High-foamability edible rapeseed protein as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of plant protein extraction, and particularly relates to high-foamability edible rapeseed protein as well as a preparation method and application thereof. According to the method, a gradient acidic-neutral pH circulating treatment technology is utilized, and membrane separation and physical field modification technologies are coupled under gradient pH control, so that the structural characteristics of the rapeseed protein are regulated and controlled in order. Primary acidity is controlled under a weak acid condition (pH is 5.0-7.0) to cooperate with an ultrafiltration step concentration process to obtain a high-concentration rapeseed protein solution (the protein concentration is greater than or equal to 6%); the secondary acidity control is to regulate and control a rapeseed protein structure under an acidic condition (pH is 2.0-4.0), couple ultrasonic, infrared and other physical field treatments, and penetrate into the internal region of the protein structure through cavitation, shearing, heat effect and other actions, so that the surface charge, hydrophobicity and other structural characteristics of the rapeseed protein structure are changed, and the interaction between polyphenol and protein is influenced; finally, the pH is circulated to be neutral, the protein is reassembled to form a controllable aggregate, and finally the rapeseed protein is endowed with the high-foam characteristic and the good color and luster.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant protein extraction, and particularly relates to an edible rapeseed protein with high foaming property, a preparation method thereof and an application thereof. Background Art

[0002] While meeting the world's growing protein demand, maintaining sustainable development is a challenge faced by the agricultural food sector. Due to the impact of animal protein on the carbon and nitrogen cycles and biodiversity, it will bring huge pressure to the environment. Therefore, excessive use of animal protein does not conform to the concept of sustainable development. In addition, the intake of a high proportion of animal protein may lead to some health problems, including an increased risk of obesity, type II diabetes and cardiovascular diseases. Therefore, finding substitutes for animal protein, such as plant, insect, fungal and microbial proteins, will be necessary to feed the estimated 10 billion people in 2050. Among them, considering the established supply chains and consumer acceptance of the above-mentioned bio-based protein sources, as well as their proven nutritional value and sustainability, plant protein is the most ideal choice.

[0003] Rapeseed is the second largest oil crop planted in the world after soybean. In 2023 - 2024, the annual output of rapeseed in China was 16.3174 million tons. It is one of the most important oil crops in China and also one of the main sources of oil supply in China. Rapeseed cake is a by-product after extracting oil from rapeseed. According to the statistics of the US Department of Agriculture and Rural Development, in the 2019 - 2020 fiscal year, China consumed about 11 million tons of rapeseed cake, accounting for one-third of the global consumption. Rapeseed cake is usually used as animal feed or agricultural fertilizer, with low economic benefits. Rapeseed cake is rich in crude protein (~40%), and rapeseed protein has the advantages of balanced amino acid composition, high nutritional value and excellent functional properties. It is a very promising new source of plant protein. Therefore, preparing edible rapeseed protein from rapeseed cake and enhancing its added value have good social and economic benefits and are conducive to the sustainable development of the rapeseed planting industry.

[0004] However, rapeseed meal has a complex composition, contains a large number of anti-nutritional factors that are closely bound to proteins (such as phenolic substances, glucosinolates, phytic acid, etc.), and the physical and chemical properties of the protein components (mainly 11S Cruciferin and 2S Napin) contained in it are very different. Therefore, the alkali dissolution and acid precipitation methods commonly used in plant protein extraction are not suitable for preparing edible rapeseed protein isolate from rapeseed meal. Existing studies have found that rapeseed protein isolate has high foaming properties, which is superior to whey protein isolate and soy protein isolate. It is a biomacromolecule-based foaming agent with great application potential in the food field. However, existing extraction methods such as alkali dissolution and acid precipitation can cause uncontrollable aggregation of rapeseed protein and produce large aggregates, resulting in its foaming properties not being fully utilized. Therefore, combining the resource advantages of rapeseed and the high nutritional value and foaming properties of rapeseed protein, it is of great significance to break through a high-foaming edible rapeseed protein isolate preparation technology. Summary of the invention

[0005] In view of this, in order to solve the problems existing in the prior art, the present invention improves the preparation method of rapeseed protein based on the obtained glucosinolate and phytic acid content that meets the EU edible rapeseed protein standards, and performs a "gradient acid-neutral" pH cycle treatment on the rapeseed protein extract, which not only avoids the irreversible denaturation of the protein that may be caused by a single pH shift technology, but also realizes the dynamic and orderly regulation of structural properties such as protein charge and surface hydrophobicity, and can also orderly regulate the interaction between protein and polyphenols, ultimately achieving structural modification, enhancement of functional properties and improvement of color of high-concentration rapeseed protein.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for preparing highly foaming edible rapeseed protein, which includes subjecting a rapeseed protein extract to a "gradient acid-neutral" pH cycle treatment and coupling membrane separation and physical field modification technology to achieve structural modification and quality improvement of high-concentration rapeseed protein.

[0008] Furthermore, the preparation method adopts a moderate salt solution synchronous scale screening technology to obtain a rapeseed protein extract; then, the acidity is controlled once, and ultrafiltration step concentration is coordinated to obtain a high-concentration rapeseed protein solution; then, the acidity is controlled twice, and physical field treatment is coupled; finally, the pH value of the high-concentration protein solution is adjusted back to a neutral environment, and freeze-dried to obtain the high-foaming edible rapeseed protein.

[0009] It should be noted that the functional properties such as solubility and foaming property of the protein obtained by the existing edible rapeseed protein preparation technology are relatively poor, which cannot meet the use requirements of food foaming agents. For plant proteins such as soybeans, peas, and flaxseeds, the modification methods based on pH shift mostly adopt alkaline condition shift. However, due to the large differences in the physicochemical properties of the protein components contained in rapeseed protein and the presence of a large amount of non-protein components such as polyphenols, the alkaline shift or the alkaline shift coupled with physical field modification technology is not applicable to rapeseed protein. Not only is the improvement degree of functional properties limited, but also because the modification process is carried out under alkaline conditions, polyphenols are easily oxidized to quinones and covalently bound to the protein, resulting in a darker color of the obtained protein and an unsatisfactory appearance. Moreover, in order to avoid irreversible denaturation of proteins caused by extreme pH, the existing pH shift technology mostly adopts a one-time weak acid / weak base shift treatment, which cannot meet the technical requirements of complex plant protein components and multiple isoelectric points, and the effect also cannot meet the needs of food industrialization.

[0010] In the present invention, after the rapeseed protein extract is centrifuged and separated after the first acid control, the high-concentration protein solution obtained after the supernatant is synergistically ultrafiltered and stepwise concentrated is in a suspended state without precipitation. At this time, the second acid control is carried out, and the rapeseed protein will undergo disaggregation and the structure will unfold. Under this condition, further coupled with physical field modification treatment, through multiple actions such as cavitation, shearing, and heating, the protein is further depolymerized and penetrates into the internal region of the protein structure, resulting in changes in the surface charge distribution, sulfhydryl-disulfide bond content, surface hydrophilicity-hydrophobicity, rigidity / flexibility and other structural characteristics of the protein. Finally, during the process of re-adjusting from the second acid control environment back to neutral, the protein undergoes re-assembly and controllable aggregation, ultimately endowing the rapeseed protein with good surface structural characteristics, enabling it to adsorb on the gas-liquid interface more quickly and form an interfacial film with viscoelastic properties, and fully exerting the high foam property of the rapeseed protein.

[0011] Furthermore, the pH range of the first acid control is 5.0 - 7.0, and the pH range of the second acid control is 2.0 - 4.0.

[0012] It should be noted that setting the pH of the first acid control system to 5.0 - 7.0 is mainly considered that under weak acid conditions, glucosinolates are easily hydrolyzed, and the binding between protein and phytic acid is weak, which is more conducive to protein enrichment and separation from substances such as phytic acid and glucosinolates. Moreover, the weak acid environment can avoid the influence of alkaline conditions on polyphenols in rapeseed protein, and can regulate the interaction between polyphenols and rapeseed protein and change the protein structural characteristics, thereby realizing the improvement of protein quality while increasing the protein yield. Combining the first acid control with ultrafiltration stepwise concentration can not only effectively remove non-protein components, but also control the degree of protein aggregation, thus breaking through the concentration limit of the existing rapeseed protein solution and obtaining a high-concentration rapeseed protein solution, making the large-scale preparation of high-foaming rapeseed protein possible.

[0013] In addition, in the second acidic control stage, the present invention couples physical field modification to treat a high-concentration rapeseed protein solution. First, the acidity of the system is further controlled to strengthen, and the pH of the high-concentration protein solution is adjusted to an acidic environment (i.e., the pH range is 2.0 - 4.0), promoting the depolymerization / partial depolymerization of rapeseed protein aggregates. At this time, the protein conformation will unfold, and the surface structural characteristics will change. Under this condition, ultrasonic and infrared physical field treatment technologies are synchronized. Through the multiple effects such as cavitation, shear, and thermal effects introduced by the physical field, the high-concentration rapeseed protein will further depolymerize and penetrate into the internal region of the protein structure, further changing the surface charge distribution, sulfhydryl-disulfide bond content, surface hydrophilicity / hydrophobicity, rigidity / flexibility, and other structural characteristics of the protein. Finally, the pH of the high-concentration rapeseed protein solution is cycled from an acidic environment to neutral. At this time, the protein depolymerization products will reassemble and controllably aggregate, ultimately endowing the rapeseed protein with good surface structural characteristics, enabling it to adsorb on the gas-liquid interface more quickly and form an interfacial film with viscoelastic properties, and fully exerting the high-foaming characteristics of the rapeseed protein.

[0014] In addition, considering that under alkaline conditions, polyphenols are easily oxidized to quinone substances, which then covalently bind to proteins, resulting in a deepening of the protein color and a decrease in functional properties such as foaming ability. The present invention uses a "gradient acidic-neutral" pH cycling treatment, which not only avoids the influence of alkaline conditions on polyphenols in rapeseed protein, but also can orderly regulate the interaction between proteins and polyphenols by controlling the pH value of the system gradient, improving the color of the protein.

[0015] Furthermore, the physical field modification treatment includes ultrasonic treatment and infrared treatment.

[0016] Furthermore, the power density of the ultrasonic treatment is 10 - 30 W / mL, and the ultrasonic treatment time is 10 - 60 min; the infrared treatment includes short-wave treatment and long-wave treatment, the temperature is 80 - 160 °C, and the infrared treatment time is 4 - 16 min.

[0017] It should be noted that by controlling the core parameters such as the physical field power and action time, the effective regulation of protein structural characteristics can be achieved, which is beneficial to exerting the foaming characteristics of rapeseed protein. The rapeseed protein prepared by the present invention has both ultra-high foaming ability, good foam stability, and excellent foam plasticity. Among them, the foaming ability can reach up to 392%, higher than common plant proteins in the food field such as soybean protein and pea protein, and superior to commonly used industrial animal foaming agents such as ovalbumin and sodium caseinate.

[0018] Furthermore, in the moderate salt solution synchronous directional sieving technology, the salt solution is a 100 mM calcium chloride or magnesium chloride solution, and the sieve mesh number for directional sieving is 60 - 200 meshes.

[0019] It should be noted that since phytic acid can chelate divalent metal ions under weak acid conditions to obtain rapeseed protein with low phytic acid content, one or a combination of CaCl2 and MgCl2 is selected as the good solvent in the present invention. Considering that the lower the salt concentration of the good solvent, the lower the subsequent salt removal difficulty, which is more in line with the concept of green and environmental protection. And the salt ion concentration will affect the interactions between protein-protein and protein-other substances, thus affecting the protein assembly and aggregation behavior. Low-concentration salts can not only enhance the cavitation and thermal effects brought by physical fields such as ultrasound and microwave; they also assist the physical fields in regulating the protein assembly and aggregation behavior, affecting the structural characteristics such as the surface charge distribution and flexibility of the protein, and can change the interfacial characteristics of the protein, which is beneficial to the improvement of foam characteristics. Therefore, the low-concentration salt ions remaining after membrane separation are beneficial to assisting the physical fields in regulating the structure of the protein, thereby endowing it with higher foaming properties. Moreover, a higher salt ion concentration will not only lead to an increase in the amount of acid used to adjust the pH of the high-concentration rapeseed protein solution after membrane separation, but also cause protein aggregation. Therefore, considering the reduction of acid usage and the difficulty of process operation, the salt solution in the present invention is defined as a 100 mM calcium chloride or magnesium chloride solution.

[0020] Furthermore, the membrane molecular weight used in the ultrafiltration stepwise concentration is 5 - 30 kDa, and the protein concentration of the high-concentration rapeseed protein solution is ≥ 6%.

[0021] It should be noted that in the present invention, the acidic control stage is coordinated with the ultrafiltration stepwise concentration, which is beneficial to removing toxic or anti-nutritional substances such as glucosinolates, phytic acid, pigments, and polyphenols. By controlling the removal rate of salt ions, the structural characteristics and aggregation behavior of the protein can be orderly controlled, preventing problems such as slow concentration or equipment blockage caused by rapid aggregation, thereby improving the protein concentration degree and obtaining a high-concentration rapeseed protein solution with a protein concentration of more than 6%.

[0022] In particular, the present invention applies the membrane separation technology to the stepwise concentration treatment. On the one hand, it can realize the concentration and purification of the dissolved protein solution in rapeseed cake and remove toxic or anti-nutritional substances such as glucosinolates, polyphenols, and phytic acid; on the other hand, it can effectively control the protein retention size, structural characteristics, and aggregation behavior, improving the yield and quality of the target protein.

[0023] The second object of the present invention is to provide a highly foaming edible rapeseed protein obtained by the preparation method as described above.

[0024] It should be noted that the highly foaming edible rapeseed protein obtained by the preparation method disclosed in the present invention not only has excellent foaming properties, but also can obtain rapeseed protein with good color without using a decolorizing agent. In particular, the highly foaming edible rapeseed protein disclosed in the present invention has a maximum foaming property of up to 392%, which is higher than that of plant proteins such as soy protein. It is 3.1 times that of ovalbumin, a commonly used industrial foaming agent, and 2.1 times that of sodium caseinate. When used, the foam is dense and has good plasticity, and has the potential to become an egg white substitute.

[0025] In addition, the third object of the present invention is to provide an application of the above-mentioned highly foaming edible rapeseed protein as a protein ingredient and a foaming agent in the food field.

[0026] It should be noted that the foaming agent is an important formula in the food field. Commonly used protein foaming agents such as whey protein and ovalbumin are mainly animal proteins, which are difficult to meet the development needs of diversified products. Therefore, it is of great significance to explore plant protein-based foaming agents. Existing research reports show that rapeseed protein has high foam characteristics and is a plant protein-based foaming agent with great application potential in the food field. However, due to its poor color and high content of anti-nutritional factors, it is not conducive to consumption, and the existing preparation process has not fully exerted its foam characteristics. The present invention improves its color, reduces its anti-nutritional factor content, improves its foaming property, prepares rapeseed protein with both high foaming property and edibility, and applies it as a food ingredient and a foaming agent in the food field.

[0027] Aiming at the problems of multi-component composition, wide isoelectric point range, and large influence of non-protein components of rapeseed protein, compared with the prior art, the preparation method of the highly foaming edible rapeseed protein disclosed in the present invention uses the "gradient acidic-neutral" pH cycling treatment technology, and couples membrane separation and physical field modification technology under gradient pH control to orderly regulate the structural characteristics such as protein charge, size, and surface hydrophobicity. It not only breaks through the concentration limit of traditional rapeseed protein solution, but also realizes the structural modification and functional property improvement of high-concentration rapeseed protein.

[0028] Among them, the primary acid control collaborative ultrafiltration step concentration technology further processes the rapeseed protein extract obtained by the synchronous directional sieving technology of moderate salt solution. Ultrafiltration is carried out step by step under weak acid conditions (pH range of 5.0 - 7.0), which is beneficial to removing small molecule substances such as glucosinolates and phytic acid, and can control the structure and aggregation behavior of proteins, obtaining a high-concentration rapeseed protein solution with a protein concentration ≥ 6%. The secondary acid control is coupled with physical field modification treatment. Under acidic conditions (pH range of 2.0 - 4.0), the high-concentration rapeseed protein solution is further acidified and modified. On the one hand, it avoids the influence of alkaline conditions on polyphenolic substances in rapeseed protein. On the other hand, through cavitation, shearing, heating and other effects, it penetrates into the internal structure of proteins, further depolymerizes the high-concentration proteins and changes the surface structure. Finally, the process of re-adjusting the pH back to neutral causes the proteins to self-assemble to form aggregates, ultimately endowing rapeseed protein with high foaming properties. The obtained high-foaming edible rapeseed protein has a maximum foaming property of up to 392%, which is higher than that of plant proteins such as soybean protein, 3.1 times that of the commonly used industrial foaming agent ovalbumin, and 2.1 times that of sodium caseinate. When used, the foam is dense and has good plasticity, and has the potential to become an egg white substitute. Brief Description of the Drawings

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0030] Figure 1 It is a schematic diagram of the preparation process of the high-foaming edible rapeseed protein of the present invention.

[0031] Figure 2 It is a macroscopic view of the foam plasticity of the rapeseed protein prepared in Comparative Examples 1 - 2 and Example 2 of the present invention.

[0032] Figure 3 It is the zeta potential of the rapeseed protein prepared in Comparative Examples 1 - 3 and Example 2 of the present invention.

[0033] Figure 4 It is the surface hydrophobicity of the rapeseed protein prepared in Comparative Examples 1 - 3 and Example 2 of the present invention.

[0034] Figure 5 It is the average particle size of the rapeseed protein prepared in Comparative Examples 1 - 3 and Example 2 of the present invention.

[0035] Figure 6 It is the particle size distribution diagram of the rapeseed protein prepared in Comparative Examples 1 - 3 and Example 2 of the present invention. Detailed Description of the Invention

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0037] The special term "embodiment" here, any embodiment described as "exemplary" does not have to be construed as superior to or better than other embodiments. For the performance index tests in the embodiments of this application, unless otherwise specified, the conventional test methods in the art are adopted. It should be understood that the terms described in this application are only for describing specific embodiments and are not used to limit the content disclosed in this application.

[0038] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the technical field to which this application belongs; the test methods and technical means not otherwise specifically noted in this application refer to the experimental methods and technical means commonly adopted by those of ordinary skill in the art.

[0039] To better illustrate the content of this application, numerous specific details are given in the following specific embodiments. Those skilled in the art should understand that this application can also be implemented without certain specific details. In the embodiments, some methods, means, instruments, devices, etc. well-known to those skilled in the art are not described in detail to highlight the gist of this application.

[0040] On the premise of no conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the obtained technical solutions belong to the content disclosed in the embodiments of this application.

[0041] The present invention discloses a highly foaming edible rapeseed protein, its preparation method and application, belonging to the technical field of plant protein extraction. First, the present invention uses a moderate salt solution synchronous directional screening technology to obtain a rapeseed protein extract, and then uses a "gradient acidic-neutral" pH cycling treatment technology. Under the control of gradient pH, it couples membrane separation and physical field modification technology. Through the first acidic control, under weak acid conditions (pH 5.0-7.0), it cooperates with the ultrafiltration stepwise concentration process to obtain a high-concentration rapeseed protein solution (protein concentration ≥ 6%); the second acidic control couples physical field modification treatment, that is, under acidic conditions (pH range 2.0-4.0), it is orderly regulated to make the rapeseed protein deaggregate and the protein conformation unfold. At this time, it couples physical field treatments such as ultrasonic and infrared. Through the effects of cavitation, shear, thermal effects, etc., it penetrates into the internal region of the protein structure, changing its surface charge distribution, hydrophobicity and other structural characteristics, and affecting the interaction between polyphenols and proteins; finally, the process of pH cycling to neutral makes the protein reassemble to form controllable aggregates, ultimately endowing the rapeseed protein with high foam characteristics and good color.

[0042] To better understand the present invention, the following examples are used to further specifically elaborate the present invention, but it should not be understood as a limitation of the present invention. For some non-essential improvements and adjustments made by those skilled in the art according to the above invention content, they are also considered to fall within the protection scope of the present invention.

[0043] The following examples and comparative examples use rapeseed variety: Qinyou 1718, Anhui.

[0044] The following experimental methods are used in the examples and comparative examples:

[0045] Determination of protein content:

[0046] The protein content of the rapeseed protein obtained in the examples and comparative examples is determined by the Kjeldahl method, specifically referring to GB5009.5-2010, and the protein conversion coefficient is 5.53.

[0047] Determination of protein whiteness value:

[0048] It is measured using a CR-410 colorimeter. Before measuring the sample, it is calibrated using a standard white board. The color parameters are expressed in L* (brightness), a* (red / green) and b* (yellow / blue) values. The calculation formula for the whiteness value (WI) of the protein is as follows:

[0049]

[0050] Determination of glucosinolate content:

[0051] It is measured using high performance liquid chromatography, specifically referring to NY / T1582-2007.

[0052] Determination of phytic acid content: Disperse 0.05 g of protein sample into 2 mL of 2.4 wt% hydrochloric acid, stir for 1 h, then centrifuge at 5000 g for 15 min. Add 0.1 g of NaCl to the supernatant and stir until dissolved. Let it stand at low temperature for 1 h and then centrifuge at 5000 g for 15 min. Take 0.5 mL of the centrifuged supernatant, and successively add 4.5 mL of deionized water and 4.0 mL of the color reagent. After standing for 20 min, measure the absorbance at a wavelength of 500 nm. Ferric chloride-sulfosalicylic acid color reagent: Mix 0.03% ferric chloride hexahydrate and 0.3% 5-sulfosalicylic acid dihydrate. Draw a standard curve using sodium phytate as the standard sample.

[0053] Analysis of the functional properties of proteins:

[0054] (1) Determination of foaming ability and foam stability

[0055] Prepare a 1% (w / v) rapeseed protein solution. Take 15 mL of the protein solution in a special cylindrical glassware and stir it at 13800 g for 2 min using an IKA high-speed homogenizer. After stirring, measure the foam height at 2 min and 60 min with a scale, and then calculate the foam volume, which are recorded as V2 and V 60 , respectively. Determine the foaming ability (FA) and foam stability (FS) of rapeseed protein through the following formula.

[0056] FA (%) = V2 / 15 × 100

[0057] FS (%) = V 60 / V2 × 100

[0058] where V2 is the foam volume at 2 min and V 60 is the foam volume at 60 min.

[0059] (2) Characterization of foam plasticity

[0060] Prepare a 1% (w / v) rapeseed protein solution, foam it with a whisk and shape it in a mold, and record the change of the foam shape over time.

[0061] Characterization of protein structure:

[0062] (1) Surface hydrophobicity

[0063] Determination was carried out with reference to the method reported by Peng et al. in the study "Foams Stabilized by β-Lactoglobulin Amyloid Fibrils: Effect of pH".

[0064] (2) Particle size and zeta potential

[0065] The concentration of the sample solution was controlled to be 1 mg / mL, and a nanoparticle size analyzer (ZetaSizer Nano-ZS, Malvern Instruments, UK) was used to measure the potential of the solution; the concentration of the sample solution was controlled to be 10 mg / mL, and a laser particle size analyzer was used to measure the particle size of the solution.

[0066] Note: (1)-(2) The pH of the solution was ~7.0 during protein structure characterization.

[0067] Sample pretreatment:

[0068] The rapeseed was cold-pressed to obtain cold-pressed rapeseed cake and meal (residual oil content < 10%), and the rapeseed cake and meal were subjected to subcritical fluid extraction for defatting to obtain defatted rapeseed meal (residual oil content < 0.3%).

[0069] Example 1

[0070] The defatted rapeseed meal was coarsely ground in a mortar and then dispersed in a 100 mM calcium chloride aqueous solution at a ratio of 1:8 (w / v). Stir at room temperature (25 °C) for 30 min, sieve through a 200-mesh sieve, and then rinse the residue on the sieve with the same volume of calcium chloride aqueous solution to obtain rapeseed protein extract. Then, the pH of the extract was adjusted to 5.8 with HCl once, stirred at room temperature for 60 min, centrifuged at 10000 g for 30 min, and the supernatant was collected. Then, it was transferred to the sample chamber of the membrane separation device, and the solution was concentrated and purified by the membrane separation stepwise concentration method (membrane with MWCO 10KDa) After concentrating to a protein concentration ≥ 6%, a high-concentration rapeseed protein solution was obtained. After the pH of the high-concentration protein solution was adjusted and stabilized at 3.0 for the second time, it was subjected to ice bath ultrasonic treatment. The ultrasonic density was set to 20 W / mL, the ultrasonic time was 30 min, the pH of the solution was adjusted back to 7.0 after ultrasonic treatment, and freeze-dried for 72 h to obtain rapeseed isolate protein, which was Example 1.

[0071] Example 2

[0072] The defatted rapeseed meal was coarsely ground in a mortar and then dispersed in a 100 mM calcium chloride aqueous solution at a ratio of 1:8 (w / v). Stir at room temperature (25 °C) for 30 min, sieve through a 200-mesh sieve, and then rinse the residue on the sieve with the same volume of calcium chloride aqueous solution to obtain rapeseed protein extract. Then, the pH of the extract was adjusted to 5.8 with HCl once, stirred at room temperature for 60 min, centrifuged at 10000 g for 30 min, and the supernatant was collected. Then, it was transferred to the sample chamber of the membrane separation device, and the solution was concentrated and purified by the membrane separation stepwise concentration method (membrane with MWCO 10KDa) The solution was concentrated and purified by a membrane, and after concentrating to a protein concentration of ≥6%, a high-concentration rapeseed protein solution was obtained. The pH of the high-concentration protein solution was adjusted and stabilized at 3.0 for ice bath ultrasonic treatment. The ultrasonic density was set at 10 W / mL, the ultrasonic time was 60 min, the pH of the solution was adjusted back to 7.0 after ultrasonic treatment, and freeze-dried for 72 h to obtain rapeseed protein isolate, which is Example 2.

[0073] Example 3

[0074] The defatted rapeseed meal was coarsely ground in a mortar and dispersed in a 100 mM calcium chloride aqueous solution at a ratio of 1:8 (w / v), stirred at room temperature (25 °C) for 30 min, sieved through a 200-mesh sieve, and then the material on the sieve was rinsed with the same volume of calcium chloride aqueous solution to obtain a rapeseed protein extract. Then the pH of the extract was adjusted to 5.8 with HCl once, stirred at room temperature for 60 min, centrifuged at 10,000 g for 30 min, and the supernatant was collected. Then it was transferred to the sample chamber of a membrane separation device, and the solution was concentrated and purified by the membrane separation stepwise concentration method (membrane with MWCO 10KDa) The solution was concentrated and purified by a membrane, and after concentrating to a protein concentration of ≥6%, a high-concentration rapeseed protein solution was obtained. The pH of the high-concentration protein solution was adjusted and stabilized at 3.0 for ice bath ultrasonic treatment. The ultrasonic density was set at 30 W / mL, the ultrasonic time was 10 min, the pH of the solution was adjusted back to 7.0 after ultrasonic treatment, and freeze-dried for 72 h to obtain rapeseed protein isolate, which is Example 3.

[0075] Example 4

[0076] The defatted rapeseed meal was coarsely ground in a mortar and dispersed in a 100 mM calcium chloride aqueous solution at a ratio of 1:8 (w / v), stirred at room temperature (25 °C) for 30 min, sieved through a 200-mesh sieve, and then the material on the sieve was rinsed with the same volume of calcium chloride aqueous solution to obtain a rapeseed protein extract. Then the pH of the extract was adjusted to 5.8 with HCl once, stirred at room temperature for 60 min, centrifuged at 10,000 g for 30 min, and the supernatant was collected. Then it was transferred to the sample chamber of a membrane separation device, and the solution was concentrated and purified by the membrane separation stepwise concentration method (membrane with MWCO 10KDa) The solution was concentrated and purified by a membrane, and after concentrating to a protein concentration of ≥6%, a high-concentration rapeseed protein solution was obtained. The pH of the high-concentration protein solution was adjusted and stabilized at 4.0 for ice bath ultrasonic treatment. The ultrasonic density was set at 20 W / mL, the ultrasonic time was 30 min, the pH of the solution was adjusted back to 7.0 after ultrasonic treatment, and freeze-dried for 72 h to obtain rapeseed protein isolate, which is Example 4.

[0077] Example 5

[0078] The defatted rapeseed meal was coarsely ground in a mortar and then dispersed in a 100 mM calcium chloride aqueous solution at a ratio of 1:8 (w / v), stirred at room temperature (25 °C) for 30 min, sieved through a 200-mesh sieve, and then the material on the sieve was rinsed with the same volume of calcium chloride aqueous solution to obtain a rapeseed protein extract. Then, the extract was adjusted to pH 5.8 with HCl once, stirred at room temperature for 60 min, centrifuged at 10,000 g for 30 min, and the supernatant was collected. Then it was transferred to the sample chamber of the membrane separation device, and the solution was concentrated and purified by the membrane separation stepwise concentration method (membrane with MWCO 10 KDa of membrane). After concentration to a protein concentration ≥ 6%, a high-concentration rapeseed protein solution was obtained. After the pH of the high-concentration protein solution was adjusted and stabilized at 3.0 for the second time, infrared treatment was carried out. The short-wave was set at 80 °C and the time was 16 min. After infrared treatment, the pH of the solution was adjusted back to 7.0, and freeze-dried for 72 h to obtain rapeseed protein isolate, which was Example 5.

[0079] Example 6

[0080] The defatted rapeseed meal was coarsely ground in a mortar and then dispersed in a 100 mM calcium chloride aqueous solution at a ratio of 1:8 (w / v), stirred at room temperature (25 °C) for 30 min, sieved through a 200-mesh sieve, and then the material on the sieve was rinsed with the same volume of calcium chloride aqueous solution to obtain a rapeseed protein extract. Then, the extract was adjusted to pH 5.8 with HCl once, stirred at room temperature for 60 min, centrifuged at 10,000 g for 30 min, and the supernatant was collected. Then it was transferred to the sample chamber of the membrane separation device, and the solution was concentrated and purified by the membrane separation stepwise concentration method (membrane with MWCO 10 KDa of membrane). After concentration to a protein concentration ≥ 6%, a high-concentration rapeseed protein solution was obtained. After the pH of the high-concentration protein solution was adjusted and stabilized at 3.0 for the second time, infrared treatment was carried out. The short-wave was set at 160 °C and the time was 4 min. After infrared treatment, the pH of the solution was adjusted back to 7.0, and freeze-dried for 72 h to obtain rapeseed protein isolate, which was Example 6.

[0081] Example 7

[0082] The defatted rapeseed meal was coarsely ground in a mortar and then dispersed in a 100 mM calcium chloride aqueous solution at a ratio of 1:8 (w / v), stirred at room temperature (25 °C) for 30 min, sieved through a 200-mesh sieve, and then the material on the sieve was rinsed with the same volume of calcium chloride aqueous solution to obtain a rapeseed protein extract. Then, the extract was adjusted to pH 5.8 with HCl once, stirred at room temperature for 60 min, centrifuged at 10,000 g for 30 min, and the supernatant was collected. Then it was transferred to the sample chamber of the membrane separation device, and the solution was concentrated and purified by the membrane separation stepwise concentration method (membrane with MWCO 10 KDa of The solution was concentrated and purified by a membrane (MWCO 10KDa). After concentrating to a protein concentration of ≥6%, a high-concentration rapeseed protein solution was obtained. The pH of the high-concentration protein solution was adjusted a second time and stabilized at 2.0, followed by infrared treatment at 120°C for 8 min in the medium wave. After the infrared treatment, the pH of the solution was adjusted back to 7.0, and freeze-dried for 72 h to obtain rapeseed protein isolate, which was Example 7.

[0083] To further prove the beneficial effects of the present invention for better understanding, the following comparative examples and experimental examples further illustrate the properties and application performance of the highly foaming edible rapeseed protein and its preparation method disclosed by the present invention. However, it should not be construed as a limitation of the present invention. For those skilled in the art, the method properties obtained from other determination experiments based on the above invention content and the applications based on the above properties are also considered to fall within the protection scope of the present invention.

[0084] Comparative Example 1

[0085] The defatted rapeseed meal was coarsely ground in a mortar and dispersed in a 100 mM calcium chloride aqueous solution at a ratio of 1:8 (w / v). Stirred at room temperature (25°C) for 30 min, sieved through a 200-mesh sieve, and then the material on the sieve was rinsed with the same volume of calcium chloride aqueous solution to obtain a rapeseed protein extract. Then the extract was adjusted to pH 5.8 with HCl once, stirred at room temperature for 60 min, centrifuged at 10000 g for 30 min, and the supernatant was collected. Then it was transferred to the sample chamber of a membrane separation device, and the solution was concentrated and purified by the membrane separation stepwise concentration method (MWCO 10KDa membrane). After concentrating to a protein concentration of ≥6%, a high-concentration rapeseed protein solution was obtained. The pH of the high-concentration protein solution was adjusted to 7.0 and freeze-dried for 72 h to obtain rapeseed protein isolate, which was Comparative Example 1.

[0086] Comparative Example 2

[0087] The defatted rapeseed meal was coarsely ground in a mortar and dispersed in a 100 mM calcium chloride aqueous solution at a ratio of 1:8 (w / v). Stirred at room temperature (25°C) for 30 min, sieved through a 200-mesh sieve, and then the material on the sieve was rinsed with the same volume of calcium chloride aqueous solution to obtain a rapeseed protein extract. Then the extract was adjusted to pH 5.8 with HCl once, stirred at room temperature for 60 min, centrifuged at 10000 g for 30 min, and the supernatant was collected. Then it was transferred to the sample chamber of a membrane separation device, and the solution was concentrated and purified by the membrane separation stepwise concentration method (MWCO 10KDa The solution was concentrated and purified by a membrane, and after concentrating to a protein concentration of ≥6%, a high-concentration rapeseed protein solution was obtained. The pH of the high-concentration protein solution was adjusted a second time and stabilized at 10.0, followed by ice bath sonication. The sonication density was set at 10 W / mL, the sonication time was 60 min, and after sonication, the pH of the solution was adjusted back to 7.0 and freeze-dried for 72 h to obtain rapeseed protein isolate, which was Comparative Example 2.

[0088] Comparative Example 3

[0089] The defatted rapeseed meal was coarsely ground in a mortar and dispersed in a 100 mM calcium chloride aqueous solution at a ratio of 1:8 (w / v). Stir at room temperature (25 °C) for 30 min, sieve through a 200-mesh sieve, and then rinse the material on the sieve with the same volume of calcium chloride aqueous solution to obtain a rapeseed protein extract. Then, the pH of the extract was adjusted to 5.8 with HCl once, stirred at room temperature for 60 min, centrifuged at 10,000 g for 30 min, and the supernatant was collected. Then it was transferred to the sample chamber of a membrane separation device, and the solution was concentrated and purified by the membrane separation stepwise concentration method (membrane with MWCO 10KDa) The solution was concentrated and purified by a membrane, and after concentrating to a protein concentration of ≥6%, a high-concentration rapeseed protein solution was obtained. The pH of the high-concentration protein solution was adjusted a second time and stabilized at 3.0, and then the pH of the solution was adjusted back to 7.0 and freeze-dried for 72 h to obtain rapeseed protein isolate, which was Comparative Example 3.

[0090] Comparative Example 4

[0091] The defatted rapeseed meal was coarsely ground in a mortar and dispersed in a 100 mM calcium chloride aqueous solution at a ratio of 1:8 (w / v). Stir at room temperature (25 °C) for 30 min, sieve through a 200-mesh sieve, and then rinse the material on the sieve with the same volume of calcium chloride aqueous solution to obtain a rapeseed protein extract. Then, the pH of the extract was adjusted to 5.8 with HCl once, stirred at room temperature for 60 min, centrifuged at 10,000 g for 30 min, and the supernatant was collected. Then it was transferred to the sample chamber of a membrane separation device, and the solution was concentrated and purified by the membrane separation stepwise concentration method (membrane with MWCO 10KDa) The solution was concentrated and purified by a membrane, and after concentrating to a protein concentration of ≥6%, a high-concentration rapeseed protein solution was obtained. The pH of the high-concentration protein solution was adjusted a second time and stabilized at 10.0, followed by infrared treatment. The infrared short wave was set at 160 °C and the time was 4 min. After infrared treatment, the pH of the solution was adjusted back to 7.0 and freeze-dried for 72 h to obtain rapeseed protein isolate, which was Comparative Example 4.

[0092] Experimental Example 1

[0093] The protein contents of the rapeseed proteins obtained in Examples 1-7 were all at a relatively high level. When the protein conversion coefficient was 5.53, the protein contents were all about 81%, meeting the requirements for concentrated protein or isolated protein in the protein products commercially available in China. In addition, after converting the data according to the conversion coefficient of 6.25, the protein contents of Examples 1-7 were about 92%, and this data can be used for comparison with the protein contents of related foreign products.

[0094] The glucosinolate and phytic acid contents of the rapeseed proteins in Examples 1-2 were detected, and the glucosinolate content was about 0.87 μmol / g and the phytic acid content was about 0.78%. Both contents met the standards of the European Union's edible rapeseed protein for glucosinolate (≤1 μmol / g) and phytic acid (≤1.5%).

[0095] Experimental Example 2

[0096] The foaming properties, foam stability and foam plasticity of the rapeseed proteins obtained in Examples 1-7 and Comparative Examples 1-4 were compared. The results are shown in Table 2 and Figure 2 as follows.

[0097] Table 1 Foaming properties and foam stability of the rapeseed proteins obtained in Examples 1-7 and Comparative Examples 1-4

[0098]

[0099]

[0100] As can be seen from Table 1, the foaming properties of the rapeseed proteins obtained in Examples 1-7 are all greater than 329%, and the highest can reach 392%. Compared with Comparative Example 1 (~210%), the foaming properties of Examples 1-7 are greatly improved, proving that the "gradient acidic-neutral" pH cycling treatment coupled with membrane separation and physical field modification technology has significant technical effects in improving the foaming properties of rapeseed proteins. Among them, the ultrasonic treatment parameters of Example 2 and Comparative Example 2 are the same, only the cycling pH is different; the infrared treatment parameters of Example 6 and Comparative Example 4 are the same, only the cycling pH is different; Examples 2 and 6 are gradient acidification (weak acid → acidic)-neutral cycling, while Comparative Examples 2 and 4 use acid-alkali-neutral adjustment. By comparing Example 2 (foaming property ~384%, foam stability ~77%) and Example 6 (foaming property ~389%, foam stability ~76%) with Comparative Example 2 (foaming property ~327%, foam stability ~74%) and Comparative Example 4 (foaming property ~239%, foam stability ~51%), it can be seen that gradient acidification-neutral cycling coupled with physical field modification can achieve dynamic and orderly regulation of structural properties such as protein charge and surface hydrophobicity, and can also orderly regulate the interaction between proteins and polyphenols. Therefore, the technical effects on foaming property and foam stability are significantly better than those of acid-alkali-neutral adjustment coupled with physical field modification; the foaming properties of Examples 1-3 and Example 5 are all greater than 384%, and the foam stabilities are all greater than 77%. By comparing them with Comparative Example 3 (foaming property ~329%, foam stability ~66%), it is found that on the basis of gradient acidification-neutral cycling, further coupling with physical field treatment can further significantly improve the foam properties of rapeseed proteins.

[0101] In addition, from Figure 2 it can be seen that the foam plasticity of Example 2 (here refers to the ability of the foam to maintain its certain shape) is significantly better than that of Comparative Example 1 and Comparative Example 2, indicating that gradient acidification-neutral cycling coupled with physical field treatment can also endow rapeseed proteins with good foam plasticity. In summary, only by coupling gradient acidification-neutral cycling with physical fields can rapeseed proteins have both ultra-high foaming properties, good foam stability and excellent foam plasticity, thereby significantly expanding the application range of rapeseed proteins as plant-based foaming agents in the food field.

[0102] From Figures 3 - 6It can be seen that the structural properties such as surface charge, particle size, and surface hydrophilic-hydrophobic group distribution of the rapeseed proteins obtained in Example 2 and Comparative Examples 1-3 are all different. In Comparative Example 1, the rapeseed protein was obtained by directly adjusting the pH to 7 and then freeze-drying after obtaining a high-concentration rapeseed protein solution without any treatment. At this time, the rapeseed protein had a wide particle size scale distribution, a relatively low surface hydrophobicity, and a relatively small surface charge. In Comparative Example 2, the rapeseed protein was obtained by freeze-drying after subjecting the obtained rapeseed protein extract to acid-alkaline-neutral adjustment coupled with ultrasonic treatment. Compared with Comparative Example 1, under the condition of moderate alkaline coupled with ultrasonic treatment of the high-concentration protein solution, large aggregates would depolymerize. During the process of readjusting to neutrality, due to the regulation of protein assembly behavior by factors such as low-concentration salt ions, polyphenols, and pH, more charged groups in the internal structure of the protein were exposed on the surface, while the hydrophobic groups were only slightly exposed, ultimately increasing the protein potential, slightly increasing the surface hydrophobicity, and reducing the scale. In Comparative Example 3, the rapeseed protein was obtained by freeze-drying after subjecting the obtained rapeseed protein extract to gradient acidification-neutral cycling treatment. During the gradient acidification-neutral cycling process of the rapeseed protein extract, the protein underwent depolymerization and re-assembled into large-scale aggregates, and when re-assembling, the charged groups on the protein surface were embedded inside the aggregate structure, ultimately reducing the protein potential and increasing the scale. Example 2 was the rapeseed protein obtained by freeze-drying after subjecting the obtained rapeseed protein extract to gradient acidification-neutral cycling coupled with ultrasonic treatment. Under the condition of acid coupled with ultrasonic treatment of the high-concentration protein solution, compared with Comparative Example 3, under the effects of cavitation and shear of ultrasonic waves, the large aggregates would undergo more intense disaggregation. During the process of readjusting to neutrality, due to the regulation of the protein re-assembly process by factors such as low-concentration salt ions, polyphenols, and pH, more hydrophobic groups in the internal structure of the protein were exposed on the surface, ultimately significantly increasing the surface hydrophobicity of the protein and reducing the scale. The above data and corresponding analyses all prove that the gradient acidification-neutral cycling coupled with physical field modification technology, the gradient acidification-neutral cycling treatment, and the acid-alkaline-neutral adjustment coupled with ultrasonic modification treatment have different regulation principles for protein structure and aggregation behavior, and thus lead to different foam properties of the protein.

[0103] Experimental Example 3

[0104] The colors of the rapeseed proteins obtained in Examples 1-7 and Comparative Examples 1-4 were compared, and the results are shown in Table 2.

[0105] Table 2 Colors of the rapeseed proteins obtained in Examples 1-7 and Comparative Examples 1-4

[0106]

[0107]

[0108] WI is the whiteness value, and the higher its value, the better the color of the sample. As can be seen from Table 2, the WI values of the rapeseed proteins prepared in Examples 1-7 are all greater than 68. By comparing Examples 1-7 with Comparative Example 1, it can be known that after the rapeseed protein is modified by gradient acidification-neutral cycle coupling physical field treatment, the color does not become worse. Moreover, when the pH and the key parameters of the physical field are appropriate, the color of the rapeseed protein is improved. For example, the WI of Example 3 is 72, higher than 68 of Comparative Example 1; by comparing Examples 1-3, Examples 5-6 with Comparative Example 1 and Comparative Example 3, it can be known that the color of the rapeseed protein can be improved only when the gradient acidification-neutral cycle is coupled with the physical field; the ultrasonic treatment parameters of Example 2 and Comparative Example 2 are the same, only the pH of the cycle is different, the infrared treatment parameters of Example 6 and Comparative Example 4 are the same, only the pH of the cycle is different. Example 2 and 6 are gradient acidification-neutral cycle, while Comparative Example 2 and 4 use acid-alkali-neutral adjustment. By comparing them with Comparative Example 1, it can be known that the acid-alkali-neutral adjustment coupling physical field modification treatment will cause the color of the rapeseed protein to become worse (the WI values of Comparative Example 2 and 4 are only 61 and 65). Only by using the gradient acidification-neutral cycle coupling physical field modification technology can the color of the rapeseed protein be improved.

[0109] In summary, the rapeseed protein prepared by the rapeseed protein preparation process created by the present invention has glucosinolate and phytic acid contents meeting the EU edible rapeseed protein standard; and the present invention breaks through the "gradient acid-neutral" pH cycle treatment coupling membrane separation and physical field modification technology, which can significantly improve the foam characteristics of rapeseed protein, and by optimizing the key parameters, the color of rapeseed protein can be improved.

[0110] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can 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 these embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of highly foaming edible rapeseed protein, characterized in that The rapeseed protein extract was subjected to a "gradient acid-neutral" pH cycle treatment, and coupled with membrane separation and physical field modification technology to achieve structural modification and quality improvement of high-concentration rapeseed protein.

2. The preparation method according to claim 1, characterized in that, The rapeseed protein extract is obtained by using a moderate salt solution synchronous directional screening technology; then, the acidity is controlled once, and ultrafiltration is coordinated with step concentration to obtain a high-concentration rapeseed protein solution; then, the acidity is controlled twice, and physical field treatment is coupled; finally, the pH value of the high-concentration protein solution is adjusted back to a neutral environment, and freeze-dried to obtain the high-foaming edible rapeseed protein.

3. The preparation method according to claim 2, wherein The pH range of the primary acid control is 5.0-7.0, and the pH range of the secondary acid control is 2.0-4.

0.

4. The preparation method according to claim 2, characterized in that, The physical field modification treatment includes ultrasonic treatment and infrared treatment.

5. The preparation method according to claim 4, wherein The power density of the ultrasonic treatment is 10-30 W / mL, and the ultrasonic treatment time is 10-60 min. The infrared treatment includes short-wave treatment and long-wave treatment, the temperature is 80-160° C., and the infrared treatment time is 4-16 min.

6. The preparation method according to claim 2, characterized in that, In the moderate salt solution synchronous directional screening technology, the salt solution is a 100mM calcium chloride or magnesium chloride solution, and the directional screening mesh number is 60-200 meshes.

7. The preparation method according to claim 2, wherein, The membrane molecular weight used in the ultrafiltration step concentration is 5-30 kDa, and the protein concentration of the high-concentration rapeseed protein solution is ≥6%.

8. An edible rapeseed protein with high foamability obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the high foaming edible rapeseed protein as claimed in claim 8 as a protein ingredient or foaming agent in the food field.

Images