Peptide for promoting synthesis of brain-derived neurotrophic factor as well as preparation method and application of peptide

Abstract

The invention belongs to the technical field of protein, and relates to a peptide for promoting synthesis of a brain-derived neurotrophic factor (BDNF) as well as a preparation method and application of the peptide. The amino acid sequence of the peptide for promoting synthesis of the brain-derived neurotrophic factor BDNF is SGFSKHF, and the peptide has the main effects that damage of CORT (corticosterone) to PC12 cells can be protected, the peptide can be spontaneously combined with a TrkB receptor, the structure of a compound formed after combination is more stable, BDNF-TrkB-CREB pathway signal transduction can be activated, generation of the BDNF is promoted, and the effect of promoting synthesis of the brain-derived neurotrophic factor BDNF is achieved. Therefore, the occurrence and development of neurodegenerative diseases can be delayed.

Description

Technical Field

[0001] The present invention belongs to the technical field of proteins and relates to a peptide for promoting the synthesis of brain-derived neurotrophic factor and a preparation method and application thereof. Background Art

[0002] Brain-derived neurotrophic factor (BDNF), a key member of the neurotrophic factor family, has been extensively studied and confirmed to play a vital role in neurogenesis, neuronal survival, differentiation, and maintenance of mature neuronal function. It promotes the synthesis and connectivity of neurons, thereby improving cognitive abilities and neural function. Furthermore, BDNF can prevent the occurrence of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. BDNF is a biologically important protein that can positively impact the health of the nervous system. Proper diet and exercise habits can effectively increase BDNF levels and prevent the occurrence of neurological diseases.

[0003] The binding of BDNF to its specific receptor, TrkB (Tropomyosin receptor kinase B), activates downstream signaling, including tyrosine phosphorylation. This in turn triggers the ERK / CREB (Extracellular signal-regulated kinase / Cyclic AMP response element-binding protein) signaling pathway, promoting the transcription of pro-BDNF mRNA. This mRNA is translated into pro-brain-derived neurotrophic factor (pro-BDNF), which is ultimately processed into mature BDNF through protein. Studies have shown that stress can lead to decreased BDNF levels, which may promote the occurrence of neurodegenerative diseases and accelerate the development of Alzheimer's disease. In addition, specific protein hydrolysates and peptides, such as tryptophan oligopeptides, ginger-degraded collagen hydrolysate, Ganoderma lucidum spore polysaccharide peptides, and kefir peptides, have been found to upregulate BDNF expression, potentially alleviating the occurrence of neurodegenerative diseases. Therefore, in-depth research and development of peptides that can activate the BDNF-TrkB-CREB signaling pathway and promote BDNF synthesis may provide new strategies for the treatment of neurodegenerative diseases (e.g., Alzheimer's disease). Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a peptide that promotes the synthesis of brain-derived neurotrophic factor, a preparation method and application thereof, and to help solve or improve the side effects caused by the use of traditional drugs to treat neurodegenerative diseases (e.g., Alzheimer's disease) by screening and preparing peptides that promote BDNF synthesis.

[0005] The purpose of the present invention and the solution to its technical problems can be achieved through the following technical solutions.

[0006] In one aspect, the present invention provides a peptide for promoting the synthesis of brain-derived neurotrophic factor, wherein the amino acid sequence of the peptide is SGFSKHF and the molecular weight is 809 Da.

[0007] On the other hand, the present invention provides a method for preparing the peptide that promotes the synthesis of brain-derived neurotrophic factor, comprising the following steps: (1) enzymatically hydrolyzing soy protein isolate with alkaline protease, papain, and flavor protease to obtain soy peptide; (2) performing in vitro simulated digestion on the soy peptide to obtain digested soy peptide; (3) performing in vitro simulated absorption of the digested soy peptide using a Caco-2 monolayer cell model to separate and obtain absorbed soy peptide; and (4) identifying the absorbed soy peptide to obtain the peptide SGFSKHF that promotes the synthesis of brain-derived neurotrophic factor.

[0008] In a third aspect, the present invention provides a composition for promoting the synthesis of brain-derived neurotrophic factor, wherein the composition comprises the peptide for promoting the synthesis of brain-derived neurotrophic factor of the present invention.

[0009] In a fourth aspect, the present invention provides use of a peptide that promotes the synthesis of brain-derived neurotrophic factor in the preparation of a medicament for delaying neurodegenerative diseases (eg, Alzheimer's disease).

[0010] Beneficial effects:

[0011] The peptide of the present invention (amino acid sequence: SGFSKHF) that promotes the synthesis of brain-derived neurotrophic factor has the following main effects: it can protect PC12 cells from damage caused by CORT (corticosterone), can spontaneously bind to the TrkB receptor, and the complex structure formed after binding is more stable, which helps to activate the BDNF-TrkB-CREB pathway signal transduction, thereby promoting the production of BDNF, thereby helping to delay the occurrence and / or development of neurodegenerative diseases (e.g., Alzheimer's disease). BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The following is a visualization diagram of the molecular docking of T1 and T2 peptides with TrkB receptor; (A) is the T1 peptide, and (B) is the T2 peptide;

[0013] Figure 2 The following are visualization diagrams of the molecular docking of T3, T4 and T5 peptides with TrkB receptor; (C) is the T3 peptide, (D) is the T4 peptide, and (E) is the T5 peptide;

[0014] Figure 3Effects of different peptides and CORT on PC12 cell viability; (A) is T1 peptide, (B) is T2 peptide, (C) is T3 peptide, (D) is T4 peptide, (E) is T5 peptide, and (F) is CORT;

[0015] Figure 4 The effects of different peptides on the cell viability of PC12 cells under CORT stimulation; (A) is the cell viability test result; (B) is the cell morphology;

[0016] Figure 5 The results of the analysis of the effects of different peptides on the expression of BDNF-TrkB-CREB pathway proteins in PC12 cells; (A) is the relative expression of BDNF; (B) is the relative expression of p-TrkB / TrkB; (C) is the relative expression of p-CREB / CREB;

[0017] Figure 6 The molecular dynamics simulation results of TrkB and TrkB-T3 complex; (A) is the RMSD fluctuation test result, (B) is the RMSF change test result; (C) is the Rg change test result. DETAILED DESCRIPTION

[0018] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the embodiments described below are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0019] The inventors obtained a peptide that promotes the synthesis of brain-derived neurotrophic factor (BDNF) by enzymatically hydrolyzing and separating soy protein isolate raw materials, thereby proposing the present invention.

[0020] In one aspect, the present invention provides a peptide for promoting the synthesis of brain-derived neurotrophic factor, wherein the amino acid sequence of the peptide is SGFSKHF and the molecular weight is 809 Da.

[0021] The peptide of the present invention that promotes the synthesis of brain-derived neurotrophic factor can protect PC12 cells from damage caused by CORT, can spontaneously bind to the TrkB receptor, and the complex structure formed after binding is more stable, which helps to activate the BDNF-TrkB-CREB pathway signal transduction, thereby promoting the production of brain-derived neurotrophic factor, thereby helping to reduce and delay the occurrence and / or development of neurodegenerative diseases (e.g., Alzheimer's disease).

[0022] On the other hand, the present invention provides a method for preparing the peptide that promotes the synthesis of brain-derived neurotrophic factor, comprising the following steps: (1) enzymatically hydrolyzing soy protein isolate with alkaline protease, papain, and flavor protease to obtain soy peptide; (2) performing in vitro simulated digestion on the soy peptide to obtain digested soy peptide; (3) performing in vitro simulated absorption of the digested soy peptide using a Caco-2 monolayer cell model to separate and obtain absorbed soy peptide; and (4) identifying the absorbed soy peptide to obtain the peptide SGFSKHF that promotes the synthesis of brain-derived neurotrophic factor.

[0023] In an embodiment of the present invention, in the process of preparing soybean peptides, soy protein isolate is obtained by enzymatic hydrolysis with alkaline protease (from Bacillus subtilis), papain (from papaya) and flavor protease (from Aspergillus oryzae), and the amount of total protease added is 0.8%-1.2% (for example, 0.8%, 0.9%, 1.0%, 1.1% or 1.2%) of the soy protein isolate in terms of mass fraction, and the mass ratio of alkaline protease, papain and flavor protease is 1:2:2-2:3:3 (for example, 1:2:2, 1:1.9:1.9, 1:8:8, 1:1.7:1.7, 1:1.6:1.6 or 2:3:3).

[0024] In the preparation process of the soybean peptide of the present invention, the soybean protein isolate is prepared into a 9%-10% (for example, 9% or 10%) protein solution, and the pH value is adjusted to 8.5; first, alkaline protease is added, and the enzymatic hydrolysis time is 30-35 minutes (for example, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes or 35 minutes), and the enzymatic hydrolysis temperature is 45-55° C. (for example, 45° C., 47° C., 49° C., 51° C., 53° C. or 55° C.); papain is added again, and the enzymatic hydrolysis is continued for 30-35 minutes (for example, 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes or 35 minutes). The product is added for 33 minutes, 34 minutes or 35 minutes, and the enzymatic hydrolysis temperature is 45-55°C (for example, 45°C, 47°C, 49°C, 51°C, 53°C or 55°C); finally, flavor protease is added and the enzymatic hydrolysis is continued for 160-175 minutes (for example, 160 minutes, 165 minutes, 170 minutes or 175 minutes), and the enzymatic hydrolysis temperature is 50-60°C (for example, 50°C, 52°C, 54°C, 56°C, 58°C or 60°C); after the enzymatic hydrolysis is completed, the product is heated to above 90°C and bathed in water for more than 10 minutes to inactivate the enzyme (for example, heated to 95°C and bathed in water for 10 minutes to inactivate the enzyme).

[0025] In an embodiment of the preparation method of the present invention, the peptide that promotes the synthesis of brain-derived neurotrophic factor is screened when it is first isolated using a method comprising the following steps: A. molecular docking the absorbed soybean peptide separated in step (2) with the TrkB receptor, and screening peptide segments based on affinity (screening peptide segments with low nucleophilic energy); B. using corticosterone to stimulate PC12 cells to establish a cell nerve injury model, and screening peptides that promote the synthesis of brain-derived neurotrophic factor by testing the effects of the peptide segments screened in step A on cell viability and / or BDNF-TrkB-CREB pathway protein expression.

[0026] In an embodiment of the preparation method of the present invention, the in vitro simulated digestion comprises: digesting the soybean peptide in sequence with simulated saliva, simulated gastric juice and simulated intestinal juice; after the in vitro simulated digestion is completed, the digestion product of the simulated intestinal juice is dried to obtain the digested soybean peptide.

[0027] In an embodiment of the preparation method of the present invention, the in vitro simulated digestion specifically comprises the following steps:

[0028] I. Dissolve the soybean peptide in water, add simulated saliva, and shake to obtain the soybean peptide oral digestive solution;

[0029] II. Adding simulated gastric fluid to the soy peptide oral digestive fluid, adjusting the pH to 3.0, then adding pepsin, and shaking the reaction to obtain soy peptide gastric digestive fluid;

[0030] III. Add simulated intestinal fluid to the soybean peptide gastric digestion fluid, adjust the pH to 7.0, then add trypsin and shake the reaction to obtain soybean peptide intestinal digestion fluid;

[0031] IV. heating the soybean peptide intestinal digestion fluid to terminate digestion, and freeze-drying to obtain digested soybean peptide;

[0032] Among them, based on 400 mL, the simulated saliva contains 15.1 mL 0.5 M KCl, 3.7 mL 0.5 M KH2PO4, 6.8 mL 1 M NaHCO3, 0.5 mL 0.15 M MgCl2(H2O)6, 0.06 mL 0.5 M (NH4)2CO3, 0.09 mL 6 M HCl, and dH2O is added to make it complete. 0.025 mL 0.3 M CaCl2(H2O)2 is added separately before use;

[0033] Based on 400 mL, simulated gastric fluid contained 6.9 mL 0.5 M KCl, 0.9 mL 0.5 M KH2PO4, 12.5 mL 1 M NaHCO3, 11.8 mL 2 M NaCl, 0.4 mL 0.15 M MgCl2(H2O)6, 0.5 mL 0.5 M (NH4)2CO3, 1.3 mL 6 M HCl, and dH2O was added to the volume. 0.005 mL 0.3 M CaCl2(H2O)2 was added separately before use.

[0034] Based on 400 mL, the simulated intestinal fluid contained 6.8 mL 0.5 M KCl, 0.8 mL 0.5 M KH2PO4, 42.5 mL 1 M NaHCO3, 9.6 mL 2 M NaCl, 1.1 mL 0.15 M MgCl2(H2O)6, 0.7 mL 6 M HCl, and was made up with dH2O. 0.04 mL 0.3 M CaCl2(H2O)2 was added separately before use.

[0035] The reason why CaCl2(H2O)2 is added to the above-mentioned simulated liquid is because: 1) the ionic strength of calcium ions in gastrointestinal fluid is simulated; 2) calcium ions in the solution can stabilize and increase the activity of enzymes; 3) calcium ions are involved in biological processes such as muscle contraction, nerve conduction and blood coagulation; and the reason for adding CaCl2(H2O)2 separately before use is: 1) calcium ions have high reactivity and are easy to react with other components or form precipitates; 2) calcium ions will affect the solubility of other components.

[0036] In an embodiment of the preparation method of the present invention, in step I, the concentration of the soybean peptide in the mixed solution of soybean peptide and simulated saliva is 1-2 g / mL (e.g., 1 g / mL, 1.5 g / mL or 2 g / mL); the reaction temperature is 35-40° C. (e.g., 35° C., 36° C., 37° C., 38° C., 39° C. or 40° C.), and the reaction time is 2-4 min (e.g., 2 min, 3 min or 4 min); in step II, pepsin is added to a final concentration of pepsin of 2000-2500 U / mL (e.g., 2000 U / mL, 2100U / mL, 2200U / mL, 2300U / mL, 2400U / mL or 2500U / mL), the reaction temperature is 35-40°C (for example, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C), and the reaction time is 2-3h (for example, 2h, 2.3h, 2.5h, 2.8h or 3h); wherein, if the amount of pepsin added is too high, it will lead to excessive enzymatic hydrolysis of the peptide and cannot simulate the actual digestion process of the human body; if the amount added is too low, it will lead to incomplete enzymatic hydrolysis of the peptide and cannot simulate the actual digestion process of the human body. In step III, trypsin is added to a final concentration of 100-150 U / mL (e.g., 100 U / mL, 110 U / mL, 120 U / mL, 130 U / mL, 140 U / mL, or 150 U / mL), the reaction temperature is 35-40°C (e.g., 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C), and the reaction time is 2-3 h (e.g., 2 h, 2.3 h, 2.5 h, 2.8 h, or 3 h); wherein, if the amount of trypsin added is too high, excessive enzymatic hydrolysis of the peptide will result and the actual digestion process of the human body cannot be simulated; if the amount added is too low, incomplete enzymatic hydrolysis of the peptide will result and the actual digestion process of the human body cannot be simulated. The volume ratio of simulated saliva, simulated gastric fluid, and simulated intestinal fluid is 1:1:2; after the trypsin hydrolysis is completed, the digestion is terminated by incubating in a water bath at 95-100°C (e.g., 95°C, 96°C, 97°C, 98°C, 99°C, or 100°C) for 10-15 minutes (e.g., 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, or 15 minutes).

[0037] In an embodiment of the preparation method of the present invention, in step IV, the freeze-drying temperature is -40 to -80°C (for example, -40°C, -50°C, -60°C, -70°C or -80°C), and the freeze-drying time is 48-72h (for example, 48h, 54h, 60h, 66h or 72h).

[0038] In an embodiment of the preparation method of the present invention, in step (2), the digested soybean peptide is dissolved in HBSS buffer and inoculated into Caco-2 monolayer cells, wherein the concentration of the soybean peptide digestion product in HBSS buffer is 4 mg / mL.

[0039] In an embodiment of the method of the present invention, in step (2), the soybean peptide digestion products are subjected to simulated absorption in vitro using a Caco-2 monolayer cell model, and the soybean peptide digestion products are separated into absorbed soybean peptides and unabsorbed soybean peptides.

[0040] In a specific embodiment, Caco-2 cells are cultured for 21 days to reduce their resistance to 400-600 Ω / cm 2 (For example, 410Ω / cm 2 , 420Ω / cm 2 , 430Ω / cm 2 , 440Ω / cm 2 、450Ω / cm 2 、480Ω / cm 2 , 490Ω / cm 2 , 500Ω / cm 2 、550Ω / cm 2 , 560Ω / cm 2 、570Ω / cm 2 , 580Ω / cm 2 , 590Ω / cm 2 、600Ω / cm 2 ) to construct a Caco-2 monolayer cell model. At this point, the cells differentiate into a monolayer structure similar to the villi of the small intestinal epithelium. After 2-4 hours (e.g., 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours) of absorption, the digested soy peptides are separated into absorbed soy peptides and unabsorbed soy peptides. If the resistance value is too high, the cells may form a multilayer structure; if the resistance value is too low, the cells may not be fully differentiated.

[0041] In a third aspect, the present invention provides a composition for promoting brain-derived neurotrophic factor synthesis, comprising the peptide for promoting brain-derived neurotrophic factor synthesis as described above. Those skilled in the art will appreciate that the composition may further comprise other substances that promote brain-derived neurotrophic factor synthesis, or active substances that synergistically increase brain-derived neurotrophic factor synthesis, as well as pharmaceutically acceptable carriers or excipients.

[0042] In a fourth aspect, the present invention provides the use of the above-mentioned peptide that promotes the synthesis of brain-derived neurotrophic factor in the preparation of a drug for delaying the occurrence and / or development of neurodegenerative diseases.

[0043] Preferably, the above-mentioned neurodegenerative diseases include but are not limited to Alzheimer's disease.

[0044] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0045] Example 1

[0046] This example provides a peptide that promotes the synthesis of brain-derived neurotrophic factor, whose amino acid sequence is SGFSKHF and molecular weight is 809 Da.

[0047] The preparation process of the peptide that promotes the synthesis of brain-derived neurotrophic factor in this example is as follows:

[0048] (1) Soy protein isolate (Shandong Yuxin Biotechnology Co., Ltd.) was prepared into a 9% soy protein isolate solution and enzymatically hydrolyzed with alkaline protease (Shanghai Yuanye Biotechnology Co., Ltd., Bacillus subtilis source), papain (Shanghai Yuanye Biotechnology Co., Ltd., papain source) and flavor protease (Shanghai Yuanye Biotechnology Co., Ltd., Aspergillus oryzae source). The amount of total protease added was 1% of the soy protein isolate by mass fraction, and the mass ratio of alkaline protease, papain and flavor protease was 1:2:2. The pH value was adjusted to 8.5. Alkaline protease was first added, and the enzymatic hydrolysis time was 35 minutes at a hydrolysis temperature of 50°C. Papain was added again and the enzymatic hydrolysis continued for 30 minutes at a hydrolysis temperature of 50°C. Finally, flavor protease was added and the enzymatic hydrolysis continued for 175 minutes at a hydrolysis temperature of 60°C. The enzyme was then inactivated in a 95°C water bath for 10 minutes to obtain a soy peptide solution. The soy peptide solution was stored at -40°C and then freeze-dried for 48 hours to obtain a soy peptide powder.

[0049] (2) Prepare simulated saliva, simulated gastric juice, and simulated intestinal juice, as shown in Table 1.

[0050] Table 1 Preparation of simulated saliva, simulated gastric fluid and simulated intestinal fluid

[0051] Reagents Concentration (M) Simulated saliva (mL) Simulated gastric fluid (mL) Simulated intestinal fluid (mL) KCl 0.5 15.1 6.9 6.8 <![CDATA[KH2PO4]]> 0.5 3.7 0.9 0.8 <![CDATA[NaHCO3]]> 1 6.8 12.5 42.5 NaCl 2 0 11.8 9.6 <![CDATA[MgCl2(H2O)6]]> 0.15 0.5 0.4 1.1 <![CDATA[(NH4)2CO3]]> 0.5 0.06 0.5 0 6MHCl 6 0.09 1.3 0.7 <![CDATA[dH2O]]> 0 373.75 365.7 338.5

[0052] (3) In vitro simulated gastrointestinal digestion of soybean peptides was performed according to the INFOGEST 2.0 method. As shown in Table 1 above, simulated salivary fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) were prepared. The following steps were included:

[0053] I. During the oral digestion stage (using simulated saliva for digestion), 10 g of soybean peptide was weighed, SSF solution was added to 10 mL, and the mixture was reacted at 37°C for 2 minutes to obtain soybean peptide oral digestion solution;

[0054] II. During the gastric digestion stage (using simulated gastric fluid for digestion), SGF was added to the soy peptide oral digestion solution to 20 mL and the pH was adjusted to 3.0. Pepsin was added to increase its activity in the gastric digestion solution to 2000 U / mL. The mixture was incubated at 37°C for 2 h to obtain the soy peptide gastric digestion solution.

[0055] III. In the intestinal digestion stage (using simulated intestinal fluid for digestion), SIF was added to 40 mL of the soybean peptide gastric digestion fluid and the pH was adjusted to 7.0. Trypsin was added to make its activity in the intestinal digestion fluid reach 100 U / mL, and the mixture was incubated at 37°C for 2 h to obtain the soybean peptide intestinal digestion fluid;

[0056] IV. After digestion is completed, the soybean peptide intestinal digestion solution is placed in boiling water and heated for 10 minutes to terminate digestion; finally, it is placed at -40°C and vacuum freeze-dried for 48 hours to obtain digested soybean peptides.

[0057] (4) A Caco-2 monolayer cell model was constructed to simulate in vitro small intestinal absorption and transport experiments: 0.5 mL of Caco-2 cells (from the Institute of Basic Medicine, Chinese Academy of Medical Sciences) were plated at 1 × 10 5 Cells were seeded at a density of 10 cells / mL on the apical (AP) side of a 12-well Transwell plate, and 1.5 mL of complete medium (MEM + 20% FBS + 1% NEAA + 1% PS) was added to the basolateral (BL) side. After overnight incubation, the medium on the AP side was removed and replaced with 0.5 mL of complete medium to remove non-adherent cells. The medium was changed every two days during the culture period, and the transepithelial electrical resistance (TEER) was measured using a resistance meter (Millipore, USA, model MERS00002). After 21 days of culture and a TEER of 400-600 Ω / cm 2When the digested soybean peptide transport experiment was carried out, the chamber was washed with preheated HBSS buffer, and 0.5 mL and 1.5 mL HBSS buffer were added to the AP side and BL side, respectively, for equilibration for 30 minutes; then the HBSS buffer on the AP side was removed, and 0.5 mL of 4 mg / mL digested soybean peptide solution (dissolved in HBSS buffer) was added and incubated in an incubator. After 2 hours, the solution on the BL side was collected and vacuum freeze-dried at -80°C for 72 hours to obtain the absorbed soybean peptide, and peptide identification was performed (if the amino acid sequence of the peptide that promotes BDNF synthesis of the present invention is known, the peptide that promotes BDNF synthesis of the present invention can be prepared according to the results of peptide identification).

[0058] LC-MS / MS was used to identify the peptide segments of the absorbed soybean peptide. First, the absorbed soybean peptide was reduced and alkylated. Dithiothreitol solution was added to the sample to make the final concentration of 10 mmol / L, and the sample was reduced in a water bath at 56°C for 1 hour. Iodoacetamide solution was added to make the final concentration of 50 mmol / L, and the reaction was carried out in the dark for 40 minutes. Desalting was performed using a desalting column, and the sample was concentrated at 45°C in a vacuum centrifugal concentrator. LC-MS / MS analysis was performed using an Easy-nLC 1200 high performance liquid chromatograph and a Q Exactive mass spectrometer. The chromatographic column was a 150 μm × 15 cm homemade column, and the Acclaim PepMap RPLC C18 (3 μm, The elution was carried out using a 4% HCl (Dr. Maisch GmbH, Germany) packing. The mobile phase consisted of 0.1% formic acid in ultrapure water (A) and 0.1% formic acid in acetonitrile (20% water + 80% acetonitrile). The gradient elution program was as follows: 0-3 min, 4% B; 3-89 min, 8%-28% B; 89-109 min, 28%-40% B; 109-110 min, 40%-95% B; 110-120 min, 95% B. The flow rate was 600 nL / min, the injection volume was 4 μL, and the sample analysis time was 120 min. The main mass spectrometry parameters were as follows: spray voltage 2.2 kV, capillary temperature 270 °C, parent ion scan range 100-1500 m / z, primary mass spectrometry resolution 70000 (400 m / z), automatic gain control 3e6, and primary maximum injection time 100 ms; HCD was used for secondary fragmentation, secondary mass spectrometry resolution 17500, automatic gain control 1e5, secondary maximum injection time 50 ms, collision energy 28 eV, and fragmentation of the first 20 ions of the collected signal. Finally, the mass spectrometry raw files were used for peptide sequence analysis using the software PEAKS Studio (8.5). The search parameters were as follows: fixed modification carbamidomethyl (C), variable modification oxidation (M), acetylation (N-term), 3 missed restriction sites, primary mass spectrometry error 20 ppm, and secondary mass spectrometry error 0.02 Da. The final peptide identification results are shown in Table 2 , which lists only the top 15 peptides with high XCorr scores and ion intensities above 2.00E+05.

[0059] XCorr (cross-correlation score) is used to measure the correlation between the experimental spectrum and the theoretical spectrum.

[0060] Formula: XCorr=Iexp,i·Itheo,i) /

[0061] Where, Iexp,i: the intensity of the i-th peak in the experimental spectrum.

[0062] Itheo,i: The intensity of the i-th peak in the theoretical spectrum.

[0063] n: number of peaks to match.

[0064] The higher the XCorr value, the better the matching quality.

[0065] Table 2 Identification results of absorbed soybean peptide segments

[0066] Serial number Peptide sequence Score strength 1 LDQNPRVF 292.6 9.00E+05 2 NALEPDHRVE 292.3 2.00E+06 3 TDPVVA 274.3 5.00E+05 4 TDLPSVVE 252.7 3.00E+06 5 SGFSKHF 247.9 9.00E+05 6 SRDPIYSN 241.0 2.00E+06 7 DQTPRVF 211.1 6.00E+06 8 AENNQRNF 205.1 2.00E+06 9 TDPNFTAA 199.3 4.00E+06 10 SGPDPFDMQ 193.7 8.00E+05 11 GEKDNVVRQ 193.5 4.00E+05 12 LDHFRSIT 178.1 9.00E+05 13 TDDDYPYRA 169.2 3.00E+05 14 TDLNPIQ 162.7 5.00E+05 15 SGFAPEF 159.5 3.00E+05

[0067] (5) Molecular docking of absorbed soybean peptide with TrkB receptor:

[0068] A1. The protein structure of TrkB (PDB ID: 1HCF) was obtained from the RCSB Protein Data Bank (https: / / www.rcsb.org / ), and the peptide structure was obtained from PEP-FOLD 4.0 (https: / / mobyle2.rpbs.univ-paris-diderot.fr / cgi-bin / portal.py#forms:PEP-FO LD4). TrkB and the peptide were dehydrated and hydrogenated using AutoDockTools 1.5.7. The center coordinates of TrkB were set to: x = 32.7, y = 15.3, z = 40.9, and the docking box size was: Molecular docking was performed using AutoDock Vina to determine the binding energies between TrkB and various peptides. DQTPRVF, LDQNPRVF, SGFSKHF, TDPNFTAA, and SRDPIYSN exhibited low affinity and numerous binding sites. Based on peptide characteristics, five peptides were selected: DQTPRVF (T1), LDQNPRVF (T2), SGFSKHF (T3), TDPNFTAA (T4), and SRDPIYSN (T5).

[0069] A2. The docking results of each peptide were imported into Pymol for visualization. The results are as follows Figure 1-2 The complex was then uploaded to the Protein-Ligand Interaction Profiler (https: / / plip-tool.biotec.tu-dresden.de / plip-web / plip / index) to obtain comprehensive information on the interaction between the receptor and the peptide. The results are shown in Table 3: The five peptides with the lowest affinity bind to the TrkB receptor primarily through hydrophobic interactions, hydrogen bonds, salt bridges, and ππ stacking, with the majority of these interactions being hydrophobic interactions and hydrogen bonds. T1-T5 primarily form hydrophobic interactions with Phe291, Pro304, and His335 residues of the TrkB receptor, hydrogen bonds with His300, Thr306, and Asn338 residues, and a salt bridge with His343 residue. Hydrophobic interactions and hydrogen bonds play a major role in the binding of T1-T5 to the TrkB receptor.

[0070] Table 3 Analysis of the interaction between different peptides and TrkB receptor

[0071]

[0072] (6) Using corticosterone to stimulate PC12 cells to establish a cell nerve injury model, by testing the effects of the peptides screened in step A on cell viability and / or BDNF-TrkB-CREB pathway protein expression, the peptides that promote brain-derived neurotrophic factor synthesis are screened:

[0073] B1. Rat adrenal pheochromocytoma well-differentiated cells PC12 (Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences) were cultured in 1640 medium (Gibco, USA) containing 10% FBS and 1% PS (penicillin-streptomycin solution) at 37°C in 5% CO2 / 95% air. When the cell density reached 80%, 0.25% TE (trypsin-EDTA solution, Gibco, USA) was used for passage, with a passage ratio of 1:3. The effects of different peptides and CORT on PC12 cell viability were evaluated by CCK-8. PC12 well-differentiated cells were cultured at 2×10 5 Cells were seeded at a density of 100 cells / mL in a 96-well plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. After cell attachment, the culture medium was removed and 100 μL of peptide solutions (0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 4 mg / mL) or CORT solutions (100 μM, 200 μM, 300 μM, 400 μM, 500 μM, and 600 μM) dissolved in 1640 medium containing 0.3% DMSO were added. After 24 hours of incubation, the solution was removed and the cells were washed with PBS. 100 μL of 10% CCK-8 solution (Shanghai Biyuntian Biotechnology Co., Ltd.) (dissolved in 1640 medium) was added and incubated for 0.5 hours. The absorbance was measured at 450 nm using a microplate reader. At the same time, a blank group (no cells were inoculated, and 1640 culture medium was added), a control group (cells were inoculated, and 1640 culture medium was added), and a solvent group (cells were inoculated, and 1640 culture medium containing 0.3% DMSO was added) were set up. Figure 3 As shown in AE, T2 and T4 had no damage to PC12 cells in the range of 0.125-4 mg / mL, and cell viability did not change significantly (P>0.05). 4 mg / mL T1 significantly reduced cell viability to 91.4±4.7% (P<0.05), 4 mg / mL T3 significantly reduced cell viability to 80.9±8.4% (P<0.05), and 2 mg / mL T5 significantly reduced cell viability to 91.0±4.9% (P<0.05). To eliminate the effect of peptides on cell damage, all peptide concentrations were unified to 1 mg / mL for subsequent experiments.

[0074] B2. PC12 cells were treated with different concentrations of CORT (corticosterone, MCE) to establish a cell nerve injury model. Figure 3 Figure F shows the effects of different CORT concentrations on PC12 cell viability. Compared with the blank group, the solvent 0.3% DMSO had no significant effect on cell viability (P>0.05), indicating that the solvent did not damage the cells. CORT significantly reduced PC12 cell viability (P<0.05). The toxicity of 100, 200, 300, 400, 500, and 600 μM CORT on PC12 cells gradually increased, with cell viability decreasing to 86.4±1.8%, 73.1±0.7%, 67.2±1.0%, 61.0±0.2%, 56.5±0.2%, and 53.3±1.3%, respectively. 400 μM CORT significantly reduced cell viability, so 400 μM CORT was selected for subsequent experiments.

[0075] B3. CCK-8 was used to detect the effects of different peptides on the viability of PC12 cells induced by CORT. PC12 highly differentiated cells were cultured at 2×10 5 Cells were seeded at a density of 100 cells / mL in a 96-well plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. After cell attachment, the culture medium was removed and 100 μL of peptide solutions (0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 4 mg / mL) or CORT solutions (100 μM, 200 μM, 300 μM, 400 μM, 500 μM, and 600 μM) dissolved in 1640 medium containing 0.3% DMSO were added. After 24 hours of incubation, the solution was removed and the cells were washed with PBS. 100 μL of 10% CCK-8 solution (dissolved in 1640 medium) was added and incubated for 0.5 hours. The absorbance was measured at 450 nm using a microplate reader. At the same time, a blank group (no cells were inoculated, and 1640 culture medium was added), a control group (cells were inoculated, and 1640 culture medium was added), and a solvent group (cells were inoculated, and 1640 culture medium containing 0.3% DMSO was added) were set up. Figure 4 A shows that compared with the CORT group, T1 had no significant effect on cell viability, while T2-T5 could significantly increase PC12 cell viability, reaching 76±0.7%, 78±1.2%, 80±1.6% and 77±2.9% respectively (P<0.05), among which T4 had the best effect. Figure 4 Figure B can more intuitively show the damage of CORT to PC12 cells and the protective effect of the peptides. The results show that T2-T5 can alleviate the damage of PC12 cells induced by CORT, among which T4 has the strongest protective effect.

[0076] B4. Select T4 and T3, which performed best in the cell viability assay, for Western blotting to detect the protein expressions of BDNF, TrkB, CREB, p-TrkB, and p-CREB. PC12 highly differentiated cells were cultured at 2×10 5 Cells were seeded at a density of 10 cells / mL in 6-well plates and incubated in an incubator for 24 hours. The control and CORT groups were treated with 1640 medium, while the experimental groups were treated with 1 mg / mL T3 and T4, respectively. Two hours after nutritional intervention, the control group was treated with 1640 medium, the CORT group was treated with 400 mM CORT, and the experimental groups were treated with 1 mg / mL T3, T4, and 400 mM CORT, respectively, and incubated for 24 hours. Following incubation, total protein was extracted from highly differentiated PC12 cells using RIPA lysis buffer (Beijing Solebold Technology Co., Ltd.) and centrifuged at 12,000 g for 20 minutes at 4°C. Protein concentration was determined using a BCA kit (Beijing Solebold Technology Co., Ltd.) and standardized. The cells were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). The PVDF membrane was blocked with QuickBlock blocking solution (Shanghai Biyuntian Biotechnology Co., Ltd.) at room temperature for 20 minutes, and incubated with specific primary antibodies at a dilution ratio of 1:1000 (BDNF Abcam, UK, CREB Abcam, UK, TrkB Abcam, UK, phospho-CREB (Ser133) Abcam, UK, phospho-TrkB (Tyr817) Shanghai Biyuntian Biotechnology Co., Ltd., and α-Tubulin Shanghai Biyuntian Biotechnology Co., Ltd.) at 4°C overnight. After incubation, the PVDF membrane was washed with TBST and incubated with HRP-secondary antibody at a dilution ratio of 1:1000 for 1 hour at room temperature. After washing with TBST, the BeyoECL Moon enhanced chemiluminescence kit (Shanghai Biyuntian Biotechnology Co., Ltd.) was used for color development and the protein bands were developed in the gel imaging analysis system. Image J software was used for grayscale analysis and the relative expression level of the target protein was calculated. Figure 5Compared with the blank group, the expression of BDNF and p-TrkB / TrkB in the CORT group (Model) was significantly decreased (P < 0.05), indicating that CORT can inhibit the BDNF-TrkB-CREB pathway. Compared with the CORT group (Model), T4 significantly increased the expression of p-TrkB / TrkB and p-CREB / CREB (P < 0.05), while T3 significantly increased the expression of BDNF, p-TrkB, and p-CREB (P < 0.05). These results suggest that both T4 and T3 can generate BDNF by acting on the BDNF-TrkB-CREB pathway, with T3 having a stronger ability to promote the phosphorylation of TrkB and CREB, further activating downstream pathways and producing more BDNF. This suggests that the SGFSKHF peptide can increase BDNF expression, thereby alleviating damage in PC12 cells and further helping to delay the onset and progression of neurodegenerative diseases such as Alzheimer's disease.

[0077] Experimental Example 1

[0078] Gromacs 2023 was used to place the complex of the peptide and TrkB receptor generated by molecular docking and the PDB file of the TrkB receptor as the initial structure in a 12-hedron water box. The force field used was Amber14sb, the water molecule model was TIP3P, and sodium ions and chloride ions were used to make the total charge of the system 0, thereby constructing the initial simulation system. The initial simulation system was subjected to two-step energy minimization, and the steepest descent method was used 10,000 times. After energy optimization, 100ps NVT simulation and NPT simulation were performed respectively, and a 100ns formal simulation was performed after the NPT simulation. The temperature control algorithm used C-rescale, and the pressure control algorithm used parrinello-rahman. The temperature was 310K, and the conformation was saved every 10ps. The simulation results were obtained by analyzing the relevant Gromacs commands. The stability of the system was evaluated by root mean square deviation (RMSD), root mean square fluctuation (RMSF) and radius of gyration (Rg). The results are shown in Figure 2. Figure 6 shown.

[0079] Depend on Figure 6 As shown in Figure A, the RMSD fluctuation range of the TrkB-T3 complex is small, indicating that the binding of T3 enhances the stability of the TrkB receptor. Figure 6Figure B shows the RMSF changes of the TrkB receptor and the TrkB-T3 complex. Both systems exhibit similar volatility across the entire residue range. However, near positions 310 and 330, the RMSF of the TrkB-T3 complex is lower than that of the TrkB receptor, indicating that peptide binding in these two regions can reduce the volatility of residues at these positions. As shown in Table 3, the binding sites of T3 to the TrkB receptor are concentrated around positions 310 (Lys312, Ala314, Leu315, Gln316) and 330 (Glu326, Thr332, Ile334). These results suggest that T3 binding can further stabilize the structure of the TrkB receptor. Figure 6 C represents the change in Rg of TrkB and the TrkB-T3 complex. The TrkB-T3 complex was higher than TrkB at most time points, indicating that peptide binding loosens the overall structure of TrkB. This is likely due to T3 binding causing a conformational change in the portion of the TrkB receptor that binds to BDNF, increasing the overall volume of the structure. T3 enhances the stability of the TrkB receptor but also loosens the overall structure of TrkB. The hypothesized mechanism is that T3 binding to the TrkB receptor stimulates signaling in the BDNF-TrkB-CREB pathway. Simultaneously, the loosened structure facilitates BDNF binding, thereby enhancing neurotrophic activity and potentially delaying the onset and progression of neurodegenerative diseases (e.g., Alzheimer's disease).

[0080] Experimental Example 2

[0081] The physicochemical properties of key peptides were analyzed using online databases. Expasy ProtParam (https: / / web.expasy.org / protparam / ) was used to retrieve peptide physicochemical properties (molecular weight, isoelectric point, atomic composition, extinction coefficient, estimated half-life, instability index, and average hydrophilicity coefficient). The hydrophobicity and hydrophilicity of peptides were determined using GRAVY, with GRAVY > 0 indicating a hydrophobic peptide and GRAVY < 0 indicating a hydrophilic peptide. Peptide stability was determined using the instability index, with an instability index > 40 indicating an unstable peptide and an instability index < 40 indicating a stable peptide. ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.html) was used to analyze peptide toxicity. AllerTOP (https: / / www.ddg-pharmfac.net / AllerTOP / index.html) was used to assess the allergenicity of peptides. The results, as shown in Table 4, show that the SGFSKHF peptide has a low molecular weight, good water solubility, strong stability, and is non-toxic and non-allergenic, which helps maintain its biological activity during processing and digestion. This indicates that the SGFSKHF peptide has good processing properties and safety, which is of great significance for the development of functional foods (or drugs) that delay the onset and progression of neurodegenerative diseases (e.g., Alzheimer's disease) and its application in related industries.

[0082] Table 4 SGFSKHF peptide analysis

[0083]

[0084] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A peptide for promoting the synthesis of brain-derived neurotrophic factor, characterized in that The amino acid sequence of the peptide is SGFSKHF and the molecular weight is 809 Da.

2. The method for preparing a peptide for promoting the synthesis of brain-derived neurotrophic factor according to claim 1, wherein: The following steps are involved: (1) Soybean peptides were obtained by enzymatically hydrolyzing soy protein isolate with alkaline protease, papain and flavor protease; (2) performing simulated digestion of soybean peptides in vitro to obtain digested soybean peptides; (3) simulating the absorption of the digestible soybean peptide in vitro using a Caco-2 monolayer cell model to separate and obtain the absorbed soybean peptide; (4) Identifying the absorbed soybean peptide to obtain the peptide SGFSKHF that promotes the synthesis of brain-derived neurotrophic factor.

3. The preparation method according to claim 2, wherein The peptide that promotes the synthesis of brain-derived neurotrophic factor is obtained by screening using a method comprising the following steps when it is first isolated: A. performing molecular docking of the absorbed soybean peptide separated in step (3) with the TrkB receptor, and screening peptide segments based on affinity; B. Use corticosterone to stimulate PC12 cells to establish a cell nerve injury model, and screen the peptide that promotes the synthesis of brain-derived neurotrophic factor by testing the effects of the peptides screened in step A on cell viability and / or BDNF-TrkB-CREB pathway protein expression.

4. The method for preparing soybean peptide according to claim 2, wherein: In step (1), the amount of the total protease of alkaline protease, papain and flavor protease added is 0.8%-1.2% by mass of the soy protein isolate; the mass ratio of the alkaline protease, papain and flavor protease is 1:2:2-2:3:3; In step (1), soy protein isolate is prepared into a 9%-10% protein solution, and the pH value is adjusted to 8.5; first, the alkaline protease is added, and the enzymatic hydrolysis time is 30-35 minutes, and the enzymatic hydrolysis temperature is 45-55°C; papain is added again, and the enzymatic hydrolysis is continued for 30-35 minutes, and the enzymatic hydrolysis temperature is 45-55°C; finally, flavor protease is added and the enzymatic hydrolysis is continued for 160-175 minutes, and the enzymatic hydrolysis temperature is 50-60°C; after the enzymatic hydrolysis is completed, the temperature is raised to above 90°C and the solution is placed in a water bath for more than 10 minutes to inactivate the enzyme.

5. The preparation method according to claim 2, characterized in that The step (2) of in vitro simulated digestion comprises: digesting the soybean peptide in sequence with simulated saliva, simulated gastric juice and simulated intestinal juice; after the in vitro simulated digestion is completed, drying the digestion product of the simulated intestinal juice to obtain the digested soybean peptide.

6. The preparation method according to claim 5, wherein the in vitro simulated digestion specifically comprises the following steps: I. dissolving the soybean peptide in water, adding simulated saliva, and shaking the reaction to obtain a soybean peptide oral digestive solution; II. Simulated gastric fluid was added to the oral digestive fluid of the soy peptide, the pH was adjusted to 3.0, and then pepsin was added and the reaction was shaken to obtain a soy peptide gastric digestive fluid; III. Adding simulated intestinal fluid to the soy peptide gastric digestion solution, adjusting the pH to 7.0, then adding trypsin, shaking the reaction to obtain a soy peptide intestinal digestion solution; IV. heating the soybean peptide intestinal digestion fluid to terminate digestion, and freeze-drying to obtain the digested soybean peptide; Wherein, based on 400 mL, the simulated saliva contains 15.1 mL 0.5 M KCl, 3.7 mL 0.5 M KH2PO4, 6.8 mL 1 M NaHCO3, 0.5 mL 0.15 M MgCl2(H2O)6, 0.06 mL 0.5 M (NH4)2CO3, 0.09 mL 6 M HCl, and dH2O is added to make it complete. 0.025 mL 0.3 M CaCl2(H2O)2 is added separately before use; Based on 400 mL, the simulated gastric fluid contained 6.9 mL 0.5 M KCl, 0.9 mL 0.5 M KH2PO4, 12.5 mL 1 M NaHCO3, 11.8 mL 2 M NaCl, 0.4 mL 0.15 M MgCl2(H2O)6, 0.5 mL 0.5 M (NH4)2CO3, 1.3 mL 6 M HCl, and was made up with dH2O. 0.005 mL 0.3 M CaCl2(H2O)2 was added separately before use. Based on 400 mL, the simulated intestinal fluid contained 6.8 mL 0.5 M KCl, 0.8 mL 0.5 M KH2PO4, 42.5 mL 1 M NaHCO3, 9.6 mL 2 M NaCl, 1.1 mL 0.15 M MgCl2 (H2O)6, 0.7 mL 6 M HCl, and was made up with dH2O. 0.04 mL 0.3 M CaCl2 (H2O)2 was added separately before use.

7. The preparation method according to claim 6, characterized in that In step I, the concentration of the soybean peptide in the mixed solution of the soybean peptide and the simulated saliva is 1-2 g / mL; the reaction temperature is 35-40° C., and the reaction time is 2-4 min; In step II, pepsin is added to a final concentration of 2000-2500 U / mL; the reaction temperature is 35-40° C., and the reaction time is 2-3 hours; In step III, trypsin is added to a final concentration of 100-150 U / mL; the reaction temperature is 35-40° C., and the reaction time is 2-3 h; The volume ratio of the simulated saliva, simulated gastric fluid and simulated intestinal fluid is 1:1:2; After the trypsin digestion is completed, the cells are placed in a water bath at 95-100° C. for 10-15 minutes to terminate the digestion.

8. The preparation method according to claim 2, characterized in that In step (3), the digested soybean peptide is dissolved in HBSS buffer and inoculated into Caco-2 monolayer cells; wherein the concentration of the soybean peptide digestion product in HBSS buffer is 4 mg / mL.

9. A composition for promoting the synthesis of brain-derived neurotrophic factor, characterized in that: The invention comprises the peptide for promoting the synthesis of brain-derived neurotrophic factor as claimed in claim 1.

10. Use of the peptide for promoting the synthesis of brain-derived neurotrophic factor according to claim 1 in the preparation of a drug for delaying the occurrence and / or development of neurodegenerative diseases.

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