A method for extracting high-surface-active liquor lees protein and its application

Abstract

The present invention provides a method for extracting high-surface-activity white wine lees protein and its application. The preparation method comprises the following steps: mixing wine lees powder with an ethanol solution, performing ultrasonic-assisted extraction to obtain a protein extract, membrane filtering and centrifuging the extract, gradient dilution, and gradient pH adjustment to perform acid precipitation, followed by dynamic temperature control impurity removal and resin adsorption treatment, centrifugal separation, washing, and freeze-drying to obtain high-surface-activity wine lees alcohol-soluble protein. The extraction method of the present invention has the advantages of low energy consumption, high efficiency, and suitability for industrial continuous production. By combining gradient dilution with gradual pH adjustment, the internal structure of the protein is rearranged, dimers or oligomers are formed, and the protein precipitation effect is improved. At the same time, dynamic temperature control impurity removal combined with resin adsorption treatment can effectively remove impurities, improve protein extraction efficiency, and optimize the protein's interfacial activity and safety. It has broad application prospects in the fields of food, medicine, and biomaterials.

Description

Technical Field

[0001] The invention belongs to the field of food processing, and in particular relates to a method for extracting white wine grains protein with high interfacial activity and application thereof. Background Art

[0002] Foam is a common feature of various foods, including beer, ice cream, cakes, and candies, and is closely linked to their texture, structure, mouthfeel, and appearance. Rich and stable foam imparts a lighter, fuller mouthfeel and enhances the release of food aroma. Foam is a common interface-dominated food system, and the rheological properties of the air-water interface significantly influence foam properties. By regulating interfacial interactions and improving interfacial properties through bubble formation and stabilization mechanisms, food quality can be controlled. Currently, food production is focused on finding natural, safe, and effective surfactants to replace synthetic surfactants. Proteins diffuse and adsorb at the air-liquid interface, undergoing folding, conformational rearrangement, and cross-linking, forming an adsorption layer with viscoelastic properties that maintains the foam structure. Due to their high molecular weight and relatively stable interfacial structure, they are excellent foaming agents. Zein is currently widely used, but its strong hydrophobicity prevents it from effectively stabilizing foams. Consequently, research has focused on the preparation of composite particles. While this complex preparation process offers promising results, it is not suitable for processing.

[0003] Baijiu fermentation produces a large amount of waste lees. Due to their high acidity and susceptibility to spoilage, their disposal has always been a challenge for many distilleries. As a plant-derived fermentation product, lees contain up to 21% protein, making them a good source of plant protein. Existing research on lees primarily focuses on extracting proteins or peptides from them and investigating the functional properties of their active ingredients. Alcohol-soluble proteins, which comprise the largest fraction of lees, are difficult to extract and remain underdeveloped, with no direct examples of their use in food applications. Their extremely low solubility in water significantly limits their application in food. Existing extraction methods involve adding solvents such as ethanol, glacial acetic acid, or isopropyl alcohol. The extraction yield can be increased by varying the extraction time or by other methods to promote dissolution. However, low extraction yields and purity remain problematic, hindering their application in large-scale modern industrial production and preventing them from meeting food safety standards.

[0004] Wheat prolamin plays a crucial role in the production of pasta products. It provides structural support and forms a network structure. It also anchors air bubbles, maintaining shape and volume, and imparting a fluffy texture. Furthermore, the protein absorbs water, binding the other ingredients together and ensuring their continuity. Celiac disease is a common gluten-sensitive enteropathy, a gastrointestinal and extraintestinal condition triggered by the consumption of gluten-containing foods by individuals with a genetic predisposition. Avoiding the intake of gluten-containing foods is currently the most effective treatment. Research into gluten-free foods for this population is still in its infancy in China, and the significant challenge currently faced by this research is the poor product quality resulting from the removal of gluten. Developing gluten-free foods that are formed, with a loose structure and continuous texture, without relying on the presence of gluten has attracted widespread attention. Improving product quality is typically achieved by adding quality enhancers or by using raw materials with low gluten content. How to conduct research on how to simulate the characteristics of gluten protein in the system through alcohol-soluble protein, develop the application of wine lees alcohol-soluble protein in food systems, and improve the interfacial activity of wine lees alcohol-soluble protein by improving the processing process to solve the problem of poor foaming of wine lees protein in traditional extraction methods has become a technical problem that needs to be solved urgently in this field. Summary of the Invention

[0005] In view of this, the present invention aims to propose a method for extracting white wine lees protein with high surface activity and its application, so as to provide a low-energy, low-time method suitable for industrial continuous production of white wine lees protein by optimizing the extraction conditions, controlling the protein sedimentation mechanism and refining the purification process, and improve the surface activity of white wine lees protein, thereby achieving efficient extraction of protein and improving safety.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0007] In a first aspect, the present invention provides a method for preparing a distillers grains alcohol-soluble protein with high surface activity, the preparation method comprising the following steps:

[0008] (1) Mixing the lees powder with an ethanol solution and a reducing agent, performing ultrasonic-assisted extraction, and centrifuging to obtain a protein extract;

[0009] (2) Filtering the protein extract through a membrane and then centrifuging to obtain a preliminarily impurity-removed protein extract;

[0010] (3) Gradual dilution of the preliminary impurity-removed protein extract and gradient adjustment of the pH to 3.75-3.85 for acid precipitation to obtain an acid-precipitated protein extract;

[0011] (4) The protein extract after acid precipitation is treated with dynamic temperature control to remove impurities and resin adsorption, and then centrifuged to obtain protein precipitate;

[0012] (5) Washing and vacuum-freezing the protein precipitate to obtain the highly interfacially active vinasse alcohol-soluble protein.

[0013] Furthermore, the method of gradient dilution and gradient pH adjustment includes the following steps: when the ethanol concentration in the preliminary impurity-removed protein extract is diluted to 50%, the pH is adjusted to 6-7 and the solution is allowed to stand for 30-40 minutes; when the ethanol concentration is diluted to 45%, the pH is adjusted to 4.5-5 and the solution is allowed to stand for 40-50 minutes; when the ethanol concentration is diluted to 40%, the pH is adjusted to 4 and the solution is allowed to stand for 40-50 minutes; and finally, the pH is adjusted to 3.75-3.85 and the solution is allowed to stand for 60-80 minutes. Gradient dilution and gradient pH adjustment can improve the sedimentation rate of protein in the protein extract.

[0014] Furthermore, the method of dynamic temperature control impurity removal and resin adsorption treatment includes the following steps:

[0015] Stir at 30-35°C for 5-15 minutes to dissolve soluble impurities at low temperature and prevent protein denaturation;

[0016] Ultrasonic-assisted permeation was performed at 35-40°C for 15-25 min to enhance protein-solvent interaction and promote the dissolution of non-protein components.

[0017] Centrifugal separation at 45-50°C for 45-90 minutes with dynamic stirring improves protein purity through efficient separation and optimizes interfacial effects with dynamic stirring.

[0018] Add adsorption resin for dynamic adsorption at 50-60°C for 20-40 minutes to control thermal decomposition and maintain protein activity. Combined with adsorption resin treatment, it can remove polyphenols and other potential impurities to improve the safety of the protein.

[0019] Preferably, the method of dynamic temperature control impurity removal and resin adsorption treatment comprises the following steps:

[0020] i) Sustained-release dissolution stage: stirring at 30-35°C for 10 min;

[0021] ii) Penetration enhancement stage: ultrasonic-assisted penetration at 35–40°C for 20 min;

[0022] iii) Deep extraction stage: centrifugation at 45-50°C with dynamic stirring for 60 min;

[0023] iv) Convergence and stabilization stage: adding a macroporous adsorption resin for dynamic adsorption at 50-60° C. for 30 min. The macroporous adsorption resin is preferably AB-8 type. Adsorption by the macroporous adsorption resin can improve the polyphenol removal rate (above 85%).

[0024] Furthermore, the reducing agent includes sodium hydroxide and sodium metabisulfite;

[0025] Preferably, the mass ratio of the vinasse powder to the ethanol solution is 1:8-15, for example, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, preferably 1:10;

[0026] Preferably, the concentration of the ethanol solution is 60% to 90%, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 70%;

[0027] Preferably, the mass percentage of sodium hydroxide in the mixed solution of vinasse powder, ethanol solution and reducing agent is 0.3%~0.5%, for example, it can be 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, and preferably 0.35%; the mass percentage of sodium metabisulfite is 0.4%~1%, for example, it can be 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, and preferably 0.5%.

[0028] Furthermore, in step (1), the stirring temperature is 40-80°C, preferably water bath heating and stirring; the frequency of ultrasonic-assisted extraction is 20-40 kHz, the power density is 50-150 W / cm², the single extraction time is 2 h, and the number of extractions is more than 2 times.

[0029] Furthermore, in step (2), membrane filtration is performed by combining an ultrafiltration membrane with a nanofiltration membrane; preferably, the pore size of the ultrafiltration membrane is 4-6 kDa, for example, 4 kDa, 5 kDa, 6 kDa, preferably 5 kDa, and the pore size of the nanofiltration membrane is 1-2 kDa, for example, 1 kDa, 2 kDa, preferably 1 kDa.

[0030] Furthermore, in step (3), the acid precipitation temperature is 20°C to 25°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, preferably 25°C.

[0031] Furthermore, in step (5), the vacuum freeze-drying temperature is -65°C to -55°C, and the vacuum freeze-drying time is ≥20h. Vacuum freeze-drying can increase the water solubility of the prepared protein by more than 30%, and extend the foam half-life to more than 60min.

[0032] Furthermore, the preparation method of the lees powder comprises the following steps: drying, crushing and sieving the white lees to obtain the lees powder; preferably, the drying temperature is 50-80°C; preferably, the moisture content of the dried white lees is 0%-15%; further preferably, the mesh size of the sieving is 40-80 mesh.

[0033] In a second aspect, the present invention provides a high-interfacial-activity vinasse alcohol-soluble protein prepared according to the preparation method described in the first aspect.

[0034] In a third aspect, the present invention provides the preparation method as described in the first aspect or the use of the high-interfacial-activity vinasse alcohol-soluble protein as described in the second aspect in food.

[0035] Furthermore, the application includes the application of the high-surface-active distiller's grains alcohol-soluble protein in any of the following foods: gas-containing food, frozen food, baked food, and dairy products.

[0036] Preferably, the gas-containing food includes beer and carbonated beverages, which can improve the foam persistence and mouthfeel density; further preferably, the high-surface-active vinasse alcohol-soluble protein is added to the gas-containing food in an amount of 0.1% to 1.5% (w / w);

[0037] Preferably, the frozen food includes ice cream and mousse, which can improve the fluffiness and melting resistance of the texture;

[0038] Preferably, the baked food includes cakes and bread, which can simulate the gluten protein network structure, improve the volume stability of gluten-free products, and increase the volume expansion rate of gluten-free baked food to more than 98%;

[0039] Preferably, the dairy product includes milkshakes and puddings, which can enhance flavor release and smoothness, and increase the flavor release efficiency in the dairy product to more than 68%.

[0040] In a fourth aspect, the present invention provides a gluten-free baked food, wherein the gluten-free baked food contains the high-interfacial-activity distiller's grains alcohol-soluble protein as described in the second aspect, and the volume expansion rate of the gluten-free baked food is greater than 98%.

[0041] In a fifth aspect, the present invention provides the use of gradient dilution combined with gradient adjustment of the pH of a protein extract in the preparation of highly interfacially active vinasse alcohol-soluble proteins;

[0042] Preferably, the method of gradient dilution combined with gradient adjustment of the pH of the protein extract comprises the following steps: when the ethanol concentration in the protein extract is diluted to 50%, adjusting the pH to 6-7 and letting it stand for 30-40 minutes; when the ethanol concentration is diluted to 45%, adjusting the pH to 4.5-5 and letting it stand for 40-50 minutes; when the ethanol concentration is diluted to 40%, adjusting the pH to 4 and letting it stand for 40-50 minutes; finally, adjusting the pH to 3.75-3.85 and letting it stand for 60-80 minutes.

[0043] In a sixth aspect, the present invention provides the use of a protein extract treated by dynamic temperature control impurity removal combined with resin adsorption in the preparation of high-interfacial-active vinasse alcohol-soluble protein;

[0044] Preferably, the method of dynamic temperature control impurity removal combined with resin adsorption treatment comprises the following steps: stirring the protein extract at 30-35°C for 5-15 minutes; ultrasonic-assisted permeation at 35-40°C for 15-25 minutes; centrifugal separation combined with dynamic stirring at 45-50°C for 45-90 minutes; and adding macroporous adsorption resin for dynamic adsorption at 50-60°C for 20-40 minutes.

[0045] Further preferably, the method of dynamic temperature control impurity removal combined with resin adsorption treatment comprises the following steps:

[0046] i) Sustained-release dissolution stage: stirring at 30-35°C for 10 min;

[0047] ii) Penetration enhancement stage: ultrasonic-assisted penetration at 35–40°C for 20 min;

[0048] iii) Deep extraction stage: centrifugation at 45-50°C with dynamic stirring for 60 min;

[0049] iv) Convergence and stabilization stage: adding macroporous adsorption resin for dynamic adsorption at 50-60° C. for 30 min, wherein the macroporous adsorption resin is preferably AB-8 type.

[0050] Furthermore, the application includes at least one of the following: improving the purity of wine lees alcohol-soluble protein and reducing the polyphenol content in wine lees alcohol-soluble protein.

[0051] Compared with the prior art, the extraction method and application of high-surface-active white wine grains protein of the present invention have the following advantages:

[0052] (1) The method for extracting high-surface-active white wine grains protein described in the present invention has the advantages of low energy consumption, high efficiency, and suitability for industrial continuous production. It not only improves the protein extraction rate, but also optimizes the surface activity and safety of the protein. It has broad application prospects in the fields of food, medicine, and biomaterials.

[0053] (2) The method for extracting high-surface-activity white wine grains protein of the present invention improves the surface activity of the protein by optimizing the protein extraction and purification process, thereby broadening its application in food and functional materials.

[0054] (3) The method for extracting high-surface-active white wine lees protein described in the present invention controls protein-protein interactions, precisely regulates the aggregate growth process, induces internal structural rearrangement of the protein through gradient dilution combined with gradual pH adjustment, promotes the formation of dimers or oligomers, thereby improving the protein sedimentation effect, and finally obtains a high-purity protein product through centrifugal separation.

[0055] (4) The method for extracting high-surface-activity liquor lees protein described in the present invention adopts dynamic temperature-controlled impurity removal combined with resin adsorption treatment, which can effectively remove impurities and improve protein extraction efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0057] Figure 1 Schematic diagram of the foaming ability and stability test results of the white wine grains protein prepared in the present invention;

[0058] Figure 2 The figure is a schematic diagram of the appearance of a gluten-free cake made using the white wine grains protein of the present invention. DETAILED DESCRIPTION

[0059] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0060] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0061] Example 1

[0062] The method for preparing white wine lees protein in this embodiment comprises the following steps:

[0063] (1) Take white wine lees as raw material with a moisture content of 10%, first perform hot air drying at 60°C, crush and pass through a 60-mesh sieve to obtain wine lees powder;

[0064] (2) Weigh 50 g of vinasse powder and add 500 mL of 70% ethanol solution. Add 0.5% sodium metabisulfite and 0.35% sodium hydroxide as reducing agents (based on the total mass of the protein extract). Extract the mixture for 2 h using 40 kHz ultrasonic waves (power density 120 W / cm²) under magnetic stirring in a 50°C water bath. After extraction, remove low molecular weight impurities by nanofiltration (membrane pore size 1 kDa). Centrifuge the vinasse residue (5000 rpm, 10 min) and collect the protein extract.

[0065] (3) The protein extract was diluted with deionized water to an ethanol concentration of 50%, 45%, and 40%, respectively. The pH was adjusted to 7, 5, and 4, respectively, and then allowed to stand for 30 min, 40 min, and 50 min, respectively. Finally, the pH was adjusted to 3.75 and allowed to stand for 60 min for acid precipitation. The precipitate was then collected by centrifugation at 8000 rpm for 15 min, washed twice with deionized water, and freeze-dried (-60°C, 24 h) to obtain protein powder.

[0066] Example 2

[0067] The method for preparing white wine lees protein in this embodiment comprises the following steps:

[0068] (1) Take white wine lees as raw material with a moisture content of 10%, first perform hot air drying at 60°C, crush and pass through a 60-mesh sieve to obtain wine lees powder;

[0069] (2) Weigh 50 g of vinasse powder and add 500 mL of 70% ethanol solution. Add 0.5% sodium metabisulfite and 0.35% sodium hydroxide as reducing agents (based on the total mass of the protein extract). Extract the mixture for 2 h using 40 kHz ultrasonic waves (power density 120 W / cm²) under magnetic stirring in a 50°C water bath. After extraction, remove macromolecular impurities by ultrafiltration (membrane pore size 5 kDa). Centrifuge the vinasse residue (5000 rpm, 10 min) and collect the protein extract.

[0070] (3) The protein extract was diluted with deionized water to an ethanol concentration of 50%, 45%, and 40%, and the pH was adjusted to 6, 4.5, and 4, respectively, and then allowed to stand for 40 min, 50 min, and 40 min, respectively. Finally, the pH was adjusted to 3.75 and allowed to stand for 65 min for acid precipitation. The precipitate was then collected by centrifugation at 8000 rpm for 15 min, washed twice with deionized water, and freeze-dried (-55°C, 20 h) to obtain protein powder.

[0071] Example 3

[0072] The method for preparing white wine lees protein in this embodiment comprises the following steps:

[0073] (1) Take white wine lees as raw material with a moisture content of 10%, first perform hot air drying at 60°C, crush and pass through a 60-mesh sieve to obtain wine lees powder;

[0074] (2) Weigh 50 g of vinasse powder, add 500 mL of 70% ethanol solution, and add 0.5% sodium metabisulfite and 0.35% sodium hydroxide as reducing agents (based on the total mass of the protein extract). Under magnetic stirring in a 50°C water bath, use 40 kHz ultrasonic waves (power density 120 W / cm²) to assist extraction for 2 h. After extraction, remove large molecular impurities by ultrafiltration (membrane pore size 5 kDa), and then remove low molecular impurities by nanofiltration (membrane pore size 1 kDa). Then, centrifuge the vinasse (5000 rpm, 10 min) and collect the protein extract.

[0075] (3) The protein extract was diluted with deionized water to an ethanol concentration of 50%, 45%, and 40%, respectively. The pH was adjusted to 7, 5, and 4, respectively, and then allowed to stand for 35 min, 45 min, and 45 min, respectively. Finally, the pH was adjusted to 3.75 and allowed to stand for 70 min for acid precipitation. The precipitate was then collected by centrifugation at 8000 rpm for 15 min, washed twice with deionized water, and freeze-dried (-58°C, 22 h) to obtain protein powder.

[0076] Example 4

[0077] The method for preparing white wine lees protein in this embodiment comprises the following steps:

[0078] (1) Take white wine lees as raw material with a moisture content of 10%, first perform hot air drying at 60°C, crush and pass through a 60-mesh sieve to obtain wine lees powder;

[0079] (2) Weigh 50 g of vinasse powder, add 500 mL of 70% ethanol solution, and add 0.5% sodium metabisulfite and 0.35% sodium hydroxide as reducing agents (based on the total mass of the protein extract). Under magnetic stirring in a 50°C water bath, use 40 kHz ultrasonic waves (power density 120 W / cm²) to assist extraction for 2 h. After extraction, remove large molecular impurities by ultrafiltration (membrane pore size 5 kDa), and then remove low molecular impurities by nanofiltration (membrane pore size 1 kDa). Then, centrifuge the vinasse (5000 rpm, 10 min) and collect the protein extract.

[0080] (3) The protein extract was diluted with deionized water to an ethanol concentration of 50%, 45%, and 40%, and the pH was adjusted to 7, 5, and 4, respectively, and then allowed to stand for 40 min, 50 min, and 50 min, respectively. Finally, the pH was adjusted to 3.75 and allowed to stand for 80 min for acid precipitation.

[0081] (4) The protein extract after acid precipitation was treated with dynamic temperature control to remove impurities and resin adsorption: stirring at 30°C for 10 minutes, low-frequency ultrasound (40kHz, 50W / cm²) assisted permeation at 40°C for 20 minutes, centrifugation at 50°C for 60 minutes, and dynamic adsorption at 60°C with AB-8 resin (solid-liquid ratio 1:15) for 30 minutes. The precipitate was then collected by centrifugation at 8000 rpm for 15 minutes, washed twice with deionized water, and freeze-dried (-60°C, 24 hours) to obtain protein powder.

[0082] Example 5

[0083] The method for preparing white wine lees protein in this embodiment comprises the following steps:

[0084] (1) Take white wine lees as raw material with a moisture content of 10%, first perform hot air drying at 60°C, crush and pass through a 60-mesh sieve to obtain wine lees powder;

[0085] (2) Weigh 50 g of vinasse powder, add 500 mL of 70% ethanol solution, and add 0.5% sodium metabisulfite and 0.35% sodium hydroxide as reducing agents (based on the total mass of the protein extract). Under magnetic stirring in a 50°C water bath, use 40 kHz ultrasonic waves (power density 120 W / cm²) to assist extraction for 2 h. After extraction, remove large molecular impurities by ultrafiltration (membrane pore size 5 kDa), and then remove low molecular impurities by nanofiltration (membrane pore size 1 kDa). Then, centrifuge the vinasse (5000 rpm, 10 min) and collect the protein extract.

[0086] (3) The protein extract was diluted with deionized water to an ethanol concentration of 50%, 45%, and 40%, and the pH was adjusted to 7, 5, and 4, respectively, and then allowed to stand for 30 min, 40 min, and 50 min, respectively. Finally, the pH was adjusted to 3.75 and allowed to stand for 60 min for acid precipitation.

[0087] (4) The protein extract after acid precipitation was treated with dynamic temperature control to remove impurities and resin adsorption: stirring at 30°C for 10 minutes, low-frequency ultrasound (40kHz, 50W / cm²) assisted permeation at 40°C for 20 minutes, centrifugation at 50°C for 60 minutes, and dynamic adsorption at 60°C with AB-8 resin (solid-liquid ratio 1:15) for 30 minutes. The precipitate was then collected by centrifugation at 8000 rpm for 15 minutes, washed twice with deionized water, and freeze-dried (-65°C, 24 hours) to obtain protein powder.

[0088] Comparative Example 1

[0089] The difference from Example 1 is that ultrasonic-assisted extraction is not used in step (2), and the remaining steps are the same as Example 1.

[0090] After testing, compared with Example 1, the protein extraction rate in Comparative Example 1 without using ultrasonic assisted extraction was reduced by 30%, the protein extract was turbid, the protein sedimentation rate decreased, and the purity of the protein powder finally obtained was reduced.

[0091] Comparative Example 2

[0092] The difference from Example 2 is that in step (3), the protein extract is directly diluted with deionized water to an ethanol concentration of 40%, the pH is directly adjusted to 3.75, and then allowed to stand for 195 minutes for acid precipitation, instead of using gradient dilution to adjust the pH. The remaining steps are the same as Example 2.

[0093] After testing, compared with Example 2, the lack of gradient dilution to adjust the pH in Comparative Example 2 resulted in insufficient protein precipitation, a 25% decrease in protein extraction rate, and reduced interfacial activity of the protein powder.

[0094] Comparative Example 3

[0095] The difference from Example 3 is that ultrafiltration and nanofiltration are not performed in step (2), and the remaining steps are the same as in Example 2.

[0096] After testing, compared with Example 3, the protein solution in Comparative Example 3 that was not filtered by membrane contained more impurities, lower protein purity, and decreased interfacial activity.

[0097] Comparative Example 4

[0098] The difference from Example 4 is that in step (4), no dynamic temperature control impurity removal and resin adsorption treatment are performed, and centrifugation is performed directly. The other steps are the same as in Example 4.

[0099] After testing, compared with Example 4, the purity of the protein obtained in Comparative Example 4 without using dynamic temperature control impurity removal and resin adsorption treatment was only 78.4%, and the polyphenol residue was 8.5 mg / g, while the purity of the protein obtained in Example 4 was 92.5%, and the polyphenol residue was 1.2 mg / g. The dynamic temperature control impurity removal and resin adsorption treatment used in this application can effectively remove impurities and improve protein purity.

[0100] Comparative Example 5

[0101] The difference from Example 4 is that in step (3), the protein extract is diluted with deionized water in a gradient manner to 50%, 45%, and 40% ethanol concentrations, and the pH is adjusted to 8, 7, and 5 respectively, and then allowed to stand for 40 minutes, 50 minutes, and 50 minutes, respectively. Finally, the pH is adjusted to 3.75 and then allowed to stand for 80 minutes for acid precipitation.

[0102] After testing, compared with Example 4, the change of pH adjustment gradient node in Comparative Example 5 resulted in a 13.4% decrease in protein purity, and the polyphenol residue was 4.6 mg / g.

[0103] Comparative Example 6

[0104] The difference from Example 4 is that in step (3), the protein extract is diluted with deionized water in a gradient manner to an ethanol concentration of 50% and 45%, respectively, and the pH is adjusted to 7 and 5 respectively, and then allowed to stand for 70 minutes and 70 minutes respectively. Finally, the pH is adjusted to 3.75 and then allowed to stand for 80 minutes for acid precipitation.

[0105] After testing, compared with Example 4, the lack of ethanol concentration gradient and pH adjustment gradient nodes in Comparative Example 6 made it difficult for the protein to aggregate and precipitate, the extraction rate dropped significantly by 80%, and the purity dropped by 35%.

[0106] Comparative Example 7

[0107] The difference from Example 5 is that freeze-drying is not used in step (4), but hot air drying (50°C, 24h) is used. The other steps are the same as those in Example 5.

[0108] After testing, compared with Example 5, the protein powder prepared in Comparative Example 7 without freeze-drying was more severely denatured, with reduced water solubility and impaired functional properties.

[0109] Performance test case

[0110] The performance tests were conducted on the white wine grains proteins obtained in Examples 1-5 and Comparative Examples 1-5. The test contents and test methods are as follows:

[0111] (1) Oil holding capacity and water absorption index: The protein sample was mixed with vegetable oil or water at a ratio of 1:5 and centrifuged at 3000 rpm for 15 min. The oil holding capacity (g / g) and water absorption index (mL / g) were calculated. The results are shown in Table 1.

[0112] (2) Water solubility: Referring to GB 5009.5-2025, the white wine lees protein sample was dispersed in deionized water (concentration 1%), magnetically stirred at room temperature for 30 min, and then centrifuged at 5000 rpm for 20 min. The supernatant was collected and the soluble protein content was determined by the Kjeldahl method. The water solubility was expressed as the percentage of soluble protein to total protein (%). The results are shown in Table 1.

[0113] (3) Swelling force: Weigh 0.5 g of protein sample (W1) and place it in a centrifuge tube. Add 10 mL of deionized water and let it stand for 24 h before centrifuging at 3000 rpm for 15 min. Discard the supernatant and measure the wet weight of the precipitate (W2). The swelling force was calculated as (W2-W1) / W1×100%. The results are shown in Table 1.

[0114] (4) Polyphenol residues: Using the Folin-Ciocalteu method, 1 g of protein sample was mixed with 10 mL of 70% ethanol solution, ultrasonically extracted for 30 min, and then centrifuged (4000 rpm, 10 min). The supernatant was added with Folin reagent and Na2CO3 solution, and the mixture was reacted in the dark for 1 h. The absorbance was measured at 765 nm, and the polyphenol content (mg / g) was calculated using the standard curve. The results are shown in Table 1.

[0115] (5) Foaming ability and stability: Prepare 1% protein solution from white wine lees protein. Pour 10 g of 1% protein solution into a measuring cup and stir at 2000 rpm for 5 min. Measure the volume of foam in the measuring cup within 0-60 min. Use the volume expansion method to test the foaming ability of protein. Foaming ability (%) = (V 泡沫 -V 初始液体 ) / V 初始液体 ×100, where V 初始液体 is the volume of protein solution, V 泡沫 is the foam volume. Foam stability is determined by the half-life method (t 1 / 2 ), foam stability = the time (min) required for the foam volume to decay from the maximum value to 50%. The results are as follows Figure 1 shown.

[0116] (6) Application performance: Gluten-free cakes were prepared according to the mass ratio of corn oil 14%, milk 25%, eggs 17%, sugar 14%, starch 27%, baking powder 1.5%, and protein powder 1%. The control group was set up without adding protein powder. The results are as follows: Figure 2 The texture characteristics of the gluten-free cake were measured using a texture analyzer, and the results are shown in Table 3.

[0117] (7) Volume expansion rate and flavor release efficiency: The volume expansion rate of gluten-free cake = the volume of gluten-free cake after baking / the volume of batter before baking × 100%. The volatile flavor compound content of gluten-free cake before and after baking was determined using headspace gas chromatography-mass spectrometry. First, the cake raw materials and the baked samples were processed and placed in a headspace bottle for equilibrium to volatilize the substances. The substances were separated by gas chromatography column, detected by mass spectrometry and compared with the spectral library to determine the substance type. Finally, the content was calculated by comparing the peak area or peak height with the standard substance to obtain the volatile flavor compound content before and after baking. Flavor release efficiency (%) = the content of volatile flavor compounds in the gluten-free cake after baking / the content of flavor compounds in the initial raw materials × 100%. The results are shown in Table 4.

[0118] Table 1 Performance test results of white wine grains protein obtained by different extraction methods

[0119]

[0120] Table 2 Effects of white wine lees protein obtained by different extraction methods on the structural characteristics of gluten-free cakes

[0121]

[0122] Table 3 Effects of white wine lees protein obtained by different extraction methods on the texture characteristics of gluten-free cake

[0123]

[0124] Table 4 Effects of white wine grains protein obtained by different extraction methods on the volume and flavor of gluten-free cakes

[0125]

[0126] according to Figure 1-2 As shown in Tables 1-4, the protein prepared by the method of the present application has better interfacial activity, and its mechanism can be attributed to the synergistic effect of multi-level structure regulation and targeted impurity removal in the process chain. The details are as follows:

[0127] (1) Ultrasonic-assisted extraction

[0128] According to the results of Example 1 and Comparative Example 1, it can be seen that the white wine lees protein prepared by ultrasonic-assisted extraction in Example 1 not only has improved protein purity, but also has significantly improved oil-retention and water-retention capacity and texture characteristics of the white wine lees protein compared to Comparative Example 1 which does not use ultrasonic-assisted extraction. This is because ultrasonic-assisted extraction destroys the wine lees cell wall (cellulose-lignin complex) through the high-frequency cavitation effect, gently releases embedded proteins, and reduces aggregation caused by mechanical shearing; selectively exposes the β-fold / random coil flexible structural domain, enhances the adjustability of the interfacial adsorption conformation, and the release of the flexible structure can improve the efficiency of hydrophobic group exposure, thereby enhancing the binding ability of water and oil.

[0129] (2) Gradient pH control

[0130] According to the results of Example 2 and Comparative Examples 2, 5, and 6, the protein purity of the white wine lees protein obtained by gradient dilution pH adjustment in Example 2 is improved compared with that obtained by not using gradient dilution pH adjustment in Comparative Example 2 or changing the pH adjustment gradient nodes in Comparative Example 5 or reducing the pH adjustment gradient nodes in Comparative Example 6. The reason is that the surface charge of the protein is adjusted in stages and the pH and number of gradient nodes are optimized, which can avoid the risk of isoelectric point aggregation; by inducing the reconstruction of the α-helix to the β-turn through pH, ​​the exposure of the hydrophobic core and the charge balance of the hydrophilic region are strengthened, and the amphiphilicity is improved, so that the hydrophilic-hydrophobic balance of the protein after gradient dilution pH adjustment is better and the interfacial hydration capacity is enhanced.

[0131] (3) Ultrafiltration + nanofiltration combined

[0132] According to the results of Example 1, Example 3 and Comparative Example 3, the water- and oil-holding properties and texture characteristics of the white wine grains protein prepared by ultrafiltration + nanofiltration in Example 3 are significantly better than those of the white wine grains protein prepared by ultrafiltration alone in Example 1, and are even better than the white wine grains protein prepared without ultrafiltration and nanofiltration in Comparative Example 3. This is because the combination of ultrafiltration + nanofiltration can grade and retain proteins with a molecular weight > 1 kDa, and remove small molecule interfering substances such as phenolic acid and tannic acid with a molecular weight < 1 kDa, reducing their competition for protein interface active sites through hydrogen bonds. The impurity removal rate reaches 89%, ensuring the purity of the protein network and improving the continuity of the interfacial membrane.

[0133] (4) Dynamic temperature control coupled with resin adsorption

[0134] According to the results of Example 4, Example 3 and Comparative Example 4, the polyphenol residues of the white wine lees protein prepared by dynamic temperature-controlled coupling resin adsorption in Example 4 were significantly reduced compared with those in Example 3 and Comparative Example 4 in which no dynamic temperature-controlled coupling resin was used for adsorption, and the textural properties such as adhesion, recovery, elasticity and chewiness were significantly improved. This is because the temperature gradient regulates the solubility difference, combined with the targeted adsorption of non-polar impurities such as sterols and esters by AB-8 resin; thermal vibration promotes the reversible rearrangement of disulfide bonds and optimizes the topological structure of the hydrophobic core and the hydrophilic surface, proving the key role of dynamic temperature-controlled coupling resin adsorption in the conformational arrangement and elasticity of proteins.

[0135] (5) Freeze-drying

[0136] According to the results of Examples 4-5 and Comparative Example 7, the water and oil holding capacity of the white wine grains protein prepared by the freeze-drying process in Examples 4-5 is significantly improved compared to that prepared by hot air drying in Comparative Example 7, and the textural properties such as hardness, recovery, elasticity, and chewiness are improved. This is because the freeze-drying process can quickly vitrify and freeze, thereby inhibiting ice crystal stress damage, retaining the flexible hinge area of ​​the protein tertiary structure (such as β-turn), and effectively maintaining the stability of the interfacial film.

[0137] In summary, the extraction method of the present invention optimizes the protein interfacial activity through the synergistic effect of "physical field assisted structure release (ultrasound) → charge directional control (pH gradient) → impurity graded removal (ultrafiltration / nanofiltration) → conformational dynamic optimization (temperature control / resin) → structural integrity protection (lyophilization)". Figure 1 As shown, the white wine grains protein obtained by the extraction method of the present invention has better foaming performance and stability, and has also been verified in the texture of gluten-free cakes, providing a standardized solution for the efficient extraction and application of functional plant proteins.

[0138] The embodiments described above are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing high-surface-active vinasse alcohol-soluble protein, characterized in that: The preparation method comprises the following steps: (1) Mixing the lees powder with an ethanol solution and a reducing agent, performing ultrasonic-assisted extraction, and centrifuging to obtain a protein extract; (2) Filtering the protein extract through a membrane and then centrifuging to obtain a preliminary impurity-removed protein extract; (3) Gradual dilution of the preliminary impurity-removed protein extract and gradient adjustment of the pH to 3.75-3.85 for acid precipitation to obtain an acid-precipitated protein extract; (4) The protein extract after acid precipitation is treated with dynamic temperature control to remove impurities and resin adsorption, and then centrifuged to obtain protein precipitate; (5) washing the protein precipitate and vacuum freeze-drying to obtain the high-interfacial-activity vinasse alcohol-soluble protein. The method of gradient dilution and gradient pH adjustment comprises the following steps: when the ethanol concentration in the preliminary impurity-removed protein extract is diluted to 50%, the pH is adjusted to 6-7 and then allowed to stand for 30-40 minutes; when the ethanol concentration is diluted to 45%, the pH is adjusted to 4.5-5 and then allowed to stand for 40-50 minutes; when the ethanol concentration is diluted to 40%, the pH is adjusted to 4 and then allowed to stand for 40-50 minutes; finally, the pH is adjusted to 3.75-3.85 and then allowed to stand for 60-80 minutes. The method for dynamic temperature-controlled impurity removal and resin adsorption treatment comprises the following steps: stirring at 30-35° C. for 5-15 minutes; ultrasonic-assisted penetration at 35-40° C. for 15-25 minutes; centrifugal separation combined with dynamic stirring at 45-50° C. for 45-90 minutes; and adding adsorption resin for dynamic adsorption at 50-60° C. for 20-40 minutes.

2. The preparation method according to claim 1, wherein: In step (2), the membrane filtration is carried out by combining an ultrafiltration membrane with a nanofiltration membrane.

3. The preparation method according to claim 1, wherein: In step (5), the vacuum freeze-drying temperature is -65°C to -55°C, and the vacuum freeze-drying time is ≥20h.

4. Use of the preparation method according to any one of claims 1 to 3 in food.

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