Methods and applications for improving the freeze-thaw stability of gluten protein

Abstract

This invention discloses a method for improving the freeze-thaw stability of gluten protein and its application. The method for improving the freeze-thaw stability of gluten protein includes the following steps: defatting gluten powder; subjecting defatted gluten to moist heat treatment; adding L-alanine and preparing a 40% w / w protein solution; and magnetic field-assisted freeze-thaw cycling. This invention uses moist heat treatment combined with static magnetic field-assisted freezing technology, supplemented with a certain amount of L-alanine, to improve the freeze-thaw stability of gluten protein and applies it to dough preparation.

Description

Technical Field

[0001] This invention specifically relates to a method for improving the freeze-thaw stability of gluten protein and its application, belonging to the field of food processing technology. Background Technology

[0002] While frozen dough technology offers advantages such as large-scale production and convenient transportation, the freezing process reduces yeast activity and damages the integrity of the gluten network. Bread made from frozen dough is prone to quality problems such as lower volume, poor water retention, and shorter shelf life.

[0003] Gluten protein, as the most important component of wheat flour, largely determines the edible quality of bread due to its physicochemical properties. Its main function in dough is to impart viscoelasticity and strength. Gluten proteins cross-link through intra- and inter-chain disulfide bonds and non-covalent interactions to form glutenin macropolymers (GMPs). However, freezing treatment damages the structural and functional properties of gluten. GMP depolymerization is the primary cause of the decline in the physicochemical properties of gluten proteins, attributed to the breaking of intermolecular disulfide bonds. In terms of gluten protein structure, GMP depolymerization mainly manifests as an increase in the content of SDS-soluble proteins and free thiol groups, and a decrease in the proportion of disulfide bonds and ordered secondary structures, reducing the ability of gluten proteins to cross-link and form the dough network framework. In addition, freezing treatment also leads to a decrease in the functional properties of gluten proteins, such as reduced water-holding capacity, foaming ability, and foam stability. These negative effects are related to the mechanical damage to the gluten network caused by ice crystal growth and recrystallization during freezing. Summary of the Invention

[0004] To address the problems of poor freeze-thaw stability, severe structural damage from ice crystals, and significantly reduced functional properties in dough prepared using traditional frozen dough techniques compared to fresh gluten protein, this invention provides a method for improving the freeze-thaw stability of gluten protein and its application. By subjecting gluten protein to wet heat treatment and adding L-alanine, and applying a static magnetic field during freezing, the secondary structure of the gluten protein prepared by this method is more stable, the content of free thiol groups is reduced, and the depolymerization of gluten macropolymers is inhibited. The reconstituted dough prepared by this method has higher viscoelasticity and shows good prospects for industrial application.

[0005] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0006] This invention provides a method for improving the freeze-thaw stability of gluten protein, comprising:

[0007] The first modified gluten protein was obtained by defatting and hot heat treatment of gluten protein.

[0008] The first modified gluten protein is mixed with L-alanine and the first solvent to form a first mixed system, which is the hydrated gluten protein suspension (which can be simply referred to as suspension or gluten protein suspension).

[0009] The first mixture is subjected to at least one freeze-thaw treatment, and a static magnetic field is applied to the first mixture during the freezing phase of the freeze-thaw treatment to obtain a second modified gluten protein.

[0010] In a more specific implementation, the method for improving the freeze-thaw stability of gluten protein includes: firstly, subjecting the gluten protein to the defatting treatment to obtain defatted gluten protein, and then subjecting the defatted gluten protein to the wet heat treatment to obtain a first modified gluten protein.

[0011] Furthermore, the method for improving the freeze-thaw stability of gluten protein includes: subjecting the gluten protein to the defatting treatment once or more with dichloromethane.

[0012] Furthermore, the defatting process includes: mixing dichloromethane with gluten protein at room temperature to defatt the gluten protein, and then separating the defatted gluten protein.

[0013] Preferably, the gluten protein can be degreased three times with dichloromethane. After the degreasing treatment, the obtained degreased gluten protein can be placed in a forced-air drying oven and air-dried overnight.

[0014] Furthermore, the mass ratio of gluten protein to dichloromethane is 1.0 to 2.0:1.

[0015] Furthermore, the method for preparing the defatted gluten protein also includes: when the gluten powder is subjected to multiple defatting treatments with dichloromethane, filtration is performed using a Buchner funnel.

[0016] Furthermore, the method also includes: after defatting the gluten powder, first air-drying the defatted gluten protein obtained from the defatting treatment, and then performing the moist heat treatment.

[0017] Furthermore, the method specifically includes: performing the damp heat treatment using a constant temperature and humidity chamber.

[0018] Furthermore, the temperature of the damp heat treatment is 60-90°C and the humidity is 70-90%.

[0019] Furthermore, the duration of the damp heat treatment is 20–40 minutes.

[0020] Preferably, the air-dried defatted gluten protein can be subjected to moist heat treatment in a constant temperature and humidity chamber.

[0021] Furthermore, the mass percentage of the first L-alanine is 0% to 1.5% (gluten-based).

[0022] Furthermore, the first solvent includes deionized water.

[0023] Furthermore, the mass percentage of the defatted gluten protein in the first mixture is 30% to 50%.

[0024] Furthermore, the freeze-thaw process includes a freezing stage and a thawing stage. The freezing stage has a freezing temperature of -18 to -20°C and a freezing time of 22 to 24 hours. The thawing stage has a thawing temperature of 4 to 25°C and a thawing time of 12 to 24 hours.

[0025] Furthermore, the magnetic field strength of the electrostatic field is 0–8 mT.

[0026] Furthermore, the method includes: continuously providing the electrostatic field during the freezing phase.

[0027] In a more specific implementation, the method for improving the freeze-thaw stability of gluten protein further includes: drying the first mixed system after freeze-thaw treatment.

[0028] Furthermore, the method includes drying the first mixed system after freeze-thaw treatment using freeze-drying.

[0029] In another aspect, the present invention provides a second modified gluten protein obtained by the method described above for improving the freeze-thaw stability of gluten protein.

[0030] In another aspect, the present invention provides a method for preparing reconstituted dough, comprising:

[0031] The second modified gluten protein obtained by the method described above for improving the freeze-thaw stability of gluten protein is mixed with starch and a second solvent to form a second mixed system.

[0032] Furthermore, the mass ratio of the second modified gluten protein to the starch and the second solvent is 14:86:59-62.

[0033] Furthermore, the second solvent includes deionized water.

[0034] Furthermore, the manufacturing method specifically includes: processing the second mixed system until a glove film is formed.

[0035] In another aspect, the present invention provides a reconstituted dough, which is obtained by the method for making the reconstituted dough described above.

[0036] Compared with the prior art, the advantages of the present invention include:

[0037] (1) The present invention provides a method for improving the freeze-thaw stability of gluten protein. The raw material used is gluten powder, also known as active gluten powder. It is in abundant supply, low in cost, and does not contain any additional chemical additives. By subjecting defatted gluten protein to wet heat treatment, it can promote the oxidation of free thiol groups to form disulfide bonds, which is beneficial to the cross-linking of gluten protein and improves the stability of the gluten network.

[0038] (2) The present invention provides a method for improving the freeze-thaw stability of gluten protein. The L-alanine used is food grade. L-alanine is cheap, readily available and beneficial to human health. Its side chain has small steric hindrance, which is conducive to the formation of helical structure. This is a characteristic of ice-binding protein, which can inhibit the growth and recrystallization of ice crystals during the freezing stage.

[0039] (3) The present invention provides a method for improving the freeze-thaw stability of gluten protein. The freeze-thaw process realistically simulates the temperature fluctuations that may occur during actual production and distribution. The static magnetic field is a non-thermal technology that is pollution-free, safe and easy to handle. The magnetic field reduces the destructive effect of ice crystals on gluten protein and effectively preserves the integrity of the structure and function of gluten protein, which is harmless to human health.

[0040] (4) The present invention provides a method for making reconstituted dough, which reconstitutes gluten protein with high freeze-thaw stability with wheat starch and mixes it with water for use in dough production. The dough preparation process is simple, reliable and easy to operate, and the prepared dough has high viscoelastic properties. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 This is a bar chart showing the secondary structure content of gluten protein finally obtained in Examples 1-6 and Comparative Examples 1-3 of the present invention;

[0043] Figure 2 This is a bar chart showing the free thiol content of gluten protein finally obtained in Examples 1-6 and Comparative Examples 1-3 of the present invention;

[0044] Figure 3 This is a bar chart showing the GMP content of gluten protein finally obtained in Examples 1-6 and Comparative Examples 1-3 of the present invention;

[0045] Figure 4a , Figure 4b , Figure 4cThese are the rheological behavior characterization curves of the recombinant doughs obtained in Examples 1-6 and Comparative Examples 1-3 of the present invention. Detailed Implementation

[0046] In view of the shortcomings of the prior art, the inventors of this invention, through long-term research and extensive practice, have proposed the technical solution of this invention. The following will further explain and illustrate this technical solution, its implementation process, and its principles.

[0047] In a typical implementation example, a method for preparing reconstituted dough includes the following steps:

[0048] 1) Defatting of gluten powder (i.e., the aforementioned gluten protein, the same below): Mix gluten powder and dichloromethane at a ratio of 100g:150mL, and defat at 25℃ with magnetic stirring for 1h. Then filter using double-layer filter paper. Repeat the above defatting process three times. Finally, air dry overnight in a 25℃ forced-air drying oven to obtain defat gluten protein.

[0049] 2) Moist heat treatment: Spread the defatted gluten protein obtained in step 1) on a petri dish or other container and place it in a constant temperature and humidity incubator. Adjust the temperature of the incubator to 60-90℃ and the humidity to 70-90%. Perform moist heat treatment on the defatted gluten protein for 30 minutes to obtain the first modified gluten protein. After the first modified gluten protein cools to room temperature, air dry it.

[0050] 3) Adding L-alanine and hydration: The first modified gluten protein obtained in step 2) is mixed with L-alanine and deionized water in proportions of 6g:0g:9g, 5.97g:0.03g:9g, 5.94g:0.06g:9g, and 5.91g:0.09g:9g and vortexed to obtain a 40% (w / w) hydrated gluten protein suspension. The amount of L-alanine added is 0%, 0.5%, 1.0%, and 1.5% (dry basis), respectively.

[0051] 4) Magnetic field-assisted freezing: The hydrated gluten protein suspension obtained in step 3) is subjected to one or more freeze-thaw cycles in a magnetic field freezer. During the freezing stage of each freeze-thaw cycle, a static magnetic field with a strength of 0-8 mT is applied to obtain the second modified gluten protein (i.e., recombinant gluten protein). Then, it is freeze-dried and passed through a 100-mesh sieve. The freezing temperature of the freezing stage of each freeze-thaw cycle is -18-20℃ and the freezing time is 22-24h. The thawing temperature of the thawing stage is 4-25℃ and the thawing time is 12-24h.

[0052] 5) Preparation of reconstituted dough: The second modified gluten protein obtained in step 4) is premixed with starch at a mass ratio of 14:86 for 3 minutes. Then the premixed powder is mixed with deionized water at a mass ratio of 100:57-62. The mixture is stirred with a dough mixer for 22 minutes until a glove film can be formed to obtain reconstituted dough. The starch in the dough can be wheat starch, etc.

[0053] Hygrothermal treatment primarily utilizes the oxidation of free thiol groups to form disulfide bonds, promoting cross-linking of gluten proteins and thus increasing the strength of the gluten network. L-alanine, being food-grade, has low steric hindrance in its side chains, which is conducive to the formation of helical structures. This is a crucial structural feature that allows ice-binding proteins to inhibit ice crystal growth and recrystallization. Considering the rotation of polar water molecules under the influence of the Lorentz force, applying a static magnetic field during the freezing stage is beneficial for the formation of small water molecule (or ice crystal) clusters. Furthermore, the presence of a static magnetic field increases the degree of water supercooling, reducing the critical radius for crystal nucleation and thus forming fine, uniform ice crystals to reduce freezing damage to gluten proteins. Magnetic fields offer advantages such as being pollution-free, easy to operate, and flexibly adjustable, reducing ice crystal damage at its source.

[0054] The following will further explain the technical solution, its implementation process and principle in conjunction with specific implementation cases. Unless otherwise specified, the gluten powder, dichloromethane, L-alanine, starch and other materials used in the embodiments of the present invention can all be obtained commercially and are all food grade. The constant temperature and humidity incubator, filtration device, magnetic field generator for providing static magnetic field and freezer used in the embodiments of the present invention are all known to those skilled in the art, and their specific equipment models are not limited here.

[0055] Example 1

[0056] A method for preparing reconstituted dough includes the following steps:

[0057] 1) Defatting of gluten powder: Gluten powder and dichloromethane were mixed magnetically at a ratio of 100g:150mL and defatted for 1 hour at room temperature. Then, the mixture was filtered using double-layer filter paper. The above defatting process was repeated three times. The mixture was then air-dried overnight in a forced-air drying oven at 25°C to obtain defatted gluten protein.

[0058] 2) Moist heat treatment: Spread the defatted gluten protein obtained in step 1) in a petri dish and place it in a constant temperature and humidity incubator. Adjust the temperature in the constant temperature and humidity incubator to 60℃ and the humidity to 80%. Perform moist heat treatment on the defatted gluten protein for 30 minutes to obtain the first modified gluten protein. After the first modified gluten protein cools to room temperature, air dry it.

[0059] 3) Add L-alanine and hydrate: Mix the first modified gluten protein obtained in step 2) with L-alanine at a ratio of 5.97g:0.03g, then dissolve in 3mL of deionized water and vortex to obtain a 40% (w / w) hydrated gluten protein suspension.

[0060] 4) Magnetic field-assisted freezing: The hydrated gluten protein suspension obtained in step 3) is subjected to three freeze-thaw cycles in a magnetic field freezer to obtain the second modified gluten protein. The freezing temperature of the freezing stage of each freeze-thaw cycle is -20℃ and the freezing time is 22h. The thawing temperature of the thawing stage is 4℃ and the thawing time is 12h. A static magnetic field with a magnetic field strength of 8mT is applied during the freezing stage of each freeze-thaw cycle. Then, it is freeze-dried and passed through a 100-mesh sieve.

[0061] 5) Preparation of reconstituted dough: The second modified gluten protein obtained in step 4) is premixed with wheat starch at a mass ratio of 14:86 for 3 minutes. Then the premixed powder is mixed with deionized water at a mass ratio of 100:57. The mixture is stirred with a dough mixer for 22 minutes until a glove membrane can be formed to obtain reconstituted dough.

[0062] Example 2

[0063] A method for preparing reconstituted dough includes the following steps:

[0064] 1) Defatting of gluten powder: Gluten powder and dichloromethane were mixed magnetically at a ratio of 100g:150mL and defatted for 1 hour at room temperature. Then, the mixture was filtered using double-layer filter paper. The above defatting process was repeated three times. The mixture was then air-dried overnight in a forced-air drying oven at 25°C to obtain defatted gluten protein.

[0065] 2) Moist heat treatment: Spread the defatted gluten obtained in step 1) in a petri dish and place it in a constant temperature and humidity incubator. Adjust the temperature in the constant temperature and humidity incubator to 70℃ and the humidity to 70%. Perform moist heat treatment on the defatted gluten protein for 30 minutes to obtain the first modified gluten protein. After the first modified gluten protein cools to room temperature, air dry it.

[0066] 3) Add L-alanine and hydrate: Mix the first modified gluten protein obtained in step 2) with L-alanine at a ratio of 5.94g:0.06g, then dissolve in 3mL of deionized water and vortex to obtain a 40% (w / w) hydrated gluten protein suspension.

[0067] 4) Magnetic field-assisted freezing: The hydrated gluten protein suspension obtained in step 3) is subjected to three freeze-thaw cycles in a magnetic field freezer to obtain the second modified gluten protein. The freezing temperature of the freezing stage of each freeze-thaw cycle is -18℃ and the freezing time is 22h. The thawing temperature of the thawing stage is 25℃ and the thawing time is 12h. A static magnetic field with a magnetic field strength of 8mT is applied during the freezing stage of each freeze-thaw cycle. Then, it is freeze-dried and passed through a 100-mesh sieve.

[0068] 5) Preparation of reconstituted dough: The second modified gluten protein obtained in step 4) is premixed with wheat starch at a mass ratio of 14:86 for 3 minutes. Then, the premixed powder and deionized water are added and mixed at a mass ratio of 100:58. The dough is then mixed with a dough mixer for 22 minutes until a glove membrane can be formed to obtain the reconstituted dough.

[0069] Example 3

[0070] A method for preparing reconstituted dough includes the following steps:

[0071] 1) Defatting of gluten powder: Gluten powder and dichloromethane were mixed magnetically at a ratio of 100g:150mL and defatted for 1 hour at room temperature. Then, the mixture was filtered using double-layer filter paper. The above defatting process was repeated three times. The mixture was then air-dried overnight in a forced-air drying oven at 25°C to obtain defatted gluten protein.

[0072] 2) Moist heat treatment: Spread the defatted gluten protein obtained in step 1) in a petri dish and place it in a constant temperature and humidity incubator. Adjust the temperature in the constant temperature and humidity incubator to 80℃ and the humidity to 90%. Perform moist heat treatment on the defatted gluten protein for 30 minutes to obtain the first modified gluten protein. After the first modified gluten protein cools to room temperature, air dry it.

[0073] 3) Add L-alanine and hydrate: Mix the first modified gluten protein obtained in step 2) with L-alanine at a ratio of 5.91g:0.09g, then dissolve in 3mL of deionized water and vortex to obtain a 40% (w / w) hydrated gluten protein suspension.

[0074] 4) Magnetic field-assisted freezing: The hydrated gluten protein suspension obtained in step 3) is subjected to three freeze-thaw cycles in a magnetic field freezer to obtain the second modified gluten protein. The freezing temperature of the freezing stage of each freeze-thaw cycle is -20℃ and the freezing time is 24h. The thawing temperature of the thawing stage is 4℃ and the thawing time is 22h. A static magnetic field with a magnetic field strength of 8mT is applied during the freezing stage of each freeze-thaw cycle. Then, it is freeze-dried and passed through a 100-mesh sieve.

[0075] 5) Preparation of reconstituted dough: The second modified gluten protein obtained in step 4) is premixed with wheat starch at a mass ratio of 14:86 for 3 minutes. Then the premixed powder is mixed with deionized water at a mass ratio of 100:59. The mixture is stirred with a dough mixer for 22 minutes until a glove membrane can be formed to obtain reconstituted dough.

[0076] Example 4

[0077] A method for preparing reconstituted dough includes the following steps:

[0078] 1) Defatting of gluten powder: Gluten powder and dichloromethane were mixed magnetically at a ratio of 100g:150mL and defatted for 1 hour at room temperature. Then, the mixture was filtered using double-layer filter paper. The above defatting process was repeated three times. The mixture was then air-dried overnight in a forced-air drying oven at 25°C to obtain defatted gluten protein.

[0079] 2) Moist heat treatment: Spread the defatted gluten protein obtained in step 1) in a petri dish and place it in a constant temperature and humidity incubator. Adjust the temperature in the constant temperature and humidity incubator to 70℃ and the humidity to 80%. Perform moist heat treatment on the defatted gluten protein for 30 minutes to obtain the first modified gluten protein. After the first modified gluten protein cools to room temperature, air dry it.

[0080] 3) Add L-alanine and hydrate: Mix the first modified gluten protein obtained in step 2) with L-alanine at a ratio of 5.94g:0.06g, then dissolve in 3mL of deionized water and vortex to obtain a 40% (w / w) hydrated gluten protein suspension.

[0081] 4) Magnetic field-assisted freezing: The hydrated gluten protein suspension obtained in step 3) is subjected to three freeze-thaw cycles in a magnetic field freezer to obtain the second modified gluten protein. The freezing temperature of the freezing stage of each freeze-thaw cycle is -20℃ and the freezing time is 22h. The thawing temperature of the thawing stage is 4℃ and the thawing time is 12h. A static magnetic field with a magnetic field strength of 8mT is applied during the freezing stage of each freeze-thaw cycle. Then, it is freeze-dried and passed through a 100-mesh sieve.

[0082] 5) Preparation of reconstituted dough: The second modified gluten protein obtained in step 4) is premixed with wheat starch at a mass ratio of 14:86 for 3 minutes. Then the premixed powder is mixed with deionized water at a mass ratio of 100:60. The mixture is stirred with a dough mixer for 22 minutes until a glove membrane can be formed to obtain reconstituted dough.

[0083] Example 5

[0084] A method for preparing reconstituted dough includes the following steps:

[0085] 1) Defatting of gluten powder: Gluten powder and dichloromethane were mixed magnetically at a ratio of 100g:150mL and defatted for 1 hour at room temperature. Then, the mixture was filtered using double-layer filter paper. The above defatting process was repeated three times. The mixture was then air-dried overnight in a forced-air drying oven at 25°C to obtain defatted gluten protein.

[0086] 2) Moist heat treatment: Spread the defatted gluten protein obtained in step 1) in a petri dish and place it in a constant temperature and humidity incubator. Adjust the temperature in the constant temperature and humidity incubator to 90℃ and the humidity to 80%. Perform moist heat treatment on the defatted gluten protein for 30 minutes to obtain the first modified gluten protein. After the first modified gluten protein cools to room temperature, air dry it.

[0087] 3) Add L-alanine and hydrate: Mix the first modified gluten protein obtained in step 2) with L-alanine at a ratio of 5.97g:0.03g, then dissolve in 3mL of deionized water and vortex to obtain a 40% (w / w) hydrated gluten protein suspension.

[0088] 4) Magnetic field-assisted freezing: The hydrated gluten protein suspension obtained in step 3) is subjected to three freeze-thaw cycles in a magnetic field freezer to obtain the second modified gluten protein. The freezing temperature of the freezing stage of each freeze-thaw cycle is -20℃ and the freezing time is 22h. The thawing temperature of the thawing stage is 4℃ and the thawing time is 12h. A static magnetic field with a magnetic field strength of 8mT is applied during the freezing stage of each freeze-thaw cycle. Then, it is freeze-dried and passed through a 100-mesh sieve.

[0089] 5) Preparation of reconstituted dough: The second modified gluten protein obtained in step 4) is premixed with wheat starch at a mass ratio of 14:86 for 3 minutes. Then the premixed powder is mixed with deionized water at a mass ratio of 100:61. The mixture is stirred with a dough mixer for 22 minutes until a glove membrane can be formed to obtain reconstituted dough.

[0090] Example 6

[0091] A method for preparing reconstituted dough includes the following steps:

[0092] 1) Defatting of gluten powder: Gluten powder and dichloromethane were mixed magnetically at a ratio of 100g:150mL and defatted for 1 hour at room temperature. Then, the mixture was filtered using double-layer filter paper. The above defatting process was repeated three times. The mixture was then air-dried overnight in a forced-air drying oven at 25°C to obtain defatted gluten protein.

[0093] 2) Moist heat treatment: Spread the defatted gluten protein obtained in step 1) in a petri dish and place it in a constant temperature and humidity incubator. Adjust the temperature in the constant temperature and humidity incubator to 70℃ and the humidity to 90%. Perform moist heat treatment on the defatted gluten protein for 30 minutes to obtain the first modified gluten protein. After the first modified gluten protein cools to room temperature, air dry it.

[0094] 3) Add L-alanine and hydrate: Mix the first modified gluten protein obtained in step 2) with L-alanine at a ratio of 5.91g:0.09g, then dissolve in 3mL of deionized water and vortex to obtain a 40% (w / w) hydrated gluten protein suspension.

[0095] 4) Magnetic field-assisted freezing: The hydrated gluten protein suspension obtained in step 3) was subjected to three freeze-thaw cycles in a magnetic field freezer to obtain the second modified gluten protein. The freezing temperature of the freezing stage in each freeze-thaw cycle was -20℃ and the freezing time was 22h, while the thawing temperature of the thawing stage was 4℃ and the thawing time was 12h. A static magnetic field with a strength of 8mT was applied during the freezing stage of each freeze-thaw cycle, and then the mixture was freeze-dried and passed through a 100-mesh sieve.

[0096] 5) Preparation of reconstituted dough: The second modified gluten protein obtained in step 4) is premixed with wheat starch at a mass ratio of 14:86 for 3 minutes. Then the premixed powder is mixed with deionized water at a mass ratio of 100:62. The mixture is stirred with a dough mixer for 22 minutes until a glove membrane can be formed to obtain reconstituted dough.

[0097] Comparative Example 1

[0098] A method for preparing reconstituted dough includes the following steps:

[0099] 1) Defatting of gluten powder: Gluten powder and dichloromethane were mixed magnetically at a ratio of 100g:150mL and defatted for 1 hour at room temperature. Then, the mixture was filtered using double-layer filter paper. The above defatting process was repeated three times. The mixture was then air-dried overnight in a forced-air drying oven at 25°C to obtain defatted gluten protein.

[0100] 2) Add L-alanine and hydrate: Mix the defatted gluten protein obtained in step 1) with L-alanine at a ratio of 5.94g:0.06g, then dissolve in 3mL of deionized water and vortex to obtain a 40% (w / w) hydrated gluten protein suspension.

[0101] 3) Magnetic field-assisted freezing: The hydrated gluten protein suspension obtained in step 2) is subjected to three freeze-thaw cycles in a magnetic field freezer to obtain modified gluten protein. The freezing temperature of the freezing stage in each freeze-thaw cycle is -20℃ and the freezing time is 22h. The thawing temperature of the thawing stage is 4℃ and the thawing time is 12h. A static magnetic field with a magnetic field strength of 8mT is applied during the freezing stage of each freeze-thaw cycle. Then, it is freeze-dried and passed through a 100-mesh sieve.

[0102] 4) Preparation of reconstituted dough: Premix the modified gluten protein obtained in step 3) with wheat starch at a mass ratio of 14:86 for 3 minutes. Then mix the premixed powder with deionized water at a mass ratio of 100:58 and use a dough mixer to stir for 22 minutes until a glove membrane can be formed to obtain reconstituted dough.

[0103] Comparative Example 2

[0104] A method for preparing reconstituted dough includes the following steps:

[0105] 1) Defatting of gluten powder: Gluten powder and dichloromethane were mixed magnetically at a ratio of 100g:150mL and defatted for 1 hour at room temperature. Then, the mixture was filtered using double-layer filter paper. The above defatting process was repeated three times. The mixture was then air-dried overnight in a forced-air drying oven at 25°C to obtain defatted gluten protein.

[0106] 2) Moist heat treatment: Spread the defatted gluten protein obtained in step 1) in a petri dish and place it in a constant temperature and humidity incubator. Adjust the temperature in the constant temperature and humidity incubator to 70℃ and the humidity to 80%. Perform moist heat treatment on the defatted gluten protein for 30 minutes to obtain the first modified gluten protein. After the first modified gluten protein cools to room temperature, air dry it.

[0107] 3) Add L-alanine and hydrate: Dissolve the first modified gluten protein obtained in step 2) in 3 mL of deionized water and vortex to obtain a 40% (w / w) hydrated gluten protein suspension.

[0108] 4) Magnetic field-assisted freezing: The hydrated gluten protein suspension obtained in step 3) is subjected to three freeze-thaw cycles in a magnetic field freezer to obtain the second modified gluten protein. The freezing temperature of the freezing stage of each freeze-thaw cycle is -18℃ and the freezing time is 22h. The thawing temperature of the thawing stage is 25℃ and the thawing time is 12h. A static magnetic field with a magnetic field strength of 8mT is applied during the freezing stage of each freeze-thaw cycle. Then, it is freeze-dried and passed through a 100-mesh sieve.

[0109] 5) Preparation of reconstituted dough: The second modified gluten protein obtained in step 4) is premixed with wheat starch at a mass ratio of 14:86 for 3 minutes. Then the premixed powder is mixed with deionized water at a mass ratio of 100:59. The mixture is stirred with a dough mixer for 22 minutes until a glove membrane can be formed to obtain reconstituted dough.

[0110] Comparative Example 3

[0111] A method for preparing reconstituted dough includes the following steps:

[0112] 1) Defatting of gluten powder: Gluten powder and dichloromethane were mixed magnetically at a ratio of 100g:150mL and defatted for 1 hour at room temperature. Then, the mixture was filtered using double-layer filter paper. The above defatting process was repeated three times. The mixture was then air-dried overnight in a forced-air drying oven at 25°C to obtain defatted gluten protein.

[0113] 2) Moist heat treatment: Spread the defatted gluten protein obtained in step 1) in a petri dish and place it in a constant temperature and humidity incubator. Adjust the temperature in the constant temperature and humidity incubator to 70℃ and the humidity to 80%. Perform moist heat treatment on the defatted gluten protein for 30 minutes to obtain the first modified gluten protein. After the first modified gluten protein cools to room temperature, air dry it.

[0114] 3) Add L-alanine and hydrate: Mix the first modified gluten protein obtained in step 2) with L-alanine at a ratio of 5.94g:0.06g, then dissolve in 3mL of deionized water and vortex to obtain a 40% (w / w) hydrated gluten protein suspension.

[0115] 4) Magnetic field-assisted freezing: The hydrated gluten protein suspension obtained in step 3) was subjected to three freeze-thaw cycles in a magnetic field freezer to obtain the second modified gluten protein. The freezing temperature of the freezing stage in each freeze-thaw cycle was -20℃ and the freezing time was 22h, while the thawing temperature of the thawing stage was 4℃ and the thawing time was 12h. Then, it was freeze-dried and passed through a 100-mesh sieve.

[0116] 5) Preparation of reconstituted dough: The second modified gluten protein obtained in step 4) is premixed with wheat starch at a mass ratio of 14:86 for 3 minutes. Then the premixed powder is mixed with deionized water at a mass ratio of 100:60. The mixture is stirred with a dough mixer for 22 minutes until a glove membrane can be formed to obtain reconstituted dough.

[0117] To further illustrate the technical effects of the present invention, measurements were performed on the reconstituted doughs obtained from Examples 1-6 and Comparative Examples 1-3, as follows:

[0118] I. Determination of the secondary structure of the gluten protein obtained after final processing (the gluten protein obtained after final processing is the gluten protein used to make reconstituted dough with wheat starch).

[0119] 1) Determination of the secondary structure of gluten protein using Fourier transform infrared spectroscopy: Gluten protein powder obtained after magnetic field-assisted freezing was mixed with potassium bromide (KBr) at a mass ratio of 1:100, ground, and then pressed into transparent sheets with a thickness of 1-2 mm. The wavelength range for spectral acquisition was 600–4000 cm⁻¹. -1 The resolution is set to 4cm. -1 A total of 32 scans were performed. Analysis software was used to analyze the amide I band (1600-1700 cm⁻¹). -1The analysis yielded the relative contents of β-sheets, random coils, β-rotations, and α-helices.

[0120] II. Determination of free sulfhydryl content in the gluten protein obtained after final treatment (the gluten protein obtained after final treatment is the gluten protein used to make reconstituted dough with wheat starch).

[0121] 50 mg of gluten protein powder obtained after magnetic field-assisted cryotherapy was completely dissolved in 5 mL of Tris-glycine buffer (2.5% sodium dodecyl sulfate (SDS), 92 mmol / L glycine, 4.1 mmol / L ethylenediaminetetraacetic acid, 86 mmol / L Tris-HCl, pH 8.0). The solution was then homogenized at high speed for 30 seconds every 10 min, repeated three times. 0.05 mL of Elman's reagent (40 mg 2-nitrobenzoic acid dissolved in 10 mL of buffer, concentration 4 mg / mL) was added, and the mixture was reacted in the dark at 25 °C for 30 min. The supernatant was centrifuged at 4000 × g for 10 min, and the absorbance was measured at 412 nm using a UV-Vis spectrophotometer. The free SH content was calculated using the following formula:

[0122]

[0123] III. Determination of GMP content in the gluten protein obtained after final processing (the gluten protein obtained after final processing is the gluten protein used to make reconstituted dough with wheat starch).

[0124] 20 mg of gluten protein powder obtained after magnetic field-assisted cryotherapy was incubated in 1.5 mL of 0.05 M phosphate-buffered saline (PBS, 2.0% (w / v) SDS, pH 6.8) at room temperature for 1 h. After centrifugation (10,000 × g) for 20 min, the supernatant was filtered through a 0.22 μm membrane to obtain SDS-extractable protein. The precipitate was extracted in 1 mL of 0.05 M PBS (2.0% SDS, 1.0% dithiothreitol (DTT), pH 6.8) for 1 h, and then centrifuged at 10,000 × g for 20 min. The collected supernatant was passed through a 0.22 μm membrane to obtain DTT-extractable protein. Two extractable gluten proteins were eluted using size exclusion high-performance liquid chromatography (HPLC) with a KW-804 column. An 2998 UV detector was used to detect the absorption peak at 214 nm. The column temperature was 30℃, the mobile phase was 50% (v / v) acetonitrile, and the flow rate was 0.7 mL / min. The GMP content was calculated as the ratio of the elution peak area of ​​the DTT-extractable protein to the total elution peak area of ​​the SDS and DTT-extractable proteins.

[0125] IV. Determination of the rheological behavior of reconstituted doughs obtained after different treatments.

[0126] The reconstituted dough was compressed to leave a 1.0 mm gap using a rheometer probe, and then the excess dough was gently scraped off. Under conditions of 0.05% strain and 25°C, the angular frequency range was 1.0-100.0 rad / s, and 6 points were recorded at each order of magnitude. Curves were plotted using storage modulus (G′), loss modulus (G″), and tanδ(G′ / G″) as functions of frequency.

[0127] Depend on Figure 1 , Figure 2 and Figure 3 It can be seen that in all embodiments, the ordered secondary structures (α-helices and β-turns) in the gluten protein after freeze-thaw cycles in Example 4 increased, while the disordered secondary structures (random coils) decreased, and the free thiol content was the lowest, thus inhibiting freeze-induced depolymerization of gluten macropolymers (GMPs). Compared with the comparative example, the ordered secondary structures and GMP content of gluten protein in the examples increased, while the free thiol content decreased, indicating that an 8mT magnetic field can alleviate the damage to the gluten protein structure caused by ice crystals during three freeze-thaw cycles, inhibit the depolymerization of gluten macropolymers (GMPs), and that wet heat treatment can promote the oxidation of free thiols to disulfide bonds, thereby inducing cross-linking of gluten protein. L-alanine can inhibit ice crystal growth and recrystallization, improving the freeze-thaw stability of gluten protein. Figure 4a , Figure 4b , Figure 4c It can be seen that the recombinant dough prepared from the gluten protein obtained in the examples has higher viscoelasticity, indicating that wet heat treatment of gluten protein, addition of an appropriate amount of L-alanine, and application of a static magnetic field during the freezing stage are all beneficial to improving the baking performance of frozen dough.

[0128] The method for improving the freeze-thaw stability of gluten protein provided by this invention produces gluten protein with a more stable secondary structure, inhibiting disulfide bond breaking and GMP depolymerization. Reconstituted dough prepared from this protein exhibits the best viscoelastic properties, which is beneficial for ensuring the quality of subsequent processing. The wet heat treatment involved in this invention is simple to operate, food-grade L-alanine is inexpensive and readily available, and the static magnetic field is efficient, environmentally friendly, and flexibly adjustable, making it suitable for industrial production of flour products.

[0129] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims.

Claims

1. A method for improving the freeze-thaw stability of gluten protein, characterized in that, include: The gluten powder was defatted and subjected to moist heat treatment at a temperature of 60-90°C and a humidity of 70-90% for 20-40 minutes in a constant temperature and humidity chamber to obtain the first modified gluten protein. A first modified gluten protein is mixed with L-alanine and a first solvent to form a first mixed system. The L-alanine is 0.5% to 1.5% by mass based on the total mass of the first modified gluten protein and the L-alanine. The first solvent is deionized water. The first mixed system is a hydrated gluten protein suspension with a mass percentage of 40%. The first mixture is subjected to at least one freeze-thaw cycle, which includes a freezing stage and a thawing stage. The freezing stage has a freezing temperature of -18 to -20°C and a freezing time of 22 to 24 hours. The thawing stage has a thawing temperature of 4 to 25°C and a thawing time of 12 to 24 hours. Furthermore, a static magnetic field with a magnetic field strength of 8 mT is applied to the first mixture during the freezing stage of the freeze-thaw cycle to obtain the second modified gluten protein.

2. The method for improving the freeze-thaw stability of gluten protein according to claim 1, characterized in that, include: The gluten powder is degreased with dichloromethane to obtain defatted gluten protein. After the gluten powder is degreased, the defatted gluten protein is first air-dried and then subjected to the wet heat treatment to obtain the first modified gluten protein.

3. The method for improving the freeze-thaw stability of gluten protein according to claim 2, characterized in that, The mass ratio of the gluten powder to dichloromethane is 1.0~2.0:

1.

4. The method for improving the freeze-thaw stability of gluten protein according to claim 2, characterized in that, The method for preparing the defatted gluten protein further includes: when the gluten powder is subjected to multiple defatting treatments with dichloromethane, filtration is performed using a Buchner funnel.

5. The method for improving the freeze-thaw stability of gluten protein according to claim 1, characterized in that, include: The first modified gluten protein, L-alanine, and the first solvent were mixed at room temperature to obtain the first mixed system.

6. The method for improving the freeze-thaw stability of gluten protein according to claim 1, characterized in that: During the freezing phase, the static magnetic field is continuously provided.

7. The method for improving the freeze-thaw stability of gluten protein according to claim 1, characterized in that, Also includes: The first mixture after freeze-thaw treatment is dried.

8. The method for improving the freeze-thaw stability of gluten protein according to claim 7, characterized in that, include: The first mixed system after freeze-thaw treatment was dried by freeze-drying.

9. The second modified gluten protein prepared by the method for improving the freeze-thaw stability of gluten protein according to any one of claims 1-8.

10. A method for preparing reconstituted dough, characterized in that, include: The second modified gluten protein obtained by the method for improving the freeze-thaw stability of gluten protein according to any one of claims 1-8 is mixed with starch and a second solvent to form a second mixed system, wherein the mass ratio of the second modified gluten protein to the starch and the second solvent is 14:86:55~62, and the second solvent is deionized water. The second mixed system is processed until a glove film is formed.

11. A reconstituted dough, characterized in that: The reconstituted dough is prepared by the method for preparing reconstituted dough according to claim 10.

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