), turn on the stirring device, set the speed to 300 r / min, and stir continuously for 20 min to prepare a protein suspension slurry.

[0036] S2. The protein suspension slurry is fed into the integrated coupling reaction chamber; the integrated coupling reaction chamber is equipped with a DBD discharge electrode and an alternating magnetic field coil;

[0037] Nitrogen gas is continuously introduced into the reaction chamber to replace all the air in the chamber; DBD discharge electrode and alternating magnetic field coil are simultaneously activated to perform DBD plasma and alternating magnetic field coupling pretreatment on the slurry. The material in the chamber is kept at a constant temperature of 26℃ throughout the process, and the treatment time is 30s.

[0038] DBD plasma uses an intermittent pulse working mode: 7s discharge, 4s interval, and 60W discharge power control.

[0039] The alternating magnetic field strength is 120 kA / m and the frequency is 300 Hz.

[0040] After simultaneous dual-field processing, the slurry is directly transported to the enzymatic hydrolysis tank via pipeline;

[0041] S3. Heat to 48℃; add acidic protease at 0.2% of the dry weight of corn gluten powder, stir for 5 minutes to mix well, and then enzymatically hydrolyze in a sealed container at a constant temperature for 1.5 hours.

[0042] After the first enzymatic hydrolysis is completed, keep stirring and adjust the pH of the system to 8.0 with 1 mol / L sodium hydroxide solution, raise the temperature to 53°C, add alkaline protease accounting for 0.4% of the dry weight of corn gluten powder, and add diatomaceous earth accounting for 2% of the total mass of the slurry system. Stir for 5 minutes and enzymatically hydrolyze at a constant temperature in a sealed tank for 2.5 hours.

[0043] S4. After enzymatic hydrolysis, heat the material to 95℃ and keep it at that temperature for 10 minutes to inactivate the enzyme. After the material cools to room temperature, place it in a centrifuge and centrifuge at 4000 r / min for 15 minutes. Collect the upper layer of enzymatic hydrolysate. Send the enzymatic hydrolysate into a vacuum drying oven, set the temperature to 50℃ and the vacuum degree to -0.08MPa, and vacuum dry until the moisture content of the material is ≤8%. After drying, the material is pulverized by a pulverizer and passed through an 80-mesh standard sieve to obtain the modified corn gluten powder product.

[0044] Example 2:

[0045] This embodiment provides a method for preparing modified proteins through coupling pretreatment, including the following steps:

[0046] S1. Add corn gluten powder and deionized protein powder to the mixing tank at a mass ratio of 1:6.5 (g / mL), turn on the stirring device, set the speed to 300 r / min, and stir continuously for 20 min to prepare a protein suspension slurry.

[0047] S2. The protein suspension slurry is fed into the integrated coupling reaction chamber; the integrated coupling reaction chamber is equipped with a DBD discharge electrode and an alternating magnetic field coil;

[0048] Nitrogen gas is continuously introduced into the reaction chamber to replace all the air in the chamber; DBD discharge electrodes and alternating magnetic field coils are simultaneously activated to perform DBD plasma and alternating magnetic field coupling pretreatment on the slurry. The material in the chamber is kept at a constant temperature of 28°C throughout the process, and the treatment time is 40 seconds.

[0049] DBD plasma uses an intermittent pulse working mode: 7s discharge, 4s interval, and 75W discharge power control.

[0050] Alternating magnetic field strength 150kA / m, frequency 400Hz;

[0051] After simultaneous dual-field processing, the slurry is directly transported to the enzymatic hydrolysis tank via pipeline;

[0052] S3. Heat to 50℃; add acidic protease at 0.35% of the dry weight of corn gluten powder, stir for 5 minutes to mix well, and then enzymatically hydrolyze at a constant temperature in a sealed container for 2 hours.

[0053] After the first enzymatic hydrolysis is completed, keep stirring and adjust the pH of the system to 9.0 with 1 mol / L sodium hydroxide solution, raise the temperature to 55°C, add alkaline protease accounting for 0.6% of the dry weight of corn gluten powder, and add diatomaceous earth accounting for 3% of the total mass of the slurry system. Stir for 5 minutes and enzymatically hydrolyze at a constant temperature in a sealed tank for 3 hours.

[0054] S4. After enzymatic hydrolysis, heat the material to 95℃ and keep it at that temperature for 10 minutes to inactivate the enzyme. After the material cools to room temperature, place it in a centrifuge and centrifuge at 4000 r / min for 15 minutes. Collect the upper layer of enzymatic hydrolysate. Send the enzymatic hydrolysate into a vacuum drying oven, set the temperature to 50℃ and the vacuum degree to -0.08MPa, and vacuum dry until the moisture content of the material is ≤8%. After drying, the material is pulverized by a pulverizer and passed through an 80-mesh standard sieve to obtain the modified corn gluten powder product.

[0055] Example 3:

[0056] This embodiment provides a method for preparing modified proteins through coupling pretreatment, including the following steps:

[0057] S1. Add corn gluten powder and deionized protein powder to the mixing tank at a mass ratio of 1:8 (g / mL), turn on the stirring device, set the speed to 300 r / min, and stir continuously for 20 min to prepare a protein suspension slurry.

[0058] S2. The protein suspension slurry is fed into the integrated coupling reaction chamber; the integrated coupling reaction chamber is equipped with a DBD discharge electrode and an alternating magnetic field coil;

[0059] Nitrogen gas is continuously introduced into the reaction chamber to replace all the air in the chamber; DBD discharge electrodes and alternating magnetic field coils are simultaneously activated to perform DBD plasma and alternating magnetic field coupling pretreatment on the slurry. The material in the chamber is kept at a constant temperature of 30°C throughout the process, and the treatment time is 50 seconds.

[0060] DBD plasma uses an intermittent pulse working mode: 7s discharge, 4s interval, and 90W discharge power control.

[0061] Alternating magnetic field strength 180kA / m, frequency 500Hz;

[0062] After simultaneous dual-field processing, the slurry is directly transported to the enzymatic hydrolysis tank via pipeline;

[0063] S3. Heat to 52℃; add acidic protease at 0.5% of the dry weight of corn gluten powder, stir for 5 minutes to mix well, and then enzymatically hydrolyze in a sealed container at a constant temperature for 2.5 hours.

[0064] After the first enzymatic hydrolysis is completed, keep stirring and adjust the pH of the system to 10.0 using 1 mol / L sodium hydroxide solution. Raise the temperature to 57°C, add alkaline protease accounting for 0.8% of the dry weight of corn gluten powder, and add diatomaceous earth accounting for 4% of the total mass of the slurry system. Stir for 5 minutes and enzymatically hydrolyze at a constant temperature in a sealed tank for 3.5 hours.

[0065] S4. After enzymatic hydrolysis, heat the material to 95℃ and keep it at that temperature for 10 minutes to inactivate the enzyme. After the material cools to room temperature, place it in a centrifuge and centrifuge at 4000 r / min for 15 minutes. Collect the upper layer of enzymatic hydrolysate. Send the enzymatic hydrolysate into a vacuum drying oven, set the temperature to 50℃ and the vacuum degree to -0.08MPa, and vacuum dry until the moisture content of the material is ≤8%. After drying, the material is pulverized by a pulverizer and passed through an 80-mesh standard sieve to obtain the modified corn gluten powder product.

[0066] Comparative Example 1:

[0067] This comparative example provides a method for preparing modified proteins through coupling pretreatment, comprising the following steps:

[0068] S1. Add corn gluten powder and deionized protein powder to the mixing tank at a mass ratio of 1:6.5 (g / mL), turn on the stirring device, set the speed to 300 r / min, and stir continuously for 20 min to prepare a protein suspension slurry.

[0069] S2. The protein suspension slurry is pretreated using the extrusion puffing treatment method in CN102864201A;

[0070] S3. Heat to 50℃; add acidic protease at 0.35% of the dry weight of corn gluten powder, stir for 5 minutes to mix well, and then enzymatically hydrolyze at a constant temperature in a sealed container for 2 hours.

[0071] After the first enzymatic hydrolysis is completed, keep stirring and adjust the pH of the system to 9.0 with 1 mol / L sodium hydroxide solution, raise the temperature to 55°C, add alkaline protease accounting for 0.6% of the dry weight of corn gluten powder, and add diatomaceous earth accounting for 3% of the total mass of the slurry system. Stir for 5 minutes and enzymatically hydrolyze at a constant temperature in a sealed tank for 3 hours.

[0072] S4. After enzymatic hydrolysis, heat the material to 95℃ and keep it at that temperature for 10 minutes to inactivate the enzyme. After the material cools to room temperature, place it in a centrifuge and centrifuge at 4000 r / min for 15 minutes. Collect the upper layer of enzymatic hydrolysate. Send the enzymatic hydrolysate into a vacuum drying oven, set the temperature to 50℃ and the vacuum degree to -0.08MPa, and vacuum dry until the moisture content of the material is ≤8%. After drying, the material is pulverized by a pulverizer and passed through an 80-mesh standard sieve to obtain the modified corn gluten powder product.

[0073] Comparative Example 2:

[0074] This comparative example provides a method for preparing modified proteins through coupling pretreatment, comprising the following steps:

[0075] S1. Add corn gluten powder and deionized protein powder to the mixing tank at a mass ratio of 1:6.5 (g / mL), turn on the stirring device, set the speed to 300 r / min, and stir continuously for 20 min to prepare a protein suspension slurry.

[0076] S2. The protein suspension slurry is fed into the integrated coupling reaction chamber. The integrated coupling reaction chamber is equipped with a DBD discharge electrode and an alternating magnetic field coil. The air environment inside the chamber is maintained, and the material inside the chamber is kept at a constant temperature of 28℃ throughout the process. First, the DBD discharge electrode is started, using an intermittent pulse working mode: discharge for 7 seconds, pause for 4 seconds, discharge power controlled at 75W, and processing time for 40 seconds. Then, the alternating magnetic field coil is started to perform alternating magnetic field pretreatment on the slurry, with an alternating magnetic field strength of 150kA / m and a frequency of 400Hz, and a processing time of 40 seconds.

[0077] After dual-field processing, the slurry is directly transported to the enzymatic hydrolysis tank via pipeline;

[0078] S3. Heat to 50℃; add acidic protease at 0.35% of the dry weight of corn gluten powder, stir for 5 minutes to mix well, and then enzymatically hydrolyze at a constant temperature in a sealed container for 2 hours.

[0079] After the first enzymatic hydrolysis is completed, keep stirring and adjust the pH of the system to 9.0 with 1 mol / L sodium hydroxide solution, raise the temperature to 55°C, add alkaline protease accounting for 0.6% of the dry weight of corn gluten powder, and add diatomaceous earth accounting for 3% of the total mass of the slurry system. Stir for 5 minutes and enzymatically hydrolyze at a constant temperature in a sealed tank for 3 hours.

[0080] S4. After enzymatic hydrolysis, heat the material to 95℃ and keep it at that temperature for 10 minutes to inactivate the enzyme. After the material cools to room temperature, place it in a centrifuge and centrifuge at 4000 r / min for 15 minutes. Collect the upper layer of enzymatic hydrolysate. Send the enzymatic hydrolysate into a vacuum drying oven, set the temperature to 50℃ and the vacuum degree to -0.08MPa, and vacuum dry until the moisture content of the material is ≤8%. After drying, the material is pulverized by a pulverizer and passed through an 80-mesh standard sieve to obtain the modified corn gluten powder product.

[0081] Comparative Example 3:

[0082] This comparative example provides a method for preparing modified proteins through coupling pretreatment, comprising the following steps:

[0083] S1. Add corn gluten powder and deionized protein powder to the mixing tank at a mass ratio of 1:6.5 (g / mL), turn on the stirring device, set the speed to 300 r / min, and stir continuously for 20 min to prepare a protein suspension slurry.

[0084] S2. The protein suspension slurry is fed into the integrated coupling reaction chamber; the integrated coupling reaction chamber is equipped with a DBD discharge electrode and an alternating magnetic field coil;

[0085] Nitrogen gas is continuously introduced into the reaction chamber to replace all the air in the chamber; DBD discharge electrodes and alternating magnetic field coils are simultaneously activated to perform DBD plasma and alternating magnetic field coupling pretreatment on the slurry. The material in the chamber is kept at a constant temperature of 28°C throughout the process, and the treatment time is 40 seconds.

[0086] DBD plasma uses an intermittent pulse working mode: 7s discharge, 4s interval, and 75W discharge power control.

[0087] Alternating magnetic field strength 150kA / m, frequency 400Hz;

[0088] After simultaneous dual-field processing, the slurry is directly transported to the enzymatic hydrolysis tank via pipeline;

[0089] S3. Heat to 50℃; add acidic protease at 0.35% of the dry weight of corn gluten powder, stir for 5 minutes to mix well, and then enzymatically hydrolyze at a constant temperature in a sealed container for 2 hours.

[0090] After the first enzymatic hydrolysis is completed, keep stirring and adjust the pH of the system to 9.0 with 1 mol / L sodium hydroxide solution, raise the temperature to 55°C, add alkaline protease accounting for 0.6% of the dry weight of corn gluten powder, stir for 5 min, and enzymatically hydrolyze at a constant temperature in a sealed tank for 3 h.

[0091] S4. After enzymatic hydrolysis, heat the material to 95℃ and keep it at that temperature for 10 minutes to inactivate the enzyme. After the material cools to room temperature, filter it through activated carbon to remove bitterness, place it in a centrifuge, and centrifuge it at 4000 r / min for 15 minutes. Collect the upper layer of enzymatic hydrolysate. Send the enzymatic hydrolysate into a vacuum drying oven, set the temperature to 50℃ and the vacuum degree to -0.08MPa, and vacuum dry it until the moisture content of the material is ≤8%. After drying, the material is pulverized by a pulverizer and passed through an 80-mesh standard sieve to obtain the modified corn gluten powder product.

[0092] Comparative Example 4:

[0093] This comparative example provides a method for preparing modified proteins using coupled pretreatment, comprising the following steps:

[0094] S1. Add corn gluten powder and deionized protein powder to the mixing tank at a mass ratio of 1:6.5 (g / mL), turn on the stirring device, set the speed to 300 r / min, and stir continuously for 20 min to prepare a protein suspension slurry.

[0095] S2. The protein suspension slurry is fed into the integrated coupling reaction chamber; the integrated coupling reaction chamber is equipped with a DBD discharge electrode and an alternating magnetic field coil;

[0096] Nitrogen gas is continuously introduced into the reaction chamber to replace all the air in the chamber; DBD discharge electrodes and alternating magnetic field coils are simultaneously activated to perform DBD plasma and alternating magnetic field coupling pretreatment on the slurry. The material in the chamber is kept at a constant temperature of 28°C throughout the process, and the treatment time is 40 seconds.

[0097] DBD plasma uses an intermittent pulse working mode: 7s discharge, 4s interval, and 75W discharge power control.

[0098] Alternating magnetic field strength 150kA / m, frequency 400Hz;

[0099] After simultaneous dual-field processing, the slurry is directly transported to the enzymatic hydrolysis tank via pipeline;

[0100] S3. Heat to 50℃; simultaneously add 0.35% acidic protease and 0.6% alkaline protease of corn gluten powder dry weight, and add 3% diatomaceous earth of the total mass of the slurry system. Stir for 5 minutes to mix evenly, then seal the tank and enzymatically hydrolyze at a constant temperature for 5 hours.

[0101] S4. After enzymatic hydrolysis, heat the material to 95℃ and keep it at that temperature for 10 minutes to inactivate the enzyme. After the material cools to room temperature, place it in a centrifuge and centrifuge at 4000 r / min for 15 minutes. Collect the upper layer of enzymatic hydrolysate. Send the enzymatic hydrolysate into a vacuum drying oven, set the temperature to 50℃ and the vacuum degree to -0.08MPa, and vacuum dry until the moisture content of the material is ≤8%. After drying, the material is pulverized by a pulverizer and passed through an 80-mesh standard sieve to obtain the modified corn gluten powder product.

[0102] Example 4:

[0103] The modified corn gluten powders prepared in Examples 1-3 and Comparative Examples 1-4 were tested for their performance indicators. The protein dissolution rate was determined according to the method described in Quan Wenji et al.'s study, "The Promoting Effect of Shear Emulsification Pretreatment on the Proteolytic Reaction of Corn Protein." The degree of hydrolysis was determined according to the method specified in GB / T 22492-2008, where the degree of hydrolysis (%) = (free amino nitrogen after hydrolysis - free amino nitrogen from raw material) / total nitrogen × 100%. The branched-chain amino acid content was determined according to the method specified in GB 5009.124-2016, and the total content of valine, leucine, and isoleucine in the samples was determined using high-performance liquid chromatography. The moisture content was determined according to the method specified in GB 5009.3-2016. The results are shown in Table 1.

[0104] Table 1. Test results of modified protein powder indicators

[0105]

[0106] As can be seen from the results of Example 2 and Comparative Example 1 in Table 1, under the premise that the subsequent processes such as enzymatic hydrolysis and debittering are exactly the same, the protein dissolution rate increased from 54.2% to 86.4% after replacing the high-temperature extrusion puffing pretreatment with DBD plasma coupling alternating magnetic field dual-field coupling pretreatment, which is a significant improvement.

[0107] Comparing the data of Example 2 and Comparative Example 2, it can be seen that, under the premise that the subsequent processes such as enzymatic hydrolysis and debittering are exactly the same, after changing the nitrogen synchronous coupling pretreatment to open air and plasma magnetic field segmented treatment, the protein dissolution rate decreased from 86.4% to 68.2%. The reason is that there is a material transfer interval in the segmented treatment, and the free thiol groups are easily re-oxidized to form disulfide bonds when they lose external force binding. The oxygen in the open environment generates a large number of oxidative free radicals that damage the protein. In this invention, nitrogen isolates oxygen and the dual field synchronous action without interval maintains the loose conformation of the protein throughout the process. The pulse discharge has no local heat accumulation and retains the structure of the enzymatic substrate to the greatest extent.

[0108] Example 2 employs a stepwise adaptive enzymatic hydrolysis mode, first using acidic low-temperature coarse hydrolysis and then adjusting the temperature for alkaline fine hydrolysis. The degree of protein hydrolysis reached 42.8%, significantly better than Comparative Example 4 (28.2%), which involved simultaneous addition of two proteases for hydrolysis without segmentation. This demonstrates that the segmented enzymatic hydrolysis process of the present invention can maximize the activation of protease activity and achieve deep hydrolysis step by step.

[0109] Comparing the data from Example 2 and Comparative Example 3, it can be seen that the in-situ synchronous addition of recyclable modified diatomaceous earth debittering process can effectively retain functional amino acids and improve flavor.

[0110] In summary, the DBD plasma-alternating magnetic field coupled pretreatment combined with gradient stepwise dual-enzyme hydrolysis and in-situ synchronous renewable diatomaceous earth debittering process provided by this invention can simultaneously achieve multiple advantages such as high protein dissolution rate, high degree of hydrolysis, high branched-chain amino acid content, and low bitterness. It solves the single defects of traditional extrusion pretreatment such as high temperature loss of nutrients, multi-enzyme synchronous hydrolysis activity antagonism, loss of functional peptides due to post-decolorization, and heavy bitterness of finished product. The overall quality of the product is superior to existing conventional modification processes, and it has higher application value in the feed and food fields.

[0111] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. A method for preparing modified proteins through coupled pretreatment, characterized in that, Includes the following steps: Protein powder is mixed with water and stirred to form a protein suspension slurry; Under an inert atmosphere, the protein suspension slurry was simultaneously pretreated by DBD plasma coupled with an alternating magnetic field; The pretreated slurry is then enzymatically hydrolyzed. Inactivate the enzyme, centrifuge, and collect the enzyme hydrolysate; The enzymatic hydrolysate was dried to obtain the modified protein.

2. The method for preparing modified proteins by coupling pretreatment as described in claim 1, characterized in that, The coupling pretreatment specifically involves: feeding the protein suspension slurry into an integrated coupling reaction chamber; the integrated coupling reaction chamber is equipped with a DBD discharge electrode and an alternating magnetic field coil; replacing the air in the integrated coupling reaction chamber with an inert gas; and simultaneously activating the DBD discharge electrode and the alternating magnetic field coil to perform coupling pretreatment on the slurry.

3. The method for preparing modified proteins by coupling pretreatment as described in claim 2, characterized in that: The discharge power of the DBD discharge electrode is 60~90W; the alternating magnetic field strength is 120~180kA / m and the frequency is 300~500Hz; the coupling pretreatment time is 30~50s.

4. The method for preparing modified proteins by coupling pretreatment as described in claim 3, characterized in that: The DBD discharge electrode adopts an intermittent pulse working mode, with a discharge time of 7 seconds and an interval of 4 seconds.

5. The method for preparing modified proteins by coupling pretreatment as described in claim 1, characterized in that, The enzymatic hydrolysis specifically involves: heating the pretreated slurry to 48-52°C; adding acidic protease and hydrolyzing at a constant temperature for 1.5-2.5 hours; adjusting the pH of the system to 8.0-10 and heating to 53-57°C; adding alkaline protease and hydrolyzing at a constant temperature for 2.5-3.5 hours.

6. The method for preparing modified proteins by coupling pretreatment as described in claim 5, characterized in that: Based on the dry weight of the protein powder raw material, the amount of acidic protease added is 0.2%~0.5%, and the amount of alkaline protease added is 0.4%~0.8%.

7. The method for preparing modified proteins by coupling pretreatment as described in claim 5, characterized in that: The enzymatic hydrolysis process also includes the addition of diatomaceous earth for debittering; the diatomaceous earth is added simultaneously with the alkaline protease.

8. The method for preparing modified proteins by coupling pretreatment as described in claim 7, characterized in that: The amount of diatomaceous earth added is 2% to 4% based on the total mass of the system.

9. A modified protein, characterized in that: The modified protein is prepared using the method for preparing modified protein by coupling pretreatment as described in any one of claims 1-8.

10. The method for preparing modified protein by coupling pretreatment as described in any one of claims 1-8, or the application of the modified protein as described in claim 9 in the preparation of functional feed, food ingredients, and bioactive peptide raw materials.

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