A peptide for promoting brain-derived neurotrophic factor synthesis and a preparation method and application thereof

Abstract

The application belongs to the technical field of proteins, and relates to a peptide for promoting synthesis of brain-derived neurotrophic factor (BDNF) and a preparation method and application thereof. The amino acid sequence of the peptide for promoting synthesis of brain-derived neurotrophic factor BDNF is SGFSKHF. The main effect of the peptide is that the peptide can protect CORT (corticosterone) from damaging PC12 cells, can spontaneously bind to a TrkB receptor, the structure of a complex formed after the binding is more stable, helps to activate a BDNF-TrkB-CREB pathway signal transduction, and then promotes generation of BDNF, thereby helping to delay occurrence and development of neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of protein technology and relates to a peptide that promotes the synthesis of brain-derived neurotrophic factor, its preparation method, and its application. Background Technology

[0002] Brain-derived neurotrophic factor (BDNF), a key member of the neurotrophic factor family, has been extensively studied and proven to play a crucial role in neurogenesis, neuronal survival, differentiation, and the maintenance of mature neuronal function. It promotes neuronal synthesis and connectivity, thereby improving cognitive abilities and neurological function. Furthermore, BDNF can help prevent neurodegenerative diseases such as Alzheimer's and Parkinson's. BDNF is a biologically important protein that has a positive impact on 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, thereby triggering the ERK / CREB (Extracellular signal-regulated kinase / Cyclic AMP response element-binding protein) signaling pathway. This promotes the transcription of pro-BDNF mRNA, which is translated into pro-BDNF, and ultimately processed into mature BDNF. Studies have shown that stress can lead to a decrease in 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 hydrolysates, 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 (BDNF), its preparation method and application. By screening and preparing peptides that promote BDNF synthesis, it is possible to help solve or improve the side effects of conventional drug treatment for neurodegenerative diseases (e.g., Alzheimer's disease).

[0005] The objectives of this invention and the solutions to its technical problems can be achieved through the following technical solutions.

[0006] On one hand, the present invention provides a peptide that promotes the synthesis of brain-derived neurotrophic factor, the amino acid sequence of which 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 soybean protein isolate with alkaline protease, papain and flavor protease to obtain soybean peptide; (2) performing in vitro simulated digestion of soybean peptide to obtain digested soybean peptide; (3) performing in vitro simulated absorption of the digested soybean peptide using a Caco-2 monolayer cell model to separate absorbed soybean peptide; (4) identifying the absorbed soybean 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 (BDNF), the composition comprising the peptide of the present invention for promoting BDNF synthesis.

[0009] In a fourth aspect, the present invention provides the use of peptides that promote the synthesis of brain-derived neurotrophic factors in the preparation of medicaments for delaying neurodegenerative diseases (e.g., Alzheimer's disease).

[0010] Beneficial effects:

[0011] The peptide (amino acid sequence SGFSKHF) of the present invention, which promotes the synthesis of brain-derived neurotrophic factor, mainly functions to protect PC12 cells from CORT (corticosterone) damage, can spontaneously bind to TrkB receptors, and the complex structure formed after binding is more stable, which helps to activate the BDNF-TrkB-CREB pathway signaling, thereby promoting the production of BDNF and thus helping to delay the occurrence and / or development of neurodegenerative diseases (e.g., Alzheimer's disease). Attached Figure Description

[0012] Figure 1 Visualization of the molecular docking of T1 and T2 peptides with the TrkB receptor; where (A) is the T1 peptide and (B) is the T2 peptide.

[0013] Figure 2 Visualization of the molecular docking of T3, T4, and T5 peptides with the TrkB receptor; where (C) is the T3 peptide, (D) is the T4 peptide, and (E) is the T5 peptide.

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

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

[0016] Figure 5 Figure 1 shows 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 level of BDNF; (B) is the relative expression level of p-TrkB / TrkB; (C) is the relative expression level of p-CREB / CREB.

[0017] Figure 6 Figure 1 shows the molecular dynamics simulation results of TrkB and the TrkB-T3 complex; (A) shows the RMSD fluctuation test results; (B) shows the RMSF change test results; (C) shows the Rg change test results. Detailed Implementation

[0018] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art should understand that the embodiments described below are only for illustrating 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] The inventors proposed this invention by enzymatically hydrolyzing and separating soybean protein isolate to obtain a peptide that promotes the synthesis of brain-derived neurotrophic factor (BDNF).

[0020] On one hand, the present invention provides a peptide that promotes the synthesis of brain-derived neurotrophic factor, the amino acid sequence of which 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 CORT-induced damage. It can spontaneously bind to the TrkB receptor, and the complex formed after binding has a more stable structure, which helps to activate the BDNF-TrkB-CREB pathway signaling, thereby promoting the production of brain-derived neurotrophic factor, thus 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 soybean protein isolate with alkaline protease, papain and flavor protease to obtain soybean peptide; (2) performing in vitro simulated digestion of soybean peptide to obtain digested soybean peptide; (3) performing in vitro simulated absorption of digested soybean peptide using a Caco-2 monolayer cell model to separate absorbed soybean peptide; (4) identifying absorbed soybean peptide to obtain the peptide SGFSKHF that promotes the synthesis of brain-derived neurotrophic factor.

[0023] In an embodiment of the present invention, during the preparation of soybean peptides, soybean protein isolate is obtained by enzymatic hydrolysis with alkaline protease (Bacillus subtilis source), papain (papaya source), and flavor protease (Aspergillus oryzae source). The total amount of protease added is 0.8%-1.2% (e.g., 0.8%, 0.9%, 1.0%, 1.1%, or 1.2%) of soybean protein isolate by mass fraction. The mass ratio of alkaline protease, papain, and flavor protease is 1:2:2-2:3:3 (e.g., 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 soybean peptides in this invention, soybean protein isolate is prepared into a 9%-10% (e.g., 9% or 10%) protein solution, and the pH value is adjusted to 8.5; firstly, alkaline protease is added, and the enzymatic hydrolysis time is 30-35 min (e.g., 30 min, 31 min, 32 min, 33 min, 34 min, or 35 min), and the enzymatic hydrolysis temperature is 45-55℃ (e.g., 45℃, 47℃, 49℃, 51℃, 53℃, or 55℃); then papain is added again, and the enzymatic hydrolysis continues for 30-35 min (e.g., 30 min, 31 min, 32 min, ...). The enzymatic hydrolysis time is 33 min, 34 min, or 35 min, at a temperature of 45-55℃ (e.g., 45℃, 47℃, 49℃, 51℃, 53℃, or 55℃). Finally, flavor protease is added and the enzymatic hydrolysis continues for 160-175 min (e.g., 160 min, 165 min, 170 min, or 175 min), at a temperature of 50-60℃ (e.g., 50℃, 52℃, 54℃, 56℃, 58℃, or 60℃). After the enzymatic hydrolysis is completed, the temperature is raised to above 90℃ and the water bath is incubated for at least 10 min to inactivate the enzyme (e.g., raised to 95℃ and the water bath is incubated for 10 min to inactivate the enzyme).

[0025] In an embodiment of the preparation method of the present invention, the peptides that promote the synthesis of brain-derived neurotrophic factor are screened during the first isolation using a method including the following steps: A. Molecular docking of the absorbable soybean peptides obtained in step (2) with the TrkB receptor, and screening peptides based on affinity (screening for peptides with low nucleophilic energy); B. Establishing a cell neural injury model by stimulating PC12 cells with corticosterone, and screening for peptides that promote 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.

[0026] In an embodiment of the preparation method of the present invention, in vitro simulated digestion includes: sequentially digesting soybean peptides 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 digested soybean peptides.

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

[0028] I. Dissolve soybean peptides in water, add simulated saliva, and shake to react, thus obtaining soybean peptide oral digestive fluid;

[0029] II. Add simulated gastric juice to the soybean peptide oral digestive fluid, adjust the pH to 3.0, then add pepsin and shake the reaction to obtain soybean 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, shake the reaction to obtain soybean peptide intestinal digestion fluid;

[0031] IV. Heat the soybean peptide intestinal digestive fluid to stop digestion, then freeze-dry to obtain digested soybean peptides;

[0032] The simulated saliva, in 400 mL volume, contains 15.1 mL of 0.5 M KCl, 3.7 mL of 0.5 M KH2PO4, 6.8 mL of 1 M NaHCO3, 0.5 mL of 0.15 M MgCl2(H2O)6, 0.06 mL of 0.5 M (NH4)2CO3, and 0.09 mL of 6 M HCl. dH2O is added to make up the difference. 0.025 mL of 0.3 M CaCl2(H2O)2 is added separately before use.

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

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

[0035] The reason for adding CaCl2(H2O)2 to the above-mentioned simulated solution is: 1) to simulate the ionic strength of calcium ions in gastrointestinal fluid; 2) calcium ions in the solution can affect the stability and activity of enzymes; 3) calcium ions participate in biological processes such as muscle contraction, nerve conduction and blood coagulation. 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 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 until the final concentration of pepsin is 2000-2500 U / mL (e.g., 2000 U). The dosages are 2100 U / mL, 2200 U / mL, 2300 U / mL, 2400 U / mL, or 2500 U / mL, and the reaction temperature is 35-40℃ (e.g., 35℃, 36℃, 37℃, 38℃, 39℃, or 40℃), and the reaction time is 2-3h (e.g., 2h, 2.3h, 2.5h, 2.8h, or 3h). However, if the amount of pepsin added is too high, it will lead to excessive peptide hydrolysis and will not be able to simulate the actual human digestive process; if the amount added is too low, it will lead to incomplete peptide hydrolysis and will not be able to simulate the actual human digestive process. 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). If the amount of trypsin added is too high, it will lead to excessive peptide hydrolysis and will not be able to simulate the actual human digestive process; if the amount added is too low, it will lead to incomplete peptide hydrolysis and will not be able to simulate the actual human digestive process. The volume ratio of simulated saliva, simulated gastric juice, and simulated intestinal juice is 1:1:2; after trypsin digestion, the digestion is terminated by 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 (e.g., -40°C, -50°C, -60°C, -70°C or -80°C), and the freeze-drying time is 48-72 h (e.g., 48 h, 54 h, 60 h, 66 h or 72 h).

[0038] In an embodiment of the preparation method of the present invention, in step (2), the digested soybean peptides are 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 product is simulated for in vitro absorption using a Caco-2 monolayer cell model, and the soybean peptide digestion product is separated into absorbed soybean peptide and unabsorbed soybean peptide.

[0040] In a specific implementation plan, Caco-2 cells are cultured for 21 days to achieve a resistivity of 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 During the process, a Caco-2 monolayer cell model is constructed. At this stage, the cells differentiate into a monolayer structure similar to small intestinal epithelial villi. After 2-4 hours (e.g., 2h, 2.5h, 3h, 3.5h, or 4h) of absorption, the digested soybean peptides are separated into absorbed soybean peptides and unabsorbed soybean peptides. If the electrical resistance is too high, the cells may form a multilayer structure; if the electrical resistance is too low, the cells may not be fully differentiated.

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

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

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

[0044] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0045] Example 1

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

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

[0048] (1) Prepare a 9% soy protein isolate solution from Shandong Yuxin Biotechnology Co., Ltd., and hydrolyze it with alkaline protease (Bacillus subtilis source, Shanghai Yuanye Biotechnology Co., Ltd.), papain (papaya source, Shanghai Yuanye Biotechnology Co., Ltd.), and flavor protease (Aspergillus oryzae source, Shanghai Yuanye Biotechnology Co., Ltd.). The 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. Adjust the pH to 8.5, first add alkaline protease, hydrolyze for 35 min at 50℃, then add papain and continue hydrolysis for 30 min at 50℃; finally add flavor protease and continue hydrolysis for 175 min at 60℃; then inactivate the enzymes by bathing in a 95℃ water bath for 10 min to obtain a soy peptide solution. Store the soy peptide solution at -40℃ and then freeze-dry for 48 h to obtain 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 juice, and simulated intestinal juice

[0051] reagents Concentration (M) Simulated saliva (mL) Simulated gastric juice (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. In the oral digestion stage (using simulated saliva for digestion), weigh 10g of soybean peptides, add SSF solution to 10mL, and then react the mixture at 37℃ for 2min to obtain soybean peptide oral digestion solution;

[0054] II. In the gastric digestion stage (using simulated gastric juice for digestion), SGF was added to 20 mL of the soybean peptide oral digestive fluid and the pH was adjusted to 3.0. Pepsin was added so that its activity in the gastric digestive fluid reached 2000 U / mL. The mixture was then cultured at 37°C for 2 h to obtain the soybean peptide gastric digestive fluid.

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

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

[0057] (4) An in vitro simulated small intestinal absorption and transport experiment was conducted by constructing a Caco-2 monolayer cell model: 0.5 mL of Caco-2 cells (from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were injected with 1 × 10⁻⁶ cells. 5 Cells were seeded at a density of 100 cells / mL on the top (Apical, AP) side of 12-well Transwell plates, and 1.5 mL of complete culture medium (MEM + 20% FBS + 1% NEAA + 1% PS) was added to the basal (BL) side. After overnight incubation, the medium on the AP side was removed and replaced with 0.5 mL of complete culture medium to remove non-adhesive cells. The culture medium was changed every two days during culture, and transepithelial electrical resistance (TEER) was measured using a Millipore MERS00002 instrument. After 21 days of culture, the TEER was measured to be 400-600 Ω / cm. 2During the digestion and transport experiment of soybean peptides, the chamber was washed with preheated HBSS buffer, and 0.5 mL and 1.5 mL of HBSS buffer were added to the AP side and BL side respectively for equilibration for 30 min. 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 h, the solution on the BL side was collected and freeze-dried at -80℃ for 72 h to obtain the absorbed soybean peptide. Peptide identification was performed (given the amino acid sequence of the peptide that promotes BDNF synthesis of the present invention, the peptide that promotes BDNF synthesis of the present invention can be prepared based on the results of peptide identification).

[0058] Peptide identification of absorbed soybean peptides was performed using LC-MS / MS. First, the absorbed soybean peptides were reductively alkylated by adding dithiothreitol solution to the sample to a final concentration of 10 mmol / L and reducing in a water bath at 56 °C for 1 h. Iodoacetamide solution was then added to a final concentration of 50 mmol / L, and the reaction was carried out in the dark for 40 min. Desalting was performed using a desalting column, followed by concentration in a vacuum centrifuge at 45 °C. 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 self-made 150 μm × 15 cm Acclaim PepMap RPLC C18 (3 μm, The packing material was Dr. Maisch GmbH, Germany. The mobile phase consisted of 0.1% formic acid, ultrapure water (A), and 0.1% formic acid, 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. Main mass spectrometry parameters: spray voltage 2.2 kV, capillary temperature 270 °C, precursor ion scan range 100-1500 m / z, primary mass spectrometry resolution 70000 (400 m / z), automatic gain control 3e6, primary maximum injection time 100 ms; secondary fragmentation was performed using HCD, 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 acquired. Finally, the raw mass spectrometry file was analyzed for peptide sequences using PEAKS Studio (8.5) software. The search parameters were as follows: fixed modification Carbamidomethyl (C), variable modification Oxidation (M), Acetylation (N-term), missed restriction site 3, primary mass spectrometry error 20 ppm, and secondary mass spectrometry error 0.02 Da. The final peptide identification results are shown in Table 2. Only the top 15 peptides with high XCorr scores and ionic strengths above 2.00E+05 are listed.

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

[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: The number of matched peaks.

[0064] A higher XCorr value indicates a better matching quality.

[0065] Table 2. Identification results of absorbed soybean peptide fragments

[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 peptides with TrkB receptors:

[0068] The protein structure of A1.TrkB (PDB ID: 1HCF) was obtained from the RCSB protein database (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-FOLD4). AutoDockTools 1.5.7 was used to dehydrate and hydrogenate TrkB and the peptide. The center coordinates of TrkB were set as: x = 32.7, y = 15.3, z = 40.9. The docking box size was: Molecular docking was performed using AutoDock Vina to obtain the binding energies of TrkB to different peptides. DQTPRVF, LDQNPRVF, SGFSKHF, TDPNFTAA, and SRDPIYSN showed lower affinity energies and a higher number of 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 for each peptide were imported into Pymol for visualization, and the results are as follows: Figure 1-2 As shown in Table 3, the complexes were 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 interactions between the receptor and peptides. The results show that the five peptides with the lowest affinity bind to the TrkB receptor primarily through hydrophobic interactions, hydrogen bonds, salt bridges, and π-π stacking, with hydrophobic interactions and hydrogen bonds being the most prevalent. T1-T5 mainly 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. Interaction analysis of different peptide segments with TrkB receptor

[0071]

[0072] (6) A cell-mediated neural injury model was established by stimulating PC12 cells with corticosterone. The peptides that promote the synthesis of brain-derived neurotrophic factor were screened by testing the effects of peptides obtained in step A on cell viability and / or BDNF-TrkB-CREB pathway protein expression.

[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 and 5% CO2 / 95% air. When the cell density reached 80%, the cells were passaged using 0.25% TE (trypsin-EDTA solution, Gibco, USA) at a passage ratio of 1:3. The effects of different peptides and CORT on PC12 cell viability were assessed using CCK-8 assay. The well-differentiated PC12 cells were cultured at a rate of 2 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of [number] cells / mL in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. After cell attachment, the culture medium was removed, and 100 μL of peptide solutions of different concentrations (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 incubation for 24 h, the solution was removed, and the cells were washed with PBS. 100 μL of 10% CCK-8 solution (Shanghai Beyotime Biotechnology Co., Ltd.) (dissolved in 1640 medium) was added, and the cells were incubated for 0.5 h. The absorbance was measured at 450 nm using a microplate reader. Simultaneously, a blank group (no cells inoculated, 1640 medium added), a control group (cells inoculated, 1640 medium added), and a solvent group (cells inoculated, 1640 medium containing 0.3% DMSO added) were set up. The results are as follows: Figure 3 As shown in the AE, T2 and T4 did not damage PC12 cells within the range of 0.125-4 mg / mL, and cell viability remained largely unchanged (P>0.05). However, 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 influence of peptides on cell damage, all peptide concentrations were standardized to 1 mg / mL for subsequent experiments.

[0074] B2. PC12 cells were treated with different concentrations of CORT (corticosterone, MCE) to establish a cellular neural injury model. Figure 3 F shows the effect of different concentrations of CORT on PC12 cell viability. Compared with the control 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 to PC12 cells gradually increased, with cell viability of 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 chosen for subsequent experiments.

[0075] B3. The effects of different peptides on CORT-induced PC12 cell viability were detected using CCK-8 assay. Highly differentiated PC12 cells were cultured at a concentration of 2 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of [number] cells / mL in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. After cell attachment, the culture medium was removed, and 100 μL of peptide solutions of different concentrations (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 incubation for 24 h, 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 the cells were incubated for 0.5 h. The absorbance was then measured at 450 nm using a microplate reader. Simultaneously, a blank group (no cells inoculated, 1640 medium added), a control group (cells inoculated, 1640 medium added), and a solvent group (cells inoculated, 1640 medium containing 0.3% DMSO added) were set up. Figure 4 As shown in Figure A, compared with the CORT group, T1 had no significant effect on cell viability, while T2-T5 significantly increased PC12 cell viability, reaching 76±0.7%, 78±1.2%, 80±1.6%, and 77±2.9%, respectively (P<0.05), with T4 showing the best effect. Figure 4 B provides a more direct view of the damage CORT causes to PC12 cells and the protective effect of its peptides. The results show that T2-T5 can alleviate CORT-induced cell damage in PC12 cells, with T4 exhibiting the strongest protective effect.

[0076] B4. T4 and T3 cells, which showed the best performance in cell viability assays, were selected for Western blotting (WB) to detect the protein expression of BDNF, TrkB, CREB, p-TrkB, and p-CREB. Highly differentiated PC12 cells were cultured at a rate of 2 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of [number] cells / mL in 6-well plates and incubated for 24 h. The control and CORT groups were treated with 1640 medium, while the experimental groups were treated with 1 mg / mL T3 and T4, respectively. After 2 h of nutrient intervention, the control group was treated with 1640 medium, the CORT group with 400 mM CORT, and the experimental groups with 1 mg / mL T3, T4, and 400 mM CORT, respectively, and incubated for 24 h. After incubation, total protein was extracted from highly differentiated PC12 cells using RIPA lysis buffer (Sole Probiotech, Beijing) and centrifuged at 12,000 g for 20 min at 4 °C. Protein concentration was determined and homogenized using a BCA kit (Sole Probiotech, Beijing), and the proteins were separated by SDS-PAGE and transferred to a PVDF membrane (Milliplatin, USA). PVDF membranes were blocked for 20 min at room temperature using QuickBlock blocking buffer (Beyotime Biotechnology Co., Ltd., Shanghai). They were then incubated overnight at 4°C with specific primary antibodies diluted 1:1000 (BDNF from Abcam, UK; CREB from Abcam, UK; TrkB from Abcam, UK; phospho-CREB (Ser133) from Abcam, UK; phospho-TrkB (Tyr817) from Beyotime Biotechnology Co., Ltd., Shanghai); and α-Tubulin from Beyotime Biotechnology Co., Ltd., Shanghai). After incubation, the PVDF membranes were washed with TBST and incubated with HRP-secondary antibody diluted 1:1000 at room temperature for 1 h. After TBST washing, color development was performed using the BeyoECL Moon Enhanced Chemiluminescence Kit (Beyotime Biotechnology Co., Ltd., Shanghai), and protein bands were visualized using a gel imaging analysis system. Grayscale analysis was performed using ImageJ software, and the relative expression levels of the target proteins were calculated. Figure 5Compared with the control group, the expression of BDNF and p-TrkB / TrkB in the CORT group (Model) was significantly reduced (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 indicate that both T4 and T3 can generate BDNF by acting on the BDNF-TrkB-CREB pathway, with T3 having a stronger ability to promote TrkB and CREB phosphorylation, further activating downstream pathways and generating more BDNF. This suggests that SGFSKHF peptide can increase BDNF expression, thereby alleviating PC12 cell damage and further contributing to delaying the occurrence and development of neurodegenerative diseases (e.g., Alzheimer's disease).

[0077] Experimental Example 1

[0078] Using Gromacs 2023, the peptide-TrkB receptor complex generated by molecular docking and the TrkB receptor PDB file were used as the initial structures and placed in a dodecahedral water box. The force field was Amber14sb, the water molecule model was TIP3P, and sodium and chloride ions were used to make the total charge of the system zero, thus constructing the initial simulation system. Two-step energy minimization was performed on the initial simulation system using the steepest descent method for 10,000 iterations. After energy optimization, 100ps NVT and NPT simulations were performed, followed by a 100ns formal simulation. The temperature control algorithm used was C-rescale, and the pressure control algorithm used was parrinello-rahman. The temperature was 310K, and the conformation was saved every 10ps. Gromacs commands were used to analyze the simulation results. The stability of the system was evaluated using root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (Rg). The results are shown below. Figure 6 As shown.

[0079] Depend on Figure 6 As shown in A, the TrkB-T3 complex exhibits a small RMSD fluctuation range, indicating that T3 binding enhances the stability of the TrkB receptor. Figure 6B represents the RMSF variation of the TrkB receptor and the TrkB-T3 complex, with both systems exhibiting similar volatility across the entire residue range. However, near positions 310 and 330, the RMSF value of the TrkB-T3 complex is lower than that of the TrkB receptor, indicating that peptide binding in these regions reduces the volatility of residues at these positions. Table 3 shows that the binding sites of T3 to the TrkB receptor are concentrated around positions 310 (Lys312, Ala314, Leu315, Gln316) and 330 (Glu326, Thr332, Ile334), suggesting that T3 binding contributes to a more stable TrkB receptor structure. Figure 6 C represents the Rg changes of TrkB and the TrkB-T3 complex. The TrkB-T3 complex was higher than TrkB at most time points, indicating that peptide binding makes the overall structure of TrkB more porous. This may be because T3 binding causes a conformational change in the part of the TrkB receptor that binds to BDNF, increasing the overall volume of the structure. T3 can enhance the stability of the TrkB receptor, but it leads to a more porous overall structure of TrkB. The speculated mechanism may be that after T3 binds to the TrkB receptor, it stimulates the BDNF-TrkB-CREB pathway signaling, and the porous structure facilitates BDNF binding, thereby enhancing neurotrophic effects and better delaying the onset and development of neurodegenerative diseases (e.g., Alzheimer's disease).

[0080] Experiment 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 physicochemical properties (molecular weight, isoelectric point, atomic composition, extinction coefficient, estimated half-life, instability index, and average hydrophilicity coefficient) of peptides. Hydrophobicity and hydrophilicity of peptides were determined using GRAVY, with GRAVY > 0 indicating hydrophobic peptides and GRAVY < 0 indicating hydrophilic peptides. Peptide stability was determined by the instability coefficient, with an instability coefficient > 40 indicating unstable peptides and an instability coefficient < 40 indicating stable peptides. 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 peptide sensitization. The results are shown in Table 4. SGFSKHF peptides have low molecular weight, good water solubility, strong stability, and are non-toxic and non-allergenic, which helps maintain their biological activity during processing and digestion. This indicates that SGFSKHF peptides have good processing performance and safety, and are of great significance for developing functional foods (or pharmaceuticals) to delay the onset of neurodegenerative diseases (e.g., Alzheimer's disease) and for their application in related industries.

[0082] Table 4. SGFSKHF Peptide Analysis

[0083]

[0084] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. The use of a peptide that promotes the synthesis of brain-derived neurotrophic factor in the preparation of a medicament for delaying the occurrence and / or development of neurodegenerative diseases, wherein the amino acid sequence of the peptide is SGFSKHF and the molecular weight is 809 Da.

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