Nut protein and extraction method thereof

Abstract

The invention belongs to a protein extraction technology in food processing, and relates to a nut protein and a preparation method thereof. According to the method, nuts are taken as raw materials, protein and grease in the nuts are synchronously separated and extracted by adopting an aqueous enzymatic method, and then nut protein is obtained by adopting acid precipitation treatment. The aqueous enzymatic method not only has the advantages of high efficiency, safety and mildness, but also has small damage to the biological activity of the protein. According to the preparation method, the protein extraction rate and the protein purity are high, and waste of a large number of protein resources is avoided.

Description

Technical Field

[0001] The present application relates to the field of protein extraction and processing, and in particular to a nut protein and an extraction method thereof. Background Art

[0002] Judging by current trends in the food industry, plant-based protein is playing an increasingly important role in dietary supplementation and food processing. To reduce saturated fatty acid intake, people are hesitant to consume excessive amounts of animal protein. Nut protein, a type of plant-based protein, not only compensates for dietary protein deficiency but also contains physiologically active substances that may have certain cardiovascular disease prevention and treatment properties. Therefore, nut protein can be used as a raw material for nutritional fortification and health foods.

[0003] To extract protein from nuts, some researchers have used an alkali-solution and acid precipitation method to extract protein from degreased nut meal. This method requires first defatting the nuts to obtain nut meal, then using an alkaline solution to destroy the protein, and then using an acidic solution to flocculate and precipitate the protein, ultimately yielding nut protein. However, the protein in the defatted meal is severely denatured, and this method suffers from low protein extraction rates and purity, resulting in a significant waste of protein resources.

[0004] In summary, there is an urgent need to develop a method for extracting protein from nuts that can simultaneously achieve high extraction rate and high protein purity. Summary of the Invention

[0005] The present application provides a nut protein and an extraction method thereof, wherein the extraction method obtains a protein extraction rate of not less than 70% and a protein purity of not less than 90%.

[0006] In a first aspect, the present application provides a method for extracting nut protein, comprising:

[0007] adding alkaline protease to the nut aqueous solution for enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0008] The enzymatic hydrolysate is subjected to acid precipitation treatment to obtain the nut protein.

[0009] In a possible embodiment, in the nut aqueous solution, the mass volume ratio of nuts to water is 1 g: (5-6) mL.

[0010] In a possible embodiment, during the enzymatic hydrolysis treatment, the pH of the nut aqueous solution is 8-9, and the enzymatic hydrolysis temperature is 45-55°C.

[0011] In one possible embodiment, the mass ratio of the alkaline protease to the nuts is (1-4):100.

[0012] In one possible embodiment, the alkaline protease has an enzyme activity of 150-250 u / g.

[0013] In a possible embodiment, the extraction method further includes: separating the enzymatic hydrolysate and performing ultrasonic treatment on the protein solution.

[0014] In one possible embodiment, the output frequency of the ultrasonic treatment is 20-25 kHz, and the output power is 300-450 W.

[0015] In a possible embodiment, the acid precipitation treatment includes: adjusting the pH of the enzymatic hydrolyzate to 4-5, and after flocculent precipitation occurs, centrifuging the solution at 1-4° C. for 15-20 minutes to obtain the nut protein.

[0016] In a possible embodiment, the extraction method further comprises: crushing the nuts to obtain nut crumbs with a particle size of 40 to 60 mesh.

[0017] In a second aspect, the present application provides a nut protein extracted by the above-mentioned extraction method.

[0018] In a third aspect, the present application provides a food additive comprising the above-mentioned nut protein.

[0019] The present application provides a nut protein and an extraction method thereof, which achieves the simultaneous extraction of oil and protein by enzymatic hydrolysis and acid precipitation of the nut raw materials using alkaline protease, obtains a protein extraction rate of not less than 70% and a protein purity of not less than 90%, and avoids waste of resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0021] Figure 1 This is the fitted secondary structure angle distribution diagram of the protein extracted in Example 1 of the present application;

[0022] Figure 2 This is the fitted secondary structure angle distribution diagram of the protein extracted in Comparative Example 1 of this application;

[0023] Figure 3 This is a data graph of the water solubility of the proteins obtained in Example 1 and Comparative Example 1 of the present application;

[0024] Figure 4 This is a graph showing the foaming properties of the proteins obtained in Example 1 and Comparative Example 1 of the present application;

[0025] Figure 5This is a digestibility data diagram of the proteins obtained in Example 1 and Comparative Example 1 of the present application.

[0026] The above drawings illustrate specific embodiments of the present application, which will be described in more detail below. These drawings and the textual description are not intended to limit the scope of the present application in any way, but rather to illustrate the concepts of the present application to those skilled in the art by reference to specific embodiments. DETAILED DESCRIPTION

[0027] Exemplary embodiments will be described in detail herein, with examples illustrated in the accompanying drawings. In the following description, when referring to the drawings, identical numerals in different figures represent identical or similar elements, unless otherwise indicated. The embodiments described in the following exemplary embodiments are not intended to represent all embodiments consistent with the present application. Rather, they are merely examples of apparatus and methods consistent with certain aspects of the present application, as detailed in the appended claims.

[0028] First, let’s explain the terms involved in this application:

[0029] α-helix: refers to the structure in a protein molecule where the peptide chain spirals around the central axis like a spring, providing structural rigidity for the protein and participating in the formation of the protein's active center;

[0030] β-fold: refers to a sheet formed by multiple peptide chains or peptide segments stretched side by side in a zigzag shape in a protein molecule, which provides high-strength support for the protein and forms a macromolecular binding plane;

[0031] β-turn: refers to the structure connecting adjacent α-helices and β-sheets in protein molecules, which can cause the peptide chain to make a sharp 180° turn, making the protein as a whole compactly folded and participating in the formation of protein-protein interaction interface.

[0032] The nut protein extraction method provided in this application solves the technical problems of low nut protein extraction rate and low protein purity by enzymatically hydrolyzing the nut raw materials with alkaline protease and obtaining nut protein through acid precipitation.

[0033] The following specific embodiments describe in detail the technical solution of the present application and how the technical solution of the present application solves the above-mentioned technical problems. The following specific embodiments can be combined with each other, and the same or similar concepts or processes may not be repeated in some embodiments. The embodiments of the present application will be described below in conjunction with the accompanying drawings.

[0034] The nut protein extraction method provided in the present application comprises: adding alkaline protease to a nut aqueous solution for enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0035] The enzymatic hydrolysate is subjected to acid precipitation treatment to obtain the nut protein.

[0036] In this application, nuts that have not been squeezed to remove oil are selected as raw materials, and alkaline protease with a specific active center structure is used to enzymatically hydrolyze nuts. Even in nut substrates that have not been degreased, alkaline protease can recognize and bind to specific peptide bonds in nut protein molecules, and can hydrolyze the macromolecular proteins in nuts into small molecule peptides and amino acids, thereby significantly improving the solubility and stability of nut protein in water, so that more protein in nuts can be hydrolyzed. Moreover, the protein in nuts that have not been squeezed to remove oil is mostly well preserved, and enzymatic hydrolysis is directly used to extract the oil and protein in nuts at the same time, which can obtain higher raw material utilization. These small molecule peptides and amino acids are then collected by acid precipitation treatment, and finally, a higher protein extraction rate and high protein purity are obtained under the condition of oil presence.

[0037] It can be understood that a too low nut content in the nut aqueous solution means that the protein substrate molecules in the nut aqueous solution are too sparse, making it difficult for the enzyme molecules to effectively "find" and contact the substrate, resulting in a low reaction rate, low enzyme utilization, and increased time and cost. A too high nut content in the nut aqueous solution means a high solid concentration, which will cause the viscosity of the aqueous solution to increase significantly. High viscosity will seriously hinder the mass transfer of alkaline protease during the enzymatic hydrolysis process, making it difficult for the enzyme to effectively diffuse to the surrounding areas of the nuts that have not been enzymatically hydrolyzed, and it will also be difficult for the nuts to diffuse away from the enzymatic hydrolysis site, thereby significantly reducing the actual enzymatic hydrolysis rate. In order to allow the nut raw materials to fully contact the alkaline protease and obtain as high a raw material utilization rate and enzymatic hydrolysis rate as possible, the mass volume ratio of nuts to water in the nut aqueous solution used in this application is 1g: (5-6)mL, for example 1g:5mL, 1g:5.5mL, 1g:6mL, etc.

[0038] Since alkaline protease requires a suitable alkaline environment and temperature to exert its enzymatic activity, specifically, when the present application performs enzymatic hydrolysis on the nut aqueous solution, an alkaline solution is used to adjust the pH of the nut aqueous solution to 8-9, for example, pH=8, pH=8.5, pH=9, etc., and the enzymatic hydrolysis temperature is controlled to 45-55°C, for example, 45°C, 50°C, 55°C, etc., to obtain better enzymatic hydrolysis effect.

[0039] During enzymatic hydrolysis, the mass ratio of alkaline protease to nut substrate directly affects the location and frequency of peptide cleavage in the nut protein, thereby affecting the molecular weight distribution of the hydrolysis product (the ratio of large peptides, small peptides, and amino acids). To achieve an ideal degree of hydrolysis without excessive hydrolysis that affects the properties of the resulting nut protein, in this application, the mass ratio of alkaline protease to nut is preferably (1-4):100, for example, 1:100, 2:100, 3:100, 4:100, etc. The inventors have discovered that excessive amounts of alkaline protease can lead to the production of a large number of short peptides with hydrophobic amino acids at the ends. These peptides not only have a strong bitter taste but also poor functionality. Therefore, strictly controlling the mass ratio of alkaline protease to nut is beneficial for obtaining nut proteins with excellent performance.

[0040] Since the enzyme preparation itself generally contains inactive ingredients such as carriers, inactivated enzymes, and microorganisms, if an alkaline protease with a lower enzyme activity is used, in order to achieve a certain enzymolysis efficiency, it is necessary to add a large amount of alkaline protease, and the addition of excessive protease will change the pH, ionic strength, or viscosity of the enzymolysis system, thereby affecting the enzymolysis efficiency. Therefore, when selecting the enzymatic activity of the alkaline protease, considering that the alkaline protease with a suitable enzyme activity is also critical for the enzymolysis process, in this application, the enzymatic activity of the alkaline protease used is preferably 150-250u / g, for example 150u / g, 170u / g, 190u / g, 200u / g, 220u / g, 250u / g, etc.

[0041] After enzymatic hydrolysis of the nut aqueous solution, in order to make the obtained nut protein have better water solubility, emulsification and digestibility, the present application also includes ultrasonic treatment of the protein solution separated from the enzymatic hydrolysis solution.

[0042] In this application, ultrasonic treatment can promote significant structural changes in proteins obtained by enzymatic hydrolysis through the physical cavitation effect. Figure 1 and Figure 2As shown in the figure, after ultrasonic treatment, the protein solution obtained by the enzymatic hydrolysis described in the present application has obvious structural differences compared to the protein extracted by the squeezing method. The proportion of β-folded structure in the protein is reduced, while the proportion of β-turn structure is increased. This is because ultrasonic treatment generates cavitation bubbles in the protein solution, and huge energy is released when the bubbles collapse instantly, forming strong mechanical shear force, local high temperature and cavitation shock wave. Under the action of shear force, the hydrogen bonds between the β-folded sheets are transversely broken, and the local high temperature causes the sheets to twist and separate. In addition, the energy of the cavitation shock wave causes the sheet plane to be torn into short peptide segments. The flexibility of the newly formed short peptide chain increases, and β-turns are spontaneously formed. The unfolding region of the β-folded sheet quickly constructs a low-energy ring structure through the β-turn, avoiding the exposure of hydrophobic residues. Finally, after ultrasonic treatment, the hydrophobic core of the protein obtained by the enzymatic hydrolysis described in the present application is partially opened, the hydrophilic groups are exposed, the interfacial adsorption capacity of the peptide chain is enhanced, the water solubility of the protein is increased, and the foaming property is improved.

[0043] The inventors found through experiments that moderate ultrasound can increase the β-turn structure in nut protein, but excessive ultrasound will cause excessive fragmentation of the protein structure. Therefore, preferably, the output frequency of the ultrasonic treatment is 20-25kHz and the output power is 300-450w.

[0044] Furthermore, in order to improve the utilization rate of nut raw materials and obtain a higher protein extraction rate, the present application can also crush the nuts and select nut crumbs with a particle size of 40-60 mesh for enzymatic hydrolysis.

[0045] The present application also provides a nut protein extracted by the above-mentioned extraction method, wherein the purity of the nut protein is not less than 90%.

[0046] The present application also provides a food additive, which includes the above-mentioned nut protein.

[0047] The technical solution of this application is described in more detail below through specific embodiments.

[0048] Example 1

[0049] Enzymatic hydrolysis: 50g of pecan kernels were crushed through a 40-mesh sieve, and 250mL of water was added and mixed to obtain a pecan aqueous solution. The pH value of the pecan aqueous solution was adjusted to 8 using a 1mol / L sodium hydroxide solution, and the pecan aqueous solution was placed in a 45°C water bath. 0.5g of alkaline protease with an enzyme activity of 150u / g was added to the solution, and the mixture was stirred evenly for 150min. After the enzymatic hydrolysis reaction was completed, the enzyme was inactivated.

[0050] Ultrasonic treatment: The enzymatic hydrolysate was centrifuged at 10,000 rpm for 15 min at 4°C to separate the upper oil layer from the lower protein solution. The lower protein solution was then ultrasonically treated for 25 min at a frequency of 20 kHz and an output power of 300 W.

[0051] Acid precipitation treatment: add 1 mol / L hydrochloric acid solution to the sonicated solution, adjust the pH value of the solution to 4 for protein acid precipitation, and after flocculent precipitation appears, centrifuge the solution at 10,000 r / min for 15 min at 4°C. Retain the precipitate for washing, neutralization, and vacuum freeze-drying to obtain pecan protein.

[0052] The pecan protein extraction rate obtained by this method was 72.85%, and the protein purity was 91.10%.

[0053] Example 2

[0054] Enzymatic hydrolysis: 50g of pecan kernels were crushed through a 50-mesh sieve and mixed with 275mL of water to obtain a pecan aqueous solution. The pH of the pecan aqueous solution was adjusted to 9 with a 1mol / L sodium hydroxide solution and the pecan aqueous solution was placed in a 50°C water bath. 1.5g of alkaline protease with an enzyme activity of 200u / g was added to the solution and stirred evenly for 150min. After the enzymatic hydrolysis reaction was completed, the enzyme was inactivated.

[0055] Ultrasonic treatment: The enzymatic hydrolysate was centrifuged at 10,000 rpm for 15 min at 4°C to separate the upper oil layer from the lower protein solution. The lower protein solution was then ultrasonically treated for 25 min at a frequency of 20 kHz and an output power of 350 W.

[0056] Acid precipitation treatment: add 1 mol / L hydrochloric acid solution to the sonicated solution, adjust the pH value of the solution to 4.5 for protein acid precipitation, and after flocculent precipitation appears, centrifuge the solution at 10,000 r / min for 15 min at 4°C. Retain the precipitate for washing, neutralization, and vacuum freeze-drying to obtain pecan protein.

[0057] The pecan protein extraction rate obtained by this method was 73.96%, and the protein purity was 91.32%.

[0058] Example 3

[0059] Enzymatic hydrolysis: 50g of pecan kernels were crushed through a 60-mesh sieve, added to 300mL of water and mixed to obtain a pecan aqueous solution; the pH value of the pecan aqueous solution was adjusted to 9 with a 1mol / L sodium hydroxide solution, and the pecan aqueous solution was placed in a 55°C water bath; 2g of alkaline protease with an enzyme activity of 250u / g was added to the solution, and the mixture was stirred evenly for 150min. After the enzymatic hydrolysis reaction was completed, the enzyme was inactivated;

[0060] Ultrasonic treatment: The enzymatic hydrolysate was centrifuged at 10,000 rpm for 15 min at 4°C to separate the upper oil layer from the lower protein solution. The lower protein solution was then ultrasonically treated for 25 min at a frequency of 20 kHz and an output power of 400 W.

[0061] Acid precipitation treatment: add 1 mol / L hydrochloric acid solution to the sonicated solution, adjust the pH value of the solution to 5 for protein acid precipitation, and after flocculent precipitation appears, centrifuge the solution at 10,000 r / min for 15 min at 4°C. Retain the precipitate for washing, neutralization, and vacuum freeze-drying to obtain pecan protein.

[0062] The pecan protein extraction rate obtained by this method was 71.79% and the protein purity was 92.10%.

[0063] Comparative Example 1

[0064] Pressing to remove oil: Put 50g of pecan kernels into the press and press at 80℃;

[0065] Alkali dissolution: The pressed cake was collected and crushed, passed through a 40-mesh sieve, and dispersed in 300 ml of deionized water to obtain a pecan meal aqueous solution. The pH of the aqueous solution was adjusted to 8 with 1 mol / L sodium hydroxide, and the solution was stirred in a 45°C water bath for 60 minutes. The aqueous solution was then centrifuged at 8000 rpm at 4°C for 20 minutes, and the supernatant was retained.

[0066] Acid precipitation: Place the supernatant in an ice bath, slowly add 1 mol / L hydrochloric acid solution, adjust the pH of the supernatant to 4.5, let it stand for 60 minutes, and then centrifuge at 10,000 r / min for 15 minutes at 4°C. Retain the precipitate for washing, neutralization, and vacuum drying to obtain pecan protein.

[0067] The extraction rate of pecan protein obtained by this method is 58.89%, and the purity of pecan protein is 78.12%.

[0068] It can be seen that the nut protein extraction method provided in this application has an extraction rate of not less than 70%, and the purity of the obtained pecan protein is not less than 90%, which is higher than the extraction rate and purity of the obtained protein by the pressing method.

[0069] When using nut protein in the food industry, it is usually necessary to consider whether some of the properties of the nut protein will affect the actual application effect. Therefore, the following tests were conducted on the pecan protein obtained in Example 1 and Comparative Example 1 of this application:

[0070] Test Example 1

[0071] The pecan protein in Example 1 and Comparative Example 1 was prepared into a solution with a mass concentration of 1 mg / mL with deionized water, and the pH of the solution was adjusted to 2, 4, 6, 8, 10, and 12. The protein solutions with different pH were stirred at room temperature for 30 minutes until they were completely rehydrated; then centrifuged at 10000 r / min at 4°C for 20 minutes, the supernatant was collected, and the solubility of the protein at different pH was determined by Kjeldahl method. The test results are shown in Table 1 and Figure 3 .

[0072] Table 1 Solubility of pecan protein obtained in Example 1 and Comparative Example 1 in water

[0073]

[0074] As can be seen from the data in Table 1, the solubility of the pecan protein obtained by the protein extraction method including ultrasonic treatment provided by the present application in water is not less than 75%, which is higher than the solubility of the pecan protein obtained by the pressing method without ultrasonic treatment in Comparative Example 1, whether in an acidic or alkaline environment.

[0075] Test Example 2

[0076] The pecan protein in Example 1 and Comparative Example 1 was prepared into a solution with a mass concentration of 10 mg / mL using deionized water. The solution was stirred at room temperature for 30 minutes, then homogenized at high speed (10,000 rpm) for 2 minutes using a homogenizer. The solution was quickly transferred to a measuring bucket, and the volume after homogenization was recorded. The ratio of the volume after homogenization to the volume before homogenization was calculated. Figure 4 .

[0077] Depend on Figure 4 It can be seen that the foaming property of the pecan protein prepared in Example 1 is better than that of the pecan protein obtained in Comparative Example 1. This is because the pecan protein obtained by the enzymatic hydrolysis method combined with ultrasonic treatment described in the present application has a higher proportion of β-turn structure, which makes the pecan protein have excellent water solubility and surface activity, can be quickly adsorbed to the gas-liquid interface, expand at the interface and form a stable interfacial film, and ultimately achieve better foaming properties.

[0078] Test Example 3

[0079] Digestibility testing was performed using an in vitro digestion simulation experiment.

[0080] First, simulated gastric digestion in vitro was performed: the pecan protein obtained in Example 1 and Comparative Example 1 was diluted with 90 mL of phosphate buffered saline (PBS) and homogenized for 60 seconds to uniformly disperse the protein. The resulting protein solution was then mixed with freshly prepared pepsin in a 37°C water bath, and the pH of the mixed solution was adjusted to 2.0 with 1 mol / L hydrochloric acid, and hydrolysis was performed for 90 minutes.

[0081] After the in vitro simulated gastric digestion is complete, the resulting sample is subjected to in vitro simulated intestinal digestion: remove the sample after simulated gastric digestion and adjust the pH to neutral using a 1 mol / L sodium hydroxide solution. Simultaneously, prepare a simulated intestinal fluid electrolyte stock solution: dissolve a predetermined amount of KCl, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6, and (NH4)2CO3 in approximately 800 ml of ultrapure water and adjust the volume to 1 L. In the obtained simulated intestinal fluid electrolyte stock solution, the concentration of KCl was 6.8 mmol / L (0.507 g / L), the concentration of KH2PO4 was 0.8 mmol / L (0.108 g / L), the concentration of NaHCO3 was 85 mmol / L (7.14 g / L), the concentration of NaCl was 38.4 mmol / L (2.24 g / L), the concentration of MgCl2(H2O)6 was 0.33 mmol / L (0.067 g / L), and the concentration of (NH4)2CO3 was 0.6 mmol / L (0.057 g / L).

[0082] Weigh a certain amount of pancreatic enzymes and bile salts, mix them with the sample after simulated gastric digestion and the above-mentioned stock solution, and ensure that the pancreatic enzyme concentration in the simulated intestinal digestion system is 100U TPAEE / mL and the bile salt concentration is 10 mmol / L. Carry out mixed culture in the intestinal stage. Still cultured at 37°C, wait for 120 minutes for complete hydrolysis. Take out 1mL of the digestion mixture at different time points in the simulated intestinal digestion, add 15% trichloroacetic acid (TCA) and place it at 4°C, and centrifuge at 4000 rpm for 15 minutes. By measuring the absorbance at 280nm, the content of hydrolyzed peptides in the digestion mixture was determined, and the mass percentage of hydrolyzed peptides and pecan protein was calculated. The test results are detailed in Tables 2 and Figure 5 :

[0083] Table 2 Digestion effects of pecan protein obtained in Example 1 and Comparative Example 1 at different digestion times

[0084]

[0085] As can be seen from the data in Table 2, the degree of digestion and decomposition of the pecan protein extracted in Example 1 of the present application is higher than that of the pecan protein obtained in Comparative Example 1. The pecan protein obtained in Example 1 is more easily digested and absorbed by the human body. This is because the enzymatic treatment temperature used in the present application can avoid thermal deformation of the pecan protein, and the alkaline protease can also specifically cut peptide bonds under mild conditions, releasing soluble small peptides, thereby avoiding the aggregation of protein peptide chains. Combined with the cavitation effect of ultrasonic treatment, the hydrogen bonds and hydrophobic interactions are temporarily destroyed, which changes the secondary structure of the pecan protein, allowing the pecan protein to unfold moderately, making it easier for digestive enzymes to access it, achieving good digestion. In the pressing method, the high temperature and high pressure environment causes the protein peptide chains to unfold and aggregate, forming a compact irreversible aggregate. In addition, the high temperature denatures the protein, hindering the protease from accessing the cleavage site of the peptide chain. During high-pressure pressing, oils and fats will also form covalent complexes with proteins, which will also reduce the contact between proteases and peptide chains, thereby obtaining pecan protein that is difficult to digest.

[0086] Finally, it should be noted that those skilled in the art will readily identify other embodiments of the present invention after considering the specification and practicing the invention disclosed herein. The present invention is intended to cover any variations, uses, or adaptations of the present invention that follow the general principles of the present invention and include common knowledge or customary techniques in the art not disclosed herein. The present invention is not limited to the precise structure described above and illustrated in the accompanying drawings, and various modifications and variations may be made without departing from the scope thereof. The scope of the present invention is limited solely by the appended claims.

Claims

1. A method for extracting nut protein, comprising: adding alkaline protease to the nut aqueous solution for enzymatic hydrolysis to obtain an enzymatic hydrolyzate; The enzymatic hydrolysate is subjected to acid precipitation treatment to obtain the nut protein.

2. The method for extracting nut protein according to claim 1, characterized in that: In the nut aqueous solution, the mass volume ratio of nuts to water is 1 g: (5-6) mL.

3. The method for extracting nut protein according to claim 1 or 2, characterized in that: During the enzymatic hydrolysis treatment, the pH of the nut aqueous solution is 8-9, and the enzymatic hydrolysis temperature is 45-55°C.

4. The method for extracting nut protein according to any one of claims 1 to 3, characterized in that: The mass ratio of the alkaline protease to the nuts is (1-4):

100.

5. The method for extracting nut protein according to any one of claims 1 to 4, characterized in that: The alkaline protease has an enzyme activity of 150-250 u / g.

6. The method for extracting nut protein according to any one of claims 1 to 5, characterized in that: Also includes: The enzymatic hydrolysate was separated and the protein solution was subjected to ultrasonic treatment.

7. The method for extracting nut protein according to claim 6, characterized in that: The output frequency of the ultrasonic treatment is 20-25 kHz, and the output power is 300-450 W.

8. The method for extracting nut protein according to any one of claims 1 to 7, characterized in that: The acid precipitation treatment comprises: Adjusting the pH of the enzymatic hydrolysate to 4-5, and after flocculent precipitation occurs, centrifuging the solution at 1-4°C for 15-20 minutes to obtain the nut protein; And / or, the extraction method further comprises: crushing the nuts to obtain nut crumbs with a particle size of 40 to 60 mesh.

9. A nut protein, characterized in that The product is obtained by extraction using the extraction method described in any one of claims 1 to 8.

10. A food additive, characterized in that The invention comprises the nut protein according to claim 9.

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