Wheat gliadin with good color and luster and glutenin with high solubility as well as preparation method and application of wheat gliadin and glutenin

Abstract

The invention belongs to the technical field of cereal protein processing, and particularly relates to wheat gliadin with good color and luster and glutenin with high solubility as well as a preparation method and application of the wheat gliadin and the glutenin. According to the method, a cold water solubility control coupling one-step freezing purification technology is broken through, the concentration of ethanol and the temperature of the solution are controlled through cold water, the dissolving state and aggregation behavior of wheat gliadin are synchronously regulated and controlled, then freezing treatment and a one-step method are coupled to achieve controllable protein aggregation, precipitation and freezing, and the purposes of separation and purification are achieved. The technology is green, efficient, simple, convenient and energy-saving, the obtained alcohol-soluble protein is good in color and luster, and industrial production is easy to realize. Furthermore, a membrane separation coupling high-pressure microjet technology is broken through, and on the basis of a technology for obtaining a glutenin concentrated solution through membrane separation, the protein concentrated solution is directly treated by adopting an alkaline condition synchronous high-pressure microjet circulating modification method, so that the structural characteristics such as the scale and the surface hydrophobicity of the target protein and the aggregation behavior are further regulated and controlled; the glutenin is endowed with better solubility.

Description

Technical Field

[0001] The invention belongs to the technical field of grain protein processing, and in particular relates to wheat alcohol-soluble protein and high-soluble glutenin with good color, and a preparation method and application thereof. Background Art

[0002] Wheat is one of the three major grain crops in the world. As one of the earliest countries in the world to grow wheat, China's wheat production has always ranked first in the world. Gluten is one of the main products after wheat starch processing. It has the advantages of abundant raw materials and low prices. Gluten is rich in protein (more than 80%), and its amino acid composition is balanced and has high nutritional value. The main components of protein contained in gluten are wheat alcohol-soluble protein (about 45-50%) and glutenin (about 30-40%). Among them, wheat alcohol-soluble protein has the function of providing adhesion and ductility, while glutenin has the function of providing elasticity and toughness. After separating the two, they can be used as food ingredients to meet the needs of different products for protein functional properties. Therefore, the development of an efficient and simple separation method is crucial to increasing the added value of gluten and realizing multi-scenario applications.

[0003] Wheat alcohol-soluble protein is a single peptide chain that forms a spherical three-dimensional structure through intramolecular disulfide bonds, hydrogen bonds and hydrophobic interactions. It is insoluble in water and easily soluble in 70-90% ethanol solution. Wheat alcohol-soluble protein is usually extracted by rotary evaporation after alcohol enrichment. Rotary evaporation is time-consuming and energy-consuming, has safety risks, and is not conducive to industrial production. Therefore, it is very important to develop a green and simple method for preparing wheat alcohol-soluble protein.

[0004] Gluten is a heterogeneous macromolecular aggregate rich in glutamine and proline. Gluten is usually prepared by alkali dissolution and acid precipitation. During the acid precipitation process, gluten will undergo structural changes and aggregation near the isoelectric point of the protein, resulting in extremely poor solubility (<10%), which in turn limits its application in food. Therefore, it is of great significance to break through the preparation method of highly soluble gluten.

[0005] In summary, considering the resource advantages and application prospects of wheat prolamin and glutenin in gluten powder, it is of great significance to develop a method that can efficiently prepare wheat prolamin and glutenin and improve their quality. Summary of the invention

[0006] In view of this, in order to solve the problems existing in the prior art, the present invention breaks through the cold water control solubility coupling one-step freezing purification technology, controls the ethanol concentration and solution temperature by cold water, realizes the synchronous regulation of the dissolution state and aggregation behavior of wheat alcohol-soluble protein in ethanol aqueous solution, and then couples the freezing treatment, and realizes the controllable aggregation, precipitation and freezing of protein in one step to obtain wheat alcohol-soluble protein. And, using membrane separation coupling high-pressure microfluidic technology, on the basis of membrane separation to obtain glutenin concentrate technology, the alkaline condition synchronous high-pressure microfluidic circulation modification method is used to directly treat the protein concentrate, further regulate the scale of the target protein, free thiol and disulfide bond content, surface hydrophobicity and other structural characteristics and aggregation behavior, and then give glutenin better solubility. The wheat alcohol-soluble protein with good color and high solubility glutenin disclosed in the present invention and the preparation method and application are simple and efficient, green and environmentally friendly, easy to realize industrial production, and help the industrial upgrading of the field of cereal protein.

[0007] In order to achieve the above objects, the first object of the present invention is to provide a method for preparing wheat prolamin and high-solubility glutenin with good color, using the following technical scheme:

[0008] A method for preparing wheat prolamin and high-soluble glutenin with good color, comprising the following steps:

[0009] (1) dispersing gluten powder in 70% ethanol aqueous solution (v / v), stirring at room temperature and centrifuging, collecting the supernatant and precipitate respectively for later use;

[0010] (2) adding cold water to the supernatant obtained in step (1) to adjust the ethanol concentration and solution temperature, mixing the solution evenly, placing the solution under freezing conditions to allow the protein to aggregate, precipitate and freeze, discarding the supernatant, and finally freeze-drying the resulting precipitate to obtain wheat alcohol-soluble protein with good color;

[0011] (3) washing the precipitate obtained in step (1) with water and dispersing it in ultrapure water, rapidly dispersing the precipitate by high-speed shearing treatment, adjusting and controlling the pH of the solution to 11.0, stirring at room temperature, centrifuging, collecting the supernatant, concentrating and purifying the supernatant by a synchronous membrane separation method under alkaline conditions, concentrating the supernatant to 20-30% of the total volume of the supernatant to obtain a concentrated solution, treating the concentrated solution by a synchronous high-pressure microfluidization circulation modification method under alkaline conditions, and finally freeze-drying the obtained protein solution to obtain highly soluble glutenin;

[0012] Furthermore, step (2) and step (3) may be performed simultaneously or asynchronously.

[0013] It is worth noting that in the existing preparation method of wheat alcohol-soluble protein, the traditional alcohol extraction rotary evaporation method needs to enrich the ethanol aqueous solution and the supernatant obtained after centrifugation for rotary evaporation. This step has high energy consumption (heating treatment is required) and long time. In addition, because wheat alcohol-soluble protein has strong foaming property, it is easy to suck back during the rotary evaporation process, and there is a safety risk. Therefore, it is difficult to achieve industrial production, which is not conducive to the industrial application of wheat alcohol-soluble protein. In addition, the wheat alcohol-soluble protein obtained by traditional extraction technology is yellowish in color and has unsatisfactory appearance quality. And glutenin is a gluten protein. Compared with the preparation of plant proteins mainly based on globulin such as soybeans, peanuts, and peas (alkali dissolution acid precipitation method, alkali dissolution pH range 8.0~10.0), the alkali dissolution pH used by the traditional alkali dissolution acid precipitation method is higher (≥11.0), and in the acid precipitation process (close to the isoelectric point of the protein), due to the surface charge of glutenin, the distribution of hydrophilic and hydrophobic groups and other structural particularities, it is easier to produce large-scale aggregates than other types of plant proteins, thereby causing the solubility of glutenin to be extremely poor, which cannot meet the actual use requirements.

[0014] In order to avoid the above problems and meet the production needs of green environmental protection, the present invention changes the processing ideas. When preparing wheat alcohol-soluble protein, firstly, 0-8°C cold water is added to the supernatant obtained after enrichment and centrifugation of the ethanol solution, and the ethanol concentration and solution temperature are controlled to achieve the synchronous regulation of the dissolution state and aggregation behavior of the wheat alcohol-soluble protein in the ethanol aqueous solution; secondly, the solution treated with cold water is left to stand for 6-24h under a freezing condition of -20°C to -35°C, which accelerates the aggregation and precipitation of the wheat alcohol-soluble protein from the ethanol aqueous solution on the one hand, and can also make the protein after precipitation quickly precipitate and freeze on the other hand, and the one-step method realizes the triple effects of aggregation, precipitation and freezing, saves energy, simplifies steps, achieves the purpose of separation and purification, and is conducive to subsequent drying treatment. In particular, the addition of cold water in the present invention can also change the freezing point of the solvent. During the freezing treatment (the freezing temperature range must be appropriate), since the absolute value of the freezing point of the solvent in the supernatant is greater than the absolute value of the temperature selected during the freezing treatment, the supernatant cannot be frozen and still maintains a solution state, while the precipitate mainly contains wheat alcohol-soluble protein and water, which can be frozen after the freezing treatment (the freezing temperature is lower than the freezing point of water). This not only speeds up the separation speed of the supernatant and the precipitate, but also helps to enrich and purify the wheat alcohol-soluble protein, and is more conducive to the industrial preparation of wheat alcohol-soluble protein.

[0015] When preparing glutenin, high-speed shear dispersion treatment is first used to break up the aggregates and accelerate their mass transfer rate, which is beneficial to the subsequent protein enrichment; secondly, the pH of the solution is adjusted to 11.0, which not only meets the minimum enrichment pH value of glutenin, but also saves the amount of alkali compared to the pH value of 12.0 in the conventional process, which is more environmentally friendly; and, membrane separation is used instead of the traditional acid precipitation process to avoid the isoelectric point of the protein, reduce protein aggregation and precipitation, and is beneficial to the improvement of the functional properties of the protein; finally, the protein extraction is integrated with the high-pressure microfluidization modification process, which can be modified simultaneously in the protein preparation process to disaggregate the protein aggregates, further regulate the protein structural characteristics and aggregation behavior, and give glutenin better solubility.

[0016] Therefore, the preparation method of wheat alcohol-soluble protein and high-soluble glutenin with good color disclosed in the present invention has a green and simple extraction process, conforms to the low-carbon concept, and is easy to realize industrial preparation. In addition, considering the differences in the compounding ratio of wheat alcohol-soluble protein and glutenin in actual use, market demand and other factors, the preparation method disclosed in the present invention can flexibly implement the preparation of wheat alcohol-soluble protein and glutenin, and flexibly select the simultaneous implementation or asynchronous implementation of the two extraction processes according to actual needs.

[0017] Furthermore, in step (2), the ethanol concentration is first adjusted to 40% to 60% by using cold water at 0-8°C, and then allowed to stand at -20°C to -35°C for 6 to 24 hours to allow the protein to aggregate, precipitate and freeze.

[0018] Furthermore, the alkaline condition simultaneous membrane separation method in step (3) is: the liquid after solid-liquid separation is moved into the sample processing chamber and concentrated using membrane separation technology, and the pH of the treatment system is maintained between 11.0±0.5, and the membrane size used for membrane separation is 30-100 kDa.

[0019] Furthermore, in step (3), high pressure microfluidics at 12000 psi to 15000 psi is used for 1 to 3 cycles.

[0020] The wheat alcohol-soluble protein extraction process disclosed in the present invention is green and simple, conforms to the low-carbon and environmental protection concepts, has excellent color, and is easy to realize industrial production; the glutenin extraction process is green and efficient, integrates protein extraction and protein modification into the preparation process, is energy-saving and environmentally friendly, and contributes to the technological upgrading of the glutenin industry.

[0021] The second object of the present invention is to provide one or a combination of wheat alcohol-soluble protein and high-soluble glutenin with good color obtained by the preparation method as described above.

[0022] And, the third object of the present invention is to provide a use of the wheat alcohol-soluble protein and high-soluble glutenin with good color as described above in food raw materials, food ingredients, foaming agents, emulsifiers or protein-based foods.

[0023] It is worth noting that the wheat alcohol-soluble protein product obtained by the preparation method disclosed in the present invention has good color and can be used as a food ingredient in the preparation of protein-based products. The high-solubility glutenin product obtained has higher solubility than conventional alkali dissolution, acid precipitation and membrane separation methods, and can be used as a food ingredient or an alternative protein for animal-derived foaming agents and emulsifiers in the preparation of cakes, breads and plant-based products.

[0024] Compared with the prior art, the preparation method of wheat alcohol-soluble protein and high-solubility glutenin with good color disclosed in the present invention breaks through the cold water control solubility coupling one-step freezing purification technology, controls the ethanol concentration and solution temperature by cold water, realizes the synchronous regulation of the dissolution state and aggregation behavior of wheat alcohol-soluble protein in ethanol aqueous solution, and then couples the freezing treatment, and realizes the controllable aggregation, precipitation and freezing of protein in one step, so as to achieve the purpose of separation and purification. The technology is green, efficient, simple and energy-saving, and the obtained alcohol-soluble protein has good color and is easy to realize industrial production. In addition, the membrane separation coupling high-pressure micro-jet technology is adopted, and on the basis of the membrane separation to obtain the protein concentrate technology, the alkaline condition synchronous high-pressure micro-jet circulation modification method is adopted to directly process the protein concentrate, and the scale, free sulfhydryl and disulfide bond content, surface hydrophobicity and other structural characteristics and aggregation behavior of the target protein are further regulated, thereby giving glutenin better solubility. The obtained wheat alcohol-soluble protein and high-solubility glutenin with good color have great application potential in the fields of food raw materials, food ingredients, foaming agents, emulsifiers or protein-based foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0026] Figure 1 These are macroscopic pictures of wheat alcohol-soluble proteins obtained by different methods in Experimental Example 1 of the present invention.

[0027] Figure 2 The present invention is a schematic diagram of the process of preparing wheat alcohol-soluble protein and high-soluble glutenin with good color. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The word "embodiment" used here as an "exemplary" does not necessarily mean that any embodiment described is superior to or better than other embodiments. Unless otherwise specified, the performance index tests in the embodiments of this application are performed using conventional test methods in the art. It should be understood that the terms described in this application are only used to describe specific implementation methods and are not used to limit the content disclosed in this application.

[0030] Unless otherwise specified, the technical and scientific terms used in this document have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs; other experimental methods and technical means not specifically specified in this application refer to experimental methods and technical means commonly used by ordinary technicians in this field.

[0031] In order to better illustrate the content of the present application, numerous specific details are provided in the specific examples below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, equipment, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0032] Under the premise of no conflict, the technical features disclosed in the embodiments of the present application can be combined arbitrarily, and the resulting technical solutions belong to the contents disclosed in the embodiments of the present application.

[0033] The present invention discloses a wheat alcohol-soluble protein and high-soluble glutenin with good color, as well as a preparation method and application, and belongs to the technical field of grain protein processing. The present invention breaks through the cold water control solubility coupling one-step freezing purification technology, controls the ethanol concentration and solution temperature by cold water, realizes the synchronous regulation of the dissolution state and aggregation behavior of wheat alcohol-soluble protein in ethanol aqueous solution, and then couples the freezing treatment, and realizes the controllable aggregation, precipitation and freezing of the protein in one step to achieve the purpose of separation and purification. The technology is green, efficient, simple, and energy-saving. The obtained alcohol-soluble protein has good color and is easy to realize industrial production. In addition, the membrane separation coupling high-pressure micro-jet technology is broken through. On the basis of the membrane separation technology to obtain glutenin concentrate, the protein concentrate is directly treated by the alkaline condition synchronous high-pressure micro-jet circulation modification method, and the structural characteristics and aggregation behavior of the target protein such as scale and surface hydrophobicity are further regulated, giving glutenin better solubility.

[0034] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as limiting the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0035] The experimental methods used in the following examples and comparative examples are as follows:

[0036] Protein content determination: The protein content of the examples and comparative examples was determined by the Kjeldahl nitrogen method, and the nitrogen conversion coefficient was determined according to the national standard GB 5009.5-2016.

[0037] Protein extraction rate determination: It is expressed as the ratio of the mass of extracted protein to the mass of gluten powder.

[0038] Extraction rate (%) = protein mass / raw material mass × 100%

[0039] Protein sample color measurement: Accurately weigh 10g of protein sample, spread it in a transparent round container, and select 3 different points to measure the chromaticity value of the protein sample with a handheld colorimeter. The chromaticity value of protein is represented by brightness value (L*), red / green value (+a* / -a*), and yellow / blue value (+b* / -b*). The whiteness value of the protein sample is calculated using the following formula.

[0040]

[0041] Protein solubility determination: prepare a 10 mg / mL protein solution, adjust the protein solution to pH 7, and then dilute it to 1 mg / mL with ultrapure water. Centrifuge at 5000 g for 10 min, collect the supernatant, and determine the protein concentration by Bradford method using BSA as the standard protein.

[0042] Solubility (%) = [(supernatant protein content × 100) / total protein content of sample before centrifugation] × 100%

[0043] Protein particle size determination: Gluten was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2h, centrifuged at 10000g for 10min, the precipitate was washed 2-3 times with water, dispersed in ultrapure water at a ratio of 1:10 (w / v), dispersed by high-speed shear for 2min, adjusted and maintained at a pH of 11.0 with NaOH at room temperature (25°C) and stirred for 2h, then centrifuged at 10000g for 20min to remove insoluble impurities, and the protein-rich supernatant was collected and then transferred to the sample chamber of the membrane separation device, and the alkaline condition synchronous membrane separation method (MWCO 30KDa) was used. The supernatant was concentrated and purified by a membrane until it was concentrated to 20-30% of the total volume. 10 mL of the above protein concentrate was taken out for standby use, and the remaining protein concentrate was treated by a basic condition synchronous high-pressure microfluidic circulation modification method (pressure 12000 psi, 1 cycle), and 10 mL of the treated protein concentrate was taken out for standby use. After the protein concentrate was diluted, the size of the protein solution was measured using a ZetaSizerNano-ZS laser particle size analyzer.

[0044] Example 1

[0045] The gluten powder was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2 hours, centrifuged at 10000g for 10 minutes, and the supernatant was collected. The ethanol concentration of the supernatant was adjusted to 40% by adding a certain volume of cold water (4°C). After stirring evenly, it was placed in a -20°C refrigerator for 12 hours and the supernatant was discarded. The obtained precipitate was freeze-dried to obtain wheat alcohol-soluble protein, which is Example 1.

[0046] Example 2

[0047] The gluten powder was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2 hours, centrifuged at 10000g for 10 minutes, and the supernatant was collected. The ethanol concentration of the supernatant was adjusted to 40% by adding a certain volume of cold water (4°C). After stirring evenly, it was placed in a -35°C refrigerator for 6 hours and the supernatant was discarded. The obtained precipitate was freeze-dried to obtain wheat alcohol-soluble protein, which is Example 2.

[0048] Example 3

[0049] The gluten powder was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2 hours, centrifuged at 10000g for 10 minutes, and the supernatant was collected. The ethanol concentration of the supernatant was adjusted to 50% by adding a certain volume of cold water (4°C). After stirring evenly, it was placed in a -20°C refrigerator for 12 hours and the supernatant was discarded. The obtained precipitate was freeze-dried to obtain wheat alcohol-soluble protein, which is Example 3.

[0050] Example 4

[0051] The gluten powder was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2 hours, centrifuged at 10000g for 10 minutes, and the supernatant was collected. The ethanol concentration of the supernatant was adjusted to 50% by adding a certain volume of cold water (4°C). After stirring evenly, it was placed in a -35°C refrigerator for 6 hours and the supernatant was discarded. The obtained precipitate was freeze-dried to obtain wheat alcohol-soluble protein, which is Example 4.

[0052] Example 5

[0053] The gluten powder was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2 hours, centrifuged at 10000g for 10 minutes, and the supernatant was collected. The ethanol concentration of the supernatant was adjusted to 60% by adding a certain volume of cold water (4°C). After stirring evenly, it was placed in a -20°C refrigerator for 24 hours and the supernatant was discarded. The obtained precipitate was freeze-dried to obtain wheat alcohol-soluble protein, which is Example 5.

[0054] Example 6

[0055] The gluten powder was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2 hours, centrifuged at 10000g for 10 minutes, and the supernatant was collected. The ethanol concentration of the supernatant was adjusted to 60% by adding a certain volume of cold water (4°C). After stirring evenly, it was placed in a -35°C refrigerator for 6 hours and the supernatant was discarded. The obtained precipitate was freeze-dried to obtain wheat alcohol-soluble protein, which is Example 6.

[0056] Example 7

[0057] The gluten powder was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2h, centrifuged at 10000g for 10min, and the obtained precipitate was washed with water 2-3 times, and then dispersed in ultrapure water at a ratio of 1:10 (w / v). After high-speed shear dispersion for 2min, the solution pH was adjusted and maintained at 11.0 with NaOH at room temperature (25°C) and stirred for 2h, and then centrifuged at 10000g for 20min to remove insoluble impurities, and the protein-rich supernatant was collected and then transferred to the sample chamber of the membrane separation device, and the alkaline condition synchronous membrane separation method (MWCO 30KDa) was used. The supernatant is concentrated and purified by a membrane until it is concentrated to 20-30% of the total volume, and the concentrated solution is treated by a synchronous high-pressure microfluidic circulation modification method under alkaline conditions (pressure 12000 psi, 1 cycle). Finally, the obtained protein solution is freeze-dried to obtain glutenin, which is Example 7.

[0058] Example 8

[0059] The gluten powder was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2h, centrifuged at 10000g for 10min, and the obtained precipitate was washed with water 2-3 times, and then dispersed in ultrapure water at a ratio of 1:10 (w / v). After high-speed shear dispersion for 2min, the solution pH was adjusted and maintained at 11.0 with NaOH at room temperature (25°C) and stirred for 2h, and then centrifuged at 10000g for 20min to remove insoluble impurities, and the protein-rich supernatant was collected and then transferred to the sample chamber of the membrane separation device, and the alkaline condition synchronous membrane separation method (MWCO 50KDa) was used. The supernatant is concentrated and purified by a membrane until it is concentrated to 20-30% of the total volume, and the concentrated solution is treated by a synchronous high-pressure microfluidic circulation modification method under alkaline conditions (pressure 12000 psi, cycle 3 times). Finally, the obtained protein solution is freeze-dried to obtain glutenin, which is Example 8.

[0060] Example 9

[0061] The gluten powder was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2h, centrifuged at 10000g for 10min, and the obtained precipitate was washed with water 2-3 times, and then dispersed in ultrapure water at a ratio of 1:10 (w / v). After high-speed shear dispersion for 2min, the solution pH was adjusted and maintained at 11.0 with NaOH at room temperature (25°C) and stirred for 2h, and then centrifuged at 10000g for 20min to remove insoluble impurities, and the protein-rich supernatant was collected and then transferred to the sample chamber of the membrane separation device, and the alkaline condition synchronous membrane separation method (MWCO 50KDa) was used. The supernatant is concentrated and purified by a membrane until it is concentrated to 20-30% of the total volume, and the concentrated solution is treated by a synchronous high-pressure microfluidic circulation modification method under alkaline conditions (pressure 15000 psi, 1 cycle). Finally, the obtained protein solution is freeze-dried to obtain glutenin, which is Example 9.

[0062] Example 10

[0063] The gluten powder was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2h, centrifuged at 10000g for 10min, and the precipitate was washed with water 2-3 times, and then dispersed in ultrapure water at a ratio of 1:10 (w / v). After high-speed shear dispersion for 2min, the solution pH was adjusted and maintained at 11.0 with NaOH at room temperature (25°C) and stirred for 2h, and then centrifuged at 10000g for 20min to remove insoluble impurities, and the protein-rich supernatant was collected and then transferred to the sample chamber of the membrane separation device, and the alkaline condition synchronous membrane separation method (MWCO 100KDa) was used. The supernatant is concentrated and purified by a membrane until it is concentrated to 20-30% of the total volume, and the concentrated solution is treated by a synchronous high-pressure microfluidic circulation modification method under alkaline conditions (pressure 12000 psi, cycle 3 times). Finally, the obtained protein solution is freeze-dried to obtain glutenin, which is Example 10.

[0064] Embodiment 11

[0065] The gluten powder was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2h, centrifuged at 10000g for 10min, and the precipitate was washed with water 2-3 times, and then dispersed in ultrapure water at a ratio of 1:10 (w / v). After high-speed shear dispersion for 2min, the solution pH was adjusted and maintained at 11.0 with NaOH at room temperature (25°C) and stirred for 2h, and then centrifuged at 10000g for 20min to remove insoluble impurities, and the protein-rich supernatant was collected and then transferred to the sample chamber of the membrane separation device, and the alkaline condition synchronous membrane separation method (MWCO 100KDa) was used. The supernatant is concentrated and purified by a membrane until it is concentrated to 20-30% of the total volume, and the concentrated solution is treated by a synchronous high-pressure microfluidic circulation modification method under alkaline conditions (pressure 15000 psi, 1 cycle). Finally, the obtained protein solution is freeze-dried to obtain glutenin, which is Example 11.

[0066] In order to further demonstrate the beneficial effects of the present invention and to better understand the present invention, the properties and application performances of the wheat alcohol-soluble protein and high-soluble glutenin with good color and the preparation method thereof disclosed in the present invention are further illustrated by the following comparative examples and experimental examples, but they should not be construed as limitations of the present invention. The properties of the methods obtained by other measurement experiments conducted by technicians in this field based on the above-mentioned invention content and the applications based on the above-mentioned properties are also deemed to fall within the protection scope of the present invention.

[0067] Comparative Example 1

[0068] The gluten powder was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2h, centrifuged at 10000g for 10min, and the supernatant was collected. The ethanol in the supernatant was removed by rotary evaporation at 40°C and then freeze-dried to obtain wheat alcohol-soluble protein. The precipitate obtained after centrifugation was washed with water 2-3 times, dispersed in ultrapure water at a ratio of 1:10 (w / v), adjusted to pH 11.0 with NaOH, and stirred at room temperature (25°C) for 2h, then centrifuged at 10000g for 20min, and the protein-rich supernatant was collected. The pH value was adjusted to 4.5 and stabilized for 30min, and then centrifuged at 10000g for 10min, and the supernatant was discarded. The precipitate was washed with water 2-3 times and freeze-dried to obtain glutenin, which is Comparative Example 1.

[0069] Comparative Example 2

[0070] The gluten powder was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2h, centrifuged at 10000g for 10min, and the supernatant was collected. Three solutions of the same volume were taken out from the supernatant, and different volumes of cold water (4°C) were added to adjust the ethanol concentration of the two supernatants to 50% and 40%, respectively, and stirred evenly. The other supernatant was not processed. At the same time, the three solutions were placed in a -20°C refrigerator for 6h and the supernatant was discarded. The obtained precipitate was freeze-dried to obtain wheat alcohol-soluble protein, which is Comparative Example 2.

[0071] Comparative Example 3

[0072] The gluten powder was dispersed in a 70% ethanol aqueous solution (v / v) at a ratio of 1:10 (w / v), stirred at room temperature (25°C) for 2h, centrifuged at 10000g for 10min, and the precipitate was washed with water 2-3 times, and then dispersed in ultrapure water at a ratio of 1:10 (w / v). After high-speed shear dispersion for 2min, the pH value of the solution was adjusted and maintained at 11.0 with NaOH at room temperature (25°C) and stirred for 2h, and then centrifuged at 10000g for 20min, the protein-rich supernatant was collected, and then transferred to the sample chamber of the membrane separation device, and the alkaline condition synchronous membrane separation method (MWCO 30KDa) was used. The supernatant is concentrated and purified by a membrane until it is concentrated to 20-30% of the total volume. Finally, the obtained protein solution is freeze-dried to obtain glutenin, which is Comparative Example 3.

[0073] Experimental Example 1

[0074] Taking the wheat alcohol-soluble proteins obtained in Examples 1-6 and Comparative Examples 1-2 as examples, the protein content, extraction rate, whiteness value and macroscopic appearance of the protein products obtained by different methods were studied. The results are shown in Tables 1, 2, 3 and Figure 1 shown.

[0075] Table 1 Protein content of wheat prolamin obtained by different methods

[0076]

[0077]

[0078] As can be seen from Table 1, the protein content of the wheat alcohol-soluble proteins prepared in Examples 1-6 of the present invention is at a relatively high level, which meets the requirements for concentrated protein or isolated protein in domestic commercial protein products. The domestic protein conversion coefficient for wheat products is 5.70. The protein content data in Table 1 are converted according to N×5.70. The protein content of Examples 1-6 is more than 82%. This data can be used for comparison of the protein content of wheat alcohol-soluble protein related products at home and abroad, and has a high promotion value. Moreover, when the extraction parameters are in a relatively optimal condition, such as Examples 4 and 6, the protein content can reach more than 88%, which is significantly higher than Comparative Example 1.

[0079] Table 2 Extraction rate of wheat alcohol-soluble protein obtained by different methods

[0080]

[0081] As shown in Table 2, in Comparative Example 2, under the same freezing temperature and freezing time, the protein extraction rate obtained without adjusting the ethanol concentration is 19%. As the ethanol concentration decreases, the protein extraction rate gradually increases. When the ethanol concentration is 40%, the extraction rate of wheat alcohol-soluble protein reaches the highest, which is 25%. In addition, although the extraction rates of Examples 1 to 6 are relatively lower than those of the alcohol-soluble rotary evaporation method in Comparative Example 1, when the extraction parameters are in a better condition, such as Example 1, the extraction rate can reach 29%, which is slightly lower than that of Comparative Example 1, and the reliance on the rotary evaporator is eliminated, and the safety problem caused by the rotary evaporation is prevented, which is more suitable for the technical requirements of large-scale production.

[0082] Table 3 Whiteness values ​​of wheat alcohol-soluble proteins obtained by different methods

[0083]

[0084] From Table 3 and Figure 1It can be seen that the whiteness values ​​of the wheat alcohol-soluble proteins obtained in Comparative Example 2 and Examples 1-6 are all above 81, which is significantly higher than that in Comparative Example 1 (~74). This may be because Examples 1-6 use cold water to control solubility and couple one-step freezing purification technology. During freezing treatment, proteins aggregate, precipitate and freeze, while polyphenols and other substances that affect color still have high solubility in ethanol aqueous solution and do not precipitate with wheat alcohol-soluble proteins; while Comparative Example 1 uses rotary evaporation, the solvent is removed, but polyphenols and other substances that affect color are still retained in the protein solution, so the color is poor relative to the embodiment. Among them, the whiteness value of Example 1 is as high as 90 or more, and the powder is more delicate, indicating that the wheat alcohol-soluble protein prepared by this patent technology has better color, which is significantly better than the traditional preparation method, and significantly broadens the application prospects of wheat alcohol-soluble protein.

[0085] Experimental Example 2

[0086] Taking the glutenin obtained in Examples 7-11 and Comparative Examples 1 and 3 as examples, the protein content, extraction rate, and protein solubility of the protein products obtained by different methods were studied. The results are shown in Tables 4, 5, and 6 below, and the protein particle size results obtained in Comparative Example 3 and Example 7 are shown in Table 7.

[0087] Glutenin belongs to gluten protein, which is soluble in alkaline solution (pH≥11). The solubility of glutenin prepared by alkaline dissolution and acid precipitation method under neutral conditions (pH~7) is extremely poor (<10%), which seriously limits its application in the field of food. Although the simultaneous membrane separation treatment under alkaline conditions can achieve purification while avoiding the isoelectric point of glutenin, so that the degree of protein aggregation is reduced, due to the particularity of the structure such as the surface charge, scale, and surface hydrophilic and hydrophobic group distribution of glutenin, the single membrane separation treatment cannot effectively control the degree of protein aggregation, and still produces large aggregates, and the scale distribution is uneven, which can only slightly improve the solubility of glutenin, and still cannot effectively improve its application value. After the membrane separation treatment, the present invention adopts alkaline conditions to synchronize high-pressure microfluidization circulation modification treatment concentrate to further regulate the scale, free sulfhydryl and disulfide bond content, surface hydrophobicity and other structural characteristics and aggregation behavior of glutenin, so that the large protein aggregates are further depolymerized, and the controllable assembly of glutenin is realized, and the solubility of the protein is further effectively improved.

[0088] Table 4 Protein content of glutenin obtained by different methods

[0089] sample Protein content (%) Comparative Example 1 Alkali dissolution and acid precipitation method (dissolution at pH 11, precipitation at pH 4.5) 84.12±1.25 Comparative Example 3 Membrane separation (30kDa) 83.99±0.23 Example 7 Membrane separation (30 kDa) coupled with high pressure microfluidization (12000 psi-1 time) 82.00±0.35 Example 8 Membrane separation (50 kDa) coupled with high pressure microfluidization (12000 psi-3 times) 81.61±0.84 Example 9 Membrane separation (50 kDa) coupled with high pressure microfluidization (15000 psi-1 time) 83.14±0.85 Example 10 Membrane separation (100 kDa) coupled with high pressure microfluidization (12000 psi-3 times) 85.29±0.65 Embodiment 11 Membrane separation (100 kDa) coupled with high pressure microfluidization (15000 psi-1 time) 85.23±1.19

[0090] As shown in Table 4, the protein content of glutenin prepared in Examples 7-11 of the present invention is at a relatively high level, which meets the requirements for concentrated protein or isolated protein in domestic commercial protein products. The protein conversion coefficient for wheat protein products in China is 5.70. The protein content data in Table 4 are converted according to N×5.70, and the protein content of Examples 7-11 is more than 80%. This data can be used to compare the protein content of glutenin-related products at home and abroad, and has a high promotion value.

[0091] Table 5 Gluten extraction rate obtained by different methods

[0092] sample Extraction rate (%) Comparative Example 1 Alkali dissolution and acid precipitation method (dissolution at pH 11, precipitation at pH 4.5) 25.6±0.75 Comparative Example 3 Membrane separation (30kDa) 33.13±0.66 Example 7 Membrane separation (30 kDa) coupled with high pressure microfluidization (12000 psi-1 time) 17.62±0.82 Example 8 Membrane separation (50 kDa) coupled with high pressure microfluidization (12000 psi-3 times) 23.38±3.24 Example 9 Membrane separation (50 kDa) coupled with high pressure microfluidization (15000 psi-1 time) 24.41±2.73 Example 10 Membrane separation (100 kDa) coupled with high pressure microfluidization (12000 psi-3 times) 25.31±1.12 Embodiment 11 Membrane separation (100 kDa) coupled with high pressure microfluidization (15000 psi-1 time) 25.86±0.94

[0093] As shown in Table 5, the glutenin extraction rate of Comparative Example 3 is higher than that of Comparative Example 1. Examples 7 to 11 are membrane separation coupled with high-pressure microfluidic circulation treatment. From the results, the extraction rates of Examples 7 to 11 are lower than those of Comparative Example 3, mainly because there will be residual solution in the high-pressure microfluidic pipeline, which leads to a decrease in the final glutenin extraction rate. However, the extraction rates of Examples 7 to 11 are equivalent to or slightly reduced from those of Comparative Example 1, and still meet the requirements for glutenin extraction rate in industrial production.

[0094] Table 6 Solubility of glutenin obtained by different methods

[0095]

[0096]

[0097] As shown in Table 6, the solubility of glutenin obtained by the alkaline dissolution and acid precipitation method in Comparative Example 1 is extremely poor, only about 5%, and although the solubility of Comparative Example 3 after a single membrane separation treatment is improved compared with the alkaline dissolution and acid precipitation method in Comparative Example 1, it is only increased by ~13%, so a single membrane separation treatment is not enough to solve the problem of low solubility. As shown in Examples 7 to 11, after coupling with high-pressure microfluidization treatment, the solubility is greatly improved, up to about 50%. This may be because the high-pressure microfluidization treatment further achieves the structural characteristics of regulating the size of the target protein, the surface hydrophilicity distribution, and the content of sulfhydryl-disulfide bonds. For example, compared with the single membrane separation treatment, the membrane separation coupled with high-pressure microfluidization treatment reduces the protein particle size by ~80nm (Table 7). Table 6 shows that the glutenin obtained by the patent technology has good solubility and significantly improves its application potential.

[0098] Table 7 Particle size of glutenin obtained by different methods

[0099] sample Particle size (nm) Comparative Example 3 272.97±4.46 Example 7 187.03±2.86

[0100] In summary, the alkali-dissolving synchronous membrane separation and purification coupled with high-pressure microfluidic modification technology covered by the present invention not only avoids protein aggregation caused by acid precipitation near the isoelectric point during the preparation process of alkali-dissolving acid precipitation, but also realizes green purification and efficient modification of glutenin by membrane separation coupled with high-pressure microfluidic circulation treatment during the extraction process, regulates the structural characteristics and aggregation behavior of glutenin, and gives glutenin good solubility. Compared with the traditional alkali-dissolving acid precipitation method, the solubility of glutenin extracted by the process of the present invention has been significantly improved, which is more suitable for industrial application.

[0101] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing wheat prolamin and high-soluble glutenin with good color, characterized in that: The steps include: (1) dispersing gluten powder in 70% ethanol aqueous solution (v / v), stirring at room temperature and centrifuging, collecting the supernatant and precipitate respectively for later use; (2) adding cold water to the supernatant obtained in step (1) to adjust the ethanol concentration and the solution temperature, mixing well, placing the solution under freezing conditions to allow the protein to aggregate, precipitate and freeze, discarding the supernatant, and finally freeze-drying the resulting precipitate to obtain wheat alcohol-soluble protein with good color; (3) washing the precipitate obtained in step (1) with water and dispersing it in ultrapure water, rapidly dispersing the precipitate by high-speed shearing treatment, adjusting and controlling the pH of the solution to 11.0, stirring at room temperature, centrifuging, collecting the supernatant, concentrating and purifying the supernatant by a synchronous membrane separation method under alkaline conditions, concentrating the supernatant to 20-30% of the total volume of the supernatant to obtain a concentrated solution, treating the concentrated solution by a synchronous high-pressure microfluidization circulation modification method under alkaline conditions, and finally freeze-drying the obtained protein solution to obtain highly soluble glutenin; Furthermore, step (2) and step (3) may be performed simultaneously or asynchronously.

2. The preparation method according to claim 1, characterized in that: In the step (2), the ethanol concentration is first adjusted to 40% to 60% with cold water at 0-8°C, and then allowed to stand at a freezing condition of -20°C to -35°C for 6 to 24 hours to allow the protein to aggregate, precipitate and freeze.

3. The preparation method according to claim 1, characterized in that: The alkaline condition simultaneous membrane separation method in step (3) is: the supernatant after solid-liquid separation is moved into the sample processing chamber, concentrated by membrane separation technology, and the pH of the treatment system is maintained between 11.0±0.

5. The membrane size used in the membrane separation is 30-100 kDa.

4. The preparation method according to claim 3, characterized in that: In the step (3), high pressure microfluidics at 12000 psi to 15000 psi is used for 1 to 3 cycles.

5. A wheat alcohol-soluble protein or a high-soluble glutenin with good color or a combination thereof obtained by the preparation method according to any one of claims 1 to 4.

6. Use of the wheat alcohol-soluble protein and high-soluble glutenin with good color as claimed in claim 5 in food raw materials, food ingredients, foaming agents, emulsifiers or protein-based foods.

Images