A walnut oligopeptide powder with the function of promoting peripheral nerve regeneration and repairing peripheral nerve injuries and a preparation method thereof

Abstract

The present invention discloses a walnut oligopeptide powder with the functions of promoting peripheral nerve regeneration and repairing peripheral nerve injuries and a preparation method thereof, belonging to the technical field of walnut oligopeptide production. The walnut oligopeptide powder uses walnut cake as a raw material, is enzymatically hydrolyzed by an enzyme preparation, and is prepared by fermentation with Kluyveromyces marxianus and Lactobacillus helveticus. The enzyme preparation is trypsin and ficin. Compared with the prior art, the yield of the walnut oligopeptide powder of the present invention is higher. Moreover, it has been proven by experiments that the walnut oligopeptide powder prepared by the present invention can effectively extend the length of peripheral motor nerves and has a good effect on nerve repair.

Description

Technical Field

[0001] The present invention relates to the technical field of walnut oligopeptide production, and particularly relates to a walnut oligopeptide powder with the function of promoting peripheral nerve regeneration and repairing peripheral nerve injury and a preparation method thereof. Background Art

[0002] Stroke refers to cerebrovascular stenosis, occlusion or rupture caused by various reasons, ultimately leading to acute cerebral blood circulation disorders. Ischemic stroke is the most common type, with relatively high mortality and disability rates. Degeneration and necrosis of nerve cells are the key factors leading to death and disability in patients with ischemic stroke. Maximally protecting normal nerve cells is an important part of the treatment. Therefore, nerve function repair is a scientific problem that urgently needs to be overcome in cerebrovascular diseases.

[0003] Walnut (Juglans regia L.) belongs to the Juglandaceae family and has a wide production range. Walnut kernels are rich in high-quality proteins, unsaturated fatty acids, carbohydrates, vitamins, dietary fiber, as well as small amounts of nutrients such as flavonoids and phenolic acids, and the content is balanced. It is an excellent choice for high-protein and high-fat nutritional foods, and also has high medicinal value. It belongs to the category of foods that are both medicine and food, and is a green functional food with great development prospects.

[0004] Research shows that: walnut oligopeptides (WOPs) are small molecular bioactive peptides extracted by hydrolyzing walnut proteins. Their absorption is better than that of walnut proteins, and they have rich amino acid content, especially glutamic acid related to the synthesis of γ-aminobutyric acid, which has a positive effect on improving sleep. WOPs have significant antioxidant functions, can effectively scavenge free radicals, and protect the brain nerves.

[0005] For example, Patent CN105936927A discloses a walnut oligopeptide and its preparation process and uses. The peptide content in the oligopeptide powder is above 70wt%, and the molecular weight of more than 85% of the walnut peptides is less than 1500 Dalton. The process mainly includes two enzymatic hydrolyses to obtain the walnut oligopeptide powder. The walnut oligopeptide powder has good antioxidant activity, has a good protective effect on neuron cells, can be used to prevent or treat related diseases such as Parkinson's disease, and relieve brain fatigue.

[0006] Patent CN117247983A discloses a method for preparing walnut peptides, and the steps are as follows: The walnut kernels are crushed to obtain walnut meal, and the walnut meal is ground with water according to a ratio. The slurry is heated at a temperature of 95 - 104 °C for 10 - 15 minutes; the pH value is adjusted to 8.5 - 9.5 with an organic base, stirred at 55 - 75 °C for 1.0 - 3.5 hours, centrifuged for 10 - 25 minutes to remove the oil layer, the supernatant is taken, and the pH value is adjusted to 4.2 - 5.1 with a 1.0 - 2.0 mol / L inorganic acid aqueous solution, centrifuged for 15 - 30 minutes, and washed with ethanol 1 - 3 times to obtain walnut protein; cooled to 15 - 20 °C, and enzymatically hydrolyzed under the condition that the pH value is 9 - 11; the material after the biochemical reaction is filtered through a nanofiltration membrane to obtain the original walnut peptide solution, and then heated to inactivate the enzyme; the inactivated walnut peptide solution is concentrated; sterilized; spray-dried to make walnut peptide powder. The walnut peptide obtained by this invention has the efficacy of protecting nerves and promoting the growth of functional neurons. However, this invention does not give the 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 clinically. As can be seen from the above prior art, the research on walnut oligopeptides mainly focuses on protecting nerves, and does not give 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 clear effects that can promote peripheral nerve regeneration and repair peripheral nerve injuries and its preparation method. Summary of the Invention

[0008] The present invention provides a walnut oligopeptide powder with the ability to promote peripheral nerve regeneration and repair peripheral nerve injuries and its preparation method. It has been experimentally proven that the walnut oligopeptide powder of the present invention can effectively extend the length of peripheral motor nerves and has a good effect on nerve repair.

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

[0010] In the first aspect, the present invention provides a walnut oligopeptide powder with the ability to promote peripheral nerve regeneration and repair peripheral nerve injuries. The walnut oligopeptide powder is prepared from walnut cake residue as a raw material, enzymatically hydrolyzed with an enzyme preparation, and then fermented with Kluyveromyces marxianus and Lactobacillus helveticus.

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

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

[0013] Second aspect, the present invention provides a preparation method of walnut oligopeptide powder, comprising the following steps:

[0014] S1. Add water to walnut cake meal and grind to obtain a slurry;

[0015] S2. Add an enzyme preparation to the slurry obtained in step S1 for enzymatic hydrolysis to obtain an enzymatic hydrolysate;

[0016] S3. After inactivating the enzyme in the enzymatic hydrolysate obtained in step S2, add Kluyveromyces marxianus and Lactobacillus helveticus for fermentation to obtain a fermentation broth;

[0017] S4. Perform solid-liquid separation, ultrafiltration, and drying on the fermentation broth obtained in step S3 to obtain the walnut oligopeptide powder.

[0018] Preferably, the walnut cake meal in step S1 is the residue obtained after removing walnut oil from walnuts by cold pressing.

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

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

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

[0022] Preferably, the addition amount of the enzyme preparation in step S2 is: 0.1-0.4% of the total mass of the walnut cake meal, and further 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 conditions for enzyme inactivation in step S3 are: maintaining at 100-105 °C for at least 10 min, and further preferably 10 min at 102 °C.

[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 further preferably 3-4%.

[0026] Preferably, the fermentation conditions in step S3 are: fermentation at 35-40 °C for 12-24 h.

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

[0028] Preferably, the ultrafiltration in step S4 is: passing through an ultrafiltration membrane after pressure filtration.

[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 an application of walnut oligopeptide powder in the preparation of a drug 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 as follows:

[0034] (1) The walnut oligopeptide powder of the present invention is prepared from walnut cake as a raw material, enzymatically hydrolyzed using an enzyme preparation, and then fermented using 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) It has been experimentally proven that the walnut oligopeptide powder prepared by the present invention can effectively extend the length of the peripheral motor nerve and has a good nerve repair effect.

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

[0037] (4) The preparation method of the present invention is simple, with low cost, and is suitable for large-scale production. Description of the Drawings

[0038] Figure 1 It is a schematic diagram for the analysis of the peripheral motor nerve of the horse-spotted fish. Detailed Embodiments

[0039] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various modifications or changes can be made to the details in this specification 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 protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific embodiments, rather than for limiting the protection scope of the present invention.

[0041] When numerical ranges are given in the embodiments, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention belongs.

[0042] It should be noted that the raw materials used in the present invention are all ordinary commercially available products, so their sources are not specifically limited.

[0043] In this embodiment, the walnut cake residue is the residue obtained after removing walnut oil by cold pressing walnuts. The enzymes and bacteria are all food-grade. Among them, 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 with the function of promoting peripheral nerve regeneration and repairing peripheral nerve injury

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

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

[0047] S2. After adjusting the pH value of the slurry to 7.5 - 8, add an enzyme preparation and enzymolyze it at 40 °C for 1 h to obtain an enzymolyzed solution.

[0048] The addition amount of the enzyme preparation is: 0.4% of the total mass of the walnut cake residue.

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

[0050] S3. After the reaction is completed, inactivate the enzyme in the enzymolyzed solution at 102 °C for 10 minutes, cool it to 35 °C, and add Kluyveromyces marxianus and Lactobacillus helveticus (mass ratio 1:1.5) at an inoculation amount of 2%, and ferment at 35 °C for 24 h to obtain a fermentation broth.

[0051] S4. Filter the fermentation broth with a plate and frame filter press, filter the filtrate through an ultrafiltration membrane (3000 Da), and dry the ultrafiltrate to obtain the described walnut oligopeptide powder.

[0052] Example 2: Walnut oligopeptide powder with the function of promoting peripheral nerve regeneration and repairing peripheral nerve injury

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

[0054] S1. According to a material-liquid ratio of 1:3, add cold water (2°C) to walnut cake meal, 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, add an enzyme preparation, and carry out enzymatic hydrolysis at 50°C for 3 h to obtain an enzymatic hydrolysate;

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

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

[0058] S3. After the reaction ends, inactivate the enzyme in the enzymatic hydrolysate at 102°C for 10 minutes, cool down to 40°C, add Kluyveromyces marxianus and Lactobacillus helveticus (mass ratio 1:0.5) according to an inoculation amount of 5%, and ferment at 40°C for 12 h to obtain a fermentation broth;

[0059] S4. Filter the fermentation broth using a plate and frame filter press, filter the filtrate through an ultrafiltration membrane (3000 Da), and dry the ultrafiltrate to obtain the described walnut oligopeptide powder.

[0060] Example 3 Walnut oligopeptide powder with the function of promoting peripheral nerve regeneration and repairing peripheral nerve injury

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

[0062] S1. According to a material-liquid ratio of 1:6, add cold water (4°C) to walnut cake meal, 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, add an enzyme preparation, and carry out enzymatic hydrolysis at 45°C for 1.5 h to obtain an enzymatic hydrolysate;

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

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

[0066] S3. After the reaction ends, inactivate the enzyme in the enzymatic hydrolysate at 102°C for 10 minutes, cool down to 37°C, add Kluyveromyces marxianus and Lactobacillus helveticus (mass ratio 1:1) according to an inoculation amount of 3%, and ferment at 37°C for 16 h to obtain a fermentation broth;

[0067] S4. Filter the fermentation broth using a plate and frame filter press, filter the filtrate through an ultrafiltration membrane (3000 Da), and dry the ultrafiltrate to obtain the described walnut oligopeptide powder.

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

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

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

[0071] S1. According to a material-liquid ratio of 1:6, add cold water (4°C) to walnut cake meal, 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, add the enzyme preparation and enzymatically hydrolyze at 45°C for 1.5 h to obtain an enzymatic hydrolysate.

[0073] The addition amount of the enzyme preparation is: 0.3% of the total mass of the walnut cake meal.

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

[0075] S3. After the reaction ends, inactivate the enzyme in the enzymatic hydrolysate at 102°C for 10 minutes, cool down to 37°C, add Lactobacillus helveticus and Lactobacillus paracasei (mass ratio 1:1) according to an inoculation amount of 3%, and ferment at 37°C for 16 h to obtain a fermentation broth.

[0076] S4. Filter the fermentation broth using a plate and frame filter press, filter the filtrate through an ultrafiltration membrane (3000 Da), and dry the ultrafiltrate to obtain the described walnut oligopeptide powder.

[0077] The comparative example 2 has walnut oligopeptide powder that promotes peripheral nerve regeneration and repairs peripheral nerve injuries

[0078] The difference between this comparative example and Example 3 lies in that the enzyme preparation is different.

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

[0080] S1. According to a material-liquid ratio of 1:6, add cold water (4°C) to walnut cake meal, 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, add the enzyme preparation and enzymatically hydrolyze at 45°C for 1.5 h to obtain an enzymatic hydrolysate.

[0082] The addition amount of the enzyme preparation is: 0.3% of the total mass of the walnut cake meal.

[0083] The enzyme preparation is trypsin, neutral protease, and flavor protease with a mass ratio of 1:1:1.

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

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

[0086] Walnut oligopeptide powder with the function of promoting peripheral nerve regeneration and repairing peripheral nerve injury in Comparative Example 3

[0087] The difference between this comparative example and Example 3 lies in: different enzyme preparations.

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

[0089] S1. According to the material-liquid ratio of 1:6, cold water (4 °C) is added to walnut cake meal, and ground 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 is added, and enzymolysis is carried out at 45 °C for 1.5 h to obtain an enzymatic hydrolysate;

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

[0092] The enzyme preparation is trypsin;

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

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

[0095] Walnut oligopeptide powder with the function of promoting peripheral nerve regeneration and repairing peripheral nerve injury in Comparative Example 4

[0096] The difference between this comparative example and Example 3 lies in: different enzyme preparations.

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

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

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

[0100] After adjusting the pH value of the slurry to 7.5 - 8, an enzyme preparation was added, and enzymatic hydrolysis was carried out at 45 °C for 1.5 h to obtain an enzymatic hydrolysate;

[0101] The addition amount of the enzyme preparation was: 0.3% of the total mass of walnut cake meal;

[0102] The enzyme preparation was ficin;

[0103] After the reaction ended, the enzymatic hydrolysate was inactivated at 102 °C for 10 minutes, cooled to 37 °C, and Kluyveromyces marxianus and Lactobacillus helveticus (mass ratio 1:1) were added at an inoculation amount of 3%, and fermentation was carried out at 37 °C for 16 h to obtain a fermentation broth;

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

[0105] Comparative Example 5

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

[0107] I. Yield

[0108] The yields of the walnut oligopeptide powder prepared in the examples and comparative examples were investigated. Among them, the calculation formula for the yield was: mass of walnut oligopeptide powder * 100 / mass of walnut cake meal, and the results are shown in Table 1.

[0109] Table 1 Yields of the walnut oligopeptide powder prepared in the examples and comparative examples

[0110] Example 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] II. Nerve repair effect

[0112] 1. Detection materials

[0113] 1.1 Sample preparation

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

[0115] Positive control: Glutathione, batch number SLCG8572, SIGMA, with the solvent being standard dilution water.

[0116] 1.2 Experimental animals

[0117] Zebrafish were all raised in fish-raising water at 28°C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, with a conductivity of 450 - 550 μS / cm; pH of 6.5 - 8.5; hardness of 50 - 100 mg / L CaCO3). They were provided by the fish-raising center of the Innovation Experiment Center of Huante Biotechnology. The experimental animal use license number is: SYXK(Zhe)2022 - 0004. The feeding management complied with the requirements of international AAALAC accreditation (accreditation number: 001458), and the IACUC ethical 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 Beilambo Biotechnology Co., Ltd., China); Electric focusing continuously variable magnification fluorescence microscope (AZ100, Nikon, Japan); Ultrasonic cleaner (JP-010T, Shenzhen Jiemeng Cleaning Equipment Co., Ltd., China).

[0120] Absolute ethanol (batch number 20240312, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Methyl cellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0121] 2. Detection Methods

[0122] 2.1 MTC Determination

[0123] Randomly select transgenic motor neuron green fluorescent zebrafish (HB9) at 6 hours post-fertilization (6 hpf) into 6-well plates, with 30 zebrafish in each well (experimental group). Set up a normal control group and a model control group, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were given absolute ethanol in water to establish a peripheral motor nerve injury model. After treatment at 28°C for 2 days, the ethanol was removed, and walnut oligopeptide powder was given in water. After treatment at 28°C for 1 day, the MTC of the samples on the model zebrafish was measured.

[0124] 2.2 Evaluation of Nerve Repair Efficacy

[0125] Randomly select 6 hpf transgenic motor neuron green fluorescent zebrafish (HB9) into a 6-well plate, with 30 zebrafish in each well (experimental group). Set up a normal control group and a model control group, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups were given absolute ethanol in water to establish a peripheral motor nerve injury model. After treatment at 28 °C for 2 days, the ethanol was removed, and walnut oligopeptide powder (concentration of 250 μg / mL) was given in water. The positive control was glutathione at a concentration of 500 μg / mL. After continuing to treat at 28 °C for 1 day, randomly select 10 zebrafish from each experimental group and place them under a fluorescence microscope for photographing. Use NIS-Elements D 3.20 advanced image processing software to analyze and collect data, and analyze the length of the peripheral motor nerve in the area of three body segments above the cloaca of the zebrafish (as Figure 1 shown), and evaluate the nerve repair efficacy of the sample based on the statistical analysis results of this index. The statistical processing results are expressed as mean ± SE. Use SPSS 26.0 software for statistical analysis, and p < 0.05 indicates that the difference is statistically significant.

[0126] 3. Detection Results

[0127] 3.1 MTC

[0128] Table 2 Experimental results of nerve repair efficacy concentration (n = 30)

[0129]

[0130] As shown in Table 2, under the experimental conditions of this experiment, the MTC of the walnut oligopeptide powder prepared in Example 3 for nerve repair efficacy was 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 control - 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 in data between groups, P < 0.05.

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

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

[0137] Compared with the model control group, there were no significant differences in the length change of the peripheral motor nerves of zebrafish in Comparative Example Groups 1-4 (P>0.05), indicating that under the experimental conditions of this experiment, the walnut oligopeptide powder prepared in Comparative Example Groups 1-4 of the present invention has no nerve repair effect.

[0138] Comparing Example 3 with Comparative Example Groups 1-4, it can be seen that after the present invention is enzymatically hydrolyzed with a specific enzyme preparation and then fermented with Kluyveromyces marxianus and Lactobacillus helveticus, the prepared walnut oligopeptide powder has a significant nerve repair effect, while the prepared walnut oligopeptide powder by the compounding of a conventional enzyme preparation and fermenting 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 limiting the protection scope of the present invention. Any simple modification or equivalent replacement made by those of ordinary skill in the art to the technical solution of the present invention does not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing walnut oligopeptide powder capable 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 with enzyme preparation, and then fermenting with Kluyveromyces marxianus and Lactobacillus helveticus, wherein the enzyme preparation is trypsin and ficin; The mass ratio of trypsin to ficin is 5-10:1; The mass ratio of Kluyveromyces marxianus to Lactobacillus helveticus is 1:0.5-1.5; The preparation method 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; 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 performed at 40-50° C. for 1-3 hours; The total inoculation amount of Kluyveromyces marxianus and Lactobacillus helveticus in step S3 is 2-5% of the mass of the enzymatic hydrolysate; The fermentation conditions in step S3 are 35-40° C. for 12-24 hours.

2. The preparation method according to claim 1, characterized in that: The mass ratio of trypsin to ficin is 8:

1.

3. Use of the walnut oligopeptide powder prepared by the preparation method according to any one of claims 1 to 2 in the preparation of a drug for repairing nerve damage.

4. The use according to claim 3, characterized in that: The nerve damage is caused by at least one of stroke, Alzheimer's disease, diabetic neuropathy, meningitis, and brain damage.

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