Walnut oligopeptide powder capable of promoting peripheral nerve regeneration and repairing peripheral nerve injury and preparation method of walnut oligopeptide powder

Abstract

The invention discloses walnut oligopeptide powder capable of promoting peripheral nerve regeneration and repairing peripheral nerve injury and a preparation method of the walnut oligopeptide powder, and belongs to the technical field of walnut oligopeptide production.The walnut oligopeptide powder is prepared by taking walnut cake meal as a raw material, conducting enzymolysis through an enzyme preparation and then conducting fermentation through kluyveromyces marxianus and lactobacillus helveticus; and the enzyme preparation is trypsin and ficin protease. Compared with the prior art, the yield of the walnut oligopeptide powder is higher. Experiments prove that the walnut oligopeptide powder prepared by the invention can effectively prolong the length of surrounding motor nerves, and has a relatively good nerve repairing effect.

Description

Technical Field

[0001] The invention relates to the technical field of walnut oligopeptide production, and in particular to walnut oligopeptide powder capable of promoting peripheral nerve regeneration and repairing peripheral nerve damage and a preparation method thereof. Background Art

[0002] Stroke refers to cerebral blood vessel stenosis, occlusion or rupture caused by various reasons, which eventually leads to acute cerebral blood circulation disorder. Ischemic stroke is the most common type, with high mortality and disability rates. Neuronal degeneration and necrosis are the key factors leading to death and disability in patients with ischemic stroke. Protecting normal neurons to the greatest extent is an important part of treatment. Therefore, neurological function repair is a scientific problem that urgently needs to be overcome in cerebrovascular disease.

[0003] Walnut (Juglans regia L.), a plant of the Juglandaceae family, is widely produced. Walnut kernels are rich in high-quality protein, unsaturated fatty acids, carbohydrates, vitamins, dietary fiber, and a small amount of flavonoids and phenolic acids and other nutrients, and the content is balanced. It is an excellent choice for high-protein, high-fat nutritious food, and also has high medicinal value. It belongs to the category of food and medicine, and is a green functional food with great development prospects.

[0004] Studies have shown that walnut oligopeptides (WOPs) are small molecule bioactive peptides extracted from hydrolyzed walnut protein. They are better absorbed than walnut protein and are rich in amino acids, especially glutamic acid, which is related to the synthesis of γ-aminobutyric acid. They have a positive effect on improving sleep. WOPs have significant antioxidant function, can effectively remove free radicals, and protect brain nerves.

[0005] For example, patent CN105936927A discloses a walnut oligopeptide and its preparation process and use, wherein the oligopeptide powder has a peptide content of more than 70wt%, of which more than 85% of the walnut peptides have a molecular weight of less than 1500 Dalton. The process mainly includes two enzymatic hydrolysis to obtain walnut oligopeptide powder. The walnut oligopeptide powder has good antioxidant activity and good protective effect on neuronal cells, and can be used to prevent or treat Parkinson's disease and other related diseases, and relieve brain fatigue.

[0006] Patent CN117247983A discloses a method for preparing walnut peptide, the steps are as follows: crushing walnut kernels to obtain walnut meal, grinding the walnut meal and water in proportion, heating the slurry at a temperature of 95-104°C for 10-15 minutes; adjusting the pH value to 8.5-9.5 with an organic base, stirring at 55-75°C for 1.0-3.5 hours, centrifuging for 10-25 minutes, removing the oil layer, taking the supernatant, adjusting the pH value to 4.2-5.1 with a 1.0-2.0 mol / L inorganic acid aqueous solution, centrifuging for 15-30 minutes, washing with ethanol 1-3 times to obtain walnut protein; cooling to 15-20°C, and performing enzymatic hydrolysis at a pH of 9-11; filtering the material after the biochemical reaction through a nanofiltration membrane to obtain a walnut peptide stock solution, and then heating to inactivate the enzyme; concentrating the walnut peptide liquid after the enzyme inactivation; sterilizing; and spray drying to obtain walnut peptide powder. The walnut peptide obtained in this invention has the effect of protecting nerves and promoting the growth of functional neurons. However, this invention does not provide any efficacy verification, and the predictability of promoting the growth of functional neurons is poor.

[0007] At present, there are relatively few drugs for nerve function repair in clinical practice. As can be seen from the above-mentioned prior art, most of the research on walnut oligopeptides focuses on protecting nerves, and does not provide its therapeutic effect on nerve repair. In view of the problems existing in the prior art, it is very necessary to find a walnut oligopeptide powder with a clear effect of promoting peripheral nerve regeneration and repairing peripheral nerve damage and a preparation method thereof. Summary of the invention

[0008] The invention provides walnut oligopeptide powder capable of promoting peripheral nerve regeneration and repairing peripheral nerve damage and a preparation method thereof. Experiments have shown that the walnut oligopeptide powder can effectively extend the length of peripheral motor nerves and has a good nerve repair effect.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] In a first aspect, the present invention provides a walnut oligopeptide powder having the function of promoting peripheral nerve regeneration and repairing peripheral nerve damage. The walnut oligopeptide powder is prepared by using walnut cake as raw material, enzymatic hydrolysis with enzyme preparation, and fermentation with Kluyveromyces marxianus and Lactobacillus helveticus.

[0011] The enzyme preparation of the present invention is trypsin and ficin. Preferably, the mass ratio of trypsin to ficin is 5-10:1, and more preferably 8:1.

[0012] The mass ratio of Kluyveromyces marxianus to Lactobacillus helveticus in the present invention is 1:0.5-1.5; preferably 1:0.8-1.2; further preferably 1:1.

[0013] In a second aspect, the present invention provides a method for preparing walnut oligopeptide powder, comprising the following steps:

[0014] S1, walnut cake is ground with water to obtain slurry;

[0015] S2, adding an enzyme preparation to the slurry obtained in step S1 for enzymolysis to obtain an enzymolysis solution;

[0016] S3, after inactivating the enzyme in the enzymatic hydrolyzate obtained in step S2, adding Kluyveromyces marxianus and Lactobacillus helveticus for fermentation to obtain a fermentation liquid;

[0017] S4, separating the fermentation liquid obtained in step S3 into solid and liquid, ultrafiltering, and drying to obtain the walnut oligopeptide powder.

[0018] Preferably, the walnut cake in step S1 is the residue after walnut oil is removed from cold-pressed walnuts.

[0019] Preferably, the temperature of the water in step S1 is 2-6°C.

[0020] Preferably, the amount of water added in step S1 is 3-8 times the mass of the walnut cake.

[0021] Preferably, the grinding in step S1 is: grinding at 3000-8000 rpm for 10-20 min.

[0022] Preferably, the amount of the enzyme preparation added in step S2 is: 0.1-0.4% of the total mass of the walnut cake, more preferably 0.2-0.3%.

[0023] Preferably, the enzymatic hydrolysis in step S2 is: enzymatic hydrolysis at 40-50° C. for 1-3 h.

[0024] Preferably, the enzyme inactivation condition in step S3 is: maintaining at 100-105°C for at least 10 minutes, more preferably at 102°C for 10 minutes.

[0025] Preferably, the total inoculation amount of Kluyveromyces marxianus and Lactobacillus helveticus in step S3 is 2-5% of the mass of the enzymatic hydrolysate, and more preferably 3-4%.

[0026] Preferably, the fermentation condition in step S3 is 35-40° C. for 12-24 h.

[0027] Preferably, the solid-liquid separation in step S4 is at least one of centrifugation, filtration and filter pressing.

[0028] Preferably, the ultrafiltration in step S4 is: pressure filtration followed by ultrafiltration membrane.

[0029] Preferably, the drying in step S4 includes at least one of freeze drying, vacuum drying and spray drying.

[0030] In a third aspect, the present invention provides the use of walnut oligopeptide powder in the preparation of a medicine for repairing nerve damage.

[0031] Preferably, the nerve damage is caused by at least one of stroke, Alzheimer's disease, diabetic neuropathy, meningitis, and brain injury.

[0032] In a fourth aspect, the present invention provides a drug for nerve repair, comprising the aforementioned walnut oligopeptide powder and pharmaceutically acceptable excipients.

[0033] The beneficial effects of the present invention are:

[0034] (1) The walnut oligopeptide powder of the present invention is prepared by using walnut cake as raw material, enzymatically hydrolyzing with an enzyme preparation, and then fermenting with Kluyveromyces marxianus and Lactobacillus helveticus. Compared with the prior art, the yield of the walnut oligopeptide powder of the present invention is higher.

[0035] (2) Experiments have shown that the walnut oligopeptide powder prepared by the present invention can effectively extend the length of peripheral motor nerves and has a good nerve repair effect.

[0036] (3) The enzyme preparation and fermentation bacteria used in the present invention meet the requirements of food raw materials and have good safety.

[0037] (4) The preparation method of the present invention is simple, low-cost, and suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the analysis of peripheral motor nerves in zebrafish. DETAILED DESCRIPTION

[0039] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0040] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0041] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0042] It is worth noting that the raw materials used in the present invention are all common commercially available products, so their sources are not specifically limited.

[0043] In this embodiment, the walnut cake is the residue added after the walnut oil is removed after cold pressing. The enzymes and bacteria are all food grade, wherein trypsin and flavor protease are purchased from Shaanxi Chenming Biotechnology Co., Ltd., ficin and neutral protease are purchased from Guangzhou Kejin Biotechnology Co., Ltd., Kluyveromyces marxianus, Lactobacillus paracasei and Lactobacillus helveticus are purchased from Shandong Xinxiong Biotechnology Co., Ltd., and the ultrafiltration membrane is 3000 Dalton.

[0044] Example 1 Walnut oligopeptide powder capable of promoting peripheral nerve regeneration and repairing peripheral nerve damage

[0045] The preparation method of the walnut oligopeptide powder is:

[0046] S1. Add cold water (6°C) to the walnut cake at a material-liquid ratio of 1:8, and grind at 8000 rpm for 10 min to obtain a slurry;

[0047] S2. After adjusting the pH value of the slurry to 7.5-8, an enzyme preparation was added and enzymolysis was performed at 40° C. for 1 h to obtain an enzymolysis solution;

[0048] The amount of the enzyme preparation added is: 0.4% of the total mass of walnut cake;

[0049] The enzyme preparation is trypsin and ficin in a mass ratio of 10:1;

[0050] S3. After the reaction is completed, the enzymatic solution is inactivated at 102° C. for 10 minutes, cooled to 35° C., Kluyveromyces marxianus and Lactobacillus helveticus are added at an inoculum amount of 2% (mass ratio 1:1.5), and fermented at 35° C. for 24 hours to obtain a fermentation solution;

[0051] S4. The fermentation liquid is filtered by a plate and frame filter press, the filtrate is filtered through an ultrafiltration membrane (3000Da), and the ultrafiltrate is dried to obtain the walnut oligopeptide powder.

[0052] Example 2 Walnut oligopeptide powder capable of promoting peripheral nerve regeneration and repairing peripheral nerve damage

[0053] The preparation method of the walnut oligopeptide powder is:

[0054] S1. Add cold water (2°C) to the walnut cake at a material-liquid ratio of 1:3, and grind at 3000 rpm for 20 min to obtain a slurry;

[0055] S2. After adjusting the pH value of the slurry to 7.5-8, an enzyme preparation was added and enzymolysis was performed at 50° C. for 3 h to obtain an enzymolysis solution;

[0056] The amount of the enzyme preparation added is: 0.1% of the total mass of walnut cake;

[0057] The enzyme preparation is trypsin and ficin in a mass ratio of 5:1;

[0058] S3. After the reaction is completed, the enzymatic solution is inactivated at 102° C. for 10 minutes, cooled to 40° C., Kluyveromyces marxianus and Lactobacillus helveticus are added at an inoculum amount of 5% (mass ratio 1:0.5), and fermented at 40° C. for 12 hours to obtain a fermentation solution;

[0059] S4. The fermentation liquid is filtered by a plate and frame filter press, the filtrate is filtered through an ultrafiltration membrane (3000Da), and the ultrafiltrate is dried to obtain the walnut oligopeptide powder.

[0060] Example 3 Walnut oligopeptide powder capable of promoting peripheral nerve regeneration and repairing peripheral nerve damage

[0061] The preparation method of the walnut oligopeptide powder is:

[0062] S1. Add cold water (4°C) to the walnut cake at a material-liquid ratio of 1:6, and grind at 5000 rpm for 15 min to obtain a slurry;

[0063] S2. After adjusting the pH value of the slurry to 7.5-8, an enzyme preparation was added and enzymolysis was performed at 45°C for 1.5 hours to obtain an enzymolysis solution;

[0064] The amount of the enzyme preparation added is: 0.3% of the total mass of walnut cake;

[0065] The enzyme preparation is trypsin and ficin in a mass ratio of 8:1;

[0066] S3. After the reaction is completed, the enzymatic solution is inactivated at 102° C. for 10 minutes, cooled to 37° C., Kluyveromyces marxianus and Lactobacillus helveticus are added at an inoculum rate of 3% (mass ratio 1:1), and fermented at 37° C. for 16 hours to obtain a fermentation solution;

[0067] S4. The fermentation liquid is filtered by a plate and frame filter press, the filtrate is filtered through an ultrafiltration membrane (3000Da), and the ultrafiltrate is dried to obtain the walnut oligopeptide powder.

[0068] Comparative Example 1 Walnut oligopeptide powder having the function of promoting peripheral nerve regeneration and repairing peripheral nerve damage

[0069] The difference between this comparative example and Example 3 is that the enzyme preparation and the fermentation bacteria are different.

[0070] Specifically, the preparation method of the walnut oligopeptide powder is:

[0071] S1. Add cold water (4°C) to the walnut cake at a material-liquid ratio of 1:6, and grind at 5000 rpm for 15 min to obtain a slurry;

[0072] S2. After adjusting the pH value of the slurry to 7.5-8, an enzyme preparation was added and enzymolysis was performed at 45°C for 1.5 hours to obtain an enzymolysis solution;

[0073] The amount of the enzyme preparation added is: 0.3% of the total mass of walnut cake;

[0074] The enzyme preparation is trypsin and ficin in a mass ratio of 3:1;

[0075] S3, after the reaction is completed, the enzymatic solution is inactivated at 102°C for 10 minutes, cooled to 37°C, and Lactobacillus helveticus and Lactobacillus paracasei are added at an inoculum amount of 3% (mass ratio 1:1), and fermented at 37°C for 16 hours to obtain a fermentation liquid;

[0076] S4. The fermentation liquid is filtered by a plate and frame filter press, the filtrate is filtered through an ultrafiltration membrane (3000Da), and the ultrafiltrate is dried to obtain the walnut oligopeptide powder.

[0077] Comparative Example 2 Walnut oligopeptide powder having the function of promoting peripheral nerve regeneration and repairing peripheral nerve damage

[0078] The difference between this comparative example and Example 3 is that the enzyme preparations are different.

[0079] Specifically, the preparation method of the walnut oligopeptide powder is:

[0080] S1. Add cold water (4°C) to the walnut cake at a material-liquid ratio of 1:6, and grind at 5000 rpm for 15 min to obtain a slurry;

[0081] S2. After adjusting the pH value of the slurry to 7.5-8, an enzyme preparation was added and enzymolysis was performed at 45°C for 1.5 hours to obtain an enzymolysis solution;

[0082] The amount of the enzyme preparation added is: 0.3% of the total mass of walnut cake;

[0083] The enzyme preparation comprises trypsin, neutral protease and flavor protease in a mass ratio of 1:1:1;

[0084] S3. After the reaction is completed, the enzymatic solution is inactivated at 102° C. for 10 minutes, cooled to 37° C., Kluyveromyces marxianus and Lactobacillus helveticus are added at an inoculum rate of 3% (mass ratio 1:1), and fermented at 37° C. for 16 hours to obtain a fermentation solution;

[0085] S4. The fermentation liquid is filtered by a plate and frame filter press, the filtrate is filtered through an ultrafiltration membrane (3000Da), and the ultrafiltrate is dried to obtain the walnut oligopeptide powder.

[0086] Comparative Example 3 Walnut oligopeptide powder having the function of promoting peripheral nerve regeneration and repairing peripheral nerve damage

[0087] The difference between this comparative example and Example 3 is that the enzyme preparations are different.

[0088] Specifically, the preparation method of the walnut oligopeptide powder is:

[0089] S1. Add cold water (4°C) to the walnut cake at a material-liquid ratio of 1:6, and grind at 5000 rpm for 15 min to obtain a slurry;

[0090] S2. After adjusting the pH value of the slurry to 7.5-8, an enzyme preparation was added and enzymolysis was performed at 45°C for 1.5 hours to obtain an enzymolysis solution;

[0091] The amount of the enzyme preparation added is: 0.3% of the total mass of walnut cake;

[0092] The enzyme preparation is trypsin;

[0093] S3. After the reaction is completed, the enzymatic solution is inactivated at 102° C. for 10 minutes, cooled to 37° C., Kluyveromyces marxianus and Lactobacillus helveticus are added at an inoculum rate of 3% (mass ratio 1:1), and fermented at 37° C. for 16 hours to obtain a fermentation solution;

[0094] S4. The fermentation liquid is filtered by a plate and frame filter press, the filtrate is filtered through an ultrafiltration membrane (3000Da), and the ultrafiltrate is dried to obtain the walnut oligopeptide powder.

[0095] Comparative Example 4 Walnut oligopeptide powder having the function of promoting peripheral nerve regeneration and repairing peripheral nerve damage

[0096] The difference between this comparative example and Example 3 is that the enzyme preparations are different.

[0097] Specifically, the preparation method of the walnut oligopeptide powder is:

[0098] The preparation method of the walnut oligopeptide powder is:

[0099] S1. Add cold water (4°C) to the walnut cake at a material-liquid ratio of 1:6, and grind at 5000 rpm for 15 min to obtain a slurry;

[0100] S2. After adjusting the pH value of the slurry to 7.5-8, an enzyme preparation was added and enzymolysis was performed at 45°C for 1.5 hours to obtain an enzymolysis solution;

[0101] The amount of the enzyme preparation added is: 0.3% of the total mass of walnut cake;

[0102] The enzyme preparation is ficin;

[0103] S3. After the reaction is completed, the enzymatic solution is inactivated at 102° C. for 10 minutes, cooled to 37° C., Kluyveromyces marxianus and Lactobacillus helveticus are added at an inoculum rate of 3% (mass ratio 1:1), and fermented at 37° C. for 16 hours to obtain a fermentation solution;

[0104] S4. The fermentation liquid is filtered by a plate and frame filter press, the filtrate is filtered through an ultrafiltration membrane (3000Da), and the ultrafiltrate is dried to obtain the walnut oligopeptide powder.

[0105] Comparative Example 5

[0106] Walnut peptide powder prepared according to Example 1 of CN117247983A.

[0107] 1. Yield

[0108] The yield of the walnut oligopeptide powder prepared in the examples and comparative examples was investigated, wherein the yield was calculated as follows: mass of walnut oligopeptide powder*100 / mass of walnut cake. The results are shown in Table 1.

[0109] Table 1 Yield of walnut oligopeptide powder prepared in Examples and Comparative Examples

[0110] Examples Yield % Example 1 32.6 Example 2 30.6 Example 3 34.2 Comparative Example 1 23.0 Comparative Example 2 24.2 Comparative Example 3 21.3 Comparative Example 4 23.7 Comparative Example 5 20.5

[0111] 2. Nerve Repair Effect

[0112] 1. Testing materials

[0113] 1.1 Sample preparation

[0114] The solvent for the walnut oligopeptide powder prepared in Examples 1-3 and Comparative Examples 1-5 is standard dilution water.

[0115] Positive control: glutathione, batch number SLCG8572, SIGMA, solvent is standard dilution water.

[0116] 1.2 Experimental animals

[0117] Zebrafish were raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, the conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO3), and were bred and provided by the Fish Farming Center of Huante Biological Innovation Experimental Center. The experimental animal use license number is: SYXK (Zhejiang) 2022-0004. The breeding and management complies with the requirements of the International AAALAC certification (certification number: 001458), and the IACUC ethics review number is: IACUC-2024-9423-01.

[0118] 1.3 Instruments, consumables and reagents

[0119] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA), 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China); electric focusing continuous zoom fluorescence microscope (AZ100, Nikon, Japan); ultrasonic cleaning machine (JP-010T, Shenzhen Jiemeng Cleaning Equipment Co., Ltd., China).

[0120] Anhydrous ethanol (Batch No. 20240312, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Methylcellulose (Batch No. C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0121] 2. Detection method

[0122] 2.1 MTC determination

[0123] Transgenic motor neuron green fluorescent zebrafish (HB9) at 6 hours post-fertilization (6hpf) were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). A normal control group and a model control group were set up, and the volume of each well was 3mL. Except for the normal control group, the other experimental groups were given anhydrous ethanol in water to establish a peripheral motor nerve injury model. After 2 days of treatment at 28℃, the ethanol was removed and walnut oligopeptide powder was given in water. After 1 day of treatment at 28℃, the MTC of the sample on the model zebrafish was measured.

[0124] 2.2 Evaluation of nerve repair efficacy

[0125] 6hpf transgenic motor neuron green fluorescent zebrafish (HB9) were randomly selected in a 6-well plate, and 30 zebrafish were treated in each well (experimental group). A normal control group and a model control group were set up, and the capacity of each well was 3mL. Except for the normal control group, the other experimental groups were given anhydrous ethanol in water to establish a peripheral motor nerve injury model. After 2 days of treatment at 28℃, the ethanol was removed, and walnut oligopeptide powder (concentration of 250μg / mL) was given in water, and the positive control glutathione concentration was 500μg / mL. After 1 day of treatment at 28℃, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope for photography. NIS-Elements D 3.20 advanced image processing software was used to analyze and collect data, and the length of the peripheral motor nerves in the three segments above the zebrafish cloaca (such as Figure 1 The results of statistical analysis of this index were used to evaluate the nerve repair efficacy of the sample. The statistical processing results were expressed as mean ± SE. SPSS26.0 software was used for statistical analysis, and p < 0.05 indicated that the difference was statistically significant.

[0126] 3. Test results

[0127] 3.1MTC

[0128] Table 2 Nerve repair efficacy concentration experimental results (n = 30)

[0129]

[0130] As shown in Table 2, under the experimental conditions, the nerve repair efficacy MTC of the walnut oligopeptide powder prepared in Example 3 is 500 μg / mL.

[0131] 3.1 Evaluation of nerve repair efficacy

[0132] Table 3 Evaluation of nerve repair efficacy (n=10)

[0133] Group Concentration (μg / mL) Peripheral motor nerve length (pixels, mean ± SE) Normal control - 325±10.7a Model comparison - 258±6.00c Positive Control 500 288±8.40b Example 1 250 290±6.68b Example 2 250 296±4.83b Example 3 250 298±7.98b Comparative Example 1 250 262±5.47c Comparative Example 2 250 260±6.13c Comparative Example 3 250 263±6.57c Comparative Example 4 250 265±7.08c Comparative Example 5 250 261±4.26c

[0134] Note: Different letters in the table indicate significant differences among the groups, P < 0.05.

[0135] As shown in Table 3, compared with the normal control group, the length of the zebrafish peripheral motor nerves in the model control group was significantly shortened, indicating that the model was successfully established.

[0136] Compared with the model control group, the changes in the length of the zebrafish peripheral motor nerves in the positive control group and Example 1-3 groups were significantly different (P < 0.05), indicating that under the experimental conditions, the walnut oligopeptide powder prepared in Examples 1-3 of the present invention has a nerve repair effect.

[0137] Compared with the model control group, there was no significant difference in the change of peripheral motor nerve length of zebrafish in comparative examples 1-4 (P>0.05), indicating that under the experimental conditions, the walnut oligopeptide powder prepared in comparative examples 1-4 of the present invention had no nerve repair effect.

[0138] By comparing Example 3 with Comparative Examples 1-4, it can be seen that the walnut oligopeptide powder prepared by the present invention using a specific enzyme preparation for enzymatic hydrolysis and then fermenting with Kluyveromyces marxianus and Lactobacillus helveticus has significant nerve repair effect, while the walnut oligopeptide powder prepared by the combination of conventional enzyme preparations and fermentation bacteria has no nerve repair effect.

[0139] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A walnut oligopeptide powder having the function of promoting peripheral nerve regeneration and repairing peripheral nerve damage, characterized in that: The walnut oligopeptide powder is prepared by using walnut cake as raw material, enzymatically hydrolyzing walnut cake with enzyme preparation, and then fermenting with Kluyveromyces marxianus and Lactobacillus helveticus. The enzyme preparation is trypsin and ficin.

2. The walnut oligopeptide powder according to claim 1, characterized in that The mass ratio of the trypsin to the ficin to the flavor protease is 5-10:

1.

3. The walnut oligopeptide powder according to claim 1, characterized in that The mass ratio of trypsin to ficin is 8:

1.

4. The method for preparing the walnut oligopeptide powder according to any one of claims 1 to 3 comprises the following steps: S1, walnut cake is ground with water to obtain slurry; S2, adding an enzyme preparation to the slurry obtained in step S1 for enzymolysis to obtain an enzymolysis solution; S3, after inactivating the enzyme in the enzymatic hydrolyzate obtained in step S2, adding Kluyveromyces marxianus and Lactobacillus helveticus for fermentation to obtain a fermentation liquid; S4, separating the fermentation liquid obtained in step S3 into solid and liquid, ultrafiltering, and drying to obtain the walnut oligopeptide powder.

5. The preparation method according to claim 4, characterized in that: The amount of the enzyme preparation added in step S2 is: 0.1-0.4% of the total mass of the walnut cake; the enzymolysis in step S2 is: enzymolysis at 40-50° C. for 1-3 hours.

6. The preparation method according to claim 4, characterized in that: The total inoculation amount of Kluyveromyces marxianus and Lactobacillus helveticus in step S3 is 2-5% of the mass of the enzymatic hydrolysate, and the mass ratio of Kluyveromyces marxianus to Lactobacillus helveticus is 1:0.5-1.

5.

7. The preparation method according to claim 4, characterized in that: The fermentation conditions in step S3 are 35-40° C. for 12-24 hours.

8. Use of the walnut oligopeptide powder according to any one of claims 1 to 3 or the walnut oligopeptide powder prepared by the preparation method according to any one of claims 4 to 7 in the preparation of a drug for repairing nerve damage.

9. The use according to claim 8, characterized in that: The nerve damage is caused by at least one of stroke, Alzheimer's disease, diabetic neuropathy, meningitis, and brain damage.

10. A drug for nerve repair, characterized in that: The invention comprises the walnut oligopeptide powder according to any one of claims 1 to 3 or the walnut oligopeptide powder prepared by the preparation method according to any one of claims 4 to 7 and pharmaceutically acceptable auxiliary materials.

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