Pea peptide with function of promoting muscle repair and growth as well as preparation method and application of pea peptide

Abstract

The invention discloses a pea peptide with the function of promoting muscle repair and growth and a preparation method and application thereof, belongs to the technical field of plant protein processing, and provides a preparation method of the pea peptide with the function of promoting muscle repair and growth, and the preparation method comprises the following steps: mixing pea protein powder with water, and soaking to obtain a pea protein solution; adding alkaline protease, and carrying out first hydrolysis to obtain first hydrolysate; adding neutral protease, and performing second hydrolysis to obtain second hydrolysate; adding compound protease, and carrying out third hydrolysis to obtain third hydrolysate; and filtering the third hydrolysate by adopting an ultrafiltration membrane to obtain an ultrafiltration component, carrying out vacuum concentration to 1 / 8 or 1 / 10 of the original volume, and drying to obtain the pea peptide. The pea peptide obtained by the preparation method disclosed by the invention is high in purity and small in molecular weight, and shows excellent performance in the aspects of promoting muscle repair and growth and the like due to high activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant protein processing, and in particular relates to pea peptide having the function of promoting muscle repair and growth, and a preparation method and application thereof. Background Art

[0002] Muscle repair and growth are critical processes for maintaining human health, enhancing athletic performance, and accelerating recovery. Whether it's minor muscle damage during daily activities or severe muscle fatigue after intense exercise, timely and effective repair mechanisms are needed to restore muscle function and promote growth. Traditionally, protein supplements such as whey protein have been widely used to support muscle repair and growth, but with consumers' growing interest in plant-based products, finding alternative sources has become increasingly important.

[0003] As a high-quality plant-based protein source, pea protein has garnered increasing attention in recent years. It's not only rich in essential amino acids but also possesses excellent solubility, foaming properties, and gel-forming abilities. More importantly, pea peptides, produced through enzymatic hydrolysis or other processing, are more readily absorbed and utilized by the body due to their small molecular weight and high bioavailability, demonstrating significant potential in promoting muscle repair and growth. Studies have shown that pea peptides can promote muscle repair and growth through multiple pathways. Certain pea peptides can activate the mammalian target of rapamycin (mTOR) signaling pathway, promoting muscle protein synthesis and inhibiting protein breakdown, thereby accelerating muscle repair and growth. Pea peptides possess anti-inflammatory properties, which can reduce muscle inflammation caused by strenuous exercise or trauma, creating favorable conditions for muscle repair. Pea peptides may also enhance the oxygen and nutrient supply to muscle tissue by dilating blood vessels and increasing blood flow, further facilitating the repair process.

[0004] While pea peptides hold great promise for promoting muscle repair and growth, their practical application still faces challenges. Selecting the right enzyme and hydrolysis conditions while maintaining high yield and purity remains a technical challenge. In summary, developing an efficient method for preparing pea peptides with muscle repair and growth-promoting properties, and applying them in food, health supplements, or pharmaceuticals, remains a challenge. Summary of the Invention

[0005] To solve the above technical problems, the present invention proposes pea peptides with the function of promoting muscle repair and growth, as well as a preparation method and application thereof. By setting a pea protein powder pretreatment step and selecting appropriate enzyme species and enzymatic hydrolysis conditions, pea peptides with a molecular weight of less than 1KDa are obtained.

[0006] To achieve the above objectives, the present invention provides a method for preparing pea peptide having the function of promoting muscle repair and growth, comprising the following steps:

[0007] (1) mixing pea protein powder with water and soaking to obtain a pea protein solution;

[0008] (2) adding alkaline protease to the pea protein solution obtained in step (1) for a first hydrolysis to obtain a first hydrolyzate;

[0009] (3) adding neutral protease to the first hydrolyzate obtained in step (2) for a second hydrolysis to obtain a second hydrolyzate;

[0010] (4) adding composite protease to the second hydrolyzate obtained in step (3), performing a third hydrolysis to obtain a third hydrolyzate;

[0011] (5) The third hydrolyzate obtained in step (4) is filtered using an ultrafiltration membrane to obtain an ultrafiltration fraction, which is vacuum concentrated to 1 / 8 or 1 / 10 of the original volume and dried to obtain pea peptide.

[0012] Preferably, the mass ratio of the pea protein powder to water in step (1) is 1:20-40; the temperature of the water in step (1) is 50-60° C.; and the soaking time in step (1) is 4-6 hours.

[0013] Preferably, the amount of alkaline protease added in step (2) is 2-4% by mass, and the enzymatic activity of the alkaline protease is 400,000-500,000 U / g; the pH of the first hydrolysis in step (2) is 8.0-9.5, the temperature of the first hydrolysis is 45-55° C., and the time of the first hydrolysis is 45-75 min.

[0014] Preferably, the amount of the neutral protease added in step (3) is 0.5-1.5% by mass, and the enzymatic activity of the neutral protease is 200,000-300,000 U / g; the pH of the second hydrolysis in step (3) is 6.5-7.5, the temperature of the second hydrolysis is 50-60° C., and the time of the second hydrolysis is 90-150 min.

[0015] Preferably, the composite protease in step (4) is prepared by mixing papain and elastase in a mass ratio of 1 to 3:1, the added amount of the composite protease is 0.4 to 0.6% by mass, the enzymatic activity of papain in the composite protease is 50,000 to 60,000 U / g, and the enzymatic activity of elastase in the composite protease is greater than 10,000 U / g.

[0016] Preferably, the temperature of the third hydrolysis in step (4) is 25-35° C., and the time of the third hydrolysis is 20-40 min.

[0017] Preferably, the pore size of the ultrafiltration membrane in step (5) is 1 KDa.

[0018] Preferably, the vacuum degree of the vacuum concentration in step (5) is 0.01-0.05 MPa, and the temperature of the vacuum concentration is 60-80°C.

[0019] The present invention also provides the pea peptide prepared by the preparation method.

[0020] The present invention also provides the use of the pea peptide prepared by the preparation method in preparing a product having the function of promoting muscle repair and growth.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects:

[0022] The present invention proposes a method for preparing pea peptides that promote muscle repair and growth. By incorporating a pretreatment step of warm water soaking, the spatial structure of pea protein can be effectively unfolded, loosening the originally compact protein molecules and significantly increasing the exposure of enzyme action sites. This pretreatment method not only improves the efficiency of the subsequent enzymatic hydrolysis process but also lays the foundation for obtaining functional peptides with higher activity. During the enzymatic hydrolysis process, the rational selection of enzyme types and their hydrolysis order are also crucial. First, alkaline protease is selected for initial hydrolysis, followed by neutral protease to gradually release small molecular weight peptides. Finally, after the neutral protease hydrolysis, without pH adjustment, a composite protease consisting of papain and elastase is used to consolidate the enzymatic hydrolysis. This step-by-step enzymatic hydrolysis strategy ensures that the entire reaction process is both efficient and thorough, maximizing the retention and enhancement of the biological activity of the resulting peptides. The selection of enzymes with similar pH values avoids excessive salt in the resulting pea peptides and ensures flavor. The resulting pea peptides are not only high in purity and low in molecular weight, but also exhibit excellent performance in promoting muscle repair and growth due to their high activity. Furthermore, precise control of conditions throughout the entire preparation process avoids problems such as excessive enzymatic hydrolysis and byproduct formation, further ensuring product safety and stability. Therefore, by optimizing parameters at each step, the present invention successfully achieves high-quality conversion from raw materials to finished products, providing reliable technical support for the development of new functional foods and health supplements. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Exhaustive swimming time was measured for mice;

[0025] Figure 2To determine the serum creatine kinase content in mice;

[0026] Figure 3 To measure leg muscle strength before and after training. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The relative molecular weight distribution determination method of the present invention is carried out with reference to the high performance gel filtration chromatography method described in Appendix A of the national standards GB / T 22492-2008 and Appendix A of GB / T 22729-208.

[0033] Example 1

[0034] (1) Pea protein powder was mixed with water at a temperature of 55° C. in a mass ratio of 1:30 and soaked for 5 h to obtain a pea protein solution;

[0035] (2) adding 3% alkaline protease (450,000 U / g) to the pea protein solution at a mass ratio, with a pH of 8.75 and a temperature of 50° C. for a first hydrolysis of 60 min to obtain a first hydrolyzate;

[0036] (3) adding 1% neutral protease (250,000 U / g) to the first hydrolyzate at a mass ratio of 1%, with a pH of 7 and a temperature of 55° C. for a second hydrolysis for 120 min to obtain a second hydrolyzate;

[0037] (4) Adding 0.5% by weight of a composite protease (papain (55,000 U / g) and elastase (>10,000 U / g) mixed in a mass ratio of 2:1) to the second hydrolyzate, performing a third hydrolysis at 30°C for 30 min to obtain a third hydrolyzate;

[0038] (5) The third hydrolyzate was filtered through an ultrafiltration membrane with a pore size of 1 kDa to obtain an ultrafiltration fraction, which was then concentrated under vacuum at a vacuum degree of 0.03 MPa and a temperature of 70°C to 1 / 10 of its original volume, and then dried to obtain pea peptide. The pea peptide prepared in this example contained 90.2% of peptide segments with a molecular weight of less than 1 kDa.

[0039] Example 2

[0040] (1) Pea protein powder was mixed with water at a temperature of 50° C. in a mass ratio of 1:20 and soaked for 6 hours to obtain a pea protein solution;

[0041] (2) adding 2% alkaline protease (500,000 U / g) to the pea protein solution at a pH of 8.0 and a temperature of 45° C. for a first hydrolysis of 75 minutes to obtain a first hydrolyzate;

[0042] (3) adding 0.5% neutral protease (300,000 U / g) to the first hydrolyzate at a pH of 6.5 and a temperature of 50° C. for a second hydrolysis of 150 min to obtain a second hydrolyzate;

[0043] (4) Adding 0.4% by weight of a composite protease (papain (60,000 U / g) and elastase (>10,000 U / g) mixed in a mass ratio of 1:1) to the second hydrolyzate, and performing a third hydrolysis at 25°C for 40 min to obtain a third hydrolyzate;

[0044] (5) The third hydrolyzate was filtered through an ultrafiltration membrane with a pore size of 1 kDa to obtain an ultrafiltration fraction, which was then concentrated under vacuum at a vacuum degree of 0.01 MPa and a temperature of 80°C to 1 / 8 of its original volume, and then dried to obtain pea peptide. The pea peptide prepared in this example contained 87.8% peptide segments with a molecular weight of less than 1 kDa.

[0045] Example 3

[0046] (1) Pea protein powder was mixed with water at a temperature of 60° C. in a mass ratio of 1:40 and soaked for 4 hours to obtain a pea protein solution;

[0047] (2) adding alkaline protease (400,000 U / g) to the pea protein solution at a mass ratio of 4%, with a pH of 9.5 and a temperature of 55° C. for a first hydrolysis of 45 minutes to obtain a first hydrolyzate;

[0048] (3) adding 1.5% neutral protease (200,000 U / g) to the first hydrolyzate at a pH of 7.5 and a temperature of 60° C. for a second hydrolysis of 90 min to obtain a second hydrolyzate;

[0049] (4) Adding 0.6% by mass of a composite protease (papain (50,000 U / g) and elastase (>10,000 U / g) mixed in a mass ratio of 3:1) to the second hydrolyzate, and performing a third hydrolysis at 35°C for 20 min to obtain a third hydrolyzate;

[0050] (5) The third hydrolyzate was filtered through an ultrafiltration membrane with a pore size of 1 kDa to obtain an ultrafiltration fraction, which was then concentrated under vacuum at a vacuum degree of 0.05 MPa and a temperature of 60°C to 1 / 10 of its original volume, and then dried to obtain pea peptide. The pea peptide prepared in this example contained 86.5% peptide segments with a molecular weight of less than 1 kDa.

[0051] Comparative Example 1

[0052] (1) Pea protein powder was mixed with water at a temperature of 55° C. in a mass ratio of 1:30 and soaked for 5 h to obtain a pea protein solution;

[0053] (2) adding 3% alkaline protease (450,000 U / g) to the pea protein solution at a mass ratio, with a pH of 8.75 and a temperature of 50° C. for a first hydrolysis of 60 min to obtain a first hydrolyzate;

[0054] (3) adding 1% neutral protease (250,000 U / g) to the first hydrolyzate at a mass ratio of 1%, with a pH of 7 and a temperature of 55° C. for a second hydrolysis for 120 min to obtain a second hydrolyzate;

[0055] (4) Adding 0.5% by weight of a composite protease (papain (55,000 U / g) and flavor protease (20,000 U / g) mixed in a mass ratio of 2:1) to the second hydrolyzate, performing a third hydrolysis at 30°C for 30 min to obtain a third hydrolyzate;

[0056] (5) The third hydrolyzate was filtered through an ultrafiltration membrane with a pore size of 1 kDa to obtain an ultrafiltration fraction, which was then concentrated under vacuum at a vacuum degree of 0.03 MPa and a temperature of 70°C to 1 / 10 of its original volume, and then dried to obtain pea peptide. The pea peptide prepared in this comparative example contained 77.1% peptide segments with a molecular weight of less than 1 kDa.

[0057] Comparative Example 2

[0058] (1) Pea protein powder was mixed with water at a temperature of 55° C. in a mass ratio of 1:30 and soaked for 5 h to obtain a pea protein solution;

[0059] (2) adding 1% neutral protease (250,000 U / g) to the pea protein solution at a mass ratio, with a pH of 7 and a temperature of 55° C. for a first hydrolysis of 120 min to obtain a first hydrolyzate;

[0060] (3) Adding 0.5% by mass of a composite protease (papain (55,000 U / g) and elastase (>10,000 U / g) mixed in a mass ratio of 2:1) to the first hydrolyzate, and performing a second hydrolysis at 30°C for 30 min to obtain a second hydrolyzate;

[0061] (4) adding 3% alkaline protease (450,000 U / g) to the second hydrolyzate at a pH of 8.75 and a temperature of 50° C. for a third hydrolysis of 60 min to obtain a third hydrolyzate;

[0062] (5) The third hydrolyzate was filtered through an ultrafiltration membrane with a pore size of 1 kDa to obtain an ultrafiltration fraction, which was then concentrated under vacuum at a vacuum degree of 0.03 MPa and a temperature of 70°C to 1 / 10 of its original volume, and then dried to obtain pea peptide. The pea peptide prepared in this example contained 68.7% of peptides with a molecular weight of less than 1 kDa.

[0063] Experimental Example 1

[0064] The experimental animals were male Kunming mice (4 weeks old). Mice that passed the 60-min swimming training were randomly divided into four groups (n=15) and kept in a quiet environment with appropriate temperature and humidity. The specific gavage dose is shown in Table 1.

[0065] Table 1 Oral gavage dosage

[0066]

[0067] Two weeks after oral administration, mice were subjected to an exhaustive swimming test. A lead bar weighing 5% of their body weight was tied to their tails. The mice were forced to swim in the water until exhaustion (they failed to return to the surface within 5 seconds). The mice were then fished out and the time was recorded. The mice were then returned to their cages to rest.

[0068] Fifteen days after gavage, mice were subjected to a 60-minute timed swim. Mice were placed in a constant-temperature water bucket. When they stopped swimming, the water surface was gently stirred with a glass rod to force them to continue swimming. After 60 minutes, the mice were fished out and allowed to rest for 30 minutes. Serum creatine kinase (U / L) was measured using a biochemical analyzer.

[0069] All experiments were repeated at least three times, and the results are presented as mean ± standard deviation. Significant differences between samples were analyzed using Duncan's multiple comparison test with a significance level of p < 0.05. Graphs were generated using Graphpad Prism 9 software.

[0070] The results are as follows Figure 1 As shown, the exhaustive swimming time of mice can reflect the exercise endurance of mice and more intuitively reflect the exercise ability and fatigue level of mice. The longer the exhaustive swimming time of mice, the better the anti-fatigue effect of the gavage peptide on mice. The average swimming time of the control group mice was 8.5 minutes, the average swimming time of the mice in the pea peptide group of Example 1 was 20.5 minutes, the average swimming time of the mice in the pea peptide group of Comparative Example 1 and the average swimming time of the mice in the pea peptide group of Comparative Example 2 were 18.2 minutes and 16.4 minutes, respectively. The results show that the pea peptide of Example 1 can significantly improve the exercise endurance of mice.

[0071] Creatine kinase (CK) exists in large quantities in the body's skeletal muscles and brain, and is very important for the body's energy metabolism. Creatine kinase can phosphorylate creatine to form high-energy phosphocreatine, which generates ATP through a series of reactions. During normal physiological metabolism, because muscle cells are not damaged, CK rarely crosses the muscle cell membrane into the blood, so the CK content in the serum remains at a stable level. High-intensity exercise can cause muscle damage, increase the permeability of the muscle cell membrane, and cause a large amount of CK in the muscle cells to extravasate into the blood, resulting in increased activity in the serum. Therefore, changes in serum CK activity can reflect the degree of damage and recovery of skeletal muscle. The higher its activity, the more severe the skeletal muscle damage and fatigue. For example Figure 2 As shown, compared with the control group, the intake of pea peptides in Example 1 can restore the CK activity of mice, promote the repair of skeletal muscle damage in mice after strenuous exercise, and have a significant anti-fatigue effect.

[0072] Experimental Example 2

[0073] 40 judo athletes were recruited as volunteers and randomly divided into 4 groups: a control group, a pea peptide group according to Example 1, a pea peptide group according to Comparative Example 1, and a pea peptide group according to Comparative Example 2, with 10 participants in each group. After daily training (the training consisted of running 10 laps around the training hall, 45-50 minutes of basic skills training, 20-30 minutes of physical training, and 5-10 minutes of relaxation exercises), the control group was supplemented with 20 g / day of commercially available protein powder (purchased from By-Health), the pea peptide group according to Example 1 was supplemented with 20 g / day of pea peptide prepared in Example 1, the pea peptide group according to Comparative Example 1 was supplemented with 20 g / day of pea peptide prepared in Comparative Example 1, and the pea peptide group according to Comparative Example 2 was supplemented with 20 g / day of pea peptide prepared in Comparative Example 2. The subjects did not use any other nutritional supplements.

[0074] Before and after the 42-day experiment, leg muscle strength was measured using a BCS-400 electronic back dynamometer (Nantong Zilang Instrument Co., Ltd.) in strict accordance with national physical fitness test requirements. Two measurements were taken, and the best value was used.

[0075] The results were expressed as mean ± standard deviation, and SPSS13.0 was used for statistical analysis. Paired sample t-test was used before and after training. One-way analysis of variance and multiple comparisons were used for changes in various indicators. P ≤ 0.05 was considered to be significantly different, and P ≤ 0.01 was considered to be very significantly different.

[0076] Table 2 Changes in leg muscle strength in each group

[0077]

[0078] Note: Compared with before training, *P<0.05, **P<0.01; compared with the control group, ##P<0.01.

[0079] From the above table 2 and Figure 3 As can be seen, compared with pre-training, the leg muscle strength of the control group subjects decreased significantly after training, while the leg muscle strength of the subjects in the pea peptide group of Example 1 increased significantly. The pea peptides prepared in Comparative Examples 1 and 2 were slightly effective in restoring leg muscle strength. The different preparation methods affected the composition and activity of the prepared pea peptides. Compared with the control group, the leg muscle strength of the subjects in the pea peptide group of Example 1 increased significantly.

[0080] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing pea peptide having the function of promoting muscle repair and growth, characterized in that: The following steps are involved: (1) mixing pea protein powder with water and soaking to obtain a pea protein solution; (2) adding alkaline protease to the pea protein solution obtained in step (1) for a first hydrolysis to obtain a first hydrolyzate; (3) adding neutral protease to the first hydrolyzate obtained in step (2) for a second hydrolysis to obtain a second hydrolyzate; (4) adding composite protease to the second hydrolyzate obtained in step (3), performing a third hydrolysis to obtain a third hydrolyzate; (5) The third hydrolyzate obtained in step (4) is filtered using an ultrafiltration membrane to obtain an ultrafiltration fraction, which is vacuum concentrated to 1 / 8 or 1 / 10 of the original volume and dried to obtain pea peptide.

2. The preparation method according to claim 1, characterized in that The mass ratio of the pea protein powder and water in step (1) is 1:20-40; the temperature of the water in step (1) is 50-60° C.; and the soaking time in step (1) is 4-6 hours.

3. The preparation method according to claim 1, characterized in that: The amount of alkaline protease added in step (2) is 2-4% by mass, and the enzymatic activity of the alkaline protease is 400,000-500,000 U / g; the pH of the first hydrolysis in step (2) is 8.0-9.5, the temperature of the first hydrolysis is 45-55° C., and the time of the first hydrolysis is 45-75 min.

4. The preparation method according to claim 1, characterized in that The amount of the neutral protease added in step (3) is 0.5-1.5% by mass, and the enzymatic activity of the neutral protease is 200,000-300,000 U / g; the pH of the second hydrolysis in step (3) is 6.5-7.5, the temperature of the second hydrolysis is 50-60° C., and the time of the second hydrolysis is 90-150 min.

5. The preparation method according to claim 1, characterized in that: The composite protease in step (4) is prepared by mixing papain and elastase in a mass ratio of 1 to 3:

1. The added amount of the composite protease is 0.4 to 0.6% by mass. The enzymatic activity of papain in the composite protease is 50,000 to 60,000 U / g, and the enzymatic activity of elastase in the composite protease is greater than 10,000 U / g.

6. The preparation method according to claim 1, characterized in that: The temperature of the third hydrolysis in step (4) is 25-35° C., and the time of the third hydrolysis is 20-40 min.

7. The preparation method according to claim 1, characterized in that: The pore size of the ultrafiltration membrane in step (5) is 1 KDa.

8. The preparation method according to claim 1, characterized in that: The vacuum degree of the vacuum concentration in step (5) is 0.01-0.05 MPa, and the temperature of the vacuum concentration is 60-80°C.

9. The pea peptide prepared by the method according to any one of claims 1 to 8.

10. Use of the pea peptide prepared by the preparation method according to any one of claims 1 to 8 in preparing a product having the function of promoting muscle repair and growth.

Images