Wheat oligopeptide with anti-fatigue effect and preparation method thereof

Abstract

The invention belongs to the technical field of protein peptide preparation, and particularly relates to wheat oligopeptide with an anti-fatigue effect and a preparation method of the wheat oligopeptide. According to the method, the raw materials are subjected to enzymolysis by using the first compound enzyme containing bacillus subtilis protease and bromelain and the second compound enzyme containing trypsin, flavourzyme and papain step by step, and treatment of centrifugation, ultrafiltration and the like is combined, so that the yield and the taste of the product are improved, and the proportion of small molecular weight peptide in the product is high. Therefore, the wheat oligopeptide prepared by the method is high in yield and good in taste, not only is beneficial to improving the endurance of the body and prolonging the exercise time, but also can improve the energy metabolism condition in the body, delay the accumulation of lactic acid, urea nitrogen and creatine kinase in the body after exercise and increase the glycogen storage amount, and macroscopically shows certain anti-fatigue ability. The technical scheme of the invention has strong practicability, is beneficial to industrial production, and can be applied to preparation of anti-fatigue functional food.

Description

Technical Field

[0001] This invention belongs to the field of protein peptide preparation technology, specifically relating to a wheat oligopeptide with anti-fatigue effects and its preparation method. Background Technology

[0002] Fatigue is a physical or psychological discomfort caused by factors such as strenuous exercise, excessive mental work, or insufficient rest. With the accelerated pace of modern life and increased work pressure, the number of people suffering from chronic fatigue syndrome (CFS) and in a sub-healthy state is increasing. Furthermore, with the growing awareness of exercise and health, how to recover after high-intensity exercise has also attracted attention. Anti-fatigue foods not only replenish energy and meet nutritional needs but also effectively relieve fatigue, thus gaining popularity among consumers.

[0003] Wheat oligopeptides can be used to delay muscle fatigue, making their preparation a hot research topic in the field. Existing methods for extracting wheat peptides include physical, chemical, and enzymatic methods. Due to safety concerns, single-stage enzymatic hydrolysis is the primary method for wheat peptide extraction. For example, Chinese patent CN 107114555 A describes a biomimetic enzymatic hydrolysis method for preparing wheat peptide powder. Wheat protein is used as the starting material, and the process involves single-stage biomimetic enzymatic hydrolysis, high-speed centrifugation, ultrafiltration, electrodialysis, concentration, and drying to obtain wheat peptide powder. This wheat peptide powder has a total protein content of over 90% and a trichloroacetic acid-soluble small molecule peptide content of over 85%, making it suitable for use in food, health food, and pharmaceuticals. While the above method can obtain wheat peptides, the yield is low, the taste is poor, and the amount of active ingredients is limited. Therefore, improving the yield, taste, and active ingredients of wheat peptides is a pressing problem to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing wheat oligopeptides. Using gluten powder as the experimental subject, wheat oligopeptides with anti-fatigue effects are prepared by a compound enzyme method. The method is simple and practical, and the product prepared by this method has a good taste and high yield, which is conducive to promoting the rapid development of wheat oligopeptides in the field of functional foods.

[0005] This invention provides a method for preparing wheat oligopeptides, comprising the following steps: The gluten powder is mixed with water, soaked, and then denatured in a water bath to obtain a denatured liquid. The liquid was subjected to a first enzymatic hydrolysis, a second enzymatic hydrolysis, enzyme inactivation, and centrifugation to obtain wheat peptide hydrolysate. The wheat peptide hydrolysate was subjected to ultrafiltration and drying to obtain wheat oligopeptides. The first enzymatic hydrolysis used included Bacillus subtilis protease and bromelain. The second enzymatic digestion uses a second complex enzyme including trypsin, papain, and flavor protease.

[0006] Preferably, in the first complex enzyme, the mass ratio of Bacillus subtilis protease to bromelain is 0.5~1.5:1.0~2.0; In the second complex enzyme, the mass ratio of trypsin, papain and flavor protease is 1.0~2.0:0.5~1.0:0.5~1.5.

[0007] Preferably, the amount of the first compound enzyme added is 0.5% to 1.5% of the dry weight of the wheat gluten; The amount of the second complex enzyme added is 1.0% to 3.0% of the dry weight of the gluten powder.

[0008] Preferably, during the first enzymatic hydrolysis, the pH of the solution is 6.0~8.0; the temperature of the first enzymatic hydrolysis is 50~80℃; and the time of the first enzymatic hydrolysis is 1~2 hours. During the second enzymatic hydrolysis, the pH of the solution is 6.5~9.0; the temperature is 50~70℃; and the time is 2~5 hours.

[0009] Preferably, during the mixed soaking, the mass ratio of the gluten powder to water is 1:5~15; The mixing and soaking process is accompanied by stirring; the mixing and soaking time is 1-2 hours.

[0010] Preferably, the water bath denaturation temperature is 80~100℃; the water bath denaturation time is 1~2h.

[0011] Preferably, ultrafiltration is performed using a 0.22-0.45 μm filter membrane to obtain components with a molecular weight <3 kDa.

[0012] Preferably, the gluten powder is the undersize material after passing through a 40-60 mesh sieve.

[0013] This invention provides wheat oligopeptides prepared by the method described in the above technical solution.

[0014] This invention provides the application of the wheat oligopeptides described in the above technical solution in the preparation of products with anti-fatigue effects.

[0015] Beneficial effects: This invention provides a method for preparing wheat oligopeptides, comprising the following steps: soaking and water-mixing gluten powder, followed by water bath denaturation to obtain a denatured liquid; subjecting the liquid to a first enzymatic hydrolysis, a second enzymatic hydrolysis, enzyme inactivation, and centrifugation to obtain a wheat peptide hydrolysate; subjecting the wheat peptide hydrolysate to ultrafiltration and drying to obtain wheat oligopeptides; wherein the first enzymatic hydrolysis uses a first complex enzyme comprising Bacillus subtilis protease and bromelain; and the second enzymatic hydrolysis uses a second complex enzyme comprising trypsin, papain, and flavor protease.

[0016] Compared with the prior art, the advantages of the present invention are as follows: 1) This invention uses a complex enzyme with specific components and performs stepwise enzymatic hydrolysis, specifically including: in the first step of enzymatic hydrolysis, Bacillus subtilis protease can directionally cleave the carboxyl terminus of hydrophobic amino acids (such as leucine and phenylalanine), while bromelain tends to act on aliphatic amino acids. The synergistic use of the two enzymes can accelerate the degradation of proteins in gluten powder; in the second step of enzymatic hydrolysis, trypsin can further cleave large peptides in the hydrolysate into smaller peptides, and the use of flavor protease can reduce the production of bitter peptides and improve the taste of the product; papain can further degrade hydrolysates the hydrolysate while also improving the unpleasant flavor of the hydrolysate.

[0017] 2) This invention utilizes centrifugation and ultrafiltration technologies to separate and retain low-molecular-weight enzyme hydrolysates containing active peptides. These active ingredients not only help improve endurance and increase exercise time, but also improve energy metabolism, delay the accumulation of lactic acid, urea nitrogen, and creatine kinase after exercise, and increase glycogen storage, exhibiting a certain anti-fatigue ability on a macroscopic scale. Therefore, the technical solution provided by this invention shows the potential to become a natural anti-fatigue food ingredient in the field of functional foods. Detailed Implementation

[0018] Unless otherwise specified, all raw materials and equipment used in this invention are conventionally purchased.

[0019] To further illustrate the present invention, the solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Example 1 A method for preparing wheat oligopeptides, comprising the following steps: (1) Homogenization: Weigh 200g of wheat gluten after passing it through a 60-mesh sieve, add water that is 10 times the weight of the wheat gluten (i.e., 2000g of water), and stir slowly during the soaking process. After stirring for 1 hour, a uniform liquid is obtained. (2) Denaturation: The homogeneous liquid is placed in a water bath for denaturation treatment at a temperature of 95°C for 1 hour to obtain the denatured liquid. (3) Enzymatic hydrolysis: Stepwise enzymatic hydrolysis using a compound enzyme: After adjusting the pH of the denatured solution to 6.8, the first complex enzyme was added for the first enzymatic hydrolysis. The first complex enzyme is composed of Bacillus subtilis protease and bromelain in a mass ratio of 1.0:1.0; the amount of the first complex enzyme added is 0.5% of the dry weight of the wheat gluten; the temperature of the first enzymatic hydrolysis is 60℃ and the time is 2h to obtain the first enzymatic hydrolysate. The pH of the first enzymatic hydrolysate was adjusted to 8.0, and then a second complex enzyme was added for a second enzymatic hydrolysis. The second complex enzyme is composed of trypsin, papain and flavor protease in a mass ratio of 1.0:0.5:1.0; the amount of the second complex enzyme added is 2.0% of the dry weight of the gluten powder; the second enzymatic hydrolysis is carried out at a temperature of 65℃ for 4 hours to obtain the second enzymatic hydrolysate. (4) Enzyme inactivation: The second enzyme hydrolysate was inactivated at a high temperature of 95℃ for 15 minutes to obtain the enzyme hydrolysate after enzyme inactivation. (5) Centrifugation: Centrifuge the enzyme hydrolysate at high speed (6000 rpm, 15 min) to obtain the supernatant, which is a preliminary wheat peptide hydrolysate; (6) Ultrafiltration: The wheat peptide hydrolysate was filtered through a nylon filter membrane with a pore size of 0.22 μm, and the components were collected; (7) Drying: Wheat oligopeptide dry powder is obtained by spray drying.

[0021] Comparative Example 1 A method for preparing wheat oligopeptides, comprising the following steps: The only difference from Example 1 is that step (3) of Comparative Example 1 only involves the first enzymatic hydrolysis, as detailed below: (3) Enzymatic hydrolysis: single enzyme hydrolysis is used: The pH of the denatured solution was adjusted to 6.8, and then a compound enzyme was added for enzymatic hydrolysis. The compound enzyme consisted of Bacillus subtilis protease and bromelain in a mass ratio of 1.0:1.0. The amount of compound enzyme added was 0.5% of the dry weight of the wheat gluten. The hydrolysis temperature was 60℃ and the time was 2 hours to obtain the first hydrolysate. The first hydrolysate was then subjected to enzyme inactivation, centrifugation, ultrafiltration, and drying.

[0022] Comparative Example 2 The only difference from Example 1 is that step (3) of Comparative Example 2 only involves a second enzymatic hydrolysis, as detailed below: (3) Enzymatic hydrolysis: single enzyme hydrolysis is used: After denaturation, the pH of the solution was adjusted to 8.0, and a compound enzyme was added for enzymatic hydrolysis. The compound enzyme consisted of trypsin, papain, and flavor protease in a mass ratio of 1.0:0.5:1.0. The amount of compound enzyme added was 2.0% of the dry weight of the gluten powder. The hydrolysis temperature was 65℃ and the time was 4 hours to obtain the hydrolysate. The hydrolysate was then subjected to enzyme inactivation, centrifugation, ultrafiltration, and drying.

[0023] Comparative Example 3 The difference from Example 1 is that the first enzymatic hydrolysis time in step (3) of Comparative Example 3 is 3 hours.

[0024] Comparative Example 4 The difference from Example 1 is that the second enzymatic hydrolysis time in step (3) of Comparative Example 4 is adjusted to 1 hour.

[0025] Comparative Example 5 The difference from Example 1 is that the enzymatic hydrolysis process in step (3) of Comparative Example 5 is as follows: After adjusting the pH of the denatured solution to 8.0, a second complex enzyme was added for the first enzymatic hydrolysis. The second complex enzyme is composed of trypsin, papain and flavor protease in a mass ratio of 1.0:0.5:1.0; the amount of the second complex enzyme added is 2.0% of the dry weight of the gluten powder; the temperature of the second enzymatic hydrolysis is 65℃ and the time is 4h to obtain the first enzymatic hydrolysate. The pH of the first enzymatic hydrolysate was adjusted to 6.8, and then the first complex enzyme was added for a second enzymatic hydrolysis. The first complex enzyme consists of Bacillus subtilis protease and bromelain in a mass ratio of 1.0:1.0; the amount of the first complex enzyme added is 0.5% of the dry weight of the wheat gluten; the first enzymatic hydrolysis is carried out at a temperature of 60℃ for 2 hours to obtain the second enzymatic hydrolysate.

[0026] Comparative Example 6 The difference from Example 1 is that in step (4) of Comparative Example 6: The second enzymatic hydrolysate was inactivated at a high temperature of 100℃ for 20 minutes.

[0027] Test Example 1 The yields of wheat oligopeptides prepared in Example 1 and Comparative Examples 1-6 were statistically analyzed, and the results are shown in Table 1. The extraction yield test formula is as follows: Wheat oligopeptide yield = Product mass (g) / Prion powder mass (g) Table 1. Extraction yield results for each embodiment and comparative example.

[0028] As can be seen from the data in Table 1, the yield of wheat oligopeptides is the highest when prepared using the technical solution provided by this invention.

[0029] Test Example 2 The procedure for determining the molecular weight of wheat oligopeptides is as follows: Preparation of standards and sample solutions: Accurately weigh 0.0100 g of each molecular weight standard, dissolve and dilute to 10 mL with the mobile phase (acetonitrile-water-trifluoroacetic acid = 45:55:0.1, v / v / v) to prepare a 1.0 mg / mL standard solution. Filter the solution through a 0.22 μm organic phase filter membrane and degas by sonication for 10 min. Weigh 0.0500 g of the standard sample and prepare a 5.0 mg / mL sample solution using the same method. The sample solution is then processed and ready for analysis.

[0030] Chromatographic conditions: Gel permeation column (300 mm × 7.8 mm, 5 μm, pore size 100 Å); mobile phase: acetonitrile-water-trifluoroacetic acid (45:55:0.1, v / v / v); flow rate: 0.5 mL / min; column temperature: 30 °C; detector temperature: 30 °C; injection volume: 20 μL; run time: 30 min.

[0031] The molecular weight of the wheat oligopeptide dry powder prepared in Example 1 was determined using the above method, and the results are shown in Table 2; the molecular weight of the wheat oligopeptide dry powder prepared in Comparative Example 1 was determined using the above method, and the results are shown in Table 3.

[0032] Table 2. Molecular weight and distribution range of samples from the examples

[0033] Table 3. Molecular weight and distribution range of comparative samples

[0034] The bioactivity of wheat oligopeptides is closely related to their molecular weight and distribution. Based on the results in Tables 2 and 3, the average molecular weight of the wheat oligopeptides prepared in Example 1 is 343 Da, with components below 3000 Da accounting for more than 90%. This will help expose more active groups, promote short-range interactions between amino acids, and enhance their functional activity. In contrast, the average molecular weight of the wheat oligopeptides prepared in Comparative Example 1 is 460 Da, with a more dispersed distribution of molecular weights below 3000 Da, which is detrimental to their bioactivity.

[0035] Test Example 3 The animal groups and administration methods are as follows: Sixty male ICR mice, weighing approximately (20±2) g, were randomly divided into six groups according to their body weight: a control group (distilled water, same dose), a positive control group (whey protein, 2 mg / g / d), and three control groups (low dose (2 mg / g / d), medium dose (6 mg / g / d), and high dose (10 mg / g / d) of wheat oligopeptides prepared in Example 1. A control group (5 mg / g / d) of wheat oligopeptides prepared in Comparative Example 1 was also included. Each group consisted of ten mice. Different doses of the test substance were administered according to the mice's weight via gavage at a rate of 0.1 mL / 10 g body weight, once daily for 30 consecutive days. During the gavage period, mice had free access to food and water. The specific animal experimental design is shown in Table 4.

[0036] Table 4 Animal Experiment Design

[0037] (1) Exhaustion Swimming Experiment Thirty minutes after the last gavage, mice in group A were washed with soapy water to remove surface oils, and a wire was tied to their tails to provide a weight of 5% of their body weight. They were then placed in a swimming tank at 25°C and approximately 30cm depth to prevent them from touching the bottom for support. Swimming time was recorded immediately when the mice were exhausted and unable to remain in the water for more than 10 seconds. The results are shown in Table 5.

[0038] Table 5. Exhaustion swimming time of mice in different treatment groups

[0039] Note: In Table 5, * indicates a highly significant difference compared to the control group (p<0.01).

[0040] The effect of wheat oligopeptides on the anti-fatigue ability of mice was assessed using the exhaustive swimming time. Based on the data in Table 5, it was found that, compared to the control group, low, medium, and high doses of wheat oligopeptides significantly increased the exhaustive swimming time of mice, with extensions of 45.76%, 74.17%, and 99.63%, respectively. In contrast, the swimming time of the control group was only extended by 26.20%. Within the dosage range of wheat oligopeptides used in this experiment, the exhaustive swimming time of mice showed a dose-dependent effect.

[0041] (2) Determination of relevant biochemical indicators Thirty minutes after the last gavage, blood was collected from the eyeballs of mice in group B of each experimental group. After standing at room temperature for 15 minutes, the supernatant was collected by centrifugation at 10000×g. Store at 80℃ for later use. Then, immediately euthanize the mice after blood collection, remove the liver and hind leg muscles, add the corresponding volumes of physiological saline at a 1:9 ratio, and homogenize to prepare a 10% tissue homogenate. Store at 80℃ for later use. The levels of lactate, blood urea nitrogen, creatine kinase, muscle glycogen, and liver glycogen in mouse serum were determined using the methods provided in the test kit. The results are shown in Table 6.

[0042] Table 6. Effects of wheat oligopeptides on anti-fatigue related biochemical indicators in mice.

[0043] Note: In Table 6, * indicates a significant difference compared to the control group. p <0.05, ** indicates a highly significant difference (p<0.01).

[0044] After strenuous exercise, energy demand is high and oxygen consumption increases, leading to enhanced anaerobic glycolysis. Muscle glycogen is continuously depleted and broken down into lactic acid, and creatine kinase levels rise. Simultaneously, to meet energy demands during exercise, protein metabolism is enhanced, resulting in increased blood urea nitrogen levels in the liver and glycogenolysis to maintain blood glucose levels. The results in Table 6 show that, compared to the control and comparative groups, different doses of wheat oligopeptides decreased lactic acid, creatine kinase, and liver urea nitrogen levels, while muscle and liver glycogen levels increased. This indicates that wheat oligopeptides can improve energy metabolism by reducing the accumulation of metabolic products and the breakdown of proteins, thereby delaying the accumulation of lactic acid, urea nitrogen, and creatine kinase, and increasing glycogen stores in the liver and skeletal muscle, thus exerting an anti-fatigue effect.

[0045] In summary, the wheat oligopeptides provided by this invention have a high yield, good taste, and a high proportion of low molecular weight active ingredients. Therefore, these wheat oligopeptides can help improve endurance, increase exercise time, improve energy metabolism, delay the accumulation of lactic acid, urea nitrogen, and creatine kinase in the body after exercise, and increase glycogen storage. Macroscopically, they exhibit a certain anti-fatigue ability and show potential as a natural anti-fatigue food ingredient in the field of functional foods.

[0046] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing wheat oligopeptides, characterized in that, Includes the following steps: The gluten powder is mixed with water, soaked, and then denatured in a water bath to obtain a denatured liquid. The liquid was subjected to a first enzymatic hydrolysis, a second enzymatic hydrolysis, enzyme inactivation, and centrifugation to obtain wheat peptide hydrolysate. The wheat peptide hydrolysate was subjected to ultrafiltration and drying to obtain wheat oligopeptides. The first enzymatic hydrolysis used included Bacillus subtilis protease and bromelain. The second enzymatic digestion uses a second complex enzyme including trypsin, papain, and flavor protease.

2. The preparation method according to claim 1, characterized in that, In the first complex enzyme, the mass ratio of Bacillus subtilis protease to bromelain is 0.5~1.5:1.0~2.0; In the second complex enzyme, the mass ratio of trypsin, papain and flavor protease is 1.0~2.0:0.5~1.0:0.5~1.

5.

3. The preparation method according to claim 1 or 2, characterized in that, Based on the dry weight of wheat gluten, the amount of the first compound enzyme added is 0.5% to 1.5% of the dry weight of wheat gluten; The amount of the second complex enzyme added is 1.0% to 3.0% of the dry weight of the gluten powder.

4. The preparation method according to claim 1, characterized in that, During the first enzymatic hydrolysis, the pH of the solution was 6.0–8.0; the temperature was 50–80℃; and the time was 1–2 hours. During the second enzymatic hydrolysis, the pH of the solution is 6.5~9.0; the temperature is 50~70℃; and the time is 2~5 hours.

5. The preparation method according to claim 1, characterized in that, When soaking the mixture, the mass ratio of the gluten powder to water is 1:5~15; The mixing and soaking process is accompanied by stirring; the mixing and soaking time is 1-2 hours.

6. The preparation method according to claim 1, characterized in that, The water bath denaturation temperature is 80~100℃; the water bath denaturation time is 1~2h.

7. The preparation method according to claim 1, characterized in that, Ultrafiltration was performed using a 0.22-0.45 μm filter membrane to obtain components with a molecular weight <3 kDa.

8. The preparation method according to claim 1, characterized in that, The gluten powder is the material that passes through a sieve of 40-60 mesh.

9. The wheat oligopeptide obtained by the preparation method according to claims 1-8.

10. The use of the wheat oligopeptide according to claim 9 in the preparation of products with anti-fatigue effects.

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