Method for preparing peanut protein from peanut meal

Abstract

The invention provides a method for preparing peanut protein from peanut meal, and belongs to the technical field of agricultural product processing. The method comprises the following steps: performing enzymolysis treatment on peanut meal powder by using alpha-amylase to obtain peanut meal enzymatic hydrolysate; and carrying out any one of the following modification treatment on the peanut meal enzymatic hydrolysate to obtain the peanut protein: ultrasonic treatment, alkali treatment and ultrasonic-assisted alkali treatment. According to the method, the problem of low solubility caused by high-temperature denaturation of the peanut protein is effectively solved through combined modification treatment by cracking the structure of the protein wrapped by the starch through the alpha-amylase, and the peanut protein prepared by the method is high in solubility, excellent in emulsifying property and high in antioxidant activity; the method can be widely applied to processing scenes of various products such as drinks, dairy products, meat products, baked foods and the like needing to be added with protein ingredients.

Description

Technical Field

[0001] This invention belongs to the field of agricultural product processing technology, specifically relating to a method for preparing peanut protein using peanut meal. Background Technology

[0002] Peanut meal is a major byproduct obtained after pressing peanuts for oil extraction, and it is rich in protein and other nutrients. However, peanut meal currently suffers from problems such as low solubility and unstable emulsification properties, which severely restricts its application in the food industry. High-temperature peanut meal, in particular, undergoes high-temperature treatment during hot pressing, resulting in severe denaturation of its protein structure and extremely low natural solubility. In the high-temperature environment of subsequent food processing, it easily aggregates and precipitates, exhibiting poor dispersibility and insufficient emulsification stability. This makes it difficult to meet the functional requirements of modern food industry for high-protein ingredients, thus limiting the high-value utilization of peanut meal.

[0003] Existing technologies mainly employ three modification methods—physical, chemical, and enzymatic—to address the low solubility of peanut protein, but all have significant drawbacks. Physical modification improves solubility by disrupting the protein's spatial structure, but its modification effect is limited, and it is energy-intensive, has high processing costs, and can further damage heat-sensitive nutrients in the protein, exacerbating nutritional value loss. Chemical modification alters the protein structure by adding chemical reagents such as acids, alkalis, and acylating agents, posing food safety risks due to reagent residues, easily leading to imbalances in protein functional properties, and increasing separation and purification costs, hindering industrial-scale promotion. Traditional enzymatic modification uses proteases to hydrolyze proteins, which can improve solubility, but it easily causes excessive protein degradation, producing bitter peptides that affect sensory quality, and the uneven molecular weight distribution of hydrolysis products makes it difficult to form a stable food functional system. Summary of the Invention

[0004] In view of this, the present invention provides a method for preparing peanut protein using peanut meal. The peanut protein obtained has high solubility and excellent emulsification properties. It is not easy to aggregate and precipitate in the subsequent food processing environment and can be widely adapted to product processing scenarios for food protein ingredients.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing peanut protein from peanut meal, comprising: enzymatically hydrolyzing peanut meal powder with α-amylase to obtain peanut meal hydrolysate; Peanut protein is obtained by subjecting the peanut meal enzymatic hydrolysate to any of the following modification treatments: ultrasonic treatment, alkali treatment, and ultrasonic-assisted alkali treatment.

[0006] Preferably, in the enzymatic hydrolysis system, the amount of water added is 5-10 mL / g peanut meal powder; and the amount of α-amylase added is 25-70 U / g peanut starch, based on the amount of peanut starch in the peanut meal powder.

[0007] Preferably, the enzymatic hydrolysis treatment is performed at a temperature of 50-60°C for 0.5-1.5 hours.

[0008] Preferably, when ultrasonic treatment is used, the power of the ultrasonic treatment is 200~400W, the intermittent pulse mode is used, and the time is 4~6min.

[0009] Preferably, when alkali treatment is used, the pH value of the alkali treatment is 11-13 and the time is 40-80 min; After the alkali treatment, the pH value is adjusted back to 6.8~7.2.

[0010] Preferably, when ultrasound-assisted alkali treatment is used, the ultrasound-assisted alkali treatment is performed during the alkali treatment process; The pH value of the alkali treatment is 11-13, and the time is 40-80 min; The ultrasonic treatment has a power of 200~400W and a duration of 4~6min, and the ultrasonic treatment is intermittent ultrasonic treatment; After the ultrasonic-assisted alkali treatment, the pH value is adjusted back to 6.8~7.2.

[0011] Preferably, after the modification treatment is completed, the process further includes freeze-drying.

[0012] This invention provides an application of the method in improving at least one of the following properties of peanut meal: solubility, emulsification properties, and antioxidant properties.

[0013] This invention provides a peanut protein prepared by the method described above.

[0014] This invention provides an application of the peanut protein in food processing.

[0015] Compared with the prior art, the present invention has the following advantages: This invention provides a method for preparing peanut protein from peanut meal, comprising: enzymatically hydrolyzing peanut meal powder with α-amylase to obtain peanut meal hydrolysate; and subjecting the peanut meal hydrolysate to any of the following modification treatments to obtain peanut protein: ultrasonic treatment, alkali treatment, and ultrasonic-assisted alkali treatment. This invention addresses the problem of insufficient solubility of peanut meal protein in existing technologies by providing a preparation method that combines α-amylase catalysis with ultrasonic-assisted alkali treatment for synergistic modification. α-amylase specifically degrades starch, eliminating its adverse effects, breaking down starch encapsulation, and fully reconstructing the protein aggregation structure, while not damaging the amino acid composition of hot-pressed peanut protein. This avoids the production of bitter peptides and uneven molecular weight distribution of hydrolysate products from protease hydrolysis, solving the problem of low solubility caused by high-temperature denaturation of peanut protein. The resulting peanut protein powder has significantly improved solubility, is less prone to aggregation and precipitation in high-temperature processing environments, and exhibits dispersibility and emulsification stability that meet the processing requirements of various food systems. The method described in this invention can effectively increase the added value of peanut meal powder, transforming peanut meal powder, which was originally limited in utilization due to its low solubility, into a high-value food protein ingredient, thus broadening its application scenarios in the food industry. It can be used in the production of high-protein ingredients in the food industry and is widely applicable to various product processing scenarios that require the addition of protein ingredients, such as beverages, dairy products, meat products, and baked goods. It conforms to the industrial development trend of resource recycling and has significant economic and social benefits. Attached Figure Description

[0016] Figure 1 The graph shows the results of peanut protein solubility determination. Figure 2 The graph shows the results of the peanut protein emulsification performance test. Figure 3 The graph shows the results of the determination of peanut protein particle size and ζ-potential. Figure 4 The image shows the results of the peanut protein antioxidant assay. Detailed Implementation

[0017] This invention provides a method for preparing peanut protein from peanut meal, comprising: enzymatically hydrolyzing peanut meal powder with α-amylase to obtain peanut meal hydrolysate; Peanut protein is obtained by subjecting the peanut meal enzymatic hydrolysate to any of the following modification treatments: ultrasonic treatment, alkali treatment, and ultrasonic-assisted alkali treatment.

[0018] In this invention, the source of the peanut meal is not specifically limited. In this embodiment, high-temperature peanut meal is used as an example to illustrate the method. High-temperature peanut meal is a major byproduct of peanut oil extraction via hot pressing. Due to the high-temperature treatment during processing, the protein structure is severely denatured, resulting in a significant decrease in solubility compared to low-temperature meal. It easily aggregates and precipitates in the high-temperature environment of subsequent food processing, exhibiting poor dispersibility and emulsification stability. Because of its poor water solubility and emulsification properties, high-temperature peanut meal is mostly used in animal feed, severely limiting its application in food. During hot pressing of peanuts for oil extraction, the temperature reaches above 120°C. In this embodiment, the high-temperature peanut meal powder is peanut cake meal after high-temperature oil extraction, pulverized using a high-speed grinder and sieved through a 60-mesh sieve. The peanut cake meal was purchased from Shandong Huayuan Food Co., Ltd. The protein content was determined to be 53.1 wt.% by the Kjeldahl method, the oil content was determined to be 3.6 wt.% by acid hydrolysis, and the residual starch content in the peanut high-temperature meal powder was determined to be 12.2 wt.% by enzymatic hydrolysis.

[0019] This invention utilizes α-amylase to enzymatically hydrolyze peanut meal powder to obtain peanut meal hydrolysate. Preferably, the peanut meal powder is preheated after mixing with water. The preheating process involves preheating the peanut protein powder and water mixture at 55°C in a water bath at 160 rpm for 15 minutes. This preheating process ensures thorough dispersion of the peanut protein powder, preventing clumping, and provides a suitable temperature environment for the subsequent enzymatic hydrolysis reaction, reducing the risk of further protein denaturation. It also preheats to the optimal temperature for the enzyme reaction, improving enzyme catalytic efficiency. After preheating, α-amylase is used for targeted enzymatic hydrolysis. In the enzymatic hydrolysis system, the preferred amount of water added is 5-10 mL / g peanut meal powder, more preferably 9 mL / g peanut meal powder; based on the amount of peanut starch in the peanut meal powder, the preferred amount of α-amylase added is 25-70 U / g peanut starch, more preferably 35-60 U / g peanut starch, and even more preferably 50 U / g peanut starch. In the embodiments of this invention, α-amylase is added at a dosage of 50 U / g peanut starch. The preferred amount of α-amylase added, based on the amount of peanut meal powder, is 200-600 U / g peanut meal powder, more preferably 300-550 U / g peanut meal powder, even more preferably 350-530 U / g peanut meal powder, and most preferably 500 U / g peanut meal powder. The preferred temperature for the enzymatic hydrolysis is 50-60℃, more preferably 55℃. The preferred time for the enzymatic hydrolysis is 0.5-1.5 h, more preferably 1 h. The core function of the α-amylase is to specifically degrade residual starch in peanut protein, specifically breaking the starch's encapsulation effect on the protein and removing starch interference. Compared to undegraded peanut meal powder, α-amylase specifically targets the α-1,4 glycosidic bonds of starch molecules, degrading the starch in peanut meal powder into smaller sugar molecules such as maltose and glucose. This effectively breaks the interaction between starch and protein, fully exposing peanut protein molecules from their encapsulated state. It also breaks down hydrogen bonds and hydrophobic interactions between starch and peanut protein, releasing the peanut protein molecules from the starch-protein complex. This allows subsequent modification treatments (ultrasonic treatment, alkali treatment, or ultrasound-assisted alkali treatment) to act more efficiently on the protein molecules, avoiding the inhibition of protein modification by starch encapsulation, thus significantly improving protein solubility. Simultaneously, the specific catalytic action of α-amylase does not damage protein molecules or high oleic acid, ensuring the nutritional integrity of the product. The specific enzymatic hydrolysis system of this invention can enzymatically hydrolyze residual starch impurities in peanut protein powder, breaking the interaction between starch and protein, eliminating the adverse effects of starch encapsulation on protein dispersibility, and mildly disrupting the starch encapsulation structure on the protein surface, laying the foundation for subsequent protein modification without damaging the amino acid composition of the denatured protein.

[0020] After obtaining peanut meal enzymatic hydrolysate, the present invention performs any one of the following modification treatments on the peanut meal enzymatic hydrolysate to obtain peanut protein: ultrasonic treatment, alkali treatment, and ultrasonic-assisted alkali treatment.

[0021] In this invention, when ultrasonic treatment is used, the ultrasonic power is preferably 200-400W, more preferably 250-350W, and most preferably 300W. The ultrasonic treatment is preferably intermittent, more preferably intermittent pulse mode, and the intermittent pulse mode is preferably a 3-second ultrasound, 2-second pause intermittent pulse mode. The ultrasonic treatment time is preferably 4-6 minutes, more preferably 5 minutes. The ultrasonic treatment temperature is preferably 0-5℃, for example, 0℃, 1℃, 2℃, 3℃, or ℃. The mechanical shearing force of the intermittent pulse ultrasound can break up protein aggregates. Temperature control using an ice-water bath during the ultrasound process effectively avoids secondary protein aggregation and denaturation caused by localized high temperatures during ultrasound, maximizing the restoration of protein solubility and improving the protein's antioxidant properties. The combination of intermittent ultrasound and an ice-water bath avoids the problem of localized high temperatures caused by continuous ultrasound, solving the problem of the heat-sensitive nature of hot-pressed peanut protein.

[0022] In this invention, when alkali treatment is used, the pH value of the alkali treatment is preferably 11-13, more preferably 12. The temperature of the alkali treatment is preferably 0-5°C, and the time is preferably 40-80 min, more preferably 60 min. After the alkali treatment, the pH value is preferably adjusted back to 6.8-7.2, more preferably to 7.0. By shifting the pH, the non-covalent bonds such as hydrogen bonds and hydrophobic interactions of hot-pressed peanut protein are disrupted, reconstructing the protein's spatial structure and maximizing its solubility and antioxidant properties. Adjusting the pH to 7.0 allows the expanded protein molecules to form a stable soluble conformation, avoiding the impact of a strongly alkaline environment on the subsequent application of the protein, while ensuring that the pH of the product meets the requirements for food processing.

[0023] In this invention, when ultrasound-assisted alkali treatment is used, the ultrasound-assisted alkali treatment is performed during the alkali treatment process; the pH value of the alkali treatment is preferably 11-13, more preferably 12. The alkali treatment time is preferably 40-80 min, more preferably 50-70 min, and most preferably 55 min. The ultrasonic power of the ultrasonic treatment is preferably 200-400 W, more preferably 250-350 W, and most preferably 300 W. The ultrasonic treatment preferably adopts an intermittent pulse mode, and the intermittent pulse mode is preferably a 3-second ultrasound followed by a 2-second pause; the ultrasonic treatment time is preferably 4-6 min, more preferably 5 min. An ice-water bath is used for temperature control during the ultrasonic treatment, and the temperature during the ultrasonic treatment is preferably 0-5℃. Ultrasonic treatment can be performed at any stage of the alkali treatment; the pH condition can be adjusted to alkali treatment and ultrasonic treatment can be performed immediately, followed by alkali treatment; or the alkali treatment can be performed first, followed by ultrasonic treatment, and then alkali treatment can continue after ultrasonic treatment. In this embodiment of the invention, the ultrasound-assisted alkaline treatment is preferably performed by adjusting the peanut meal enzymatic hydrolysate to pH 12, followed by ultrasound treatment for 5 minutes under alkaline conditions, and then continuing alkaline treatment for 55 minutes after ultrasound treatment. The strongly alkaline pH shift structure is responsible for disrupting the disulfide bonds, hydrogen bonds, and other forces within the protein, allowing the protein spatial structure to expand; intermittent ultrasound breaks down protein aggregates through cavitation effects, improving the uniformity of modification; the ultrasound-assisted alkaline treatment preferably uses an ice-water bath to control the reaction temperature, avoiding protein denaturation and high oleic acid oxidation. In ultrasound-assisted alkali treatment, strong alkaline pH shift, intermittent ultrasound, and ice-water bath cooling work synergistically. Compared to traditional single pH shift or continuous ultrasound structures, this combined structure achieves a balance between improved modification efficiency and nutrient retention. On the one hand, the cavitation effect of ultrasound enhances the destructive effect of pH shift on protein structure, making the protein more likely to form a soluble conformation, with a solubility increase of over 20% compared to single pH shift modification. On the other hand, the combination of intermittent ultrasound and ice-water bath avoids the local high temperature problem caused by continuous ultrasound, effectively preventing protein denaturation and high oleic acid oxidation due to excessively high local temperatures. Compared to conventional ultrasound and high-temperature physical modification, energy consumption is lower, and secondary protein aggregation and denaturation are avoided. After ultrasound-assisted alkali treatment, the pH value is preferably adjusted back to 6.8~7.2, more preferably to 7.0. Adjusting the pH to 7.0 allows the stretched protein molecules to form a stable soluble conformation, avoiding the impact of a strongly alkaline environment on subsequent protein applications, while ensuring that the product's pH meets food processing requirements.

[0024] When this invention employs a synergistic modification method combining starch enzymatic hydrolysis, ultrasonic treatment, and alkali treatment, the solubility, emulsifying properties, and antioxidant activity of peanut meal protein are significantly improved, resulting in peanut protein with superior overall performance. The embodiments of this invention compare the performance of peanut protein prepared by different modification methods. The results show that, compared with unmodified peanut meal, the peanut protein prepared by the method of this invention has significantly improved solubility, emulsifying properties, and antioxidant properties. Compared with modified peanut protein using only ultrasonic combined with alkali treatment, introducing a starch enzymatic hydrolysis step can further improve its solubility, emulsifying properties, and antioxidant properties. Particle size and Zeta potential detection results indicate that the method of this invention can be dually controlled, effectively reducing the average particle size of peanut protein powder and increasing the Zeta potential, inhibiting the aggregation behavior of high-temperature denatured peanut protein, and laying a structural foundation for the long-term dispersion stability of peanut protein powder in food systems such as emulsions and protein beverages.

[0025] In this invention, after the modification treatment is completed, freeze-drying is preferably further included. Preferably, the modified peanut protein is pre-frozen and then freeze-dried under vacuum to obtain peanut protein powder with high solubility. The pre-freezing temperature is preferably -70~-90℃, more preferably -80℃. The pre-freezing time is preferably 12 hours or more, more preferably 12~48 hours, and most preferably 12 hours. The pre-freezing can completely freeze the solution. The vacuum degree of the vacuum freeze-drying is preferably ≤10 Pa, more preferably 1~10 Pa, and in this embodiment of the invention, it is 1 Pa. The temperature is preferably ≤-50℃, more preferably -80℃~-50℃, and most preferably -60℃. In this embodiment of the invention, it is -60℃. The vacuum freeze-drying time is preferably until the sample moisture content is ≤5%, more preferably 1%~5%. In this embodiment of the invention, it is dried until the sample moisture content is 3%. After drying, the sample is stored in a desiccator for later use and its indicators are tested as soon as possible to avoid moisture absorption and structural rebound. The function and advantages of vacuum freeze-drying: This structure replaces the traditional hot air drying structure, removing moisture from protein solutions under low-temperature vacuum conditions. Compared to hot air drying, freeze-drying allows the protein solution to sublimate and dehydrate in a frozen state, maximizing the preservation of the protein's soluble conformation and heat-sensitive nutrients such as oleic acid. It avoids the damage to the protein's soluble conformation caused by the high temperatures generated during water evaporation in traditional hot air drying, while effectively preserving heat-sensitive nutrients such as oleic acid.

[0026] This invention utilizes a combined process structure of "α-amylase-catalyzed degradation - ultrasound-assisted alkali treatment - freeze-drying" to achieve precise control over the structure of peanut protein, thereby improving its solubility. Simultaneously, the process conditions are mild, maximizing the retention of remaining heat-sensitive nutrients in the protein. The product is odorless, has excellent sensory quality, and compensates for some nutrient loss during hot pressing. The process steps of this invention form a continuous structure, from raw material dispersion, enzyme catalysis, ultrasound-assisted pH modification to freeze-drying, with each step tightly integrated to form a complete modification process. It eliminates the need for high temperature and high pressure, avoiding further high-temperature denaturation of the protein powder. The entire process leaves no chemical reagent residues and eliminates the need for complex subsequent separation and purification steps, preventing sample contamination and nutrient loss in intermediate stages. Compared to traditional segmented and discontinuous modification processes, this continuous structure improves product quality stability, reduces the risk of impurity introduction during production, shortens the production cycle, controls processing costs, facilitates industrial-scale production, and improves production efficiency.

[0027] Based on the fact that the method can effectively improve the solubility, emulsification properties and antioxidant properties of peanut meal, the present invention provides the application of the method in improving at least one of the following properties of peanut meal: solubility, emulsification properties and antioxidant properties.

[0028] This invention provides peanut protein prepared by the method described above. The peanut protein prepared by the method of this invention exhibits high solubility, emulsifying properties, and antioxidant activity. The antioxidant activity includes DPPH free radical scavenging rate. The peanut protein prepared by this invention achieves a solubility as high as 65.17% and an emulsifying activity index of 12.92 m. 2 / g, the emulsification stability index was extended to 119.81 min, and the DPPH free radical scavenging rate reached 72.81%. The particle size and zeta potential measurements showed that the average particle size of the peanut protein powder prepared by the method of the present invention was reduced to 1543 nm, and the zeta potential reached -21.2 mV. The minimization of protein particles and the maximization of surface charge can effectively inhibit the aggregation behavior of high-temperature denatured peanut protein, laying a structural foundation for the long-term dispersion stability of peanut protein powder in food systems such as emulsions and protein beverages.

[0029] This invention provides the application of the peanut protein in food processing. The peanut protein of this invention has high solubility, excellent emulsifying properties, and strong antioxidant activity, and can be widely adapted to various product processing scenarios requiring the addition of protein ingredients, such as beverages, dairy products, meat products, and baked goods.

[0030] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0031] In all embodiments and comparative examples of this invention, the high-temperature peanut meal powder is peanut cake meal after high-temperature oil pressing, which is pulverized by a high-speed pulverizer and sieved through a 60-mesh sieve. The peanut cake meal was purchased from Shandong Huayuan Food Co., Ltd., and the protein content was determined to be 53.1 wt.% by the Kjeldahl method, the oil content was determined to be 3.6 wt.% by acid hydrolysis, and the residual starch content in the high-temperature peanut meal powder was determined to be 12.2 wt.% by enzymatic hydrolysis.

[0032] Example 1 A method for preparing peanut protein by enzymatic hydrolysis of starch combined with ultrasound and alkali treatment, comprising the following steps: Step 1: Raw material pretreatment and dispersion. Weigh 10g of high-temperature peanut meal powder and place it in a 250mL Erlenmeyer flask. Add 90mL of deionized water (mass ratio of 10% (w / w)). After shaking and mixing evenly, place the Erlenmeyer flask on a 55℃ constant temperature water bath shaker and preheat it at 160r / min for 15min to obtain a high-temperature peanut meal dispersion.

[0033] Step 2: α-Amylase hydrolysis. α-Amylase was added to the above dispersion at a rate of 50 U / g starch (calculated based on the 12.2 wt.% starch content in the peanut high-temperature meal powder). The mixture was kept at a water bath temperature of 55℃ and a shaking speed of 160 r / min for 1 hour to obtain the peanut high-temperature meal hydrolysate.

[0034] Step 3: Ultrasonic-Assisted Alkali Treatment. After the enzymatic hydrolysis reaction, quickly transfer the peanut meal hydrolysate solution from the Erlenmeyer flask to a beaker with a magnetic stirrer. Place the beaker in a pre-cooled ice-water bath and start the magnetic stirrer (100 rpm). After stirring for 60 minutes, slowly add 2 mol / L NaOH solution dropwise while stirring, controlling the drop rate to 1 drop / second, and precisely adjust the pH of the solution to 12.0. After the pH stabilizes, start the ultrasonic equipment and perform pulse ultrasonic treatment according to the preset parameters (ultrasonic power 300W, treatment time 5 minutes, using a 3-second ultrasonic and 2-second intermittent pulse mode). During the ultrasonic treatment, use an ice-water bath to control the temperature, continuously replenishing ice water. Monitor the solution temperature in real time with a thermometer to ensure it is maintained at 0~5℃ throughout the process, avoiding secondary protein aggregation and denaturation caused by localized high temperatures during ultrasonic treatment. After 5 minutes of ultrasonic treatment, continue with alkali treatment for 55 minutes at a temperature of 25℃, accompanied by magnetic stirring (100 rpm).

[0035] Step 4: pH Adjustment. Slowly add 2 mol / L HCl solution dropwise to the alkaline-treated mixture to adjust the pH of the mixture to 7.0, thus obtaining the peanut protein solution.

[0036] Step 5: Freeze-drying. Transfer the peanut protein solution to a freeze-drying tray and pre-freeze it in an ultra-low temperature freezer at -80℃ for 12 hours to ensure complete freezing. Then, place the pre-frozen sample in a vacuum freeze dryer and freeze-dry it under a vacuum of 1 Pa and a shelf temperature of -60℃ until the sample moisture content is 3%.

[0037] Step Six: Storage. The freeze-dried peanut protein powder is pulverized through an 80-mesh sieve, placed in a sealed bag, and stored in a desiccator away from light. Peanut protein prepared by enzymatic hydrolysis of starch combined with ultrasonic treatment and alkali treatment is designated as PPH-EUPH.

[0038] Comparative Example 1 A type of high-temperature peanut meal powder, denoted as PPH.

[0039] Comparative Example 2 A method for preparing peanut protein by alkali treatment, comprising the following steps: Step 1: Raw material pretreatment and dispersion. Weigh 10g of peanut high-temperature meal powder and place it in a 250mL beaker. Add 90mL of deionized water (mass ratio 10% (w / w)), add a rotor, and start magnetic stirring (speed 100r / min). After stirring for 120min, let it stand overnight in a 4℃ refrigerator to allow the protein to fully hydrate and obtain a peanut high-temperature meal dispersion.

[0040] Step 2: Alkali Treatment. After restoring the hydrated peanut meal dispersion to 25°C, magnetically stir for 10 minutes, then slowly add 2 mol / L NaOH solution dropwise while stirring continuously. Control the dropping rate to 1 drop / second, and precisely adjust the pH of the solution to 12.0. After the pH stabilizes, perform alkali treatment for 1 hour at 25°C, accompanied by magnetic stirring (100 rpm).

[0041] After alkali treatment, the peanut protein prepared by alkali treatment was subjected to pH adjustment, freeze drying and storage according to steps four to six in Example 1. The peanut protein prepared by alkali treatment is denoted as PPH-PH.

[0042] Comparative Example 3 A method for preparing peanut protein by ultrasonic treatment, comprising the following steps: Step 1: Raw material pretreatment and dispersion. Weigh 10g of peanut high-temperature meal powder and place it in a 250mL beaker. Add 90mL of deionized water (mass ratio 10% (w / w)), add a rotor, and start magnetic stirring (speed 100r / min). After stirring for 120min, let it stand overnight in a 4℃ refrigerator to allow the protein to fully hydrate and obtain a peanut high-temperature meal dispersion.

[0043] Step 2: Ultrasonic treatment. Perform pulsed ultrasonic treatment according to preset parameters (ultrasonic power 300W, treatment time 5min, using a pulse mode of 3s ultrasound and 2s interval). During the ultrasonic treatment, use an ice water bath for temperature control, continuously replenish ice water during the ultrasonic process, and monitor the solution temperature in real time with a thermometer to ensure that it is maintained at 0~5℃ throughout the process, so as to avoid secondary protein aggregation and denaturation caused by local high temperature during ultrasound.

[0044] After ultrasonic treatment, pH adjustment, freeze drying and storage were performed according to steps four to six in Example 1. The peanut protein prepared by ultrasonic treatment was denoted as PPH-U.

[0045] Comparative Example 4 A method for preparing peanut protein by ultrasound-assisted alkali treatment comprises the following steps: Step 1: Raw material pretreatment and dispersion. Weigh 10g of peanut high-temperature meal powder and place it in a 250mL beaker. Add 90mL of deionized water (mass ratio 10% (w / w)), add a rotor, and start magnetic stirring (speed 100r / min). After stirring for 120min, let it stand overnight in a 4℃ refrigerator to allow the protein to fully hydrate and obtain a peanut high-temperature meal dispersion.

[0046] Step Two: Ultrasonic-Assisted Alkali Treatment. After restoring the hydrated peanut meal dispersion to 25°C, magnetically stir for 10 minutes, then slowly add 2 mol / L NaOH solution dropwise while stirring continuously, controlling the drop rate to 1 drop / second. Precisely adjust the solution pH to 12.0. Once the pH stabilizes, start the ultrasonic equipment and perform pulsed ultrasonic treatment according to preset parameters (ultrasonic power 300W, treatment time 5 minutes, using a 3-second ultrasonic pulse mode with 2-second intervals). Continuously replenish ice water during ultrasonication, and monitor the solution temperature in real time with a thermometer to ensure it is maintained between 0 and 5°C throughout the process, avoiding secondary protein aggregation and denaturation caused by localized high temperatures during ultrasonication. After 5 minutes of ultrasonic treatment, continue with alkali treatment for 55 minutes at 25°C, accompanied by magnetic stirring (100 rpm).

[0047] After ultrasonic-assisted alkali treatment, the peanut protein prepared by ultrasonic-assisted alkali treatment was subjected to pH adjustment, freeze drying and storage according to steps four to six in Example 1. The peanut protein prepared by ultrasonic-assisted alkali treatment is denoted as PPH-UPH.

[0048] Example 2 A method for preparing peanut protein by enzymatic hydrolysis of starch combined with alkali treatment comprises the following steps: Step 1: Raw material pretreatment and dispersion. Weigh 10g of peanut high-temperature meal powder and place it in a 250mL Erlenmeyer flask. Add 90mL of deionized water and shake to mix evenly. Then place the Erlenmeyer flask on a 55℃ constant temperature water bath shaker and preheat it at 160r / min for 15min to obtain a peanut high-temperature meal dispersion.

[0049] Step 2: α-Amylase hydrolysis. Add α-amylase to the above dispersion at a dosage of 50 U / g starch (calculated based on the 12.2% starch content in the high-temperature peanut meal powder), maintain a water bath temperature of 55℃ and a shaking speed of 160 r / min, and react for 1 hour to obtain the high-temperature peanut meal hydrolysate.

[0050] Step 3: Alkali Treatment. After the enzymatic hydrolysis reaction is complete, quickly transfer the peanut meal hydrolysate from the Erlenmeyer flask to a beaker equipped with a magnetic stirrer. Place the beaker in a pre-cooled ice-water bath and start the magnetic stirrer (100 rpm). After stirring for 10 minutes, slowly add 2 mol / L NaOH solution dropwise while stirring, controlling the dropping rate to 1 drop / second, and precisely adjust the pH of the solution to 12.0. After the pH stabilizes, perform alkali treatment for 1 hour at 25°C, accompanied by magnetic stirring (100 rpm).

[0051] After alkali treatment, the peanut protein prepared by enzymatic hydrolysis of starch combined with alkali treatment was pH adjusted, freeze-dried and stored according to steps four to six in Example 1. The peanut protein was denoted as PPH-EPH.

[0052] Example 3 A method for preparing peanut protein by enzymatic hydrolysis of starch combined with ultrasonic treatment, comprising the following steps: Step 1: Raw material pretreatment and dispersion. Weigh 10g of peanut high-temperature meal powder and place it in a 250mL Erlenmeyer flask. Add 90mL of deionized water and shake to mix evenly. Then place the Erlenmeyer flask on a 55℃ constant temperature water bath shaker and preheat it at 160r / min for 15min to obtain a peanut high-temperature meal dispersion.

[0053] Step 2: α-Amylase hydrolysis. Add α-amylase to the above dispersion at a dosage of 50 U / g starch (calculated based on the 12.2% starch content in the high-temperature peanut meal powder), maintain a water bath temperature of 55℃ and a shaking speed of 160 r / min, and react for 1 hour to obtain the high-temperature peanut meal hydrolysate.

[0054] Step 3: Ultrasonic Treatment. Quickly transfer the peanut meal hydrolysate from the Erlenmeyer flask to a beaker equipped with a magnetic stirrer (with a rotor). Place the beaker in a pre-cooled ice-water bath to 25°C. Start the magnetic stirrer (100 rpm) and stir for 10 minutes. Then, perform pulse ultrasonic treatment according to the preset parameters (ultrasonic power 300W, treatment time 5 minutes, using a 3-second ultrasonic and 2-second intermittent pulse mode). Use an ice-water bath to control the temperature during ultrasonication, continuously replenishing ice water. Monitor the solution temperature in real time with a thermometer to ensure it is maintained between 0 and 5°C throughout the process, avoiding secondary protein aggregation and denaturation caused by localized high temperatures during ultrasonication.

[0055] After ultrasonic treatment, pH adjustment, freeze drying and storage were carried out according to steps four to six in Example 1. The peanut protein prepared by enzymatic hydrolysis of starch combined with ultrasonic treatment was designated as PPH-EU.

[0056] Example 4 Peanut protein index detection Within 72 hours of obtaining the peanut protein, the solubility, high oleic acid content, amino acid composition, and other indicators of the peanut protein prepared in Example 1 and Comparative Examples 1-5 were detected, and the particle size and ζ-potential of the peanut protein were also detected.

[0057] Protein solubility assay: Centrifuge the dissolved PPH solution (10 mg / mL) at 5000 r / min for 15 min using a refrigerated centrifuge. Add 1 mL of the supernatant to 5 mL of Folin-phenol reagent A, mix well, and incubate at 25°C for 10 minutes. Then add 0.5 mL of reagent B (Folin-phenol), shake immediately, and store at 25°C for 30 minutes. Measure the color at 500 nm. Use 1 mL of water as a blank control instead of the sample. Calculate the concentration of soluble protein based on the standard curve. Solubility is calculated using formula I. Formula I Emulsificability was determined using the following method: A certain amount of hot-pressed peanut meal protein was weighed and dissolved in distilled water to prepare a 1% (w / w) protein solution. Then, 15 mL of the protein solution and 5 mL of corn oil from a 50 mL centrifuge tube were mixed at 12,000 rpm for 2 minutes at 25°C using an FJ-200 laboratory high-speed homogenizer (Shanghai, China). At the end of homogenization, 50 μL of the sample was removed from the bottom of the centrifuge tube and mixed with 5 mL of 0.1% (w / w) SDS solution. After shaking, the absorbance of the sample at 500 nm was immediately measured and recorded as A0. Distilled water was used as the reference solution instead of protein. The emulsion was allowed to stand at 25°C for 10 minutes, and the absorbance was measured again and recorded as A1. The emulsifying activity index was calculated according to Formula II, and the emulsifying stability index was calculated according to Formula III.

[0058] Formula II Where EAI is the emulsifying activity index, m 2 / g; N is the dilution factor; is the oil phase volume fraction, v / v; C is the concentration of the protein solution before emulsification, g / mL.

[0059] Formula III In the formula, ESI is the emulsion stability index, min; A0 is the absorbance value at 0 min; and A1 is the absorbance value at 10 min.

[0060] Antioxidant activity (DPPH free radical scavenging rate) was assessed using the following method: A sample solution with a concentration of 10 mg / mL was prepared and centrifuged (3000 r / min; 10 min). The supernatant was then mixed with 0.20 M DPPH solution (dissolved in ethanol) at a 1:1 ratio. The mixture was then placed in a water bath (37℃) and reacted in the dark for 30 min. The absorbance at 517 nm was measured, and the absorbance of the sample solution after the reaction (517 nm) was recorded as X1. Similarly, the absorbance measured under the same conditions using distilled water was used as a control (X0). Anhydrous ethanol was used for zeroing, and the results were calculated according to Formula IV.

[0061] Formula IV Methods for determining particle size and zeta potential: Samples were prepared into solutions with a concentration of 1 mg / mL, and the zeta potential and particle size distribution were measured using a nanoparticle size analyzer and a ZETA potential analyzer, respectively. All measurements were performed at 25°C, and each sample was repeated three times.

[0062] The test results are shown in Table 1 and Figures 1-4 As shown. PPH-E represents peanut protein prepared according to the method of Example 1, omitting steps three and four.

[0063] Table 1. Detection results of peanut protein indicators

[0064] 1. Protein solubility The protein solubility results showed that the solubility of unmodified peanut high-temperature meal (PPH) in neutral water was only 8.75%, indicating that the protein was severely denatured, the aggregate structure was dense, and the surface hydrophilic groups were buried after high-temperature pressing.

[0065] In the single modification treatment, alkali treatment (PPH-PH) can increase the solubility to 48.46%, which is 5.5 times that of unmodified peanut high-temperature meal (PPH) (P<0.05). The mechanism is that the strong alkaline environment (pH 12.0) induces protein molecules to unfold and disulfide bonds to rearrange. After returning to neutral, some unfolded structures are "frozen", thereby enhancing protein-water interaction. The solubility of ultrasonic treatment (PPH-U) is 24.35%, which is significantly lower than that of alkali treatment, indicating that although the cavitation effect has a certain breaking effect on aggregates, it is not enough to fully expose hydrophilic groups.

[0066] When ultrasound-assisted alkaline treatment (PPH-UPH) was performed, the solubility further increased to 58.53%, which was higher than the predicted value of the sum of the individual effects of the two factors, indicating that the two have a synergistic effect: the microjets generated by ultrasound in the alkaline environment and the local high temperature can inhibit protein reaggregation and allow more hydrophobic regions to be permanently expanded, thereby synergistically improving the solubility.

[0067] Enzymatic starch pretreatment significantly amplified the aforementioned modification effects. Comparing PPH-UPH and PPH-EUPH, the solubility of the sample after starch removal increased from 58.53% to 65.17%, an increase of 6.64 percentage points (P < 0.05). This is because the disappearance of the starch-protein complex reduced steric hindrance, allowing OH-... - The cavitation effect acts more directly on the protein itself, while reducing the viscosity of the system, which is conducive to the formation and collapse of ultrasonic cavitation bubbles.

[0068] In summary, the combined modification strategy of enzymatic starch hydrolysis coupled with ultrasound-alkali treatment can increase the solubility of high-temperature denatured peanut protein by 7.4 times, laying a structural foundation for subsequent improvement of its food functional properties such as emulsification and foaming.

[0069] 2. Emulsification data Emulsification data shows ( Figure 2 The emulsifying activity index (EAI) of unmodified peanut high-temperature meal (PPH) was only 5.56m. 2 / g, with an emulsion stability index (ESI) of only 45.07 min, indicating that high-temperature denaturation leads to weak adsorption capacity at the protein interface and insufficient density of the protein film on the surface of oil droplets, making the emulsion extremely prone to flocculation and aggregation.

[0070] In single modification treatments, alkali treatment (PPH-PH) and ultrasonic treatment (PPH-U) increased the EAI to 7.42 and 9.47 m, respectively. 2 / g, ESI increased to 51.01 and 53.54 min, with limited increase; among them, ultrasound promoted EAI better than pH shift, indicating that particle size reduction and interface exposure contributed more to emulsifying activity.

[0071] When ultrasound was combined with pH shift (PPH-UPH), the EAI increased to 10.58 m. 2 / g, ESI rose to 57.98 min, both higher than the predicted value of the sum of the individual effects of the two factors, indicating that the cavitation effect under alkaline conditions can inhibit protein reaggregation and synergistically promote the exposure of hydrophobic groups and interfacial adsorption.

[0072] Enzymatic starch pretreatment significantly amplified the aforementioned synergistic effect. After starch removal, the EAI of PPH-EUPH reached 12.92 m. 2 / g, which is 2.3 times that of the unmodified variety; ESI was significantly extended to 119.81 min, which is about 2.7 times that of the unmodified variety. This is because the disappearance of the starch-protein complex reduces steric hindrance and interfacial competitive adsorption, allowing the protein to form a denser and more viscoelastic interfacial film on the oil droplet surface.

[0073] 3. Particle size and ζ-potential data Results of particle size and ζ-potential measurements ( Figure 3 The results showed that the average particle size of unmodified peanut high-temperature meal (PPH) was 2494 nm, and the Zeta potential was -13.9 mV. This indicates that high-temperature denaturation promotes the formation of dense aggregates of protein molecules with low surface charge density. The electrostatic repulsion within the system is insufficient to maintain dispersion stability, thus making flocculation highly likely. In single modification treatments, alkali treatment (PPH-PH) and ultrasonic treatment (PPH-U) reduced the average particle size of PPH to 1857 nm and 2273 nm, respectively, while slightly increasing the Zeta potential to -14.5 mV and -15.6 mV, respectively. The protein aggregation tendency was somewhat alleviated, but the modified system was still weakly stable. When using a combined ultrasonic and pH shift modification process (PPH-UPH), the average particle size of the sample was further reduced to 1761 nm, and the Zeta potential increased to -16.2 mV. This indicates that the ultrasonic cavitation shearing effect under alkaline conditions can effectively inhibit the reaggregation behavior of protein molecules, and the two modification methods showed a significant synergistic effect. After introducing enzymatic starch pretreatment, the average particle size of the sample modified by enzymatic starch hydrolysis-ultrasound-pH shift (PPH-EUPH) decreased to 1543 nm, and the Zeta potential reached -21.2 mV. Compared with unmodified PPH, the particle size decreased by 38%, and the absolute value of the Zeta potential increased by 53%, achieving high stability. This may be because the enzymatic starch pretreatment removed the starch-protein complex in the system, reduced steric hindrance, and significantly improved the shear efficiency of subsequent ultrasonic cavitation; at the same time, the alkaline environment not only induced the spatial structure unfolding of protein molecules, but also promoted their partial hydrolysis, exposing more charged groups (–COO). -This process significantly increases the surface charge density of the protein. Correlation analysis showed that the particle size of the high-temperature peanut meal was significantly negatively correlated with the zeta potential (r≈–0.92). This indicates that the combined modification strategy of enzymatic starch hydrolysis coupled with ultrasound-pH shift can effectively inhibit the aggregation behavior of high-temperature denatured peanut protein by achieving dual regulation of minimizing protein particles and maximizing surface charge. This lays the structural foundation for the long-term dispersion stability of this protein in food systems such as emulsions and protein beverages.

[0074] 4. Antioxidant data Antioxidant data results ( Figure 4 The results showed that PPH-EUPH had the highest DPPH free radical scavenging rate, reaching 72.81%. The combined modification strategy of ultrasound-pH shift can effectively improve the antioxidant properties of high-temperature peanut meal.

[0075] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing peanut protein using peanut meal, characterized in that, include: Peanut meal powder was enzymatically hydrolyzed using α-amylase to obtain peanut meal hydrolysate; Peanut protein is obtained by subjecting the peanut meal enzymatic hydrolysate to any of the following modification treatments: ultrasonic treatment, alkali treatment, and ultrasonic-assisted alkali treatment.

2. The method according to claim 1, characterized in that, In the enzymatic hydrolysis system, the amount of water added is 5~10mL / g peanut meal powder; Based on the amount of peanut starch in peanut meal powder, the amount of α-amylase added is 25~70 U / g peanut starch.

3. The method according to claim 1, characterized in that, The enzymatic hydrolysis treatment is carried out at a temperature of 50~60℃ for a time of 0.5~1.5h.

4. The method according to claim 1, characterized in that, When ultrasonic treatment is used, the power of the ultrasonic treatment is 200~400W, the intermittent pulse mode is adopted, and the time is 4~6min.

5. The method according to claim 1, characterized in that, When alkali treatment is used, the pH value of the alkali treatment is 11-13, and the time is 40-80 min; After the alkali treatment, the pH value is adjusted back to 6.8~7.

2.

6. The method according to claim 1, characterized in that, When ultrasound-assisted alkali treatment is used, the ultrasound-assisted alkali treatment means performing ultrasound treatment during the alkali treatment process; The pH value of the alkali treatment is 11-13, and the time is 40-80 min; The ultrasonic treatment has a power of 200~400W and a duration of 4~6min, and the ultrasonic treatment is intermittent ultrasonic treatment; After the ultrasonic-assisted alkali treatment, the pH value is adjusted back to 6.8~7.

2.

7. The method according to any one of claims 1 to 6, characterized in that, After the modification process is completed, the process also includes freeze-drying.

8. The application of the method according to any one of claims 1 to 7 in improving at least one of the following properties of peanut meal: solubility, emulsification properties and antioxidant properties.

9. Peanut protein prepared by the method according to any one of claims 1 to 7.

10. The application of the peanut protein according to claim 9 in food processing.

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