Pea peptides with functions of promoting muscle repair and growth, and preparation method and application thereof

Abstract

The application discloses a pea peptide with the functions 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 functions of promoting muscle repair and growth, which comprises the following steps: mixing pea protein powder with water, soaking to obtain a pea protein solution; adding alkaline protease for first hydrolysis to obtain a first hydrolysis liquid; adding neutral protease for second hydrolysis to obtain a second hydrolysis liquid; adding compound protease for third hydrolysis to obtain a third hydrolysis liquid; the third hydrolysis liquid is filtered by using an ultrafiltration membrane to obtain an ultrafiltration component; the ultrafiltration component is vacuum concentrated to 1 / 8 or 1 / 10 of the original volume; and drying is performed to obtain the pea peptide. The pea peptide obtained by the preparation method has high purity, small molecular weight, high activity, and excellent performance in promoting muscle repair and growth.

Description

Technical Field

[0001] This invention belongs to the field of plant protein processing technology, and particularly relates to pea peptides with muscle repair and growth promotion functions, their preparation methods and applications. Background Technology

[0002] Muscle repair and growth are crucial processes for maintaining health, improving athletic performance, and accelerating recovery. Whether it's minor muscle injury during daily activities or severe muscle fatigue after strenuous 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 increasingly focusing on plant-based products, finding alternative sources has become particularly important.

[0003] Pea protein, as a high-quality plant protein source, has received increasing attention in recent years. It is not only rich in essential amino acids but also possesses excellent solubility, foaming properties, and gel-forming ability. More importantly, pea peptides, produced from pea protein through enzymatic hydrolysis or other processing methods, are more easily absorbed and utilized by the human body due to their small molecular weight and high bioavailability, thus showing significant potential in promoting muscle repair and growth. Existing research has shown that pea peptides can promote muscle repair and growth through multiple pathways. Some pea peptides can accelerate muscle repair and growth by activating the mammalian target of rapamycin (mTOR) signaling pathway, promoting muscle protein synthesis and inhibiting protein breakdown. Pea peptides also have anti-inflammatory properties, reducing muscle inflammation levels caused by strenuous exercise or injury, creating favorable conditions for muscle repair. Furthermore, pea peptides may enhance the supply of oxygen and nutrients to muscle tissue by dilating blood vessels and increasing blood flow, further promoting the repair process.

[0004] Although pea peptides show great promise in promoting muscle repair and growth, several challenges remain in their practical application. Selecting suitable enzymes and hydrolysis conditions while maintaining high yield and purity remains a technical hurdle. In conclusion, developing an efficient method for preparing pea peptides with muscle-repairing and growth-promoting functions, and applying them in the food, health product, or pharmaceutical fields, is still a problem to be solved. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes pea peptides with muscle repair and growth promotion functions, along with their preparation method and applications. By setting a pea protein powder pretreatment step and selecting appropriate enzymes and enzymatic hydrolysis conditions, pea peptides with a molecular weight of less than 1 kDa are obtained.

[0006] To achieve the above objectives, this invention provides a method for preparing pea peptides with muscle repair and growth promotion functions, comprising the following steps:

[0007] (1) Mix pea protein powder with water and soak to obtain pea protein solution;

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

[0009] (3) Add neutral protease to the first hydrolysate obtained in step (2) and perform a second hydrolysis to obtain the second hydrolysate;

[0010] (4) The second hydrolysate obtained in step (3) is added to the complex protease and hydrolyzed for the third time to obtain the third hydrolysate;

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

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

[0013] Preferably, the amount of alkaline protease added in step (2) is 2-4% by mass, and the enzyme 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℃, and the time of the first hydrolysis is 45-75 min.

[0014] Preferably, the amount of neutral protease added in step (3) is 0.5-1.5% by mass, and the enzyme 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℃, and the time of the second hydrolysis is 90-150 min.

[0015] Preferably, the complex protease in step (4) is prepared by mixing papain and elastase in a mass ratio of 1 to 3:1, and the amount of the complex protease added is 0.4 to 0.6% by mass. The enzyme activity of papain in the complex protease is 50,000 to 60,000 U / g, and the enzyme activity of elastase in the complex protease is >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 vacuum concentration in step (5) is 0.01 to 0.05 MPa, and the vacuum concentration temperature is 60 to 80°C.

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

[0020] This invention also provides the application of the pea peptides prepared by the aforementioned method in the preparation of products that promote muscle repair and growth.

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

[0022] This invention proposes a method for preparing pea peptides with muscle repair and growth promotion functions. By combining a pretreatment step of warm water soaking, the spatial structure of pea protein can be effectively unfolded, making the originally compact protein molecules loose, thereby significantly increasing the exposure of enzyme action sites. This pretreatment method not only improves the efficiency of subsequent enzymatic hydrolysis but also lays the foundation for obtaining functional peptides with higher activity. During enzymatic hydrolysis, the rational selection of enzyme types and their hydrolysis sequence is equally crucial. First, alkaline protease is used for initial hydrolysis, followed by neutral protease to gradually release small molecular weight peptides. Finally, after neutral protease hydrolysis, without pH adjustment, a complex protease composed of papain and elastase is used for consolidation hydrolysis. This stepwise hydrolysis strategy ensures that the entire reaction process is both efficient and thorough, maximizing the preservation and enhancement of the bioactivity of the obtained peptides. Selecting enzymes with similar pH levels avoids excessive salt content in the obtained pea peptides, ensuring flavor. The final obtained pea peptides are not only high in purity and small in molecular weight but also exhibit excellent performance in promoting muscle repair and growth due to their high activity. Furthermore, because the conditions were precisely controlled throughout the entire preparation process, problems such as excessive enzymatic hydrolysis or byproduct formation were avoided, further ensuring the safety and stability of the product. Therefore, by optimizing parameters at each stage, this invention successfully achieved high-quality transformation from raw materials to the final product, providing reliable technical support for the development of novel functional foods and health products. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The exhaustive swimming time of mice was determined.

[0025] Figure 2For the determination of serum creatine kinase levels in mice;

[0026] Figure 3 For the purpose of training, the strength of the front and back leg muscles was measured. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0032] The method for determining the relative molecular weight distribution in this invention is performed in accordance with the high-performance gel filtration chromatography method described in Appendix A of national standards GB / T 22492-2008 and GB / T22729-208.

[0033] Example 1

[0034] (1) Mix pea protein powder with water at 55℃ at a mass ratio of 1:30 and soak for 5 hours to obtain pea protein solution.

[0035] (2) Add 3% by mass of alkaline protease (450000U / g) to the pea protein solution, set the pH to 8.75, set the temperature to 50℃, and perform the first hydrolysis for 60 min to obtain the first hydrolysate.

[0036] (3) Add 1% by mass of neutral protease (250000U / g) to the first hydrolysate, set the pH to 7, set the temperature to 55℃, and perform the second hydrolysis for 120 min to obtain the second hydrolysate.

[0037] (4) Add 0.5% by mass of a complex protease (papain (55000U / g) and elastase (>10000U / g) mixed at a mass ratio of 2:1) to the second hydrolysate, and perform a third hydrolysis at 30℃ for 30 min to obtain the third hydrolysate.

[0038] (5) The third hydrolysate was filtered using an ultrafiltration membrane with a pore size of 1 kDa to obtain the ultrafiltration fraction. The fraction was then concentrated under vacuum at 0.03 MPa and 70°C to 1 / 10 of its original volume, and dried to obtain pea peptides. In this example, the content of peptides with a molecular weight less than 1 kDa in the prepared pea peptides was 90.2%.

[0039] Example 2

[0040] (1) Mix pea protein powder with water at 50°C at a mass ratio of 1:20 and soak for 6 hours to obtain pea protein solution.

[0041] (2) Add 2% by mass of alkaline protease (500000U / g) to the pea protein solution, set the pH to 8.0, set the temperature to 45℃, and perform the first hydrolysis for 75 min to obtain the first hydrolysate.

[0042] (3) Add 0.5% by mass of neutral protease (300000U / g) to the first hydrolysate, set the pH to 6.5, set the temperature to 50℃, and perform a second hydrolysis for 150 min to obtain the second hydrolysate;

[0043] (4) Add 0.4% by mass of a complex protease (papain (60000U / g) and elastase (>10000U / g) mixed in a mass ratio of 1:1) to the second hydrolysate, and perform a third hydrolysis at 25℃ for 40 min to obtain the third hydrolysate.

[0044] (5) The third hydrolysate was filtered using an ultrafiltration membrane with a pore size of 1 kDa to obtain the ultrafiltration fraction. The fraction was then concentrated under vacuum at 0.01 MPa and 80°C to 1 / 8 of its original volume, and dried to obtain pea peptides. In this example, the content of peptides with a molecular weight less than 1 kDa in the prepared pea peptides was 87.8%.

[0045] Example 3

[0046] (1) Mix pea protein powder with water at 60℃ at a mass ratio of 1:40 and soak for 4 hours to obtain pea protein solution.

[0047] (2) Add 4% by mass of alkaline protease (400000U / g) to the pea protein solution, set the pH to 9.5, and the temperature to 55℃. Perform the first hydrolysis for 45 minutes to obtain the first hydrolysate.

[0048] (3) Add 1.5% by mass of neutral protease (200000U / g) to the first hydrolysate, set the pH to 7.5, set the temperature to 60℃, and perform the second hydrolysis for 90 min to obtain the second hydrolysate;

[0049] (4) Add 0.6% by mass of a complex protease (papain (50000U / g) and elastase (>10000U / g) mixed in a mass ratio of 3:1) to the second hydrolysate, and perform a third hydrolysis at 35℃ for 20 min to obtain the third hydrolysate;

[0050] (5) The third hydrolysate was filtered using an ultrafiltration membrane with a pore size of 1 kDa to obtain the ultrafiltration fraction. The fraction was then concentrated under vacuum at 0.05 MPa and 60°C to 1 / 10 of its original volume, and dried to obtain pea peptides. In this example, the content of peptides with a molecular weight less than 1 kDa in the prepared pea peptides was 86.5%.

[0051] Comparative Example 1

[0052] (1) Mix pea protein powder with water at 55℃ at a mass ratio of 1:30 and soak for 5 hours to obtain pea protein solution.

[0053] (2) Add 3% by mass of alkaline protease (450000U / g) to the pea protein solution, set the pH to 8.75, set the temperature to 50℃, and perform the first hydrolysis for 60 min to obtain the first hydrolysate.

[0054] (3) Add 1% by mass of neutral protease (250000U / g) to the first hydrolysate, set the pH to 7, set the temperature to 55℃, and perform the second hydrolysis for 120 min to obtain the second hydrolysate.

[0055] (4) Add 0.5% by mass of a complex protease (papain (55000U / g) and flavor protease (20000U / g) mixed at a mass ratio of 2:1) to the second hydrolysate, and perform a third hydrolysis at 30℃ for 30 min to obtain the third hydrolysate.

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

[0057] Comparative Example 2

[0058] (1) Mix pea protein powder with water at 55℃ at a mass ratio of 1:30 and soak for 5 hours to obtain pea protein solution.

[0059] (2) Add 1% by mass of neutral protease (250000U / g) to the pea protein solution, set the pH to 7, the temperature to 55℃, and perform the first hydrolysis for 120 min to obtain the first hydrolysate.

[0060] (3) Add 0.5% by mass of a complex protease (papain (55000U / g) and elastase (>10000U / g) mixed at a mass ratio of 2:1) to the first hydrolysate, and perform a second hydrolysis at 30℃ for 30 min to obtain the second hydrolysate;

[0061] (4) Add 3% by mass of alkaline protease (450000U / g) to the second hydrolysate, set the pH to 8.75, set the temperature to 50℃, and perform the third hydrolysis for 60 min to obtain the third hydrolysate.

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

[0063] Experimental Example 1

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

[0065] Table 1. Gavage Dosage

[0066]

[0067] Two weeks after gavage, the mice underwent an exhaustive swimming test. A lead bar, representing 5% of the mouse's body weight, was attached to the mouse's tail. The mouse was forced to swim in the water until it was exhausted (the mouse failed to return to the surface within 5 seconds). The mice were then removed from the water, the time was recorded, and they were returned to their cages to rest.

[0068] Fifteen days after gavage, the mice were given a 60-minute timed swimming exercise. The mice were placed in a temperature-controlled water tank, and when they stopped swimming, the water surface was gently stirred with a glass rod to encourage them to continue swimming. After 60 minutes, the mice were removed and allowed to rest for 30 minutes. Serum creatine kinase (U / L) levels were measured using a biochemical analyzer.

[0069] All experiments were repeated at least three times, and results are expressed as mean ± standard deviation. Duncan's multiple comparison test was used in SPSS 22 to determine the significance of differences between samples, with a significance level set at p < 0.05. GraphpadPrism 9 software was used for plotting.

[0070] The results are as follows Figure 1 As shown, the time to exhaustion while swimming in mice reflects their exercise endurance and provides a relatively direct indication of their athletic ability and fatigue level. The longer the time to exhaustion while swimming in mice, the better the anti-fatigue effect of the gavage peptides. The average swimming time of the control group mice was 8.5 min, while the average swimming time of the mice in the Example 1 pea peptide group was 20.5 min. The average swimming times of the mice in the Comparative Example 1 pea peptide group and the Comparative Example 2 pea peptide group were 18.2 min and 16.4 min, respectively. These results indicate that the pea peptides in Example 1 can significantly improve the exercise endurance of mice.

[0071] Creatine kinase (CK) is abundant in skeletal muscle and the brain, playing a crucial role in energy metabolism. CK phosphorylates creatine to form high-energy phosphocreatine, which then generates ATP through a series of reactions. During normal physiological metabolism, because muscle cells are not damaged, CK rarely crosses the cell membrane into the bloodstream, thus maintaining a stable CK level in serum. High-intensity exercise leads to muscle damage, increasing the permeability of muscle cell membranes and causing a large amount of CK to leak from muscle cells into the bloodstream, resulting in increased CK activity in serum. Therefore, changes in serum CK activity can reflect the degree of skeletal muscle damage and recovery; higher activity indicates more severe skeletal muscle damage and fatigue. Figure 2 As shown, compared with the control group, the intake of pea peptides in Example 1 can restore CK activity in mice, promote the repair of skeletal muscle damage in mice after strenuous exercise, and has a significant anti-fatigue effect.

[0072] Experimental Example 2

[0073] Forty judo athletes were recruited as volunteers and randomly divided into four groups: a control group, the Example 1 pea peptide group, the Comparative Example 1 pea peptide group, and the Comparative Example 2 pea peptide group, with 10 participants in each group. After daily training (which consisted of 10 laps around the training hall, 45-50 minutes of basic skills practice, 20-30 minutes of physical conditioning, and 5-10 minutes of relaxation exercises), the control group supplemented with 20g / day of commercially available protein powder (purchased from By-Health), the Example 1 pea peptide group supplemented with 20g / day of pea peptide prepared in Example 1, the Comparative Example 1 pea peptide group supplemented with 20g / day of pea peptide prepared in Comparative Example 1, and the Comparative Example 2 pea peptide group supplemented with 20g / day of pea peptide prepared in Comparative Example 2. No other nutritional supplements were used by the participants.

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

[0075] Results are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 13.0. Paired-samples t-tests were used before and after training. Changes in each indicator were analyzed using one-way ANOVA and multiple comparisons. P ≤ 0.05 was considered statistically significant, and P ≤ 0.01 was considered highly statistically significant.

[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 It can be seen that, compared with before training, the leg muscle strength of the control group subjects was significantly reduced after training, while the leg muscle strength of the subjects in the pea peptide group of Example 1 was significantly increased. 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 was significantly increased.

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

Claims

1. A method for preparing pea peptides with muscle repair and growth promotion functions, characterized in that, Includes the following steps: (1) Mix pea protein powder with water and soak to obtain pea protein solution; (2) Add alkaline protease to the pea protein solution obtained in step (1) for the first hydrolysis to obtain the first hydrolysate; (3) Add neutral protease to the first hydrolysate obtained in step (2) and perform a second hydrolysis to obtain the second hydrolysate; (4) Add the compound protease to the second hydrolysate obtained in step (3) and perform a third hydrolysis to obtain the third hydrolysate; (5) The third hydrolysate obtained in step (4) is filtered by ultrafiltration membrane to obtain ultrafiltration component, which is then concentrated under vacuum to 1 / 8 or 1 / 10 of the original volume and dried to obtain pea peptide. The amount of alkaline protease added in step (2) is 3% by mass, the enzyme activity of alkaline protease is 450,000 U / g, and the first hydrolysis time is 60 min; The amount of neutral protease added in step (3) is 1% by mass, the enzyme activity of the neutral protease is 250,000 U / g, and the second hydrolysis time is 120 min; The complex protease mentioned in step (4) is prepared by mixing papain and elastase at a mass ratio of 2:

1. The amount of the complex protease added is 0.5% by mass. The enzyme activity of papain in the complex protease is 55000 U / g, the enzyme activity of elastase in the complex protease is >10000 U / g, and the time of the third hydrolysis is 30 min. The pore size of the ultrafiltration membrane in step (5) is 1 kDa.

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

3. The preparation method according to claim 1, characterized in that, In step (2), the pH of the first hydrolysis is 8.0~9.5, and the temperature of the first hydrolysis is 45~55℃.

4. The preparation method according to claim 1, characterized in that, In step (3), the pH of the second hydrolysis is 6.5~7.5, and the temperature of the second hydrolysis is 50~60℃.

5. The preparation method according to claim 1, characterized in that, The temperature of the third hydrolysis in step (4) is 25~35℃.

6. The preparation method according to claim 1, characterized in that, The vacuum degree of vacuum concentration in step (5) is 0.01~0.05MPa, and the temperature of vacuum concentration is 60~80℃.

7. Pea peptides prepared by the preparation method according to any one of claims 1 to 6.

8. The use of pea peptides prepared by the preparation method according to any one of claims 1 to 6 in the preparation of anti-fatigue products.

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