High-functionality plant protein and preparation method and application thereof

Abstract

The application belongs to the technical field of plant protein extraction, and discloses a high-functionality plant protein as well as a preparation method and application thereof.A membrane separation coupling physical field modification technology is disclosed in the application, after the protein concentrate obtained through membrane separation is controlled to be moderately alkaline, the protein concentrate is coupled with physical field modification, not only the protein aggregation caused by acid precipitation near the isoelectric point in the preparation process of alkali extraction and acid precipitation is avoided, but also the green purification and efficient modification of the protein are realized through the membrane separation coupling physical field in the extraction process, the structural characteristics of the plant protein are improved, the plant protein is endowed with good functional characteristics, compared with the existing industrial commonly used plant protein extraction technology, the plant protein component obtained by the application is richer, and has the advantages of better solubility, foaming property and emulsifying property, and the protein preparation process involved in the application combines the purification and modification processes, is simple to operate, saves energy and reduces consumption, and has great application potential in the preparation and food utilization of plant protein.

Description

Technical Field

[0001] This invention belongs to the field of plant protein extraction technology, specifically relating to a high-functionality plant protein, its preparation method, and its application. Background Technology

[0002] Protein is one of the three major nutrients for the human body. my country has abundant plant resources such as oilseeds and legumes, which are rich in protein (mostly ≥20%), such as rapeseed, flaxseed, walnuts, peas, and mung beans. In particular, the by-products of oilseed processing, namely oil cakes, contain a large amount of protein. For example, rapeseed cake and flaxseed cake have a protein content >35%, and walnut cake has a protein content of over 50%. Moreover, plant proteins from oilseeds and legumes have the advantages of relatively balanced amino acid composition and high nutritional value. Therefore, enriching the protein in legume and oilseed cakes can not only improve the resource utilization rate of raw materials, but also contribute to the effective supply of protein in my country. Furthermore, plant proteins from oilseeds and legumes have a variety of functional properties, such as solubility, emulsification, and foaming properties. These properties allow plant proteins to be used as emulsifiers, foaming agents, and other additives in food processing, effectively enhancing their market value.

[0003] Currently, alkaline extraction and acid precipitation is the most commonly used method in industry for preparing plant proteins from oilseeds, legumes, and other sources. However, this method easily leads to the aggregation of plant proteins, resulting in a deterioration of their functional properties. Specifically, when the pH of the solution after alkaline extraction is adjusted to the pH required for acid precipitation (close to the isoelectric point of the protein), the protein, with a net charge close to 0, easily aggregates and precipitates through hydrophobic interactions and other forces. This process may cause changes in the higher-order structure of the protein, ultimately resulting in poor functional properties of the plant protein, such as a solubility generally <30%, which greatly limits the application potential of plant proteins. At the same time, this extraction method also has problems such as water and energy waste and environmental pollution, which means that traditional protein extraction technology cannot well meet the needs of the rapidly developing food industry.

[0004] Therefore, it is of great significance to develop a method for preparing plant proteins with high functional properties by combining the resource advantages of oilseeds, beans and other plant proteins. Summary of the Invention

[0005] In view of this, in order to solve the problem that acid precipitation near the isoelectric point in the traditional alkaline extraction and acid precipitation process can easily lead to protein aggregation, which in turn limits the functional properties, the present invention adopts membrane separation coupled with physical field modification technology to obtain a green preparation method for high-functionality plant proteins.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A green method for preparing high-functional plant protein involves controlling the protein concentrate obtained by membrane separation to a moderately alkaline state, and then coupling it with a physical field for modification to obtain a high-functional plant protein.

[0008] It is worth noting that in the traditional alkaline extraction and acid precipitation process, acid precipitation near the isoelectric point can easily lead to protein aggregation, limiting the functional properties of plant proteins. To avoid the impact of acid precipitation on the functional properties of plant proteins, a method was developed to obtain a protein concentrate by simultaneous ultrafiltration of defatted oilseed cake with a weak alkali, followed by reconstitution with water and then freeze-drying to obtain plant proteins. However, although this method has advantages such as high yield and good functional properties, its functional properties still cannot meet the current needs of the food industry for protein ingredients.

[0009] Therefore, based on the protein concentrate technology obtained by membrane separation, this invention directly controls the physical field modification of the protein concentrate under moderately alkaline conditions without drying or reconstitution steps. By adding a physical field, the size, surface properties, and aggregation state of the target protein can be further controlled, thereby endowing the plant protein with better solubility, foaming ability, and emulsifying capacity. Compared with obtaining the target protein, reconstitution, and then pH shifting combined with ultrasonic modification, the target protein preparation process involved in this invention combines green purification and modification processes, eliminating steps such as drying and reconstitution of the target protein. The process is simple, efficient, green, energy-saving, environmentally friendly, and easier to industrialize, with a more significant improvement in the functional properties of the target protein. Furthermore, the new process disclosed in this invention can be seamlessly integrated with existing processes containing membrane separation. The key indicator of the original process—the protein extraction rate—is maintained with the support of the new physical field treatment step. This facilitates the effective integration of physical field treatment within the original process framework, contributing to the upgrading of protein product production processes and effectively enhancing the industrial value of deep-processed oilseed cakes and soybean flour products.

[0010] Furthermore, the moderate alkalinity of the protein concentrate is controlled within the range of 8.0-11.0, the physical field modification is selected from ultrasonic treatment or infrared treatment, and the plant protein includes pea protein, mung bean protein, soybean protein, chickpea protein, rapeseed protein, flaxseed protein, sesame protein, walnut protein, perilla protein, wheat protein, or rice protein.

[0011] It is worth noting that although directly applying pH shifting combined with ultrasonic treatment to plant proteins can further improve their functional properties, this method involves complex steps, and the resolution effect of ultrasonically treated proteins is limited, which is time-consuming and labor-intensive and cannot meet the industrial needs of deep processing of oilseed cakes and soybean flour.

[0012] This invention utilizes the property of protein structure unfolding / partial unfolding under alkaline conditions. After controlling the pH of the protein concentrate, it is directly subjected to physical field treatments such as ultrasound and infrared. Under the action of cavitation and thermal effects, the protein can be depolymerized and its internal structure can be further changed, such as the breaking of disulfide bonds and the exposure of internal hydrophobic groups. This regulates the surface structural characteristics of plant proteins, thereby improving the functional properties of plant proteins such as solubility, foaming, and emulsification.

[0013] Furthermore, the ultrasonic power density of the ultrasonic treatment is 5-30 W / mL, and the ultrasonic time is 10-60 min.

[0014] Furthermore, the infrared temperature of the infrared treatment is 80-160℃, the infrared treatment time is 8-32 minutes, and the infrared wavelength includes medium wave and short wave.

[0015] Furthermore, the preparation method includes the following steps:

[0016] S1. Process defatted oilseed cake or soybean flour using a membrane separation method to obtain a protein concentrate;

[0017] S2. Adjust the pH of the protein concentrate to a stable value of 8.0-11.0, and then perform physical field modification;

[0018] S3. Freeze-dry the solution modified by the physical field to obtain the high-functionality plant protein.

[0019] Furthermore, the membrane separation method for processing defatted oilseed cake or soybean flour includes the following steps:

[0020] 1) Disperse defatted oilseed meal or soybean flour in water, stir at room temperature, and then perform alkali dissolution treatment;

[0021] 2) Centrifuge to remove insoluble impurities and collect the supernatant;

[0022] 3) The supernatant obtained in step 2) was concentrated and purified by membrane separation to obtain protein concentrate.

[0023] It is worth noting that compared with the existing industrial plant protein extraction techniques, such as alkaline extraction and acid precipitation, membrane separation technology can effectively increase the protein content in the protein concentrate. This invention, by coupling physical field treatment technology with the original membrane separation technology, not only avoids adversely affecting the plant protein extraction rate but also achieves a high-efficiency enhancement of the plant protein's functionality through physical field treatment. The new process has been validated for compatibility within the original membrane separation technology framework, effectively maintaining the key performance indicator of protein extraction rate, demonstrating significant technological optimization.

[0024] Furthermore, the pH value of the alkali dissolution treatment in step 1) is 8.5-11.0.

[0025] Considering the different compositions of plant proteins, the pH at which proteins dissolve from oilseed cakes or soybean flour varies during alkali dissolution treatment. Flaxseed and rapeseed proteins mainly consist of globulins and albumins, which can be effectively dissolved at around pH 9. Walnut protein, on the other hand, is mainly gluten, and most proteins can only dissolve at pH 11. Therefore, this invention limits the pH value for alkali dissolution treatment to 8.5-11.0. That is, considering the requirements of green environmental protection, this invention selects weakly alkaline conditions of pH 8.5-10.0 for plant proteins mainly composed of globulins and albumins, preferably pH 8.5-9.0; while for plant proteins mainly composed of gluten, the preferred alkali dissolution pH value is 11.0.

[0026] Furthermore, the membrane used in step 3) of the membrane separation method is 5-30 kDa.

[0027] The membrane separation technology used in this invention can, on the one hand, purify and concentrate the protein solution dissolved in oilseed cake, removing non-protein components with small molecular weights, such as polyphenols, pigments, and small molecule sugars; on the other hand, it can also effectively control the protein cutoff size. If the membrane size is too large (e.g., greater than 30 kDa), small molecular weight proteins (e.g., albumin) will also be removed, resulting in low protein yield and affecting the functional properties of the target protein.

[0028] In particular, unlike existing plant protein extraction technologies which are mostly applicable to the preparation of globulin-rich pea protein, mung bean protein, soybean protein, chickpea protein, rapeseed protein, perilla protein, flaxseed protein, or sesame protein, the membrane separation coupling physical field modification technology disclosed in this invention is also applicable to the preparation of gluten-rich walnut protein, wheat protein, rice protein, and other proteins, as well as protein products containing albumin.

[0029] The membrane separation coupled with physical field modification technology disclosed in this invention, after controlling the protein concentrate obtained from membrane separation to a moderately alkaline state, is then coupled with physical field modification. This not only avoids protein aggregation caused by acid precipitation near the isoelectric point during alkaline extraction and acid precipitation, but also achieves green purification and efficient modification of proteins during extraction through membrane separation coupled with physical fields. This improves the structural characteristics of plant proteins and endows them with excellent functional properties. Compared with simple weak-alkali simultaneous ultrafiltration and pH shifting combined with ultrasonic treatment, plant proteins prepared using the membrane separation coupled with physical field modification technology of this invention have advantages in terms of better solubility, foaming properties, and emulsifying properties.

[0030] Furthermore, in existing alkaline extraction and acid precipitation processes for obtaining plant proteins, most albumin remains in the supernatant after the acid precipitation step, while globulins / glutenin precipitate out, ultimately resulting in plant proteins dominated by globulins / glutenin components, making albumin enrichment difficult. This invention, based on the consideration of enriching protein components, achieves the enrichment of multiple components such as albumin, globulins, and glutenin by controlling the molecular size of the membrane, thus providing technical support for the enrichment and utilization of multi-component proteins.

[0031] The present invention also aims to provide a highly functional plant protein prepared by the preparation method described above, and the application of the highly functional plant protein in food preparation.

[0032] It is worth noting that foaming agents and emulsifiers are important food additives. The most commonly used protein products include ovalbumin, sodium caseinate, and whey protein, most of which are animal-derived proteins. While these proteins possess good foaming and emulsifying properties, they still struggle to meet the requirements of most food processing. In actual food processing, they are often used in combination with other food additives with similar or auxiliary functions. With the rapid development of the food industry and consumers' increasing pursuit of dietary quality, reducing the amount and types of food additives is a trend. Therefore, developing plant-based protein ingredients with higher foaming and emulsifying properties is of great significance. The high-functionality plant protein disclosed in this invention combines the resource advantages and nutritional value of oilseed meal protein and legume protein, and its ultra-high foaming and emulsifying properties have enormous application potential in food preparation.

[0033] Compared with existing technologies, the green preparation method for highly functional plant proteins disclosed in this invention omits the steps of drying and then reconstituted and modified plant proteins. That is, green purification and efficient modification of proteins are achieved during the extraction process, thereby improving the structural characteristics of plant proteins and giving them advantages such as good solubility, foaming properties and emulsifying ability. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0035] Figure 1 This is a process flow diagram of the green preparation method of highly functional plant protein disclosed in this invention.

[0036] Figure 2The images show circular dichroisms of flaxseed protein isolates obtained by different methods in Experimental Examples 1-2 and Comparative Examples 3-5 of this invention.

[0037] Figure 3 The images show the non-reducing and reducing SDS-PAGE spectra of flaxseed protein isolates obtained in Comparative Example 6, Example 1, and Example 3 of this invention.

[0038] Figure 4 The average particle size of the walnut protein isolate obtained in Example 8 and Comparative Examples 7-8 of this invention.

[0039] Figure 5 The images show the endogenous fluorescence of walnut protein isolates obtained in Example 8 and Comparative Examples 7-8 of this invention.

[0040] Figure 6 The surface hydrophobicity of the walnut protein isolate obtained in Example 8 and Comparative Examples 7-8 of this invention.

[0041] Figure 7 The free thiol and disulfide bond content of walnut protein isolate obtained in Example 8 and Comparative Examples 7-8 of this invention.

[0042] Figure 8 The solubility of flaxseed protein isolate obtained in Examples 1-3, Example 5 and Comparative Examples 1-5 of this invention.

[0043] Figure 9 The emulsifying properties of flaxseed protein isolate obtained in Example 2 and Comparative Examples 3-5 of this invention.

[0044] Figure 10 The foaming properties of flaxseed protein isolates obtained in Examples 1-4 and Comparative Examples 1 and 3-5 of this invention.

[0045] Figure 11 The solubility of walnut protein isolate obtained in Examples 6-9, Example 11 and Comparative Examples 7-8 of this invention.

[0046] Figure 12 The emulsifying properties of walnut protein isolates obtained in Examples 6-9, Example 11, and Comparative Examples 7-8 of this invention.

[0047] Figure 13 The solubility of rapeseed protein obtained in Examples 12-17 and Comparative Example 9 of this invention is shown.

[0048] Figure 14 The emulsifying properties of rapeseed protein obtained in Examples 12-14, Example 16 and Comparative Example 9 of the present invention.

[0049] Figure 15 This refers to the emulsification stability of rapeseed protein obtained in Examples 12-15, Example 17, and Comparative Example 9 of the present invention.

[0050] Figure 16 The foaming properties of rapeseed protein obtained in Examples 12-17 and Comparative Example 9 of this invention. Detailed Implementation

[0051] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in the embodiments of this application, unless otherwise specified, employs conventional testing methods in the art. It should be understood that the terminology used in this application is merely for describing particular implementations and is not intended to limit the scope of this disclosure.

[0053] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; other experimental methods and technical means not specifically mentioned herein refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0054] To better illustrate the content of this application, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this application can be implemented even without certain specific details. In the embodiments, some methods, means, instruments, and devices well-known to those skilled in the art are not described in detail in order to highlight the main points of this application.

[0055] Without conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the resulting technical solution belongs to the content disclosed in the embodiments of this application.

[0056] This invention discloses a high-functionality plant protein, its preparation method, and its applications, belonging to the field of plant protein extraction technology. The membrane separation coupled with physical field modification technology disclosed in this invention, after controlling the protein concentrate obtained from membrane separation to a moderately alkaline state, couples physical field modification. This not only avoids protein aggregation caused by acid precipitation near the isoelectric point during alkaline extraction and acid precipitation, but also achieves green purification and efficient modification of the protein during extraction through membrane separation coupled with physical fields. This improves the structural characteristics of the plant protein and endows it with excellent functional properties. Compared with alkaline dissolution and acid precipitation, weak alkali simultaneous ultrafiltration, and pH shifting combined with ultrasonic treatment, the plant protein extracted using this invention has a richer composition and better solubility, foaming properties, and emulsifying properties. Furthermore, the protein preparation process involved in this invention combines purification and modification processes, is simple to operate, energy-saving, and has great application potential in plant protein preparation and its food utilization.

[0057] To better understand the present invention, the following embodiments are provided for further detailed description of the present invention, but they should not be construed as limiting the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also considered to fall within the protection scope of the present invention.

[0058] The experimental and characterization methods involved in this invention are as follows:

[0059] 1. Methods for determining protein content and protein extraction rate:

[0060] (1) Protein content

[0061] The protein content of the examples and comparative examples was determined by the Kjeldahl method, and the nitrogen conversion coefficient was determined in accordance with the national standard GB5009.5-2016.

[0062] (2) Protein extraction rate

[0063] The protein extraction rate (%) was calculated according to the method reported by Shen et al. in their study "The impact of hempseed dehulling on chemical composition, structure properties and aromatic profile of hemp protein isolate". The extraction rate was calculated by dividing the mass of the lyophilized protein sample by the mass of the defatted meal before extraction.

[0064] 2. Protein structure characterization methods:

[0065] (1) Particle size and potential

[0066] The concentration of the sample solution was controlled at 1 mg / mL, and the potential and particle size of the solution were determined using a nanoparticle size analyzer (ZetaSizerNano-ZS, Malvern Instruments, UK).

[0067] (2) Circular dichroism

[0068] The sample solution concentration was controlled at 0.2 mg / mL, and far-ultraviolet circular dichroism (CD) spectra were obtained at 190–260 nm using a circular dichroism (CD) under conditions of 1.0 nm bandwidth and 100 mm optical path.

[0069] (3) Polyacrylamide gel electrophoresis (SDS-PAGE)

[0070] The sample solution concentration was 2 mg / mL, prepared using Omni-Easy TM The assay was performed using a one-step PAGE gel rapid preparation kit.

[0071] (4) Intrinsic fluorescence

[0072] The sample solution concentration was controlled at 1 mg / mL, and analysis was performed using an F7000 fluorescence spectrophotometer. A quartz cuvette with a path length of 1 cm was used. The excitation wavelength was 290 nm, and the emission wavelength was 300–400 nm. The excitation and emission slit widths were both set to 2.5 nm, the voltage to 750 V, and the scan rate to 1200 nm / min.

[0073] (5) Surface hydrophobicity

[0074] The determination was performed according to the method reported by Peng et al. in their study "Foams Stabilized by β-Lactoglobulin Amyloid Fibrils: Effect of pH".

[0075] (6) Content of free thiol groups and disulfide bonds

[0076] The determination was performed according to the method reported by Yang et al. in their study "Ultrasound coupled with weak alkali cycling-induced exchange of free sulfhydryl-disulfide bond for remodeling interfacial flexibility of flaxseed protein isolates".

[0077] Note: The solution pH for protein structure characterization in (1) to (5) is ~7.0.

[0078] 3. Characterization of protein functional properties:

[0079] (1) Solubility determination

[0080] The protein sample was diluted with ultrapure water to a concentration of 1 mg / mL and centrifuged at 5000g for 10 min. After centrifugation, the supernatant was collected, and the protein concentration was determined using the Bradford method with BSA as the standard protein.

[0081] Wherein, solubility (%) = [(supernatant protein content × 100) / total protein content of the sample before centrifugation] × 100%

[0082] (2) Determination of emulsifying properties and emulsion stability

[0083] Prepare a 1% (w / v) protein solution. Mix 15 mL of the protein solution with 5 mL of walnut oil at 13800 rpm for 2 min to homogenize. Take 50 μL of the freshly prepared emulsion and mix it with 5 mL of 0.1% SDS. Measure the absorbance at 500 nm and record it as A0. Let it stand at room temperature for 10 min, and measure the absorbance at 500 nm again, recording it as A0. 10 The formulas for calculating emulsifying property (EAI) and emulsifying stability (ESI) are as follows:

[0084] EAI(m 2 / g)=(2×2.303×A0×DF) / [θ×C×10000]

[0085] ESI(min)=(A0×ΔT) / ΔA×100%

[0086] In the formula, DF is the dilution factor; C is the protein concentration (mg / mL); θ is the oil volume fraction; ΔT = 10 min; and ΔA is the difference between A0 and A2. 10 difference.

[0087] (3) Determination of foaming properties and foam stability

[0088] Prepare a 1% (w / v) protein solution. Take 15 mL of the protein solution into a specially designed cylindrical glass dish and homogenize using an IKA high-speed homogenizer at 13800 rpm for 2 min. After homogenization, measure the foam height at 2 min and 60 min using a ruler, and record them as V2 and V, respectively. 60 Calculate foaming property (FA) and foam stability (FS).

[0089] FA(%) = V² / 15 × 100

[0090] FS(%) = V 60 / V2×100

[0091] Where V2 is the foam volume over 2 minutes, V 60 It is the foam volume over 60 minutes.

[0092] Note: The solution pH for (1) to (3) protein functional characterization is ~7.0.

[0093] The oilseed varieties used in the following examples and comparative examples are: flaxseed: Zhangye Huangzi, Gansu; walnut: Wen 185, Xinjiang; rapeseed: Qinyou 1718, Anhui.

[0094] Example 1

[0095] Defatted flaxseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 9.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 30kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH was adjusted to a stable value of approximately 10.0, and ultrasonic treatment was performed at a power of 5 W / mL for 60 min. An ice bath was used to prevent overheating of the solution during ultrasonication. The resulting solution was freeze-dried to obtain flaxseed protein isolate, which is Example 1, named: Membrane Separation (30 kDa) Coupled with Ultrasonic Treatment (5 W / mL - 60 min).

[0096] Example 2

[0097] Defatted flaxseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 9.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 30kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH was adjusted to a stable value of approximately 10.0, and infrared treatment was performed at a short-wavelength of 160°C for 4 minutes. The resulting solution was freeze-dried to obtain flaxseed protein isolate, which is Example 2, named: Membrane Separation (30 kDa) Coupled with Infrared Short-Wave Treatment (160°C - 4 min).

[0098] Example 3

[0099] Defatted flaxseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was then adjusted to 9.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 5kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH was adjusted to a stable value of approximately 10.0, and infrared treatment was performed at a short-wavelength temperature of 80°C for 16 minutes. The resulting solution was freeze-dried to obtain flaxseed protein isolate, which is Example 3, named: Membrane Separation (5kDa) Coupled with Infrared Short-Wave Treatment (80°C - 16 min).

[0100] Example 4

[0101] Defatted flaxseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 9.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 5kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH was adjusted to a stable value of approximately 10.0, and ultrasonic treatment was performed at a power of 20 W / mL for 20 min, using an ice bath to prevent overheating. The resulting solution was freeze-dried to obtain flaxseed protein isolate, which is Example 4, named: Membrane Separation (5 kDa) Coupled with Ultrasonic Treatment (20 W / mL - 20 min).

[0102] Example 5

[0103] Defatted flaxseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 10.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 10 kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH was adjusted to a stable value of approximately 10.0, and ultrasonic treatment was performed at a power of 10 W / mL for 40 min. An ice bath was used to prevent overheating of the solution during ultrasonication. The resulting solution was freeze-dried to obtain flaxseed protein isolate, which is Example 5, named "Membrane Separation (10 kDa) Coupled with Ultrasonic Treatment (10 W / mL - 40 min)".

[0104] Example 6

[0105] Defatted walnut meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 11.0 with NaOH. Next, the mixture was centrifuged at 10000g for 20 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 30kDa). The supernatant was concentrated and purified using a membrane to 25% of its total volume. Subsequently, the pH was adjusted to a stable value of ~11.0, and ultrasonic treatment was performed with an ultrasonic power of 10 W / mL and an ultrasonic time of 60 min. An ice bath was used to prevent overheating of the solution during ultrasonication. The resulting solution was freeze-dried to obtain walnut protein isolate, which is Example 6, named "Membrane Separation (30 kDa) Coupled with Ultrasonic Treatment (10 W / mL - 60 min)".

[0106] Example 7

[0107] Defatted walnut meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 11.0 with NaOH. Next, the mixture was centrifuged at 10000g for 20 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 10 kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH was adjusted to a stable value of approximately 11.0, and ultrasonic treatment was performed with an ultrasonic power of 20 W / mL and an ultrasonic time of 35 min. An ice bath was used to prevent overheating of the solution during ultrasonication. The resulting solution was freeze-dried to obtain walnut protein isolate, which is Example 7, named: Membrane Separation (10 kDa) Coupled with Ultrasonic Treatment (20 W / mL - 35 min).

[0108] Example 8

[0109] Defatted walnut meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 11.0 with NaOH. Next, the mixture was centrifuged at 10000g for 20 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 10 kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH was adjusted to a stable value of approximately 11.0, and ultrasonic treatment was performed at a power of 10 W / mL for 30 min. An ice bath was used to prevent overheating of the solution during ultrasonication. The resulting solution was freeze-dried to obtain walnut protein isolate, which is Example 8, named: Membrane Separation (10 kDa) Coupled with Ultrasonic Treatment (10 W / mL - 30 min).

[0110] Example 9

[0111] Defatted walnut meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 11.0 with NaOH. Next, the mixture was centrifuged at 10000g for 20 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 10 kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH was adjusted to a stable value of approximately 11.0, and infrared treatment was performed at 80°C for 32 minutes. The resulting solution was freeze-dried to obtain walnut protein isolate, which is Example 9, named: Membrane Separation (10kDa) Coupled with Infrared Mid-Wave Treatment (80°C - 32 min).

[0112] Example 10

[0113] Defatted walnut meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 11.0 with NaOH. Next, the mixture was centrifuged at 10000g for 20 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 10 kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH was adjusted to a stable value of approximately 11.0, and infrared treatment was performed at 120°C for 16 minutes. The resulting solution was freeze-dried to obtain walnut protein isolate, which is Example 10, named: Membrane Separation (10kDa) Coupled with Infrared Mid-Wave Treatment (120°C - 16 min).

[0114] Example 11

[0115] Defatted walnut meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 11 with NaOH. Next, the mixture was centrifuged at 10000g for 20 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 30kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH was adjusted to a stable value of approximately 11.0, and infrared treatment was performed at 160°C for 8 minutes. The resulting solution was freeze-dried to obtain walnut protein isolate, which is Example 11, named: Membrane Separation (30 kDa) Coupled with Infrared Mid-Wave Treatment (160°C - 8 min).

[0116] Example 12

[0117] Defatted rapeseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 8.5 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 5kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH was adjusted to a stable value of approximately 8.2, and ultrasonic treatment was performed at a power of 10 W / mL for 60 min. An ice bath was used to prevent overheating of the solution during ultrasonication. The resulting solution was freeze-dried to obtain rapeseed protein, which is Example 12, named: Membrane Separation (5 kDa) Coupled with Ultrasonic Treatment (10 W / mL - 60 min).

[0118] Example 13

[0119] Defatted rapeseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 9.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 10kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH was adjusted to a stable value of approximately 8.7, and ultrasonic treatment was performed with an ultrasonic power of 20 W / mL and an ultrasonic time of 35 min. An ice bath was used to prevent overheating of the solution during ultrasonication. The resulting protein solution was freeze-dried to obtain rapeseed protein, which is Example 13, named: Membrane Separation (10 kDa) Coupled with Ultrasonic Treatment (20 W / mL - 35 min).

[0120] Example 14

[0121] Defatted rapeseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 9.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 10kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH was adjusted to a stable value of approximately 8.7, and ultrasonic treatment was performed at a power of 30 W / mL for 10 min. An ice bath was used to prevent overheating of the solution during ultrasonication. The resulting solution was freeze-dried to obtain rapeseed protein, which is Example 14, named: Membrane Separation (10 kDa) Coupled with Ultrasonic Treatment (30 W / mL - 10 min).

[0122] Example 15

[0123] Defatted rapeseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 8.5 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 5kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. The pH was then adjusted to a stable value of approximately 8.2 before infrared treatment at 80°C for 16 minutes. The resulting solution was freeze-dried to obtain rapeseed protein, which is Example 15, named: Membrane Separation (5kDa) Coupled with Infrared Mid-Wave Treatment (80°C - 16 min).

[0124] Example 16

[0125] Defatted rapeseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 9.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 10kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. The pH was then adjusted to a stable value of approximately 8.7 before infrared treatment at a short-wave temperature of 120°C for 8 minutes. The resulting solution was freeze-dried to obtain rapeseed protein, which is Example 16, named: Membrane Separation (10 kDa) Coupled with Infrared Short-Wave Treatment (120°C - 8 min).

[0126] Example 17

[0127] Defatted rapeseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 9.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a membrane separation method (MWCO 30kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. The pH was then adjusted to a stable value of approximately 8.7 before infrared treatment at 160°C for 4 minutes. The resulting solution was freeze-dried to obtain rapeseed protein, which is Example 17, named: Membrane Separation (30 kDa) Coupled with Infrared Mid-Wave Treatment (160°C - 4 min).

[0128] To further demonstrate the beneficial effects of the present invention and to better understand the present invention, the following comparative examples and experimental examples further illustrate the properties and application performance of the preparation method of the high-functional plant protein disclosed in the present invention. However, these should not be construed as limiting the present invention. Other methods and applications obtained by those skilled in the art based on the above-described invention and their applications are also considered to fall within the protection scope of the present invention.

[0129] Comparative Example 1

[0130] Defatted flaxseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was then adjusted to 9.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and subjected to simultaneous weak-alkali ultrafiltration (MWCO 5 kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH of the concentrate was adjusted to approximately 10.0, and the solution was freeze-dried to obtain flaxseed protein isolate, which is Comparative Example 1, named: Membrane Separation (5 kDa).

[0131] Comparative Example 2

[0132] Defatted flaxseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was then adjusted to 10.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and subjected to weak-alkali simultaneous ultrafiltration (MWCO 10 kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH of the concentrate was adjusted to approximately 10.0, and the concentrate was freeze-dried to obtain flaxseed protein isolate, which is Comparative Example 2, named: Membrane Separation (10 kDa).

[0133] Comparative Example 3

[0134] Defatted flaxseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 9.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and subjected to weak-alkali simultaneous ultrafiltration (MWCO 30 kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH of the concentrate was adjusted to approximately 10.0, and the concentrate was freeze-dried to obtain flaxseed protein isolate, which is Comparative Example 3, named: Membrane Separation (30 kDa).

[0135] Comparative Example 4

[0136] Defatted flaxseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 9.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and subjected to weak-alkali simultaneous ultrafiltration (MWCO 30 kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. The concentrated solution was then adjusted to a pH of approximately 10.0 and freeze-dried to obtain flaxseed protein isolate. Next, the flaxseed protein isolate was reconstituted, the pH was adjusted to 10, and then it was sonicated at a power of 5 W / mL for 60 min, with an ice bath to prevent overheating. The resulting solution is Comparative Example 4, named: Membrane Separation (30 kDa) - Freeze-Drying - Reconstituted - Sonication (5 W / mL - 60 min).

[0137] Comparative Example 5

[0138] Defatted flaxseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 9.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and separated using a weak-base simultaneous membrane separation method (MWCO 30 kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. The concentrated solution was then adjusted to a pH of approximately 10.0 and freeze-dried to obtain flaxseed protein isolate. Next, the flaxseed protein isolate was reconstituted, the pH was adjusted to 10, and then subjected to infrared treatment at a short-wavelength temperature of 160°C for 4 minutes. The resulting solution is Comparative Example 5, named: (Membrane Separation (30 kDa) - Freeze-Drying - Reconstituted - Infrared Short-Wave Treatment 160°C - 4 min).

[0139] Comparative Example 6

[0140] Defatted flaxseed meal powder was dispersed in ultrapure water at a ratio of 1:15 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was then adjusted to 9.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. Subsequently, the pH was adjusted to 4.2. The mixture was centrifuged again under the same conditions, the precipitate was collected, and the solution was reconstituted with ultrapure water, maintaining the pH at 7.0. Finally, the resulting solution was freeze-dried to obtain flaxseed protein isolate, which is Comparative Example 6, named: Alkali Extraction and Acid Precipitation Method.

[0141] Comparative Example 7

[0142] Defatted walnut meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 11.0 with NaOH. Next, the mixture was centrifuged at 10000g for 20 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and subjected to weak-alkaline simultaneous ultrafiltration (MWCO 10 kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH of the concentrate was adjusted to approximately 11.0, and the solution was freeze-dried to obtain walnut protein isolate, which is Comparative Example 7, named: Membrane Separation (10 kDa).

[0143] Comparative Example 8

[0144] Defatted walnut meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 11.0 with NaOH. Next, the mixture was centrifuged at 10000g for 20 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and subjected to weak-alkali simultaneous ultrafiltration (MWCO 10 kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. The pH of the concentrate was then adjusted to approximately 7.0, followed by freeze-drying to obtain walnut protein isolate. Next, the walnut protein isolate was reconstituted, the pH was adjusted to 11.0, and then subjected to sonication at a power of 10 W / mL for 30 min. An ice bath was used to prevent overheating during sonication. The resulting solution was freeze-dried to obtain Comparative Example 8, named: Membrane Separation (10 kDa) - Freeze-Drying - Reconstituted - Sonication (10 W / mL - 30 min).

[0145] Comparative Example 9

[0146] Defatted rapeseed meal powder was dispersed in ultrapure water at a ratio of 1:10 (w / v) and stirred at room temperature (25°C) for 2 hours. The pH was adjusted to 9.0 with NaOH. Next, the mixture was centrifuged at 10000g for 30 minutes to remove insoluble impurities, and the protein-rich supernatant was collected. This supernatant was then transferred to the sample chamber of a membrane separation device and subjected to simultaneous weak-alkali ultrafiltration (MWCO 10kDa). The supernatant was concentrated and purified using a membrane to approximately 25% of its total volume. Subsequently, the pH of the concentrate was adjusted to approximately 8.7, and the solution was freeze-dried to obtain rapeseed protein, which is Comparative Example 9, named: Membrane Separation (10 kDa).

[0147] Experimental Example 1

[0148] Taking the flaxseed protein isolates obtained in Examples 1-5 and Comparative Examples 1-6 as examples, the protein content, functional properties, and structural differences of protein products obtained by different methods were studied. The results are shown in Table 1. Figures 8-10 and Figure 2 , Figure 3 As shown in Table 2.

[0149] Table 1. Content of flaxseed protein isolate obtained by different methods

[0150]

[0151] As shown in Table 1, the flaxseed protein isolates prepared in Examples 1-5 of this invention all have high protein content, meeting the requirements for concentrated or isolated protein products in the domestic market. The protein conversion factor for flaxseed protein products in China is 6.25, and most other countries also use a protein conversion factor of 6.25. The protein content data in Table 1 is converted using N×6.25. The protein content of Examples 1-5 all exceeds 90%. This data can be used to compare the protein content of flaxseed protein-related products both domestically and internationally, and has high promotional value.

[0152] Furthermore, research has found that the membrane separation coupled with physical field modification technology disclosed in this invention not only does not adversely affect the protein content in the protein concentrate obtained from membrane separation (the extraction rates of flaxseed protein isolates prepared in Examples 1-5 and Comparative Examples 1-3 are approximately 28%-32%), but also improves the protein extraction rate based on existing alkaline extraction and acid precipitation techniques (compared to the extraction rate of ~20% in Comparative Example 6, the extraction rates of Examples 1-5 are all improved). Therefore, the introduction of physical field modification technology achieves a smooth transition with the original membrane separation protein concentrate technology, effectively integrating and coordinating within the original process framework, and jointly promoting the industrial upgrading of high-yield, high-functionality plant protein products.

[0153] The functional properties of flaxseed protein isolate were further analyzed. For example... Figure 8 As shown, the solubility of Examples 1-3 and 5 all exceeded 80%, reaching a maximum of 98.24%, which is significantly higher than the solubility of flaxseed protein isolate obtained in Comparative Examples 1-5. Figure 9 As shown, in terms of emulsifying performance, Example 2 has a viscosity of 9.61 m. 2 / g, which is superior to the 7.72, 7.99, and 6.08m of comparative examples 3–5. 2 / g. For example... Figure 10 As shown, in terms of foaming performance, Example 1 had 249.85% and Example 2 had 228.63%, which were better than Comparative Examples 3-5 (180.42%, 189.71%, and 205.19%, respectively); Example 3 had 228.63% and Example 4 had 210.00%, which were better than Comparative Example 1 (133.78%).

[0154] Table 2. Particle size and potential of flaxseed protein isolate obtained by different methods

[0155]

[0156] Depend on Figure 2It can be seen that the circular dichroism spectra of Examples 1, 2, and Comparative Examples 3-5 are different, representing differences in their secondary structures. Combined with the particle size and potential measurement results in Table 2: compared with Comparative Example 3, the particle size of Examples 1 and 2 is reduced, and the potential also changes, indicating that ultrasonic and infrared treatment alter the structural characteristics of flaxseed protein isolate. By comparing Examples 1 and Comparative Example 4, and Examples 2 and Comparative Example 5 respectively, it is shown that the effects of membrane separation coupled with ultrasonic / infrared treatment and membrane separation freeze-drying followed by reconstitution under alkaline conditions with ultrasonic / infrared treatment on the structural characteristics of flaxseed protein isolate are different, further verifying the differences in the structural characteristics of flaxseed protein isolate treated by different methods.

[0157] Depend on Figure 3 The non-reducing and reduced SDS-PAGE spectra of flaxseed protein isolate show that the molecular weight distribution of Comparative Example 6, Example 3, and Example 1 ranges from 10 kDa to 55 kDa. The main bands are albumin in the 10-17 kDa range, as well as globulins in the 17-26 kDa, 35 kDa, and 55 kDa range. Compared with Comparative Example 6, the molecular weight bands in the 10-17 kDa range of Example 3 and Example 1 are darker, indicating that Example 3 and Example 1 can enrich more albumin from flaxseed meal into the target flaxseed protein isolate.

[0158] Experimental Example 2

[0159] Taking the walnut protein obtained in Examples 6-11 and Comparative Example 6 as examples, the protein content, functional properties, and structural differences of protein products obtained by different methods were studied. The results are shown in Table 3. Figures 11-12 and Figures 4-7 As shown.

[0160] Table 3. Content of walnut protein isolate obtained by different methods

[0161]

[0162] As shown in Table 3, the protein content of the walnut protein isolates prepared in Examples 6-11 and Comparative Examples 7 and 8 of the present invention is all above 80%.

[0163] Specifically, the protein extraction rates of the walnut protein isolates prepared in Examples 6-11 and Comparative Examples 7 and 8 were approximately 46%-48%. Therefore, the membrane separation coupled physical field modification technology disclosed in this invention does not adversely affect the protein content in the protein concentrate obtained from membrane separation. It can achieve a smooth transition with existing membrane separation protein concentrate technologies. The two technologies are effectively integrated and coordinated within the framework of existing protein extraction processes involving membrane separation, jointly promoting the industrial upgrading of high-yield, high-functionality plant protein products.

[0164] The protein properties of walnut protein isolate were further analyzed. For example... Figure 11 As shown, the solubility of Examples 6-9 and Example 11 all exceeded 58%, reaching a maximum of 69.26%, which is significantly higher than the solubility of walnut protein isolate obtained in Comparative Examples 7 and 8. Figure 12 As shown, in terms of emulsifying performance, Examples 6-9 and 11 have a concentration of 15.07 m. 2 / g, 9.94m 2 / g, 11.92m 2 / g, 11.15m 2 / g, 15.05m 2 / g, which is superior to 7.62m in Comparative Example 7. 2 / g and 8.95m of Comparative Example 8 2 / g.

[0165] Depend on Figures 4-7 It can be seen that, compared with Comparative Examples 7 and 8, Example 8 has a smaller average particle size (~200 nm), higher intrinsic fluorescence intensity and surface hydrophobicity (~36000), and less disulfide bond content (~22 μg / mL). This further illustrates that there are significant differences in the effects of membrane separation coupled with ultrasonic treatment, membrane separation followed by freeze-drying, and membrane separation followed by reconstitution of walnut protein isolate under alkaline conditions with ultrasonic treatment on the structural characteristics of walnut protein isolate. It also further verifies that membrane separation coupled with ultrasonic treatment can endow walnut protein isolate with smaller size, fewer disulfide bonds and higher surface hydrophobicity. These improvements in structural characteristics are beneficial for the protein to disperse in water and quickly adsorb at the interface between two phases, forming an interfacial membrane structure with viscoelastic properties, thereby improving the solubility and emulsification properties of walnut protein.

[0166] Experimental Example 3

[0167] Taking the rapeseed protein obtained in Examples 12-17 and Comparative Example 9 as examples, the protein content and functional characteristics of protein products obtained by different methods were studied. The results are shown in Table 4 and... Figures 13-16 As shown.

[0168] Table 4. Rapeseed protein content obtained by different methods

[0169]

[0170] As shown in Table 4, the protein content of the rapeseed protein prepared in Examples 12-17 and Comparative Example 9 of this invention is all above 60%.

[0171] Specifically, the protein extraction rates of Examples 12-17 and Comparative Example 9 were approximately 15.5%-17.5%. Therefore, the membrane separation coupled physical field modification technology disclosed in this invention does not adversely affect the protein content in the protein concentrate obtained from membrane separation, and can achieve a smooth transition with existing membrane separation protein concentrate technologies. The two technologies are effectively integrated and coordinated within the framework of existing protein extraction processes involving membrane separation, jointly promoting the industrial upgrading of high-yield, high-functionality plant protein products.

[0172] The functional characteristics of rapeseed protein were further analyzed. For example... Figure 13 As shown, the solubility of Examples 12-17 all exceeded 40%, reaching a maximum of 65.36%, which is significantly higher than the solubility of rapeseed protein obtained in Comparative Example 9. Figure 14 As shown, in terms of emulsifying performance, Examples 12-14 and 16 have a viscosity of 9.49 m³ / s. 2 / g, 8.43m 2 / g, 11.15m 2 / g, 12.59m 2 / g, all of which are superior to the 6.21m of Comparative Example 9. 2 / g, and as Figure 15 As shown, in terms of emulsification stability, Examples 12-15 and 17 have emulsification times of 61.29 min, 57.57 min, 78.40 min, 72.75 min, and 131.24 min, respectively, all of which are superior to the 42.02 min of Comparative Example 9. Figure 16 As shown, in terms of foaming performance, Examples 12-17 have foaming properties of 403.69%, 376.89%, 348.31%, 373.32%, 369.75%, and 385.82%, respectively, all of which are superior to Comparative Example 9's 294.73%.

[0173] In summary, the membrane separation coupled with physical field modification technology disclosed in this invention, after controlling the protein concentrate obtained from membrane separation to a moderately alkaline state, coupled with physical field modification, not only avoids protein aggregation caused by acid precipitation near the isoelectric point during alkaline extraction and acid precipitation, but also achieves green purification and efficient modification of proteins during extraction through membrane separation coupled with physical fields. This improves the structural characteristics of plant proteins and endows them with excellent functional properties. On the one hand, compared with alkaline extraction and acid precipitation, it can achieve multi-component enrichment of albumin, globulin, and glutenin in plant proteins; on the other hand, compared with weak alkali simultaneous ultrafiltration and pH shifting combined with ultrasonic treatment, the plant proteins obtained by the membrane separation coupled with physical field modification process of this invention have better solubility, foaming properties, and emulsifying properties.

[0174] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A green preparation method for high-functionality plant protein, characterized in that, After controlling the protein concentrate obtained from membrane separation to a moderately alkaline state, it is then coupled with a physical field for modification to obtain a highly functional plant protein, including the following steps: S1. Plant protein is processed by membrane separation to obtain protein concentrate. The membrane used in the membrane separation method is 5-30 kDa. The plant protein includes soybean protein, rapeseed protein, flaxseed protein or walnut protein. S2. Adjust the pH of the protein concentrate to a stable value of 8.0-11.0, and then perform physical field modification. The physical field modification is selected from ultrasonic treatment or infrared treatment, and the ultrasonic treatment time is 10-60 min, the infrared treatment time is 8-32 min; the ultrasonic power density of the ultrasonic treatment is 5-30 W / mL, and the infrared temperature of the infrared treatment is 80-160 ℃. S3. Freeze-dry the solution modified by the physical field to obtain the high-functionality plant protein.

2. The preparation method according to claim 1, characterized in that, Infrared wavelengths include medium and short waves.

3. The highly functional plant protein prepared by the preparation method according to any one of claims 1 to 2.

4. The application of the highly functional plant protein as described in claim 3 in food processing.

Images