Extraction method and application of white spirit vinasse protein with high interfacial activity

Abstract

The invention provides an extraction method and application of high-interfacial-activity white spirit vinasse protein, and the preparation method comprises the following steps: mixing vinasse powder with an ethanol solution, carrying out ultrasonic-assisted extraction to obtain a protein extracting solution, carrying out membrane filtration and centrifugation on the extracting solution, then carrying out gradient dilution, and carrying out gradient adjustment on the pH value for acid precipitation to obtain the high-interfacial-activity white spirit vinasse protein. And then carrying out dynamic temperature control impurity removal and resin adsorption treatment, centrifugal separation, washing and freeze-drying to obtain the vinasse prolamin with high interfacial activity. The extraction method has the advantages of being low in energy consumption, high in efficiency and suitable for industrial continuous production, the protein sedimentation effect is improved by combining gradient dilution with pH step-by-step adjustment, inducing rearrangement of the internal structure of the protein, promoting formation of dimers or oligomers, and meanwhile, impurities can be effectively removed by adopting dynamic temperature control impurity removal combined with resin adsorption treatment, and the extraction efficiency is improved. The protein extraction efficiency is improved, the interfacial activity and safety of the protein are optimized, and the method has wide application prospects in the fields of food, medicine, biological materials and the like.

Description

Technical Field

[0001] The present invention belongs to the field of food processing, and particularly relates to a method for extracting high-surface activity distillers' grains protein and its application. Background Art

[0002] Foams widely exist in foods in different states such as beer, ice cream, cakes, and candies, and are closely related to the texture, structure, taste, and appearance of foods. Rich and stable foams give foods a lighter and fuller taste and better release the aroma of foods. Foam is a common interface-dominated food system, and the rheological properties of the air-water interface have a great impact on foam properties. According to the formation and stabilization mechanism of bubbles, the interaction between interfaces is adjusted to improve its interfacial properties, so as to achieve the goal of regulating food quality. At present, in food production, efforts have been made to find natural and safe substances with good surface activity to replace synthetic surfactants. Proteins can diffuse and adsorb onto the air-liquid interface, fold, rearrange their conformations and crosslink to form an adsorption layer with viscoelastic properties, maintaining its foam structure. Due to its large molecular weight and relatively more stable interface system, it can be used as a good foaming agent. Zein is currently widely used. Due to its strong hydrophobicity, it cannot effectively stabilize foams, so its research focus is on the preparation of composite particles. Although the complex preparation process has good effects, it is not suitable for processing and production.

[0003] A large amount of distillers' grains waste is generated during liquor fermentation. Due to its perishability and high acidity, the treatment of distillers' grains has always been a difficult problem faced by many wineries. As a fermentation product of plant origin, distillers' grains have a protein content as high as 21% and are a good source of plant protein. The existing research on distillers' grains mainly focuses on the extraction of proteins or polypeptides in distillers' grains and the exploration of the functions of their active ingredients. As the largest proportion of zein, there are problems such as difficult extraction and low development level, and there is no direct food application case. Due to the extremely low solubility of distillers' grains zein in water, its application in foods is greatly restricted. The existing extraction methods are extraction methods using solvents such as ethanol, glacial acetic acid, and isopropanol, and the extraction rate is increased by changing the extraction time or other ways to promote its dissolution. However, there are still problems such as low extraction rate and low extraction purity at present, which do not meet the requirements for large-scale modern industrial production and are also difficult to meet the food safety standards.

[0004] Wheat alcohol-soluble proteins play an important role in the production of pasta products. On the one hand, they play a role in structural support and forming a network structure. Fixed bubbles maintain the shape and volume, giving a soft taste. On the other hand, protein absorbs water to bond other ingredients together to ensure the continuity of the components. Celiac disease is a common gluten-sensitive enteropathy, which is a gastrointestinal and extraintestinal disease induced by individuals carrying hereditary genes who ingest gluten-containing foods. Avoiding the intake of gluten-containing foods is currently the most effective method. Gluten-free foods studied for this group of people are still in their infancy in China. The poor product quality caused by the removal of gluten protein is the biggest difficulty in current research. How to make gluten-free foods shape, with loose structure and continuous organization without relying on the action of gluten protein has received widespread attention. Generally, product quality is improved by adding quality improvers or 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 distiller's grains alcohol-soluble protein in food systems, and improve the interfacial activity of distiller's grains alcohol-soluble protein by improving the processing process to solve the problem of poor foaming of distiller's grains protein in traditional extraction methods has become a technical problem that needs to be urgently solved in this field. Summary of the invention

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

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows: In a first aspect, the present invention provides a method for preparing a high-surface-active vinasse alcohol-soluble protein, the preparation method comprising the following steps: (1) mixing lees powder with ethanol solution and reducing agent, performing ultrasonic-assisted extraction, and centrifuging to obtain a protein extract; (2) filtering the protein extract by membrane filtration and centrifuging to obtain a preliminary impurity-free protein extract; (3) gradient diluting the preliminary impurity-removed protein extract and gradiently adjusting 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 subjected to dynamic temperature control to remove impurities and resin adsorption treatment, and then centrifuged to obtain protein precipitate; (5) Washing and vacuum-freezing the protein precipitate to obtain the highly interfacially active wine lees alcohol-soluble protein.

[0007] Further, the method for gradient dilution and gradient pH adjustment includes the following steps: when the ethanol concentration in the preliminarily impurity-removed protein extract is diluted to 50%, adjust the pH to 6 - 7 and let it stand for 30 - 40 min; when the ethanol concentration is diluted to 45%, adjust the pH to 4.5 - 5 and let it stand for 40 - 50 min; when the ethanol concentration is diluted to 40%, adjust the pH to 4 and let it stand for 40 - 50 min; finally, adjust the pH to 3.75 - 3.85 and let it stand for 60 - 80 min. Gradient dilution and gradient pH adjustment can increase the sedimentation rate of proteins in the protein extract.

[0008] Further, the method for impurity removal by dynamic temperature control and resin adsorption treatment includes the following steps: Stir at 30 - 35 °C for 5 - 15 min to dissolve soluble impurities at low temperature and prevent protein denaturation; Perform ultrasonic-assisted penetration at 35 - 40 °C for 15 - 25 min to enhance the protein-solvent interaction and promote the dissolution of non-protein components; Carry out dynamic stirring and centrifugal separation at 45 - 50 °C for 45 - 90 min. Through efficient separation, the purity of the protein can be improved, and at the same time, the interface effect can be optimized by combining dynamic stirring; Add adsorption resin for dynamic adsorption at 50 - 60 °C for 20 - 40 min to control the pyrolysis phenomenon, maintain protein activity, and combine with adsorption resin treatment to remove polyphenols and other potential impurities and improve the safety of the protein.

[0009] Preferably, the method for impurity removal by dynamic temperature control and resin adsorption treatment includes the following steps: i) Slow-release dissolution stage: Stir at 30 - 35 °C for 10 min; ii) Penetration enhancement stage: Combine ultrasonic-assisted penetration at 35 - 40 °C for 20 min; iii) Deep extraction stage: Carry out dynamic stirring and centrifugal separation at 45 - 50 °C for 60 min; iv) Convergence and stabilization stage: Add macroporous adsorption resin for dynamic adsorption at 50 - 60 °C for 30 min. The macroporous adsorption resin is preferably AB-8 type. The polyphenol removal rate can be increased (more than 85%) through the adsorption of the macroporous adsorption resin.

[0010] Further, the reducing agent includes sodium hydroxide and sodium metabisulfite; Preferably, the mass ratio of the distiller's grains powder to the ethanol solution is 1:8 - 15, for example, it can be 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, and preferably 1:10; Preferably, the concentration of the ethanol solution is 60% - 90%, for example, it can be 60%, 65%, 70%, 75%, 80%, 85%, 90%, and preferably 70%. Preferably, the mass percentage of sodium hydroxide in the mixed solution of distillers' grains 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%.

[0011] 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.

[0012] Furthermore, in step (2), membrane filtration is carried out by combining ultrafiltration membrane and nanofiltration membrane; preferably, the pore size of the ultrafiltration membrane is 4 - 6 kDa, for example, it can be 4 kDa, 5 kDa, 6 kDa, and preferably 5 kDa, and the pore size of the nanofiltration membrane is 1 - 2 kDa, for example, it can be 1 kDa, 2 kDa, and preferably 1 kDa.

[0013] Furthermore, in step (3), the acid precipitation temperature is 20°C - 25°C, for example, it can be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, and preferably 25°C.

[0014] Furthermore, in step (5), the vacuum freeze - drying temperature is - 65°C - - 55°C, the vacuum freeze - drying time ≥20 h. Through vacuum freeze - drying, the water solubility of the prepared protein can be increased by more than 30%, and the foam half - life can be extended to more than 60 min.

[0015] Furthermore, the preparation method of the distillers' grains powder includes the following steps: drying, pulverizing, and sieving the distillers' grains to obtain the distillers' grains powder; preferably, the drying temperature is 50 - 80°C; preferably, the water content of the dried distillers' grains is 0% - 15%; further preferably, the mesh number of sieving is 40 - 80 meshes.

[0016] In the second aspect, the present invention provides highly surface - active distillers' grains prolamine prepared by the preparation method according to the first aspect.

[0017] In the third aspect, the present invention provides the application of the preparation method according to the first aspect or the highly surface - active distillers' grains prolamine according to the second aspect in food.

[0018] Further, the application includes the application of the highly surface-active distillers' soluble protein in any of the following foods: aerated foods, frozen foods, baked foods, and dairy products.

[0019] Preferably, the aerated foods include beer and carbonated beverages, which can improve the foam persistence and the mouthfeel density; More preferably, the addition amount of the highly surface-active distillers' soluble protein in the aerated foods is 0.1% - 1.5% (w / w); Preferably, the frozen foods include ice cream and mousse, which can improve the texture fluffiness and anti-melting property; Preferably, the baked foods include 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 foods to more than 98%; Preferably, the dairy products include milkshakes and puddings, which can enhance the flavor release and smoothness, and increase the flavor release efficiency in dairy products to more than 68%.

[0020] In the fourth aspect, the present invention provides a gluten-free baked food, which contains the highly surface-active distillers' soluble protein as described in the second aspect, and the volume expansion rate of the gluten-free baked food is more than 98%.

[0021] In the fifth aspect, the present invention provides the application of gradient dilution combined with gradient adjustment of the pH of the protein extraction solution in the preparation of the highly surface-active distillers' soluble protein; Preferably, the method of gradient dilution combined with gradient adjustment of the pH of the protein extraction solution includes the following steps: when the ethanol concentration in the protein extraction solution is diluted to 50%, adjust the pH to 6 - 7 and then let it stand for 30 - 40 min; when the ethanol concentration is diluted to 45%, adjust the pH to 4.5 - 5 and then let it stand for 40 - 50 min; when the ethanol concentration is diluted to 40%, adjust the pH to 4 and then let it stand for 40 - 50 min; finally, adjust the pH to 3.75 - 3.85 and then let it stand for 60 - 80 min.

[0022] In the sixth aspect, the present invention provides the application of dynamic temperature control for impurity removal combined with resin adsorption treatment of the protein extraction solution in the preparation of the highly surface-active distillers' soluble protein; Preferably, the method of dynamic temperature control for impurity removal combined with resin adsorption treatment includes the following steps: stir the protein extraction solution at 30 - 35 °C for 5 - 15 min; perform ultrasonic-assisted penetration at 35 - 40 °C for 15 - 25 min; combine dynamic stirring and centrifugal separation at 45 - 50 °C for 45 - 90 min; add macroporous adsorption resin for dynamic adsorption at 50 - 60 °C for 20 - 40 min.

[0023] More preferably, the method of dynamic temperature control for impurity removal combined with resin adsorption treatment includes the following steps: i) Sustained-release dissolution stage: Stir for 10 min at 30 - 35 °C; ii) Permeation enhancement stage: Combine ultrasonic-assisted permeation for 20 min at 35 - 40 °C; iii) Deep extraction stage: Combine dynamic stirring and centrifugal separation for 60 min at 45 - 50 °C; iv) Convergence and stabilization stage: Add macroporous adsorption resin for dynamic adsorption for 30 min at 50 - 60 °C, and the macroporous adsorption resin is preferably of AB-8 type.

[0024] Furthermore, the application includes at least one of the following: improving the purity of zein in distillers' grains and reducing the polyphenol content in zein of distillers' grains.

[0025] Compared with the prior art, the extraction method and application of the high interfacial activity distillers' grains protein of the present invention have the following advantages: (1) The extraction method of the high interfacial activity distillers' grains protein of 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 interfacial activity and safety of the protein, and has broad application prospects in the fields of food, medicine, and biological materials.

[0026] (2) The extraction method of the high interfacial activity distillers' grains protein of the present invention improves the interfacial activity of the protein and broadens its application in food and functional materials by optimizing the protein extraction and purification process.

[0027] (3) The extraction method of the high interfacial activity distillers' grains protein of the present invention controls the protein-protein interaction, precisely regulates the aggregate growth process, induces the rearrangement of the internal structure of the protein through gradient dilution combined with stepwise pH adjustment, promotes the formation of dimers or oligomers, thereby improving the protein sedimentation effect, and finally obtaining a high-purity protein product through centrifugal separation.

[0028] (4) The extraction method of the high interfacial activity distillers' grains protein of the present invention adopts dynamic temperature control for impurity removal combined with resin adsorption treatment, which can effectively remove impurities and improve the protein extraction efficiency. Brief Description of the Drawings

[0029] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings: Figure 1 It is a schematic diagram of the foaming ability and stability test results of the distillers' grains protein prepared by the present invention; Figure 2 It is a schematic diagram of the appearance of a gluten-free cake prepared with the distillers' grains protein of the present invention. Detailed Embodiments

[0030] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0031] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0032] Example 1 The method for preparing distillers' grains protein in this example includes the following steps: (1) Take distillers' grains as raw materials, with a moisture content of 10%. First, perform hot air drying at 60 °C, crush and pass through a 60-mesh sieve to obtain distillers' grains powder; (2) Weigh 50 g of distillers' grains 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 extraction solution). Under magnetic stirring in a 50 °C water bath, use 40 kHz ultrasonic waves (power density 120 W / cm²) to assist in extraction for 2 h. After extraction, remove low-molecular impurities by nanofiltration (membrane pore size 1 kDa), and then use centrifugal separation of distillers' grains residue (5000 rpm, 10 min) to collect the protein extraction solution; (3) Gradient dilute the protein extraction solution with deionized water until the ethanol concentration is 50%, 45%, and 40% in sequence, and adjust the pH to 7, 5, and 4 respectively and let it stand for 30 min, 40 min, and 50 min in sequence. Finally, adjust the pH to 3.75 and let it stand for 60 min for acid precipitation. Then centrifuge at 8000 rpm for 15 min to collect the precipitate, wash it twice with deionized water, and perform freeze-drying treatment (-60 °C, 24 h) to finally obtain protein powder.

[0033] Example 2 The method for preparing distillers' grains protein in this example includes the following steps: (1) Take distillers' grains as raw materials, with a moisture content of 10%. First, perform hot air drying at 60 °C, crush and pass through a 60-mesh sieve to obtain distillers' grains powder; (2) Weigh 50 g of distillers' grains 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 extraction solution). Under magnetic stirring in a 50 °C water bath, use 40 kHz ultrasonic waves (power density 120 W / cm²) to assist in extraction for 2 h. After extraction, remove macromolecular impurities by ultrafiltration (membrane pore size 5 kDa), and then use centrifugal separation of distillers' grains residue (5000 rpm, 10 min) to collect the protein extraction solution; (3) Gradiently dilute the protein extract with deionized water to ethanol concentrations of 50%, 45%, and 40% in sequence, and adjust the pH to 6, 4.5, and 4 respectively, then let it stand for 40 min, 50 min, and 40 min respectively. Finally, adjust the pH to 3.75 and let it stand for 65 min for acid precipitation. Then centrifuge at 8000 rpm for 15 min to collect the precipitate, wash it twice with deionized water, and perform freeze-drying treatment (-55 °C, 20 h) to finally obtain protein powder.

[0034] Example 3 The method for preparing distiller's grains protein in this example includes the following steps: (1) Take distiller's grains as raw materials with a moisture content of 10%. First, perform hot air drying treatment at 60 °C, crush and pass through a 60-mesh sieve to obtain distiller's grains powder; (2) Weigh 50 g of distiller's grains powder, add 500 mL of 70% ethanol solution, and add 0.5% sodium metabisulfite and 0.35% sodium hydroxide as reducing agents (calculated 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 macromolecular impurities by ultrafiltration (membrane pore size 5 kDa), then remove low-molecular impurities by nanofiltration (membrane pore size 1 kDa), and then use centrifugation to separate the distiller's grains residue (5000 rpm, 10 min) to collect the protein extract; (3) Gradiently dilute the protein extract with deionized water to ethanol concentrations of 50%, 45%, and 40% in sequence, and adjust the pH to 7, 5, and 4 respectively, then let it stand for 35 min, 45 min, and 45 min respectively. Finally, adjust the pH to 3.75 and let it stand for 70 min for acid precipitation. Then centrifuge at 8000 rpm for 15 min to collect the precipitate, wash it twice with deionized water, and perform freeze-drying treatment (-58 °C, 22 h) to finally obtain protein powder.

[0035] Example 4 The method for preparing distiller's grains protein in this example includes the following steps: (1) Take distiller's grains as raw materials with a moisture content of 10%. First, perform hot air drying treatment at 60 °C, crush and pass through a 60-mesh sieve to obtain distiller's grains powder; (2) Weigh 50 g of distiller's grains powder, add 500 mL of 70% ethanol solution, and add 0.5% sodium metabisulfite and 0.35% sodium hydroxide as reducing agents (calculated 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 macromolecular impurities by ultrafiltration (membrane pore size 5 kDa), then remove low-molecular impurities by nanofiltration (membrane pore size 1 kDa), and then use centrifugation to separate the distiller's grains residue (5000 rpm, 10 min) to collect the protein extract; (3) Gradiently dilute the protein extract with deionized water to ethanol concentrations of 50%, 45%, and 40% in sequence, and adjust the pH to 7, 5, and 4 correspondingly. Then let it stand for 40 min, 50 min, and 50 min respectively. Finally, adjust the pH to 3.75 and let it stand for 80 min for acid precipitation.

[0036] (4) Perform dynamic temperature-controlled impurity removal and resin adsorption treatment on the acid-precipitated protein extract: Stir at 30 °C for 10 min, combine with low-frequency ultrasonic waves (40 kHz, 50 W / cm²) for assisted penetration at 40 °C for 20 min, centrifuge at 50 °C for 60 min, add AB-8 resin (solid-liquid ratio 1:15) for dynamic adsorption at 60 °C for 30 min. Then centrifuge at 8000 rpm for 15 min to collect the precipitate, wash it twice with deionized water, and perform freeze-drying treatment (-60 °C, 24 h) to finally obtain protein powder.

[0037] Example 5 The method for preparing distiller's grains protein in this example includes the following steps: (1) Take distiller's grains as raw materials with a moisture content of 10%. First, perform hot air drying treatment at 60 °C, crush and pass through a 60-mesh sieve to obtain distiller's grains powder; (2) Weigh 50 g of distiller's grains 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) for assisted extraction for 2 h. After extraction, remove macromolecular impurities by ultrafiltration (membrane pore size 5 kDa), then remove low-molecular impurities by nanofiltration (membrane pore size 1 kDa), and then perform centrifugal separation of distiller's grains residue (5000 rpm, 10 min) to collect the protein extract; (3) Gradiently dilute the protein extract with deionized water to ethanol concentrations of 50%, 45%, and 40% in sequence, and adjust the pH to 7, 5, and 4 correspondingly. Then let it stand for 30 min, 40 min, and 50 min respectively. Finally, adjust the pH to 3.75 and let it stand for 60 min for acid precipitation.

[0038] (4) Perform dynamic temperature-controlled impurity removal and resin adsorption treatment on the acid-precipitated protein extract: Stir at 30 °C for 10 min, combine with low-frequency ultrasonic waves (40 kHz, 50 W / cm²) for assisted penetration at 40 °C for 20 min, centrifuge at 50 °C for 60 min, add AB-8 resin (solid-liquid ratio 1:15) for dynamic adsorption at 60 °C for 30 min. Then centrifuge at 8000 rpm for 15 min to collect the precipitate, wash it twice with deionized water, and perform freeze-drying treatment (-65 °C, 24 h) to finally obtain protein powder.

[0039] Comparative Example 1 The difference from Example 1 is that ultrasonic-assisted extraction was not used in step (2), and the remaining steps are the same as those in Example 1.

[0040] It was detected that, compared with Example 1, the protein extraction rate in Comparative Example 1 without ultrasonic-assisted extraction decreased by 30%, the protein extraction solution was turbid, the protein sedimentation rate decreased, and the purity of the finally obtained protein powder decreased.

[0041] Comparative Example 2 The difference from Example 2 is that in step (3), the protein extraction solution was directly diluted with deionized water to an ethanol concentration of 40%, the pH was directly adjusted to 3.75, and then left standing for 195 min for acid precipitation, instead of using gradient dilution to adjust the pH. The remaining steps are the same as those in Example 2.

[0042] It was detected that, compared with Example 2, the lack of gradient dilution to adjust the pH in Comparative Example 2 led to insufficient protein sedimentation, a 25% reduction in the protein extraction rate, and a decrease in the surface activity of the protein powder.

[0043] Comparative Example 3 The difference from Example 3 is that ultrafiltration and nanofiltration were not carried out in step (2), and the remaining steps are the same as those in Example 2.

[0044] It was detected that, compared with Example 3, the protein solution without membrane filtration in Comparative Example 3 had more impurities, the protein purity decreased, and the surface activity decreased.

[0045] Comparative Example 4 The difference from Example 4 is that in step (4), dynamic temperature-controlled impurity removal and resin adsorption treatment were not carried out, and centrifugation was directly carried out. Other steps are the same as those in Example 4.

[0046] It was detected that, compared with Example 4, the protein purity prepared in Comparative Example 4 without using dynamic temperature-controlled impurity removal and resin adsorption treatment was only 78.4%, and the polyphenol residue was 8.5 mg / g, while the protein purity prepared in Example 4 was 92.5%, and the polyphenol residue was 1.2 mg / g. The dynamic temperature-controlled impurity removal and resin adsorption treatment adopted in this application can effectively remove impurities and improve the protein purity.

[0047] Comparative Example 5 The difference from Example 4 is that in step (3), the protein extraction solution was gradient-diluted with deionized water to ethanol concentrations of 50%, 45%, and 40% in sequence, and the pH was adjusted to 8, 7, and 5 respectively and left standing for 40 min, 50 min, and 50 min in sequence, and finally the pH was adjusted to 3.75 and left standing for 80 min for acid precipitation.

[0048] After detection, compared with Example 4, the change in the pH adjustment gradient nodes in Comparative Example 5 led to a 13.4% decrease in protein purity, and the polyphenol residue was 4.6 mg / g.

[0049] Comparative Example 6 The difference from Example 4 is that in step (3), the protein extract was gradient-diluted with deionized water to ethanol concentrations of 50% and 45% in sequence, and the pH was adjusted to 7 and 5 in sequence and allowed to stand for 70 min and 70 min respectively, and finally the pH was adjusted to 3.75 and allowed to stand for 80 min for acid precipitation.

[0050] After detection, compared with Example 4, the lack of ethanol concentration gradient and pH adjustment gradient nodes in Comparative Example 6 made it difficult for proteins to aggregate and precipitate, and the extraction rate decreased significantly by 80%, and the purity decreased by 35%.

[0051] Comparative Example 7 The difference from Example 5 is that in step (4), freeze-drying treatment was not used, but hot air drying (50 °C, 24 h) was used, and the other steps were the same as in Example 5.

[0052] After detection, compared with Example 5, the protein powder prepared without using freeze-drying in Comparative Example 7 was more severely denatured, with reduced water solubility and impaired functional properties.

[0053] Performance test examples Perform performance tests on the distillers' grains proteins prepared in Examples 1-5 and Comparative Examples 1-5. The test contents and test methods are as follows: (1) Oil-holding capacity and water absorption index: Determined by the centrifugation method of GB 5009.3-2025. The protein sample was mixed with vegetable oil or water at a ratio of 1:5, and after centrifugation 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.

[0054] (2) Water solubility: Referring to the standard of GB 5009.5-2025, the distillers' grains protein sample was dispersed in deionized water (concentration 1%). After magnetic stirring at room temperature for 30 min, it was centrifuged at 5000 rpm for 20 min. The supernatant was taken and the soluble protein content was determined by the Kjeldahl method. The water solubility was expressed as the percentage of soluble protein in the total protein mass (%), and the results are shown in Table 1.

[0055] (3) Swelling power: Weigh 0.5 g of the protein sample (W 1 ) and place it in a centrifuge tube, add 10 mL of deionized water, let it stand for 24 h, and then centrifuge at 3000 rpm for 15 min. After discarding the supernatant, measure the wet weight of the precipitate (W 2 ). The swelling power calculation formula is (W 2 - W 1 ) / W1 × 100%, and the results are shown in Table 1.

[0056] (4) Polyphenol residue: 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 taken and added with Folin reagent and Na 2 CO 3 solution, reacted in the dark for 1 h, and the absorbance was measured at 765 nm. The polyphenol content (mg / g) was calculated using the standard curve, and the results are shown in Table 1.

[0057] (5) Foaming ability and stability: The protein from distillers' grains was made into a 1% protein solution. 10 g of the 1% protein solution was poured into a measuring cup and stirred at 2000 rpm for 5 min. The foam volume in the measuring cup was measured within 0 - 60 min. The volume expansion method was used to detect the foaming property of the protein. Foaming property (%) = (V 泡沫 - V 初始液体 ) / V 初始液体 × 100, where V 初始液体 is the volume of the protein solution and V 泡沫 is the foam volume. The foam stability was measured 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%, and the results are as Figure 1 shown.

[0058] (6) Application performance: Gluten-free cakes were prepared according to the mass ratio of 14% corn oil, 25% milk, 17% eggs, 14% sugar, 27% starch, 1.5% baking powder, and 1% protein powder. The group without adding protein powder was used as the control group, and the results are as Figure 2 well as in Table 2. The texture properties of the prepared gluten-free cakes were measured using a texture analyzer, and the results are shown in Table 3.

[0059] (7) Volume expansion rate and flavor release efficiency: The volume expansion rate of the gluten-free cake = the volume of the baked gluten-free cake / the volume of the batter before baking × 100%. The content of volatile flavor substances in the gluten-free cake before and after baking was determined by headspace-gas chromatography-mass spectrometry. First, the cake raw materials and the baked samples were processed, placed in a headspace bottle to balance and volatilize the substances, separated by a gas chromatography column, detected by a mass spectrometer, and compared with a spectral library to determine the substance types. Finally, the content was calculated by comparing the peak area or peak height with the standard substance, and the content of volatile flavor substances before and after baking was obtained. Flavor release efficiency (%) = the content of volatile flavor substances in the baked gluten-free cake / the content of flavor substances in the initial raw materials × 100%. The results are shown in Table 4.

[0060] Table 1 Performance test results of the protein from distillers' grains prepared by different extraction methods

[0061] Table 2 Effects of distillers' grains protein prepared by different extraction methods on the structural characteristics of gluten-free cakes

[0062] Table 3 Effects of distillers' grains protein prepared by different extraction methods on the texture properties of gluten-free cakes

[0063] Table 4 Effects of distillers' grains protein prepared by different extraction methods on the volume and flavor of gluten-free cakes

[0064] According to Figure 1-2 and the results in Tables 1-4, it can be seen that the protein prepared by the method of this application has better interfacial activity, and its mechanism can be attributed to the synergistic effect of multi-level structure regulation and impurity targeted removal in the process chain. Specifically as follows: (1) Ultrasonic-assisted extraction According to the results of Example 1 and Comparative Example 1, it can be seen that the distillers' grains protein prepared by ultrasonic-assisted extraction in Example 1 not only has higher protein purity compared with Comparative Example 1 without ultrasonic-assisted extraction, but also has significantly improved oil-holding and water-holding capacity and texture properties of the distillers' grains protein. This is because ultrasonic-assisted extraction destroys the cell wall of distillers' grains (cellulose-lignin complex) through high-frequency cavitation effect, gently releases the embedded protein, and reduces the aggregation caused by mechanical shear; selectively exposes the β-sheet / random coil flexible domain, enhances the conformational tunability of interfacial adsorption, and the release of the flexible structure can improve the efficiency of hydrophobic group exposure, thereby enhancing the binding ability of water and oil.

[0065] (2) Gradient pH regulation According to the results of Example 2 and Comparative Examples 2, 5, and 6, it can be seen that the distillers' grains protein prepared by gradient dilution pH regulation in Example 2 has higher protein purity and significantly improved water absorption performance compared with Comparative Example 2 without gradient dilution pH regulation, Comparative Example 5 with changed pH regulation gradient nodes, or Comparative Example 6 with reduced pH regulation gradient nodes. This is because the surface charge of the protein is adjusted in stages and the pH and number of nodes of the gradient are optimized, which can avoid the risk of isoelectric point aggregation; by pH-induced reconstruction of α-helix to β-turn, it strengthens the exposure of the hydrophobic core and the charge balance in the hydrophilic region, improves amphiphilicity, makes the hydrophilic-hydrophobic balance of the protein after gradient dilution pH regulation better, and enhances the interfacial hydration ability.

[0066] (3) Ultrafiltration + nanofiltration combination According to the results of Example 1, Example 3 and Comparative Example 3, it can be seen that the water-holding and oil-holding properties and texture characteristics of the distillers' grains protein prepared by the combined use of ultrafiltration and nanofiltration in Example 3 are significantly better than those of the distillers' grains protein prepared by only ultrafiltration in Example 1, and are even better than those of the distillers' grains protein prepared without ultrafiltration and without nanofiltration in Comparative Example 3. This is because the combined use of ultrafiltration and nanofiltration can fractionally retain proteins with a molecular weight > 1 kDa and remove small molecule interfering substances such as phenolic acids and tannic acids with a molecular weight < 1 kDa, reducing their competition for protein interfacial active sites through hydrogen bonds. The impurity clearance rate reaches 89%, ensuring the purity of the protein network and improving the continuity of the interfacial membrane.

[0067] (4)Dynamic temperature control coupled with resin adsorption According to the results of Example 4, Example 3 and Comparative Example 4, it can be seen that the polyphenol residues of the distillers' grains protein prepared by dynamic temperature control coupled with resin adsorption in Example 4 are significantly reduced compared with those in Example 3 and Comparative Example 4 without dynamic temperature control coupled with resin adsorption, and the texture characteristics such as adhesiveness, resilience, elasticity and chewiness are significantly improved. This is because the temperature gradient regulates the solubility difference, combined with the targeted adsorption of AB-8 resin for non-polar impurities such as sterols and esters; thermal vibration promotes the reversible rearrangement of disulfide bonds, optimizing the topological structure of the hydrophobic core and hydrophilic surface, demonstrating the key role of dynamic temperature control coupled with resin adsorption in protein conformation arrangement and elasticity.

[0068] (5)Low-temperature freeze-drying According to the results of Examples 4-5 and Comparative Example 7, it can be seen that the water-holding and oil-holding capacities of the distillers' grains protein prepared by the freeze-drying process in Examples 4-5 are significantly improved compared with those of Comparative Example 7 using hot air drying, and the texture characteristics such as hardness, resilience, elasticity and chewiness are improved. This is because the freeze-drying process can quickly vitrify and freeze, thereby inhibiting ice crystal stress damage and retaining the flexible hinge region (such as β-turn) of the protein tertiary structure, effectively maintaining the stability of the interfacial membrane.

[0069] In summary, the extraction method of the present invention through the synergistic effect of "physical field-assisted structure release (ultrasound) → charge-directed regulation (pH gradient) → impurity fractional clearance (ultrafiltration / nanofiltration) → conformational dynamic optimization (temperature control / resin) → structural integrity protection (freeze-drying)" makes the protein interfacial activity optimal. As Figure 1 shown, the distillers' grains protein prepared 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.

[0070] The above-described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing high-interfacial active vinasse alcohol-soluble protein, characterized in that: The preparation method comprises the following steps: (1) mixing lees powder with ethanol solution and reducing agent, performing ultrasonic-assisted extraction, and centrifuging to obtain a protein extract; (2) filtering the protein extract by membrane filtration and centrifuging to obtain a preliminary impurity-free protein extract; (3) gradient diluting the preliminary impurity-removed protein extract and gradiently adjusting 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 subjected to dynamic temperature control to remove impurities and resin adsorption treatment, and then centrifuged to obtain protein precipitate; (5) Washing and vacuum-freezing the protein precipitate to obtain the highly interfacially active wine lees alcohol-soluble protein.

2. The preparation method according to claim 1, characterized in that: The method for 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; when the ethanol concentration is diluted to 45%, the pH is adjusted to 4.5-5; when the ethanol concentration is diluted to 40%, the pH is adjusted to 4; and finally the pH is adjusted to 3.75-3.

85.

3. The preparation method according to claim 1, characterized in that: The method for dynamic temperature control 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.

4. The preparation method according to claim 1, characterized in that: In step (2), membrane filtration is performed by combining ultrafiltration membrane and nanofiltration membrane.

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

6. A high-interfacial-activity vinasse alcohol-soluble protein prepared according to the preparation method described in any one of claims 1 to 5.

7. The preparation method according to any one of claims 1 to 5 or the use of the high-surface-active vinasse alcohol-soluble protein according to claim 6 in food.

8. A gluten-free baked food, characterized in that The gluten-free baked food contains the high-surface-active distiller's grains alcohol-soluble protein according to claim 7, and the volume expansion rate of the gluten-free baked food is greater than 98%.

9. Application of gradient dilution combined with gradient adjustment of pH value of protein extract in the preparation of highly interfacially active wine lees alcohol-soluble protein.

10. Application of dynamic temperature control impurity removal combined with resin adsorption to treat protein extract in the preparation of highly interfacially active wine lees alcohol-soluble protein.

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