A pea peptide, its preparation method and application

Abstract

This invention discloses a pea peptide, its preparation method, and its application, belonging to the technical field of plant polypeptide preparation and application. A mixture of sodium bicarbonate and sodium alginate is sprayed onto pea protein powder, and then dried at 70-80℃ for 3-5 hours. The dried pea protein powder is mixed with water to obtain a mixed solution, which is then heated to 45-55℃. Alkaline protease is added for enzymatic hydrolysis, and papain is added to the hydrolysate for further hydrolysis. After hydrolysis, enzyme inactivation is performed. The hydrolysate is filtered, concentrated, and spray-dried to obtain the pea peptide. The pea peptide prepared by this invention has a high recovery rate, low free amino acid content, and more than 85% of the molecules with a molecular weight less than 600 Da. It also exhibits high enzymatic hydrolysis efficiency, short hydrolysis time, and good antioxidant activity.

Description

Technical Field

[0001] This invention belongs to the field of preparation and application technology of plant polypeptides, specifically relating to a pea peptide, its preparation method and application. Background Technology

[0002] Peas are a major edible crop with a wide cultivation range. Rich in protein, vitamins, and carbohydrates, peas play an important role in human health as a protein supplement or food component. Pea protein is a crucial component of peas, possessing high nutritional value, a balanced amino acid profile, and abundant phytochemicals including saponins, flavonoids, tannins, butyraldehyde, and acetic acid. Pea peptides (PP) are amino acid sequences extracted from pea protein. Research on pea peptides has garnered significant attention in food science and nutrition, as they exhibit various biological activities such as hypoglycemic, hypotensive, anti-inflammatory, antioxidant, and antibacterial effects. Much research on pea peptides focuses on extraction, preparation, structural analysis, and bioactivity evaluation.

[0003] Patent CN108220371A discloses a method for preparing pea peptides with prebiotic effects and their application in food. Pea peptides are obtained by enzymatic hydrolysis with alkaline protease and neutral protease. The prepared pea peptides have a protein content of up to 92%, a polypeptide content of up to 87%, a peptide content of up to 90% with a molecular weight of <1000Da, and a free amino acid content as low as 4.7%. They have prebiotic effects. The preparation method does not require manual adjustment of pH value, simplifies the operation, effectively shortens the preparation time, and reduces production costs. However, the yield of pea peptides extracted using this method is relatively low. Patent CN112626155A discloses a method for preparing pea peptides. The method involves pretreatment with high-pressure steam sterilization to unfold the structure of pea protein, making it more conducive to enzymatic hydrolysis. Then, a combined enzymatic hydrolysis of Alcalase and proteinase P is used to reduce the bitterness of the peptide powder. The total amount of enzyme used is lower than in existing technologies. The prepared pea peptide powder has a protein content ≥90%, a peptide yield of 73%, and a free amino acid content of less than 15%. However, this method requires a high-pressure steam sterilization temperature of 121℃, which can damage the protein peptide structure. Furthermore, the method uses limited enzyme sources, resulting in high costs and a low yield of pea peptides.

[0004] Therefore, improving the yield of pea protein peptides, reducing the amount of enzyme used, and lowering the cost of enzymatic hydrolysis are of great significance. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a pea peptide, its preparation method, and its application. The pea peptide protein prepared by this invention has a high recovery rate, low content of free amino acids, and more than 85% of the content of molecules with a molecular weight of less than 600 Da. It also has high enzymatic hydrolysis efficiency and short enzymatic hydrolysis time, and the prepared pea peptide has good antioxidant activity.

[0006] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for preparing pea peptides, comprising the following steps: (1) Spray a mixture of sodium bicarbonate and sodium alginate onto pea protein powder, and dry it at 70-80℃ for 3-5 hours after spraying. (2) Mix the dried pea protein powder with water to obtain a mixed solution and heat it to 45~55℃. Add alkaline protease for enzymatic hydrolysis. Continue to add papain to the hydrolysate for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, perform enzyme inactivation treatment. (3) The enzymatic hydrolysate from step (2) is filtered, concentrated, and spray-dried to obtain pea peptides.

[0007] Furthermore, in step (1), the mass percentage concentration of sodium bicarbonate in the mixture is 5-8%, and the mass percentage concentration of sodium alginate is 2-4%.

[0008] Furthermore, in step (1), the mass ratio of pea protein powder to the mixture is 1:0.05~0.1. During the spraying process, the mixture is stirred continuously to ensure uniform mixing and avoid large-area clumping.

[0009] Furthermore, in step (2), the amount of alkaline protease added is 2-5% of the mass of pea protein powder, and the enzymatic hydrolysis time is 60-90 min.

[0010] Furthermore, in step (2), the amount of papain added is 0.5-1.5% of the mass of pea protein powder, and the enzymatic hydrolysis time is 60-120 min.

[0011] Furthermore, in step (2), the mass ratio of pea protein powder to water is 1:8~20, and in step (3), the enzyme inactivation treatment conditions are 90~100℃ for 5~10min.

[0012] In a second aspect, the present invention provides pea peptides prepared by the aforementioned preparation method.

[0013] In a third aspect, the present invention provides the application of the pea peptide in the preparation of products with antioxidant functions.

[0014] Furthermore, the products with antioxidant functions mentioned above are food, pharmaceuticals, or cosmetics.

[0015] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: (1) In this invention, a mixture of sodium bicarbonate and sodium alginate and a heating and drying method are used to process pea protein powder. Sodium bicarbonate slowly decomposes during the drying process at 70-80℃, producing carbon dioxide gas, which forms tiny pores inside the protein particles, causing the protein to expand in volume. The alkaline environment destroys the hydrogen bonds and disulfide bonds between protein molecules, allowing the protein molecules to fully extend, further promoting a loose structure and exposing more enzyme cleavage sites. The macromolecular chains of sodium alginate can wrap around the protein surface, guiding the protease to act on specific peptide segments and improving the enzymatic hydrolysis efficiency. The macromolecular chains of sodium alginate form a network structure at high temperature, stabilizing the carbon dioxide bubbles in the protein matrix and preventing them from escaping quickly. Sodium bicarbonate and sodium alginate form a rigid structure with pea protein during the drying process, maintaining the expanded state of the protein.

[0016] (2) In this invention, sodium bicarbonate and sodium alginate cause pea protein to be in an expanded state. The porous structure makes it easier for protease to penetrate into the protein, significantly increasing the contact area between the enzyme and the substrate. The extended state of the protein molecules fully exposes the peptide bonds, increasing the number of enzyme cleavage sites. The network structure formed by sodium alginate can guide the protease to act on specific peptide segments, improving the selectivity of enzymatic hydrolysis. During the enzymatic hydrolysis process, some undecomposed sodium bicarbonate can provide a weakly alkaline environment, matching the enzymatic hydrolysis environment of alkaline protease. The carboxyl group of sodium alginate can buffer the pH fluctuation of the system, avoiding the decrease in enzyme activity caused by pH fluctuation during the enzymatic hydrolysis process. In addition, the carbon dioxide in the expanded structure can promote the uniform dispersion of enzyme in the system, avoiding the enzyme from being inactivated due to excessively high local concentration. This shortens the enzymatic hydrolysis time, improves the enzymatic hydrolysis efficiency, and increases the yield of pea peptides.

[0017] (3) The present invention uses alkaline protease and papain to enzymatically hydrolyze pea peptides. The enzymes are widely available and low in cost, which further reduces the cost of preparing pea peptides from pea protein. The pea peptides prepared by the present invention have a higher yield (high protein recovery rate), smaller molecular weight, higher solubility, and are easier to absorb. When applied to food, medicine, and cosmetics, they are more easily absorbed by the matrix, showing a better free radical scavenging effect and good application prospects. Detailed Implementation

[0018] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions.

[0020] The alkaline protease and papain used in this invention were commercially available.

[0021] Example 1 (1) Add sodium bicarbonate to water and stir thoroughly to dissolve it. Then add sodium alginate and stir to dissolve it to obtain a mixed solution. The mass percentage concentration of sodium bicarbonate in the mixed solution is 6%, and the mass percentage concentration of sodium alginate is 3%. (2) Spray the mixture from step (1) onto the pea protein powder. The mass ratio of pea protein powder to the mixture is 1:0.08. Stir constantly during the spraying process to ensure even mixing and avoid clumping. After spraying, dry at 75°C for 4 hours. (3) Mix the dried pea protein powder with 15 times the mass of deionized water to obtain a mixed solution and heat it to 50°C. Add 3% of the mass of pea protein powder alkaline protease for 80 min of enzymatic hydrolysis. After the enzymatic hydrolysis is completed, continue to add 1% of the mass of pea protein powder papain to the hydrolysate for 90 min of enzymatic hydrolysis. After the enzymatic hydrolysis is completed, heat it to 100°C and inactivate the enzyme for 5 min. (4) Filter the enzymatic hydrolysate from step (3), concentrate the filtrate to 10% of the original volume, and spray dry to obtain pea peptides.

[0022] Example 2 (1) Add sodium bicarbonate to water and stir thoroughly to dissolve it. Then add sodium alginate and stir to dissolve it to obtain a mixed solution. The mass percentage concentration of sodium bicarbonate in the mixed solution is 8%, and the mass percentage concentration of sodium alginate is 3%. (2) Spray the mixture from step (1) onto the pea protein powder. The mass ratio of pea protein powder to the mixture is 1:0.5. Stir constantly during the spraying process to ensure even mixing and avoid clumping. After spraying, dry at 75°C for 4 hours. (3) Mix the dried pea protein powder with 15 times the mass of deionized water to obtain a mixed solution and heat it to 50°C. Add 2% of the mass of pea protein powder alkaline protease for 90 min. After the enzymatic hydrolysis, continue to add 1.5% of the mass of pea protein powder papain to the hydrolysate for 60 min. After the enzymatic hydrolysis, heat it to 100°C and inactivate the enzyme for 5 min. (4) Filter the enzymatic hydrolysate from step (3), concentrate the filtrate to 10% of the original volume, and spray dry to obtain pea peptides.

[0023] Example 3 (1) Add sodium bicarbonate to water and stir thoroughly to dissolve it. Then add sodium alginate and stir to dissolve it to obtain a mixed solution. The mass percentage concentration of sodium bicarbonate in the mixed solution is 5%, and the mass percentage concentration of sodium alginate is 2%. (2) Spray the mixture from step (1) onto the pea protein powder. The mass ratio of pea protein powder to the mixture is 1:0.1. Stir continuously during the spraying process to ensure even mixing and avoid clumping. After spraying, dry at 70°C for 5 hours. (3) Mix the dried pea protein powder with 15 times the mass of deionized water to obtain a mixed solution and heat it to 50°C. Add 5% of the mass of pea protein powder alkaline protease for 60 min of enzymatic hydrolysis. After the enzymatic hydrolysis is completed, continue to add 0.5% of the mass of pea protein powder papain to the hydrolysate for 120 min of enzymatic hydrolysis. After the enzymatic hydrolysis is completed, heat it to 100°C and inactivate the enzyme for 5 min. (4) Filter the enzymatic hydrolysate from step (3), concentrate the filtrate to 10% of the original volume, and spray dry to obtain pea peptides.

[0024] Example 4 (1) Add sodium bicarbonate to water and stir thoroughly to dissolve it. Then add sodium alginate and stir to dissolve it to obtain a mixed solution. The mass percentage concentration of sodium bicarbonate in the mixed solution is 6%, and the mass percentage concentration of sodium alginate is 2%. (2) Spray the mixture from step (1) onto the pea protein powder. The mass ratio of pea protein powder to the mixture is 1:0.1. Stir constantly during the spraying process to ensure even mixing and avoid clumping. After spraying, dry at 80°C for 3 hours. (3) Mix the dried pea protein powder with 15 times the mass of deionized water to obtain a mixed solution and heat it to 50°C. Add 4% of the mass of pea protein powder alkaline protease for 60 min of enzymatic hydrolysis. After the enzymatic hydrolysis is completed, continue to add 1% of the mass of pea protein powder papain to the hydrolysate for 90 min of enzymatic hydrolysis. After the enzymatic hydrolysis is completed, heat it to 100°C and inactivate the enzyme for 5 min. (4) Filter the enzymatic hydrolysate from step (3), concentrate the filtrate to 10% of the original volume, and spray dry to obtain pea peptides.

[0025] Comparative Example 1 Unlike Example 1, sodium alginate was not added to the mixture in step (1) of Comparative Example 1. The remaining steps and conditions were the same as in Example 1, and pea peptides were finally prepared.

[0026] Comparative Example 2 Unlike Example 1, Comparative Example 2 did not include a mixture containing sodium alginate and sodium bicarbonate in the pea protein powder. Specifically: (1) Mix pea protein powder with 15 times the mass of deionized water to obtain a mixed solution and heat it to 50°C. Add 3% of the mass of pea protein powder alkaline protease for 80 min. After the enzymatic hydrolysis, continue to add 1% of the mass of pea protein powder papain to the hydrolysate for 90 min. After the enzymatic hydrolysis, heat it to 100°C and inactivate the enzyme for 5 min. (2) The enzymatic hydrolysate in step (1) is filtered, the filtrate is concentrated to 10% of the original volume, and spray-dried to obtain pea peptides.

[0027] Comparative Example 3 Unlike Example 1, Comparative Example 3 does not perform drying treatment after spraying in step (2), but the remaining steps are the same as in Example 1.

[0028] Performance testing (1) The protein recovery rate (the ratio of the nitrogen content in the enzymatic hydrolysate filtrate to the protein in the substrate before enzymatic hydrolysis), peptide content, percentage of peptides with a molecular weight less than 600 Da, free amino acid content, and peptide yield of the pea peptides prepared in Examples 1-4 and Comparative Examples 1-3 were tested. The results are shown in Table 1 below: Table 1. Performance test results of pea peptides prepared in Examples 1-4 and Comparative Examples 1-3 .

[0029] As shown in Table 1, the pretreatment of pea protein powder using a mixed solution of sodium bicarbonate and sodium alginate, combined with high-temperature (70-80℃) heat treatment, causes the protein structure to swell and maintains this swelled state. The porous structure allows proteases to penetrate more easily into the protein, exposing more cleavage sites, thereby increasing the enzymatic activity of both alkaline and neutral proteases. This reduces the amount of enzyme used while increasing the hydrolysis rate, resulting in lower molecular weight pea peptides that are more easily absorbed and have improved activity. Comparative Example 3, which added a mixed solution of sodium bicarbonate and sodium alginate, did not undergo heat treatment and therefore did not release carbon dioxide to cause protein swelling. It only provided a weakly alkaline environment during enzymatic hydrolysis, and its effect was not significantly different from Comparative Example 2, which did not add the mixed solution of sodium bicarbonate and sodium alginate.

[0030] (2) In vitro antioxidant activity test 1) DPPH free radical scavenging activity assay: Pea peptide sample solution (4 mg / mL) was mixed with an equal volume of 0.04 mg / mL DPPH·ethanol solution, and incubated for 30 min in the dark. The absorbance was measured at 517 nm and the DPPH scavenging activity was calculated. The results are shown in Table 2 below.

[0031] 2) ABTS free radical scavenging activity assay: 7 mM ABTS aqueous solution and 2.45 mM potassium persulfate aqueous solution were mixed in equal volumes and left to stand in the dark for 12 h to generate ABTS. + Free radicals. Pea peptide sample solution (4 mg / mL, 1.0 mL) with ABTS +The free radical solution (2 mL) was mixed and reacted in the dark for 10 min. The absorbance was measured at 734 nm and the free radical scavenging activity of ABTS was calculated. The results are shown in Table 2 below.

[0032] Table 2 Results of in vitro antioxidant activity test of pea peptides .

[0033] Table 2 shows that the addition of sodium bicarbonate and sodium alginate has little effect on the antioxidant activity of the peptides. The antioxidant activity of the pea peptides prepared in the comparative method is slightly lower than that of the pea peptides prepared by the method of this application, which may be related to the high content of high molecular weight peptides. The pea peptides prepared in combination with the present invention have a higher yield (high protein recovery rate), smaller molecular weight, higher solubility, and are more easily absorbed. When applied to food, pharmaceuticals, and cosmetics, they are more easily absorbed by the matrix, exhibiting better free radical scavenging effects and possessing antioxidant activity. They can be applied to the preparation of products with antioxidant functions, such as food, pharmaceuticals, or cosmetics.

Claims

1. A method for preparing pea peptides, characterized in that, Includes the following steps: (1) Spray a mixture of sodium bicarbonate and sodium alginate onto pea protein powder, and dry it at 70-80℃ for 3-5 hours after spraying. (2) Mix the dried pea protein powder with water to obtain a mixed solution and heat it to 45~55℃. Add alkaline protease for enzymatic hydrolysis. Continue to add papain to the hydrolysate for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, perform enzyme inactivation treatment. (3) The enzymatic hydrolysate from step (2) is filtered, concentrated, and spray-dried to obtain pea peptides.

2. The method for preparing pea peptides according to claim 1, characterized in that, In step (1), the mass percentage concentration of sodium bicarbonate in the mixture is 5-8%, and the mass percentage concentration of sodium alginate is 2-4%.

3. The method for preparing pea peptides according to claim 1, characterized in that, In step (1), the mass ratio of pea protein powder to the mixture is 1:0.05~0.

1. During the spraying process, the mixture is stirred continuously to ensure even mixing and to avoid clumping.

4. The method for preparing pea peptides according to claim 1, characterized in that, In step (2), the amount of alkaline protease added is 2-5% of the mass of pea protein powder, and the enzymatic hydrolysis time is 60-90 min.

5. The method for preparing pea peptides according to claim 1, characterized in that, In step (2), the amount of papain added is 0.5-1.5% of the mass of pea protein powder, and the enzymatic hydrolysis time is 60-120 min.

6. The method for preparing pea peptides according to claim 1, characterized in that, In step (2), the mass ratio of pea protein powder to water is 1:8~20, and in step (3), the enzyme inactivation treatment conditions are 90~100℃ for 5~10min.

7. A pea peptide prepared by the preparation method according to any one of claims 1 to 6.

8. The use of a pea peptide prepared by the preparation method according to any one of claims 1 to 6 or the pea peptide according to claim 7 in the preparation of a product with antioxidant function.

9. The application according to claim 8, characterized in that, The products with antioxidant functions mentioned above are food, pharmaceuticals, or cosmetics.