AU2021106760A4

AU2021106760A4 - Walnut peptide capable of assisting in improving memory and preparation method and use thereof

Info

Publication number: AU2021106760A4
Application number: AU2021106760A
Authority: AU
Inventors: Wei Wei; Jiuxun Zhang; Xiping Zhang; Xuejun Zhang; Zhao Zhang
Original assignee: Zhongshiduqing Shandong Biotech Co Ltd
Current assignee: Zhongshiduqing Shandong Biotech Co Ltd
Priority date: 2021-08-24
Filing date: 2021-08-24
Publication date: 2021-11-18
Anticipated expiration: 2029-08-24

Abstract

OF THE DISCLOSURE The present disclosure discloses a walnut peptide and a preparation method thereof, and belongs to the technical field of bioactive peptide preparation. In the present disclosure, a walnut residue pulp is subjected to enzymolysis with a cellulase and a pectinase, enzyme deactivation is conducted, and an obtained product is filtered and dried to obtain a high-protein walnut protein. An alkaline protease and a papain are added to a solution of the walnut protein for enzymolysis, and enzyme deactivation is conducted. An activated carbon is added to a walnut protein pulp enzymatic hydrolyzate for heat preservation, filtered and dried to obtain a walnut peptide with high protein content and moderate molecular weight. The present disclosure further provides a compound walnut peptide composition containing the walnut peptide, including the walnut peptide, a whey protein isolate, a DHA, a multivitamin, a mineral, and a lecithin. The walnut peptide and the compound walnut peptide composition provided by the present disclosure have the effects of neuroprotection and auxiliary memory improvement, can significantly up-regulate a brain-derived neurotrophic factor gene (BDNF gene) and a glial cell line-derived neurotrophic factor gene (GDNF gene), and increase expression of a brain-derived neurotrophic factor and a glial cell line-derived neurotrophic factor.

Description

WALNUT PEPTIDE CAPABLE OF ASSISTING IN IMPROVING MEMORY AND PREPARATION METHOD AND USE THEREOF TECHNICAL FIELD

[01] The present disclosure relates to the technical field of the bioactive peptide preparation, in particular to a walnut peptide and a preparation method and use thereof.

BACKGROUNDART

[02] A walnut is the nut of any tree of the genus Juglans, particularly the Persian or English walnut, Juglans regia.. Walnut, almond, cashew, and hazelnut are known as the world-famous "Four Dry Fruits". Walnut kernels are rich in nutrients, with relatively-high fat content, 15-20 grams of protein, and 10 grams of carbohydrates per 100 grams; the walnut kernels also contains many trace elements and minerals such as calcium, phosphorus, and iron, etc., as well as many vitamins such as carotene and riboflavin that are necessary for human body. The walnut kernels are well-liked.

[03] Walnut residue is a by-product of walnut oil extraction and is currently only used as a feed or fertilizer. As a result, nutrient substances in the walnut residue, especially the bioactive substances, are underutilized and neglected. The low utilization of a walnut residue with high nutritional value leads to low product additional value. In recent decades, the development of bioactive peptides has brought new ideas to the deep processing of the walnut residue. The enzymatic hydrolysate of the walnut residue protein has relatively-high biological activity. The diverse biological activity and function and good processing characteristics of a polypeptide have gradually become a research hotspot.

[04] Memory decline as a clinical symptom is manifested in many neurodegenerative diseases. Currently, there are more studies on Alzheimer's disease, vascular dementia, Parkinson's disease, aluminum poisoning, and epilepsy, etc. The memory decline has attracted wide attention as a prominent feature in the occurrence and development of neurodegenerative diseases. There are many causes of neurodegenerative diseases, including p-amyloid peptide (AP) accumulation, cholinergic system dysfunction, excessive glutamic acid content, mitochondrial function disorder, oxidative stress, and inflammation release, etc. It can be seen that the regulation trend of memory ability in neurodegenerative diseases is multi-directional and multi-targeted. Therefore, the development of an active substance that can prevent and ameliorate memory decline and cognitive function degradation has become the focus of scientific workers.

[05] Walnut protein is a high-quality vegetable protein containing 18 kinds of amino acids, of which 8 kinds of essential amino acids have complete range and reasonable content. Glutamic acid (Glu), arginine (Arg) and aspartic acid (Asp) in the walnut peptide are important in maintaining the human nervous system. There are only few studies on the structure of a polypeptide sequence in the walnut peptide that has the effects of neuroprotection and auxiliary memory improvement. The existing walnut peptide extraction process has high cost and large waste, and is not conducive to industrialization. In addition, various proteases are usually used to participate in the entire enzymolysis, and differences between the proteases are ignored. Therefore, the enzymolysis effect is unsatisfactory, and the prepared walnut peptide has low quality.

SUMMARY

[06] In order to solve the problems in the prior art, the present disclosure provides a walnut peptide and a preparation method thereof. Due to relatively-high protein content and a molecular weight mainly concentrated below 1,000 Da, the walnut peptide has the functions of neuroprotection and auxiliary memory improvement.

[07] The present disclosure further provides a compound walnut peptide composition. The composition can significantly up-regulate a brain-derived neurotrophic factor gene (BDNF gene) and a glial cell line-derived neurotrophic factor gene (GDNF gene), and increase expression of a brain-derived neurotrophic factor and a glial cell line-derived neurotrophic factor. Accordingly, the composition has the functions of neuroprotection and auxiliary memory improvement.

[08] In order to realize the above objective, the present disclosure provides the following technical solutions:

[09] The present disclosure provides a preparation method of a walnut peptide, including the following steps:

[10] (1) subjecting a walnut residue pulp to enzymolysis with a cellulase and a pectinase, conducting enzyme deactivation, filtering and drying an obtained product to obtain a walnut protein;

[11] (2) preparing a walnut protein pulp, heating the walnut protein pulp at a constant temperature of 50-60°C, adjusting the pH, conducting enzymolysis using an alkaline protease and a papain sequentially to obtain a walnut protein enzymatic hydrolysate; and

[12] (3) adding an activated carbon to the walnut protein enzymatic hydrolysate for heat preservation, filtering and drying to obtain the walnut peptide; where

[13] in step (1), the cellulase has an addition amount of 1,200-1,800 U/mL, the pectinase has an addition amount of 400-600 U/mL, and the walnut residue pulp is subjected to enzymolysis at a pH of 6-7 at 50-55°C for 3-5 h.

[14] Preferably, the alkaline protease may have an addition amount of 450-550 U/mL, and the papain may have an addition amount of 200-300 U/mL; the enzymolysis of the alkaline protease and the papain may be conducted at a pH of 8-8.5 at 50-60°C; the alkaline protease may be subjected to enzymolysis for 4.5-5.5 h, and then for 3.5-4.5 h after adding the papain.

[15] The present disclosure further provides a walnut peptide prepared by the preparation method.

[16] The present disclosure further provides a compound walnut peptide composition containing the walnut peptide, including the following components by mass percentage: the walnut peptide 48-60%, a whey protein isolate 28-40%, a DHA 5-6%, a multivitamin 0.2-0.4%, a mineral 34%, and a lecithin 24%.

[17] The present disclosure further provides use of the walnut peptide or the compound walnut peptide composition in the preparation of a medicine for achieving neuroprotection and/or improving memory.

[18] Compared with the prior art, the technical solutions of the present disclosure have the following beneficial effects:

[19] In the present disclosure, the walnut residue is processed into the walnut peptide by a specific biological enzymolysis technology. The walnut peptide has relatively-high protein content, reaching 91.7%, of which the glutamic acid (Glu), the arginine (Arg) and the aspartic acid (Asp) have a content of 16.27%, 10. 7 %, and 7.62%, respectively. Moreover, the walnut peptide has a molecular weight mainly concentrated below 1,000 Da, accounting for 92.16%.

[20] The walnut peptide and the compound walnut peptide composition provided by the present disclosure can significantly up-regulate the BDNF gene and the GDNF gene, and increase expression of the brain-derived neurotrophic factor and the glial cell line-derived neurotrophic factor. Accordingly, the walnut peptide and the compound walnut peptide composition can achieve neuroprotection and improve memory.

[21] The preparation method of the walnut peptide in the present disclosure is simple, has low cost and less waste, and can be used in industrial production.

BRIEF DESCRIPTION OF THE DRAWINGS

[22] FIG. 1 is a gel permeation chromatogram of a walnut peptide.

[23] FIG. 2 is a typical diagram of a zebrafish trajectory after sample treating.

[24] FIG. 3 is a percentage comparison chart of a movement distance in a blue area accounting for a total movement distance in an entire area of the zebrafish.

[25] FIG. 4 is a relative expression of a BDNF gene.

[26] FIG. 5 is a relative expression of a GDNF gene.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[27] The present disclosure provides a preparation method of a walnut peptide, including the following steps:

[28] (1) subjecting a walnut residue pulp to enzymolysis with a cellulase and a pectinase, conducting enzyme deactivation, filtering and drying an obtained product to obtain a walnut protein;

[29] (2) preparing a walnut protein pulp, heating the walnut protein pulp at a constant ) temperature of 50-60°C, adjusting the pH, conducting enzymolysis using an alkaline protease and a papain sequentially to obtain a walnut protein enzymatic hydrolysate; and

[30] (3), adding an activated carbon to the walnut protein enzymatic hydrolysate for heat preservation, filtering and drying to obtain the walnut peptide.

[31] In the present disclosure, the walnut residue is a defatted walnut residue obtained by dehulling a walnut and cold pressing to remove the oil. There is no limitation on the specific source of the defatted walnut residue.

[32] In the present disclosure, the defatted walnut residue and water are mixed to obtain a walnut residue pulp at a material-to-liquid ratio of 1:(8-12) g/mL, preferably 1:(9-11) g/mL, more preferably 1:10 g/mL. The water is preferably deionized water.

[33] In the present disclosure, the walnut residue pulp is adjusted to a pH of 6-7, preferably 6.5-6.8, and heated to 50-55°C, preferably 52-54°C. There is no limitation on the specific pH regulators used, and conventional acid-base pH regulators can be used. There is no limitation on the specific heating method. The activity of the cellulase and the pectinase can be improved by adjusting the pH and temperature of the walnut residue pulp.

[34] In the present disclosure, the walnut residue pulp is subjected to enzymolysis by adding the cellulase and the pectinase for 3-5 h, preferably 4 h. The cellulase has an addition amount of 1,200-1,800 U/mL, preferably 1,500-1,600 U/mL; and the pectinase has an addition amount of 400-600 U/mL, preferably 500-550 U/mL. The walnut protein is compositely extracted by simultaneously adding the cellulase and the pectinase to the walnut residue to conduct double enzymolysis, which can effectively improve the extraction efficiency of walnut protein. There is no limitation on the specific source of the cellulase and the pectinase.

[35] In the present disclosure, an enzymatic hydrolyzate of the walnut residue pulp is treated with enzyme deactivation at 100-110°C for 8-10 min.

[36] In the present disclosure, the enzymatic hydrolyzate of the walnut residue pulp after enzyme deactivation is filtered and dried. In an optional implementation, the enzymatic hydrolyzate is filtered using a plate and frame filter press, and a filter cake is collected; the filter cake is dried in a tube bundle dryer to obtain the walnut protein. There is no limitation on the specific filtering and drying method. The prepared walnut protein has a protein content reaching 82-89%.

[37] In the present disclosure, the prepared walnut protein is mixed with water to obtain a walnut protein pulp at a material-to-liquid ratio of 1:(18-22) g/mL, preferably 1:(19-21) g/mL, more preferably 1:20 g/mL. The water is preferably deionized water.

[38] In the present disclosure, the pH of walnut protein pulp is adjusted to 8.8-9.2, preferably 8.9-9.0. There is no limitation on the specific pH regulators used, and conventional acid-base pH regulators can be used. In an optional implementation, the pH of walnut protein pulp is adjusted to 9.0 with a NaOH solution in a concentration of 9-10 mol/L. The present disclosure finds that the walnut protein has a better solubility under this pH.

[39] In the present disclosure, the walnut protein pulp is homogenized. In an optional implementation, the walnut protein pulp is treated in a colloid mill (at 5000-6000 rpm, with a filter screen of 50-60 mesh) for 10-15 min, and then in a high-pressure homogenizer (at 6-7 Kpa) for -10 min. There is no limitation on the specific homogenizing method. The pulp can have a consistent particle size, and a fine and unlayered texture through homogenizing, which is beneficial to improve the stability of the pulp.

[40] In the present disclosure, the homogenized walnut protein pulp is heated to 50-60°C, preferably 55-58°C, and kept for 1-2 h, preferably 1.5 h. The heating and heat preservation is conducive to the decolorization and deodorization of the walnut protein pulp.

[41] In the present disclosure, the pH of the walnut protein pulp after heat preservation is further adjusted to 8-8.5, preferably 8.2-8.4. There is no limitation on the specific pH regulators used, and conventional acid-base pH regulators can be used. In an optional implementation, the pH of walnut protein pulp is adjusted to 8.5 with an HCl solution in a concentration of 0.5-1 mol/L. The pH adjusting is conducted at a constant temperature of 50-60°C. The activity of the alkaline protease and the papain in a subsequent enzymolysis can be improved by adjusting the pH and temperature of the walnut protein pulp.

[42] In the present disclosure, the walnut protein pulp is subjected to enzymolysis by adding the alkaline protease for 4.5-5.5 h, preferably 5 h. The alkaline protease has an addition amount of 450-550 U/mL, preferably 500 U/mL. There is no limitation on the specific source of the alkaline protease.

[43] In the present disclosure, after finishing the enzymolysis by alkaline protease, the papain is added to continue the enzymolysis for 3.5-4.5 h, preferably 4 h. The papain has an addition amount of 200-300 U/mL, preferably 250 U/mL. There is no limitation on the specific source of the papain.

[44] In the present disclosure, more bioactive walnut polypeptides can be obtained by enzymolysis through the relatively-more specific enzymolysis sites of alkaline protease and papain. The glutamic acid (Glu), arginine (Arg) and aspartic acid (Asp) that are important in maintaining the human nervous system, can reach 16.27%, 10.7%, and 7.62%, respectively. This is beneficial to improve the efficacy of the obtained walnut peptide in neuroprotection and memory improvement.

[45] In the present disclosure, the enzymatic hydrolyzate of the walnut protein pulp is heated to 100-110°C and kept for 5-7 min to inactivate the protease therein.

[46] In the present disclosure, the enzymatic hydrolyzate of the walnut protein pulp after enzyme deactivation is cooled to 45-55°C, preferably 50°C and kept for 5-7 h, preferably 6 h by adding the activated carbon, and deodorized and decolorized. The activated carbon has an addition amount of

7-9 wt%, preferably 8 wt%. The taste of the enzymatic hydrolyzate of the protein pulp can be improved by adding the activated carbon to remove the different degrees of bitterness therein.

[47] In the present disclosure, the enzymatic hydrolyzate of the walnut protein pulp is filtered and dried to obtain the walnut peptide. In an optional implementation, the debittered enzymatic hydrolyzate of the walnut protein pulp is passed through a 28,000-30,000 Da ceramic membrane, and then a 1-1.2 KDa organic membrane; an obtained filtrate is spray-dried to obtain the walnut peptide. There is no limitation on the specific filtering and drying method.

[48] The present disclosure further provides a compound walnut peptide composition, including a walnut peptide prepared by the preparation method of the present disclosure.

[49] In the present disclosure, the compound walnut peptide composition includes the following components by mass ratio: the walnut peptide 48-60%, a whey protein isolate 28-40%, a DHA -6%, a multivitamin 0.2-0.4%, a mineral 34%, and a lecithin 24%. Preferably, the composition includes: the walnut peptide 50-58%, the whey protein isolate 30-38%, the DHA 5.5-6%, the multivitamin 0.3-0.4%, the mineral 3.5-4%, and the lecithin 2.5-3%. The relative content of protein can be increased by adding the whey protein isolate, the DHA, and the lecithin to walnut peptide, thereby further improving the effects of neuroprotection and memory improvement.

[50] In the present disclosure, the multivitamin in the compound walnut peptide composition includes: vitamin A, a-tocopherol, y-tocopherol, -tocopherol, vitamin E, vitamin D, vitamin KI, vitamin BI, vitamin B2, pyridoxine, vitamin B6, vitamin B12, vitamin C, niacin, folic acid, pantothenic acid, and biotin. There is no limitation on the specific ratio and content of various vitamins in the multivitamin.

[51] In the present disclosure, the mineral in the compound walnut peptide composition includes: iodine, chloride, calcium, copper, iron, potassium, manganese, sodium, zinc, arsenic, mercury, lead, nitrate, phosphorus, magnesium, and selenium. There is no limitation on the specific ratio and content of various minerals in the mineral.

[52] In the present disclosure, there is no limitation on a preparation method of the compound walnut peptide composition, and each component can be simply mixed according to a proportion.

[53] The walnut peptide and the compound walnut peptide composition provided by the present disclosure can significantly up-regulate the BDNF gene and the GDNF gene, and increase expression of the brain-derived neurotrophic factor and the glial cell line-derived neurotrophic factor. Accordingly, the walnut peptide and the compound walnut peptide composition can achieve neuroprotection and improve memory, and can be used in the preparation of a medicine for achieving neuroprotection and/or improving memory.

[54] The technical solutions in the present disclosure will be clearly and completely described below in conjunction with the Examples of the present disclosure. Apparently, the described examples are merely some rather than all of the examples of the present disclosure. All other examples obtained by a person of ordinary skill in the art based on the examples of the present disclosure without creative efforts shall fall within the protection scope of the present disclosure.

[55] Example 1

[56] A preparation method of a walnut peptide included:

[57] (1) Double-enzyme composite extraction of a walnut protein: 1,200 kg of a defatted walnut residue was mixed with deionized water according to a material-to-liquid ratio of 1:10 g/mL, pH was adjusted to 6.5, heated to 54°C, a cellulase (1,500 U/mL) and a pectinase ( 500 U/mL) were added, enzymolysis was conducted for 4 h, enzyme deactivation was conducted at 100°C for 10 min, a filter cake was collected using a plate and frame filter press, and dried using a tube bundle dryer to obtain the walnut protein.

[58] (2) Production process of a walnut peptide:

[59] a, 500 kg of the walnut protein was mixed with 10,000 L of deionized water to obtain a walnut protein solution;

[60] b, the walnut protein solution in step a was stirred evenly, the pH was adjusted to 9.0 with a NaOH in a concentration of 10 mol/L, treated using a colloid mill (at 6,000 rpm, with a filter screen of 60 mesh) for 10 min, and then using a high-pressure homogenizer (at 7 Kpa ) for 6 min;

[61] c, a homogenate obtained from high-pressure homogenization was fed, stirred, heated to °C and kept for 1 h;

[62] d, the pH of the solution in step c was adjusted to 8.5 with a 1 mol/L HCl at a constant temperature;

[63] e, the solution in step d was subjected to enzymolysis for 5 h by adding an alkaline protease (500 U/mL), and then for 4 h by adding a papain (250 U/mL);

[64] f, the solution in step e was heated to 110°C and keep for 5 min to inactivate the protease;

[65] g, the solution after enzyme deactivation in step f was cooled to 50°C, an 8% activated carbon (40 kg) was added, and a resulting mixture was kept at the temperature for 6 h; the mixture was passed through a 30,000 Da ceramic membrane and then through a 1 KDa organic membrane; and a resulting filtrate was spray-dried to obtain the walnut peptide.

[66] Example 2

[67] This example was different from Example 1 only in that in step (1), a defatted walnut residue was mixed with deionized water at a material-to-liquid ratio of 1:12 g/mL.

[68] Example 3

[69] This example was different from Example 1 only in that in step (1), an enzymolysis was conducted for 5 h by adding a cellulase (1,800 U/mL) and a pectinase (400 U/mL).

[70] Example 4

[71] This example was different from Example 1only in that in step a, 500 kg of a walnut protein was mixed with 11,000 L of deionized water to obtain a walnut protein solution.

[72] Example 5

[73] This example was different from Example 1only in that in step e, a solution in step d was subjected to an enzymolysis for 4.5 h by adding an alkaline protease (300 U/mL), and then for 4.5 h by adding a papain (400 U/mL).

[74] Example 6

[75] This example was different from Example 1 only in that in step g, a 7% activated carbon was added.

[76] The physical and chemical properties of the walnut peptide prepared in Example 1 were determined as follows: a protein content determination (GB5009.9), a moisture content determination (GB5009.3), and an ash content determination (GB5009.4). Determination results were shown in Table 1.

[77] Table 1 Determination results of basic physical and chemical properties of the walnut peptide Protein Moisture content Ash content Example 1 91.7% 3.1% 5.2%

[78] The molecular weight of the walnut peptide prepared in Example 1 was determined by gel permeation chromatography, and the results were shown in FIG. 1. It can be seen from FIG. 1 that the walnut peptide prepared in Example 1 has a molecular weight mainly concentrated below 1,000 Da, accounting for about 92.16%.

[79] Example 7

[80] A compound walnut peptide composition and a preparation method thereof were provided:

[81] The compound walnut peptide composition included the following components by mass ratio: a walnut peptide 55%, a whey protein isolate 33%, a DHA 5%, a multivitamin 0.2%, a mineral 3.8%, and a lecithin 3%.

[82] The multivitamin included: vitamin A 417 g/100 g, a-tocopherol 32.8 mg/100 g, y-tocopherol 2.82 mg/100 g, 6-tocopherol 0.624 mg/100 g, vitamin E 36.2mg/100 g, vitamin D 6.43 pg/100 g, vitamin KI 50.6 g/100 g, vitamin BI 1.32 mg/100 g, vitamin B2 1.20 mg/100 g, pyridoxine 2.08 mg/100 g, vitamin B6 2.08 mg/100 g, vitamin B12 3.3 g/100 g, vitamin C 54.7 mg/100 g, niacin 2088 g/100 g, folic acid 216 g/100 g, pantothenic acid 3.97 mg/100 g, and biotin 23.3 g/100 g.

[83] The mineral included: iodine 0.89 mg/kg, chloride 0.62 g/100g, calcium 2920 mg/kg, copper 5.73 mg/kg, iron 56.4 mg/kg, potassium 8660 mg/kg, manganese 3.73 mg/kg, sodium 1690 mg/kg, zinc 43.8 mg/ kg, arsenic 0.0113 mg/kg, mercury 0.0182 mg/kg, lead 0.0534 mg/kg, nitrate 8.9 mg/kg, phosphorus 3.55x102 mg/100 g, magnesium 1.22x103 mg/kg, and selenium 0.37 mg/kg.

[84] A preparation method of the compound walnut peptide composition included:

[85] (1) pre-mixing: the four components walnut peptide, DHA, multivitamin and mineral were weighed in proportions, and passed through a 30-mesh sieve to obtain a primary mixture;

[86] (2) mixing: the primary mixture obtained in step (1) was slowly added to warm water (at pH 6.0) at 45°C in a weight ratio to water of 1:5, uniformly stirred at a rate of 70 rpm, and the whey protein isolate and lecithin were added for mixing evenly;

[87] (3) a product obtained in step (2) was frozen and vacuum-dried (using a YRDS-15010 Chinese-style freeze dryer for health products and foods) at -40°C to -45°C and a vacuum degree of 220 Pa for 46 h.

[88] (4) a product obtained in step 3) was pulverized through a 40-mesh sieve with a WN-500A pulverizer to obtain the compound walnut peptide composition.

[89] Example 8

[90] This example was different from Example 7 only in that the compound walnut peptide composition included the following components by mass ratio: a walnut peptide 50%, a whey protein isolate 38%, a DHA 6%, a multivitamin 0.4%, a mineral 3%, and a lecithin 2.6%.

[91] Example 9

[92] The walnut peptide prepared in Example 1 and the compound walnut peptide composition prepared in Example 7 were tested for the efficacy on memory improvement (promoting brain development).

[93] (1) Preparation of samples

[94] The walnut peptide prepared in Example 1 was prepared into a mother liquor of 2.00 mg/mL for immediate use with standard dilution water.

[95] The compound walnut peptide composition prepared in Example 7 was prepared into a mother liquor of 2.00 mg/mL for immediate use with standard dilution water.

[96] Positive control was: edaravone, yellow powder, batch number K1820098, Shanghai Aladdin Biochemical Technology Co., Ltd., stored in a cool and dark place. The edaravone was prepared into a mother liquor of 5.00 mg/mL with DMSO, and stored at -20°C.

[97] (2) Experimental animals

[98] Zebrafishes were fed in fish farming water (water quality: 200 mg of instant sea salt was added to per 1 L reverse osmosis water, with a conductivity of 450-550 [S/cm; a pH of 6.5-8.5; and a hardness of 50-100 mg/L CaCO3) at 28°C; and use license of experimental animals was: SYXK (Zhejiang) 2012-0171. The feeding management met the requirements of international AAALAC certification (certification number: 001458).

[99] Wild-type AB strain zebrafishes were subjected to a natural paired mating reproduction mode. Zebrafishes aged 3 days post-fertilization (3 dpf) were used to determine the maximum test concentration (MTC)of samples. Zebrafishes aged 5 dpf were used to evaluate the memory improvement of samples.

[100] (3) Instruments, consumables and reagents

[101] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertAl, Shanghai Tusen Vision Technology Co., Ltd.); precision electronic balance (CP214, OHAUS, USA); behavior analyzer (V3.11, View Point Life Sciences, France).

[102] Dimethyl sulfoxide (DMSO, batch number BCBZ1685, Sigma, USA); bisphenol AF (batch number J1925142, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[103] (4) Determination items:

[104] 1. MTC determination

[105] Zebrafishes (3 dpf wild-type AB strain) were randomly added to a 6-well plate, with 30 per well (as an experimental group). The zebrafishes were given water-dissolved samples separately (Table 2 for the specific concentration of each sample), and meanwhile a normal control group was set up with a volume of 3 mL per well. After being treated at 28°C for 72 h, the MTC of the sample to normal zebrafishes was determined.

[106] Evaluation of memory improvement

[107] Zebrafishes (5 dpf wild-type AB strain) were randomly added to beakers, with 30 per beaker (as an experimental group). The zebrafishes were given water-dissolved samples separately (Table 3 for the specific concentration of each sample), a positive control edaravone with a concentration of 5.00 g/mL was set up, and meanwhile a normal control group and a model control group were set up with a volume of 20 mL per beaker. Except for the normal control group, all other experimental groups were given water-dissolved bisphenol AF to establish a zebrafish memory impairment model. After treatment at 28°C for 1 day, 5 zebrafishes were randomly selected from each experimental group and placed in cross-shaped modules. The modules were divided into four areas: yellow, blue, red and green, and each group was placed in 6 modules. Data was collected with the behavior analyzer; a percentage (%) of a total movement distance in a blue area accounting for a total movement distance in an entire area of the zebrafish within 20 minutes was analyzed to obtain an index. The memory improvement of the sample was evaluated using a statistical analysis result of the index. A statistical processing result was expressed as mean SE. Statistical analysis was conducted with SPSS26.0 software, and p<0.05 indicated that the difference was statistically significant.

[108] (5) Test result

[109] 1. MTC determination results were shown in Table 2.

[110] Table 2 Experimental results of the concentration of walnut peptide and compound walnut peptide composition for memory improvement Group Concentration Death count (per Mortality Genotype (tg/mL) fish) Normal control / 0 0 No significant abnormalities group Walnut peptide 125 0 0 Similar to normal control group l1

250 0 0 Similar to normal control group 500 0 0 Similar to normal control group 1000 0 0 Similar to normal control group 2000 30 100 Compound 125 0 0 Similar to normal control walnut peptide group composition 250 0 0 Similar to normal control group 500 0 0 Similar to normal control group 1000 30 100 2000 30 100

[111] It can be seen from Table 2 that under the experimental conditions, the MTC of walnut peptide and compound walnut peptide composition to normal zebrafish are 1,000 g/mL and 500

[tg/mL, separately. The final evaluation concentration was determined to be: 125 g/mL and 250

[g/mL of walnut peptide, and 250 g/mL and 500 g/mL of compound walnut peptide composition.

[112] 2. The memory improvement evaluation was shown in Table 3, FIG. 2 and FIG. 3.

[113] Table 3 Experimental results of memory improvement evaluation of samples (n=6) Concentration Proportion of blue (%, mean Group (tg/mL) ±SE)

Normal control group 43 4**

Model control group - 23 2

Edaravone 5.00 36 2*** Walnut peptide 125 35 4*

250 38 3** Compound walnut peptide composition 250 40 2**

500 43 4***

[114] Compared with the model control group,* p <0.05, ** p <0.01, and*** p <0.001.

[115] It can be seen from Table 3 that under the experimental conditions, the walnut peptide and the compound walnut peptide composition have obvious memory improvement, and there is no statistical difference in the improvement at the same concentration.

[116] An area in a dashedbox in FIG. 2 is ablue area of aplus maze, namely a quantitative area. It can be seen from FIG. 3 that the walnut peptide and the compound walnut peptide composition (also known as walnut peptide compound) groups have a proportion of blue higher than that in the model control group. The compound walnut peptide composition with a concentration of 500 pg/mL has a proportion of blue significantly higher than that in the model control group. It shows that in the present disclosure, the prepared walnut peptide and the compound walnut peptide composition have a significant efficacy on the memory improvement of memory-impaired zebrafishes.

[117] Example 10

[118] The effects of the walnut peptide prepared in Example 1 and the compound walnut peptide composition prepared in Example 7 on a brain-derived neurotrophic factor gene (BDNF gene) and a glial cell-derived neurotrophic factor gene (GDNF gene) were tested.

[119] (1) Information of sample preparation

[120] The walnut peptide prepared in Example 1 was prepared into a mother liquor of 2.00 mg/mL for immediate use with standard dilution water.

[121] The compound walnut peptide composition prepared in Example 7 was prepared into a mother liquor of 2.00 mg/mL for immediate use with standard dilution water.

[122] (2) Experimental animals

[123] The zebrafishes were fed in fish-farming water (water quality: 200 mg of instant sea salt was added to per 1 L reverse osmosis water, with a conductivity of 450-550 [S/cm; a pH of 6.5-8.5; and a hardness of 50-100 mg/L CaCO3) at 28°C, and the experimental animal use license number was: SYXK (Zhejiang) 2012-0171. The feeding management met the requirements of international AAALAC certification (certification number: 001458).

[124] Wild-type AB strain zebrafishes were subjected to a natural paired mating reproduction mode. Zebrafishes aged 3 days post-fertilization (3 dpf) were used to evaluate the effects of samples on BDNF gene and GDNF gene.

[125] (3) Instruments, consumables and reagents

[126] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertAl, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); 6-well plate (Nest Biotech, China); ordinary PCR amplification instrument (T100, Bio-Rad, Singapore); fluorescence quantitative PCR instrument (CFX Connect, Bio-Rad, Singapore); high-speed refrigerated centrifuge (Heraeus Fresco17, Thermo Fisher, Germany); ultraviolet-visible spectrophotometer (Nanodrop2000, Thermo, Austria); microplate mini centrifuge (BE-6100, Haimen Kylin-Bell Lab Instruments Co.,Ltd., China); 96-well plate with low skirt (transparent) (HSP9601, Bio-Rad, USA); and optical adhesive sealing film B (MSB1001, Bio-Rad, USA).

[127] Absolute ethanol (batch number 20200302, Sinopharm Chemical Reagent Co., Ltd., China); iTaq Universal SYBR Green Supermix (Cat. No. 1725124, Bio-Rad, USA); FastQuant RT Kit (With gDNase) (Cat. No. KR106, TIANGEN, China); and RNA-Quick Purification Kit (Cat. No. RNOO1, YiShan Biotech, China).

[128] (4) Determination method

[129] Zebrafishes (3 dpf wild-type AB strain) were randomly added to a 6-well plate, with 30 per well (as an experimental group). The zebrafishes were given water-dissolved samples separately

(Table 4 for the concentration), and meanwhile a normal control group was set up with a volume of 3 mL per well and three parallel experiments were set up. After being treated at 28°C for 72 h, the total RNA of each group of zebrafishes was extracted using the RNA-Quick Purification Kit, and concentration and purity of the total RNA were determined by the ultraviolet-visible spectrophotometer. 20.0 tL of cDNA was synthesized using 2.00 tg of zebrafish sample total RNA according to the instructions of cDNA first-strand synthesis kit, and the expression of p-actin, BDNF gene and GDNF genes was detected by quantitative polymerase chain reaction (q-PCR). A relative RNA expression of BDNF gene and GDNF gene was calculated using the p-actin as an internal reference for gene expression. A statistical processing result was expressed as mean SE. Statistical analysis was conducted with SPSS26.0 software, and p<0.05 indicated that the difference was statistically significant.

[130] (5) Test result

[131] 1. RNA extraction results and primer sequence information

[132] After the sample was treated, the zebrafish total RNA was extracted, and the RNA concentration and an A260/A280 ratio were measured with the ultraviolet-visible spectrophotometer, shown in Table 4 for details.

[133] Table 4 Concentration of total RNA and A260/A280 ratio (n=3)

Concentratio RNA concentration (pg/ pL) A260/A280 Group n (pg/mL) Sample 1 Sample 2 Sample 3 Sample 1 Sample 2 Sample 3

Normal control group - 0.630 0.398 0.527 2.01 2.00 1.97

Walnut peptide 125 0.671 0.409 0.401 2.02 2.00 2.00

250 0.530 0.487 0.547 1.97 2.00 2.03

Compound walnut peptide 250 0.533 0.586 0.543 1.95 2.04 2.03

composition 500 0.610 0.463 0.438 2.03 2.00 2.01

[134] It can be seen from Table 4 that the A260/A280 ratio is 1.8-2.2, indicating that the extracted zebrafish total RNA has relatively desirable quality and can be used in subsequent q-PCR experiments. The primer sequences in the q-PCR experiment were shown in Table 5.

[135] Table 5 Primer sequence information Gene Primer sequence Forward 5'-TCGAGCAGGAGATGGGAACC-3' p-actin Reverse 5'-CTCGTGGATACCGCAAGATTC-3' BDNF Forward 5'-TCGAGCAGGAGATGGGAACC-3'

Reverse 5'-CTCGTGGATACCGCAAGATTC-3'

GDNF Forward 5'-AGCCATCCAAGAGAGCTGTG-3'

Gene Primer sequence

Reverse 5'-GTCCCGCTTCATCTGAGGTT-3'

[136] In the Table 5: for the p-actin gene, the primer F is shown in SEQ ID NO: 1, and the primer R is shown in SEQ ID NO: 2; for the BDNF gene, the primer F is shown in SEQ ID NO: 3, and the primer R is shown in SEQ ID NO: 4; and for the GDNF gene, the primer F is shown in SEQ ID NO: , and the primer R is shown in SEQID NO: 6.

[137] 2. The effects of samples on BDNF gene and GDNF gene were shown in Table 6, FIG. 4 and FIG. 5 for details.

[138] Table 6 Effects of samples on expression of BDNF gene and GDNF gene (n=3).

Concentration Relative expression of BDNF Relative expression of Group (tg/mL) (mean SE) GDNF (mean SE)

Normal control group - 1.00 0.056 1.00 0.022

Walnut peptide 125 1.28 0.063* 1.20 0.040

250 1.38 0.020** 1.25 0.091 Compound walnut peptide 250 1.54 0.083** 1.19 0.008

composition 500 1.15 0.023 1.26 0.046*

[139] Compared with the normal control group, *p <0.05, and **p <0.01.

[140] It can be seen from Table 6, FIG. 4 and FIG. 5 that, under the experimental conditions, both walnut peptide and compound walnut peptide composition can significantly up-regulate the BDNF gene and GDNF gene. The compound walnut peptide composition has a highest relative expression of the BDNF gene in a 250 pg/mL group, and a highest relative expression of the GDNF gene in a 500 pg/mL group. It indicates that the walnut peptide and the compound walnut peptide composition provided by the present disclosure can significantly up-regulate the BDNF gene and the GDNF gene, and increase expression of the brain-derived neurotrophic factor and the glial cell line-derived neurotrophic factor. Accordingly, the walnut peptide and the compound walnut peptide composition can achieve neuroprotection and improve memory.

[141] The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.

Claims

WHAT IS CLAIMED IS:

1. A preparation method of a walnut peptide, comprising the following steps: (1) subjecting a walnut residue pulp to enzymolysis with a cellulase and a pectinase, conducting enzyme deactivation, filtering and drying an obtained product to obtain a walnut protein; (2) preparing a walnut protein pulp, heating the walnut protein pulp at a constant temperature of -60°C, adjusting the pH, conducting enzymolysis using an alkaline protease and a papain sequentially to obtain a walnut protein enzymatic hydrolysate; and (3) adding an activated carbon to the walnut protein enzymatic hydrolysate for heat preservation, filtering and drying to obtain the walnut peptide; wherein in step (1), the cellulase has an addition amount of 1,200-1,800 U/mL, the pectinase has an addition amount of 400-600 U/mL, and the walnut residue pulp is subjected to enzymolysis at a pH of 6-7 at 50-55°C for 3-5 h.

2. The preparation method according to claim 1, wherein the alkaline protease has an addition amount of 450-550 U/mL, and the papain has an addition amount of 200-300 U/mL; the enzymolysis of the alkaline protease and the papain is conducted at a pH of 8-8.5 at 50-60°C; and the alkaline protease is subjected to enzymolysis for 4.5-5.5 h, and then for 3.5-4.5 h after adding the papain.

3. A walnut peptide prepared by the preparation method according to any one of claims 1-2.

4. A compound walnut peptide composition containing the walnut peptide according to claim 3, comprising the following components by mass percentage: the walnut peptide 48-60%, a whey protein isolate 28-40%, a DHA 5-6%, a multivitamin 0.2-0.4%, a mineral 34%, and a lecithin 2-4%.

5. Use of the walnut peptide according to claim 3 or the compound walnut peptide composition according to claim 4 in the preparation of a medicine for achieving neuroprotection and/or improving memory.

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DRAWINGS 24 Aug 2021

Automatic scale chromatogram 2021106760

Minute GPC result Retention Peak Distribution Polydis time Dalton Dalton Dalton Dalton % Area code name persity (Minute) (Da) (Da) (Da) (Da)

FIG. 1

－2/5－ 24 Aug 2021 2021106760

Normal control group

Model control group Edaravone 5.00 μg/mL

Walnut peptide 125 μg/mL Walnut peptide 250 μg/mL

Walnut peptide Walnut peptide compound 250 μg/mL compound 500 μg/mL FIG. 2

N or m al Blue proportion (%) co nt M ro lg od el ro co up nt ro lg ro up Ed W a al ra nu vo tp ne ep W tid e

FIG. 3 －3/5－

al 12 nu tp 5 ep tid e W 25 0

μg/mL a co ln m u po t p un ep W d tid co a l n 2 5 0 e m u po t p un ep d tid 50 e

N or m GDNF relative expression al co nt ro W lg al nu ro up tp ep tid W e 12 al nu 5 tp ep tid e

FIG. 4 －4/5－

25

μg/mL W 0 a co ln m u po t p un ep d tid W 25 e 0 co aln m u po t p un ep d tid 50 e

－5/5－ 24 Aug 2021

GDNF relative expression 2021106760

up 12 5 0 e e o 25 id 0 id 0 gr e e t t ep 50 l id id ep 25 tro pt pt p d p ut un d o n p e p e n ut un n lc ut ut al po al po a n n m W m m al al W o co or W W c N μg/mL

FIG. 5