Preparation method of walnut anti-fatigue peptide

Abstract

The invention discloses a preparation method of walnut anti-fatigue peptide, which comprises the following steps: S1, crushing cold-pressed walnut meal, and sieving with a 20-60-mesh sieve to obtain walnut meal powder; s2, adding water into the walnut meal powder according to a solid-liquid ratio of 1: 0.8-1: 1.2 (g / mL), and uniformly stirring to obtain a semi-solid walnut meal matrix; s3, uniformly mixing bacterial liquid of at least one fungus and bacterial liquid of at least two bacteria according to a certain proportion, adding the mixture into the semi-solid walnut meal matrix, and carrying out synergistic fermentation at 28-37 DEG C for 48-72 hours to obtain a fermented product; and S4, performing fractionation on the fermented product by adopting an ultrafiltration membrane method to obtain polypeptide components of different molecular weight segments, and obtaining the anti-fatigue polypeptide from the polypeptide components. The preparation method of the walnut anti-fatigue peptide is simple and convenient to operate, green, environmentally friendly and low in production cost, efficient utilization of walnut protein resources is achieved, and the anti-fatigue effect of the anti-fatigue peptide is remarkable.

Description

Technical Field

[0001] The present disclosure relates to the technical field of preparation of functional active peptides, and in particular to a method for preparing a walnut anti-fatigue peptide. Background Art

[0002] In recent years, with the accelerated pace of life and increased work pressure, the research and development of anti-fatigue functional foods has received widespread attention. Anti-fatigue peptides, as bioactive polypeptides, have become a research hotspot in the functional food field due to their easy absorption and rapid onset of action. Walnuts, a traditional nutritious food, are rich in protein, unsaturated fatty acids, and various bioactive substances. Their proteins can be hydrolyzed by enzymes or fermented by microorganisms to produce bioactive peptides with specific functions. However, current methods for preparing walnut peptides suffer from low efficiency, high cost, and unstable functional activity, limiting their application in functional foods.

[0003] Specifically, current existing technologies often use enzymatic hydrolysis or fermentation with a single bacterial strain to produce walnut peptides. These methods fail to fully utilize the protein resources in walnut meal, resulting in low peptide yields and relatively limited biological activity, most of which rarely involves anti-fatigue activity. Therefore, it is necessary to develop an economical, stable, and efficient method for producing walnut anti-fatigue peptides.

[0004] The disclosure of the above background technology content is only used to assist in understanding the inventive concept and technical solution of the present invention. It does not necessarily belong to the prior art of this patent application. In the absence of clear evidence that the above content has been disclosed on the filing date of this patent application, the above background technology should not be used to evaluate the novelty and creativity of this application. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing walnut anti-fatigue peptides to solve at least one of the above-mentioned background technical problems.

[0006] To solve the above problems, the technical solution of the present invention is achieved as follows: A method for preparing walnut anti-fatigue peptide comprises the following steps: S1, the cold-pressed walnut meal crushed, through a 20-60 mesh sieve to obtain walnut meal powder; S2. adding water to the walnut meal powder at a material-liquid ratio of 1:0.8 to 1:1.2 (g / mL), and stirring evenly to obtain a semi-solid walnut meal matrix; S3, mixing bacterial liquids of at least one fungus and at least two bacteria in a certain proportion, adding the mixture to the semi-solid walnut meal matrix, and co-fermenting at 28-37° C. for 48-72 hours to obtain a fermentation product; S4. The fermentation product is fractionated by an ultrafiltration membrane method to obtain polypeptide components of different molecular weight segments, and an anti-fatigue polypeptide is obtained from the polypeptide components.

[0007] In some embodiments, in step S2, water is added to the walnut meal powder so that the moisture content of the walnut meal is 44%-54%, and the mixture is stirred evenly to obtain a semi-solid walnut meal matrix.

[0008] In some embodiments, in step S3, the fungus Rhizopus chinensis and the bacterial solutions of Bacillus subtilis and Lactobacillus plantarum are mixed in a ratio of 2:1:1, added to the semi-solid walnut meal, and cultured at a constant temperature of 28-37°C for 48-72 hours to obtain a fermentation product.

[0009] In some embodiments, step S3 includes: S30, inoculate Rhizopus chinensis on potato dextrose agar (PDA) medium, wait for spores to mature, wash off the Rhizopus chinensis spores on the PDA medium with sterile water, count them with a hemocytometer, and adjust the spore concentration to 10 7 / mL, for future use; S31, inoculate Bacillus subtilis into 100mL of sterilized nutrient broth medium, then inoculate Lactobacillus plantarum into 100mL of sterilized MRS broth medium, culture at 37°C and 180r / min for 24h, adjust the bacterial solution concentration to 10 with sterile saline. 7 cfu / mL, spare; S32. The three bacterial suspensions prepared in steps S30 and S31 are mixed in a ratio of 2:1:1, 5%-10% of the mixed bacterial suspension is inoculated into the sterilized semi-solid walnut meal matrix, and cultured at a constant temperature of 28-37° C. for 48-72 h.

[0010] In some embodiments, in step S4, the fermentation product is fractionated by ultrafiltration membrane method to obtain walnut polypeptide components with four molecular weight ranges: MW>5 kDa, MW=3-5 kDa, MW=1-3 kDa and MW<1 kDa.

[0011] In some embodiments, in step S4, the anti-fatigue effects of the walnut polypeptide components in the four molecular weight ranges are measured to screen out polypeptide components with good anti-fatigue effects.

[0012] In some embodiments, the polypeptide sequences of the polypeptide components screened out with good anti-fatigue effects are identified, and then biological activity evaluation and molecular docking are performed, and novel peptides with high scores and good molecular docking binding energy are screened out for solid phase synthesis.

[0013] In some embodiments, the amino acid sequences of the screened novel peptides are RESWPGSR and PLWRLF, respectively.

[0014] In some embodiments, components with MW < 1 kDa are selected as determined by anti-fatigue effects.

[0015] In some embodiments, polypeptide components with MW <1 kDa are identified and analyzed using liquid chromatography-mass spectrometry, using a Hola C18 column as the separation column and 0.05% formic acid aqueous solution and 0.05% formic acid-acetonitrile solution as the mobile phases. The relative molecular mass information and amino acid composition of the enzymatically hydrolyzed polypeptides are determined based on the primary and secondary fragment ion information of the mass spectrometer.

[0016] The beneficial effects of the technical solution of the present invention are: Compared with the existing technology, the preparation method of the walnut anti-fatigue peptide of the present invention is simple to operate, green and environmentally friendly, has low production cost, realizes the efficient utilization of walnut protein resources, and the anti-fatigue effect of the anti-fatigue peptide is significant.

[0017] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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 the embodiments or the description of the prior art. 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 inventive work. The drawings herein are incorporated into and constitute a part of this specification, illustrate embodiments consistent with the present invention, and together with the specification, are used to explain the principles of the present invention.

[0019] Figure 1 Exhaustive swimming time of mice treated with mixed peptides; Figure 2 Serum urea nitrogen content of mice treated with mixed peptides; Figure 3 The activity of lactate dehydrogenase in the serum of mice treated with mixed peptides; Figure 4 The liver glycogen content of mice treated with mixed peptides; Figure 5 The muscle glycogen content of mice treated with mixed peptides; Figure 6 The molecular docking results of the novel peptide ESWPGSR from fermented walnut meal and LDH protein; Figure 7 The molecular docking results of the novel peptide PLWRLF from fermented walnut meal and LDH protein are shown; Figure 8Exhaustive swimming time for mice treated with two novel peptides; Figure 9 Serum urea nitrogen content in mice treated with two novel peptides; Figure 10 The activity of lactate dehydrogenase in the serum of mice treated with two novel peptides; Figure 11 The liver glycogen content of mice treated with two novel peptides; Figure 12 Muscle glycogen content in mice treated with two novel peptides. DETAILED DESCRIPTION

[0020] In order to make the technical problems, technical solutions and beneficial effects to be solved by the embodiments of the present invention more clear and understandable, and to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0021] It should be noted that the terms "first," "second," and the like in the description and claims of the present invention are used to distinguish similar objects and are not necessarily used to describe a specific order or precedence. It should be understood that the terms used in this manner are interchangeable where appropriate so that the embodiments of the present invention described herein can be implemented in an order other than those described herein. In addition, the terms "including" and "having," as well as any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to these processes, methods, products, or apparatus.

[0022] The cold-pressed walnut meal involved in the embodiments can be directly purchased on the market.

[0023] The present invention provides a method for preparing a walnut anti-fatigue peptide, comprising the following steps: S1, the cold-pressed walnut meal crushed, through a 20-60 mesh sieve to obtain walnut meal powder; S2, adding water to the walnut meal powder at a material-liquid ratio of 1:0.8 to 1:1.2 (g / mL), stirring evenly to obtain a semi-solid walnut meal matrix; In some embodiments, water is added to the walnut meal powder so that the moisture content of the walnut meal is 44%-54%, and the mixture is stirred to obtain a semi-solid walnut meal matrix; Specifically, in one embodiment, 10 g of walnut meal powder was weighed into a conical flask, 8-12 mL of sterile water was added, and the mixture was stirred evenly to obtain a semi-solid walnut meal matrix.

[0024] S3, mixing bacterial liquids of at least one fungus and at least two bacteria in a certain proportion, adding the mixture to the semi-solid walnut meal matrix, and co-fermenting at 28-37° C. for 48-72 hours to obtain a fermentation product; Specifically, in an embodiment of the present invention, the bacterial liquid of the fungus Rhizopus chinensis CICC3142 and the bacteria Bacillus subtilis CICC10732 and Lactobacillus plantarum CICC24936 are mixed in a ratio of 2:1:1, added to semi-solid walnut meal, and cultured at a constant temperature of 28-37°C for 48-72 hours to obtain a fermentation product.

[0025] S4. The fermentation product is fractionated by ultrafiltration membrane method to obtain polypeptide components of different molecular weight segments, and anti-fatigue polypeptides with amino acid sequences of RESWPGSR and PLWRLF are obtained from the polypeptide components.

[0026] By combining the fungus (Rhizopus chinensis) with bacteria (Bacillus subtilis and Lactobacillus plantarum) to ferment cold-pressed walnut meal, a collaborative fermentation method was developed to not only increase the utilization of walnut protein but also yield a novel walnut peptide with anti-fatigue activity. Using cold-pressed walnut meal as the raw material not only reduces production costs but also enables efficient recycling of walnut protein resources. The collaborative fermentation of Rhizopus chinensis, Bacillus subtilis, and Lactobacillus plantarum increased walnut peptide yield, yielding a walnut peptide with anti-fatigue activity, providing new insights for the industrial production and application of walnut peptides.

[0027] In some embodiments, step S3 includes: S30, inoculate Rhizopus chinensis CICC3142 on potato dextrose agar (PDA) medium. After the spores mature, wash the Rhizopus chinensis spores on the PDA medium with sterile water, count them with a hemocytometer, and adjust the spore concentration to 10 7 / mL, for future use; S31. Inoculate Bacillus subtilis CICC10732 into 100 mL of sterilized nutrient broth medium, and then inoculate Lactobacillus plantarum CICC24936 into 100 mL of sterilized MRS broth medium. Incubate at 37°C and 180 rpm for 24 h. Adjust the bacterial solution concentration to 10 with sterile saline. 7 cfu / mL, spare; S32. Mix the three bacterial suspensions prepared in steps S30 and S31 in a ratio of 2:1:1, inoculate 5%-10% of the mixed bacterial suspension into the sterilized semi-solid walnut meal matrix, and culture at a constant temperature of 28-37° C. for 48-72 hours.

[0028] In some embodiments, in step S4, the fermentation product is fractionated by ultrafiltration membrane method to obtain walnut polypeptide components with four molecular weight ranges: MW>5 kDa, MW=3-5 kDa, MW=1-3 kDa and MW<1 kDa.

[0029] In some embodiments, step S4 includes: By measuring the anti-fatigue effects of the walnut polypeptide components in the four molecular weight segments, polypeptide components with good anti-fatigue effects are screened out, and the polypeptide sequences therein are identified. Then, biological activity evaluation and molecular docking are performed, and new peptides with high scores and good molecular docking binding energy are screened out for solid-phase synthesis.

[0030] In some embodiments, the amino acid sequences of the screened novel peptides are RESWPGSR and PLWRLF, respectively.

[0031] The present invention prepares anti-fatigue peptides by synergistically fermenting walnut meal with Rhizopus chinensis, Bacillus subtilis, and Lactobacillus plantarum. This synergistic fermentation method for preparing anti-fatigue peptides is novel, simple to operate, low-cost, environmentally friendly, and has significant anti-fatigue effects. Furthermore, it should be noted that the anti-fatigue peptides focus on regulating energy metabolism and intervening in fatigue signaling pathways, directly improving physiological function.

[0032] The following describes the determination of the anti-fatigue effect of the walnut peptide of the present invention.

[0033] Anti-fatigue effect measurement experiment example 1 Fifty healthy, specific pathogen-free (SPF) ICR male mice, weighing 18-22 g initially, were housed for one week at a temperature of 23 ± 2°C, a humidity of 50-60%, a 12-h light / dark cycle, and free access to food and water. They were then randomly divided into five groups of 10 mice each.

[0034] Group 1: Control; Group 2: MW > 5kDa; Group 3: MW = 3-5kDa; Group 4: MW = 1-3kDa; Group 5: MW < 1kDa. Each group received 200 mg / kg / day of walnut polypeptide by gavage, while the control group received sterile water. The gavage cycle lasted four weeks.

[0035] (1) Weighted swimming test During the mice's housing period, they underwent weekly swimming training. In the first week, they underwent unloaded swimming training. In the second week, a metal block weighing 2% of their body weight was placed on the posterior third of their tail. In the third week, a metal block weighing 4% of their body weight was placed on the tail. In the fourth week, a metal block weighing 5% of their body weight was placed on the tail. Each training session was conducted to exhaustion, with exhaustion defined as the mouse sinking for 3 seconds without surfacing. The weighted swimming time was recorded.

[0036] The exhaustive swimming time of mice in each group is shown in Figure 1 Compared to the control group, the exhaustive swimming time of mice in all four groups receiving walnut peptides was increased. The group receiving peptides with a MW < 1 kDa had the longest exhaustive swimming time of 32 minutes, 22 minutes longer than the control group. This suggests that the walnut peptides obtained by this method have a strong anti-fatigue effect.

[0037] (2) Serum biochemical factor detection After the mice completed the last weight-bearing swim, they were killed, blood was collected from the eyeballs, and the biochemical factors in the serum were measured using enzyme-linked detection kits. Excessive fatigue or strenuous exercise can increase the level of urea nitrogen (BUN) in the serum. The BUN test results are as follows: Figure 2 As shown, the serum BUN level of mice in the different molecular weight polypeptide treatment groups was significantly reduced, especially the polypeptide group with MW < 1kDa had the most significant reduction effect, which was 35.4% lower than the control group.

[0038] Lactate dehydrogenase is an enzyme that catalyzes the reversible reaction between lactate and pyruvate. It plays an important role in energy metabolism. During strenuous exercise or prolonged activity, muscle cells produce a large amount of lactic acid. Lactate dehydrogenase can promote the metabolism of lactic acid, maintain the acid-base balance in cells, and prevent acidosis caused by lactic acid accumulation. The results of lactate dehydrogenase activity test in each group of mice are as follows: Figure 3 The test results show that the lactate dehydrogenase activity of the control group was 859.3U / L, while the lactate dehydrogenase activity of the mice in the walnut polypeptide-treated groups was significantly enhanced. The lactate dehydrogenase activity of the MW<1kDa group was 1371.67U / L, which was 59.62% higher than that of the control group.

[0039] (3) Determination of liver glycogen and muscle glycogen content Liver glycogen and muscle glycogen play an important role in anti-fatigue. They are both important forms of energy storage in the body and can quickly release energy when needed, helping to maintain normal body functions and delay the occurrence of fatigue. The liver and muscle of each group of mice were taken, sterile 0.9% sodium chloride solution was added, and homogenized at 4°C. The content of liver glycogen and muscle glycogen was determined according to the instructions of the relevant ELISA kit. The results are as follows Figure 4 and Figure 5 The results showed that compared with the control group, all four peptide groups increased the content of liver glycogen and muscle glycogen. Among them, the peptide group with MW < 1KDa had the best effect, with the content of liver glycogen and muscle glycogen reaching 20.79mg / g and 7.09mg / g, respectively, which were 2.08 times and 1.88 times that of the control group.

[0040] In summary, walnut polypeptides of different molecular weights prepared by this method all have a certain degree of anti-fatigue effect, among which the component with MW < 1 kDa has the best anti-fatigue effect.

[0041] As an embodiment of the present invention, the method further comprises: screening anti-fatigue peptides to obtain novel peptides RESWPGSR and PLWRLF.

[0042] Specifically, peptide components with a MW <1 kDa were identified. Liquid chromatography-mass spectrometry (LC-MS / MS) analysis was performed using a Hola C18 column (100 mm × 2.1 mm, 2.7 μm) with 0.05% formic acid in water and 0.05% formic acid in acetonitrile as the mobile phases. The relative molecular mass and amino acid composition of the enzymatically digested peptides were determined based on the primary and secondary fragment ion information from the mass spectrometer. LC-MS / MS analysis yielded 556 peptide sequences with ≤10 amino acids.

[0043] Peptide sequences with a bioactivity score > 0.5 were screened (screened through the bioactive peptide website http: / / distilldeep.ucd.ie / PeptideRanker). The crystal structure of the anti-fatigue-related LDH protein (PDB ID: 7EPM) was downloaded from the PDB database (http: / / www.rcsb.org). PyMOL2.3.0 software was used to delete water molecules, irrelevant protein chains and original ligands in the protein structure for molecular docking. The 3D structure file of the ligand small molecule compound was drawn by ChemDraw 2022 software, and the molecular force field optimization of the small molecule structure was performed using Chem3D 2022 software to finally obtain the optimal molecular structure with the lowest energy state. Molecular docking was performed using AutoDock Vina1.2.0 software to obtain the docking binding free energy and molecular docking schematic diagram. Two new peptides RESWPGSR and PLWRLF with low docking binding energy and not yet reported were screened for solid-phase synthesis for subsequent animal experimental verification. Among them, the results of the docking of the two new peptides with the LDH protein molecule are shown in Figure 6 and Figure 7 .

[0044] To validate the novel peptide's anti-fatigue properties, 40 healthy, specific pathogen-free (SPF) ICR male mice, initially weighing 18-22 g, were housed for one week at a temperature of 23 ± 2°C, 50-60% humidity, a 12-hour light / dark cycle, and free access to food and water. The mice were then randomly divided into four groups, each containing 10 mice. Group 1: control group, receiving sterile water by gavage; Group 2: low-dose (L) group, receiving 50 mg / kg / day by gavage; Group 3: medium-dose (M) group, receiving 100 mg / kg / day; and Group 4: high-dose (H) group, receiving 200 mg / kg / day. The gavage cycle lasted four weeks.

[0045] (1) Weighted swimming test The specific method is the same as the weight-bearing swimming test in Example 1, which will not be repeated here. The exhaustive swimming time of mice in each group treated with the novel peptide RESWPGSR and PLWRLF is shown in Figure 8 The results showed that the exhaustive swimming time of the control group was 10.3 minutes. The exhaustive swimming time of mice treated with the novel peptide RESWPGSR was significantly prolonged with increasing peptide doses, with the exhaustive swimming time of mice in the high-dose group reaching 27.3 minutes. Similarly, after treatment with the novel peptide PLWRLF, the exhaustive swimming time of mice was also prolonged, showing a dose-dependent increase, with the exhaustive swimming time of mice at the highest dose reaching 24.7 minutes. This indicates that the two novel peptides have a good anti-fatigue effect.

[0046] (2) Serum biochemical factor detection After the mice completed the last weight-bearing swimming test, they were killed, blood was collected from the eyeballs, and the levels of urea nitrogen (BUN) and lactate dehydrogenase (LDH) in the serum were determined using ELISA kits. Figure 9 and Figure 10 As shown. Figure 9 It can be seen that compared with the control group, the BUN content in the mouse serum was significantly reduced after treatment with peptide RESWPGSR and peptide PLWRLF, which decreased by 27.8% and 25.4%, respectively.

[0047] The results of lactate dehydrogenase activity assay were as follows Figure 10 The results showed that the lactate dehydrogenase activity of the control group was 859.3U / L, while the lactate dehydrogenase activity was significantly increased after the mice were treated with the novel peptides RESWPGSR and PLWRLF, with statistically significant differences compared with the control group. The lactate dehydrogenase activity in the high-dose group increased by 1.55 times and 1.42 times, respectively.

[0048] (3) Determination of liver glycogen and muscle glycogen content The sample preparation and determination method of liver glycogen and muscle glycogen are described in the above Example 1 and will not be repeated here. Figure 11 and Figure 12 The results showed that compared with the control group, the content of liver glycogen and muscle glycogen in mice increased after treatment with low, medium and high doses of peptide RESWPGSR, with the high dose group increasing by 57.16% and 94.7% respectively. Similarly, treatment with peptide PLWRLF also increased the content of liver glycogen and muscle glycogen. Figure 11 B and Figure 12 B. The high-dose group had the best effect, which was increased by 51.35% and 84.61% respectively compared with the control group.

[0049] In summary, the two new peptides (RESWPGSR and PLWRL) prepared by this method both have good anti-fatigue effects.

[0050] The present invention prepares anti-fatigue peptides by synergistically fermenting walnut meal with Rhizopus chinensis, Bacillus subtilis, and Lactobacillus plantarum. This method of preparing anti-fatigue peptides through microbial synergistic fermentation is novel, simple to operate, low-cost, environmentally friendly, and has significant anti-fatigue effects.

[0051] Other embodiments of the present invention will readily occur to those skilled in the art after considering the specification and practicing the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the present invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The description and examples are to be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

[0052] It is understandable that the above content is a further detailed description of the invention in combination with specific / preferred embodiments, and it cannot be determined that the specific implementation of the invention is limited to these descriptions. For ordinary technicians in the technical field to which the invention belongs, without departing from the concept of the invention, they can also make several substitutions or modifications to these described embodiments, and these substitutions or modifications should be deemed to fall within the scope of protection of this patent. In the description of this specification, the reference terms "one embodiment", "some embodiments", "preferred embodiments", "examples", "specific examples", or "some examples" are intended to mean that the specific features, structures, materials or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the invention.

[0053] In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction. Although the embodiments and advantages of the present invention have been described in detail, it should be understood that various changes, substitutions and modifications can be made herein without departing from the scope defined by the appended claims.

[0054] Furthermore, the scope of the present invention is not intended to be limited to the particular embodiments of the processes, machines, manufactures, compositions of matter, means, methods, and steps described in the specification. One of ordinary skill in the art will readily appreciate that any of the above-described disclosures, processes, machines, manufactures, compositions of matter, means, methods, or steps, currently existing or later developed, may be utilized to perform substantially the same functions or achieve substantially the same results as the corresponding embodiments described herein. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufactures, compositions of matter, means, methods, or steps.

Claims

1. A method for preparing a walnut anti-fatigue peptide, characterized in that: The following steps are involved: S1, the cold-pressed walnut meal crushed, through a 20-60 mesh sieve to obtain walnut meal powder; S2. adding water to the walnut meal powder at a material-liquid ratio of 1:0.8 to 1:1.2 (g / mL), and stirring evenly to obtain a semi-solid walnut meal matrix; S3, mixing bacterial liquids of at least one fungus and at least two bacteria in a certain proportion, adding the mixture to the semi-solid walnut meal matrix, and co-fermenting at 28-37° C. for 48-72 hours to obtain a fermentation product; S4. The fermentation product is fractionated by an ultrafiltration membrane method to obtain polypeptide components of different molecular weight segments, and an anti-fatigue polypeptide is obtained from the polypeptide components.

2. The method for preparing the walnut anti-fatigue peptide according to claim 1, wherein In step S2, water is added to the walnut meal powder so that the moisture content of the walnut meal is 44%-54%, and the mixture is stirred evenly to obtain a semi-solid walnut meal matrix.

3. The method for preparing the walnut anti-fatigue peptide according to claim 1, wherein: In step S3, the fungus Rhizopus chinensis and the bacterial solutions of Bacillus subtilis and Lactobacillus plantarum are mixed in a ratio of 2:1:1, added to the semi-solid walnut meal, and cultured at a constant temperature of 28-37° C. for 48-72 hours to obtain a fermentation product.

4. The method for preparing the walnut anti-fatigue peptide according to claim 1, wherein: Step S3 includes: S30, inoculate Rhizopus chinensis on potato dextrose agar (PDA) medium, wait for spores to mature, wash off the Rhizopus chinensis spores on the PDA medium with sterile water, count them with a hemocytometer, and adjust the spore concentration to 10 7 / mL, for future use; S31, inoculate Bacillus subtilis into 100mL of sterilized nutrient broth medium, then inoculate Lactobacillus plantarum into 100mL of sterilized MRS broth medium, culture at 37°C and 180r / min for 24h, adjust the bacterial solution concentration to 10 with sterile saline. 7 cfu / mL, spare; S32. The three bacterial suspensions prepared in steps S30 and S31 are mixed in a ratio of 2:1:1, 5%-10% of the mixed bacterial suspension is inoculated into the sterilized semi-solid walnut meal matrix, and cultured at a constant temperature of 28-37° C. for 48-72 h.

5. The method for preparing the walnut anti-fatigue peptide according to claim 1, wherein: In step S4, the fermentation product is fractionated by ultrafiltration membrane method to obtain walnut polypeptide components with four molecular weight ranges: MW>5 kDa, MW=3-5 kDa, MW=1-3 kDa and MW<1 kDa.

6. The method for preparing the walnut anti-fatigue peptide according to claim 5, wherein: In step S4, the anti-fatigue effects of the walnut polypeptide components in the four molecular weight ranges are measured to screen out polypeptide components with good anti-fatigue effects.

7. The method for preparing the walnut anti-fatigue peptide according to claim 6, wherein: The polypeptide sequence of the polypeptide component with good anti-fatigue effect screened out is identified, and then biological activity evaluation and molecular docking are performed, and new peptides with high scores and good molecular docking binding energy are screened out for solid phase synthesis.

8. The method for preparing the walnut anti-fatigue peptide according to claim 6, wherein: The amino acid sequences of the screened novel peptides were RESWPGSR and PLWRLF.

9. The method for preparing the walnut anti-fatigue peptide according to claim 6, wherein: The fractions with MW < 1 kDa were selected based on the anti-fatigue effect assay.

10. The method for preparing the walnut anti-fatigue peptide according to claim 9, characterized in that: Peptide components with MW < 1 kDa were identified and analyzed by liquid chromatography-mass spectrometry using a Hola C18 column as the separation column and 0.05% formic acid in water and 0.05% formic acid in acetonitrile as the mobile phases. The relative molecular mass information and amino acid composition of the enzymatically hydrolyzed peptides were determined based on the primary and secondary fragment ion information of the mass spectrometer.

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