Emotion-regulating peptide, preparation method and application thereof

Abstract

The application belongs to the technical field of proteins, and particularly relates to a mood regulation peptide as well as a preparation method and application thereof. The amino acid sequence of the mood regulation peptide is TDLPSVVE, and the molecular weight is 859 Da. The mood regulation peptide can spontaneously combine with a StAR receptor, enhance the stability of the StAR receptor, and reduce the level of CORT (corticosterone) produced by Y1 cells, so as to play a mood regulation role.

Description

Technical Field

[0001] This invention belongs to the field of protein technology and relates to a mood-regulating peptide, its preparation method, and its application. Background Technology

[0002] In modern society, the fast pace of life and intense competitive pressure commonly lead to an increase in negative emotions such as mood swings, depression, and anger. Emotion regulation has become crucial, as it not only relates to individual mental health but also directly impacts interpersonal relationships and work efficiency. Food-derived peptides, as an emerging means of emotion regulation, have seen research primarily focused on anti-excitement and antidepressant peptides, which are mainly isolated and purified from raw materials such as milk, fish, eggs, and soybeans. However, current research on emotion-regulating peptides is relatively limited, and their preparation processes and mechanisms of action are not explored in sufficient depth.

[0003] Corticosterone, a key stress hormone, plays a crucial role in mood regulation. Excessive corticosterone secretion can lead to mood problems such as depression. Long-term high corticosterone levels are closely associated with mood and mental health issues. StAR protein, a membrane-bound protein, is primarily responsible for promoting the transport of cholesterol to the steroid hormone synthesis pathway and plays a key role in corticosterone synthesis and secretion. Inhibiting StAR expression can reduce cholesterol transport to mitochondria, lower steroid levels, and consequently reduce corticosterone levels, thus achieving mood regulation. Therefore, exploring peptides that can inhibit StAR receptor expression may provide new approaches to mood regulation.

[0004] Currently, mood regulation mainly relies on drug therapy and psychotherapy. However, exploring new approaches to mood regulation from the perspective of nutritional intervention has become a research hotspot in academia. Food-derived peptides, due to their diverse biological activities, are considered to have potential applications in the field of mood regulation. Nevertheless, current research on mood-regulating peptides is relatively limited, and their preparation processes and mechanisms of action are not yet fully explored. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a mood-regulating peptide, its preparation method and application, so as to help provide a new pathway for mood regulation.

[0006] The objectives of this invention and the solutions to its technical problems can be achieved through the following technical solutions.

[0007] On one hand, this invention provides a mood-regulating peptide with the amino acid sequence TDLPSVVE and a molecular weight of 859 Da. The molecular formula of this peptide is C0. 37 H 62 N8O 15It is hydrophilic and stable, with an isoelectric point of 3.7. The peptide has an extinction coefficient of 0, an estimated half-life of 7.2 h, and is non-toxic and non-sensitizing.

[0008] On the other hand, the present invention provides a method for preparing the mood-regulating peptide, comprising the following steps:

[0009] (1) Soybean peptides are subjected to in vitro simulated digestion using pepsin and trypsin to obtain soybean peptide digestion products; the soybean peptides are obtained by enzymatic hydrolysis of soybean protein isolate using alkaline protease, papain and flavor protease.

[0010] (2) Using the Caco-2 monolayer cell model, the digested soybean peptide products were subjected to in vitro simulated absorption to obtain absorbed soybean peptides.

[0011] (3) The absorbed soybean peptide was identified to obtain the mood-regulating peptide TDLPSVVE.

[0012] In a third aspect, the present invention provides a mood-regulating composition comprising the mood-regulating peptides of the present invention.

[0013] In a fourth aspect, the present invention provides the use of the mood-regulating peptide of the present invention in the preparation of mood-regulating drugs.

[0014] Beneficial effects:

[0015] The mood-regulating peptide of this invention can spontaneously bind to the StAR receptor, enhance the stability of the StAR receptor, and reduce the level of CORT (corticosterone) produced by Y1 cells, thereby exerting a mood-regulating effect.

[0016] The mood-regulating peptide (TDLPSVVE) of this invention has a low molecular weight, good water solubility, strong stability, is non-toxic and non-allergenic, and is beneficial to maintaining its biological activity during processing and digestion. Attached Figure Description

[0017] Figure 1 Visualization of the molecular docking of P1 and P2 peptides with the StAR receptor; where (A) is the P1 peptide and (B) is the P2 peptide.

[0018] Figure 2 Visualization of the molecular docking of P3, P4 and P5 peptides with the StAR receptor; where (C) is P3 peptide, (D) is P4 peptide, and (E) is P5 peptide.

[0019] Figure 3 Figure showing the results of the analysis of the effects of different peptides and ACTH on Y1 cell viability;

[0020] Figure 4Figure showing the results of the analysis of the effects of different peptide segments on ACTH-induced CORT production in Y1 cells;

[0021] Figure 5 Figure showing the results of the analysis of the effects of different peptide segments on ACTH-induced StAR gene expression in Y1 cells;

[0022] Figure 6 Figure 1 shows the molecular dynamics simulation results of the StAR and StAR-P2 complex; (A) shows the RMSD fluctuation test results, (B) shows the RMSF change test results, and (C) shows the Rg change test results. Detailed Implementation

[0023] 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.

[0024] Food-derived peptides are considered to have potential applications in mood regulation due to their diverse biological activities. However, current research on mood-regulating peptides is relatively limited. This invention proposes a method to obtain mood-regulating peptides by processing soybean peptides.

[0025] On the one hand, the present invention provides a mood-regulating peptide with the amino acid sequence TDLPSVVE and a molecular weight of 859 Da.

[0026] The mood-regulating peptide of this invention can spontaneously bind to the StAR receptor, enhance receptor stability, and reduce the level of CORT produced by Y1 cells, thereby exerting a mood-regulating effect.

[0027] On the other hand, the present invention provides a method for preparing the mood-regulating peptide, comprising the following steps:

[0028] (1) Soybean peptides were digested in vitro using pepsin and trypsin to obtain soybean peptide digestion products; soybean peptides were obtained by enzymatic hydrolysis of soybean protein isolate using alkaline protease, papain and flavor protease.

[0029] (2) Using the Caco-2 monolayer cell model, the digestion products of soybean peptides were simulated in vitro to obtain absorbed soybean peptides.

[0030] (3) The absorbed soybean peptides were identified to obtain the mood-regulating peptide TDLPSVVE of the present invention.

[0031] In an embodiment of the present invention, the mood-regulating peptide is screened during the initial isolation using a method comprising the following steps: I. Molecular docking of the absorbable soybean peptide obtained in step (2) with the StAR receptor, and screening peptides based on affinity; II. Establishing a cell model by stimulating Y1 cells with adrenocorticotropic hormone, and screening the mood-regulating peptide by testing the effects of the peptides screened in step A on cell viability, CORT content and StAR gene expression.

[0032] 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).

[0033] 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. First, alkaline protease is added, and the enzymatic hydrolysis time is 30-35 minutes (e.g., 30 minutes, 31 minutes, 32 minutes, 33 minutes, 34 minutes, or 35 minutes) at a temperature of 45-55°C (e.g., 45°C, 47°C, 49°C, 51°C, 53°C, or 55°C). Then, papain is added again, and enzymatic hydrolysis continues for another 30-35 minutes (e.g., 30 minutes, 31 minutes, 32 minutes, ...). 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).

[0034] In an embodiment of the present invention, step (1) specifically includes the following steps:

[0035] A. Soybean peptides are mixed with simulated saliva and reacted to obtain a soybean peptide oral digestive fluid.

[0036] B. Add simulated gastric juice to the soybean peptide oral digestive fluid, adjust the pH to 3.0, then add pepsin, react, and obtain soybean peptide gastric digestive fluid;

[0037] C. Add simulated intestinal fluid to the soybean peptide gastric digestive fluid, adjust the pH to 7.0, then add trypsin, react, and obtain soybean peptide intestinal digestive fluid;

[0038] D. Heat the soybean peptide digest solution to terminate digestion, then freeze-dry to obtain the soybean peptide digest product;

[0039] 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.

[0040] 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.

[0041] 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 added to make up the difference. 0.04 mL of 0.3 M CaCl2(H2O)2 is added separately before use.

[0042] 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 ensure 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.

[0043] In an embodiment of the preparation method of the present invention, in step A, 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℃ (e.g., 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃); and the reaction time is 2-4 min (e.g., 2 min, 3 min or 4 min). In step B, pepsin is added until the final concentration of pepsin is 2000-2500 U / mL (e.g., 2000 U / mL, 2100 U / mL, 2200 U / mL, 2300 U / mL, 2400 U / mL, or 2500 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 hours (e.g., 2 hours, 2.3 hours, 2.5 hours, 2.8 hours, or 3 hours). If the amount of pepsin added is too high, it will lead to excessive enzymatic hydrolysis of peptides and will not be able to simulate the actual human digestive process; if the amount added is too low, it will lead to incomplete enzymatic hydrolysis of peptides and will not be able to simulate the actual human digestive process. In step C, trypsin is added until the final concentration is 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℃ (e.g., 35℃, 36℃, 37℃, 38℃, 39℃, or 40℃), and the reaction time is 2-3 hours (e.g., 2 hours, 2.3 hours, 2.5 hours, 2.8 hours, or 3 hours). However, if the amount of trypsin added is too high, it will lead to… The addition of too little peptides will result in incomplete peptide digestion 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).

[0044] In an embodiment of the preparation method of the present invention, in step D, 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 48-72h (e.g., 48h, 54h, 60h, 66h or 72h).

[0045] 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.

[0046] In an embodiment of the method of the present invention, in step (2), the soybean peptide digestion products are simulated for in vitro absorption using a Caco-2 monolayer cell model, separating the soybean peptide digestion products into absorbed soybean peptides and unabsorbed soybean peptides. In a specific embodiment, Caco-2 cells are cultured for 21 days to achieve a resistivity of 400-600 Ω / cm. 2 During this process, a Caco-2 monolayer cell model is constructed. 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 and unabsorbed soybean 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. In specific implementations, the resistance value of the Caco-2 monolayer cells can be 410, 420, 430, 440, 450, 480, 490, 500, 550, 560, 570, 580, 590, or 600 Ω / cm. 2 .

[0047] In a third aspect, the present invention provides a mood-regulating composition comprising the aforementioned mood-regulating peptide. Those skilled in the art will appreciate that the composition may also comprise other mood-regulating substances, or active substances that synergistically enhance the mood-regulating effect, as well as pharmaceutically acceptable carriers or excipients.

[0048] In a fourth aspect, the present invention provides the application of the mood-regulating peptide of the present invention in the preparation of mood-regulating drugs.

[0049] 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.

[0050] Example 1

[0051] This embodiment provides an emotion peptide with the amino acid sequence TDLPSVVE and a molecular weight of 859 Da.

[0052] The preparation process of the mood-regulating peptide in this embodiment is as follows:

[0053] (1) Prepare a 9% soy protein isolate solution from Shandong Yuxin Biotechnology Co., Ltd., and enzymatically 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 amount of 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℃. 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.

[0054] (2) Prepare simulated saliva, simulated gastric juice and simulated intestinal juice, as shown in Table 1.

[0055] Table 1. Preparation of simulated saliva, simulated gastric juice, and simulated intestinal juice

[0056] 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

[0057] (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. In the oral digestion stage, 10g of soybean peptides were weighed and added to 10mL of SSF solution, and the mixture was reacted at 37℃ for 2min. In the gastric digestion stage, SGF was added to 20mL of oral digestive fluid and the pH was adjusted to 3.0. Pepsin was added to achieve an activity of 2000U / mL in the gastric digestive fluid, and the mixture was incubated at 37℃ for 2h. In the intestinal digestion stage, SIF was added to 40mL of gastric digestive fluid and the pH was adjusted to 7.0. Trypsin was added to achieve an activity of 100U / mL in the intestinal digestive fluid, and the mixture was incubated at 37℃ for 2h. After digestion, the digestive fluid was heated in boiling water for 10min to terminate digestion. Finally, the digestive fluid was placed at -40℃ and then freeze-dried under vacuum for 48 hours to obtain digested soybean peptides.

[0058] (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 (USA). After 21 days of culture, the TEER was measured to be 400-600 Ω / cm. 2 To conduct 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 peptides, which were then identified as peptides.

[0059] 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.

[0060] XCorr (cross-correlation score) is used to measure the correlation between experimental and theoretical spectra.

[0061] formula:

[0062]

[0063] Iexp,i: The intensity of the i-th peak in the experimental spectrum.

[0064] Itheo,i: The intensity of the i-th peak in the theoretical spectrum.

[0065] n: The number of matched peaks.

[0066] A higher XCorr value indicates a better matching quality.

[0067] Table 2. Identification results of absorbed soybean peptide fragments

[0068]

[0069]

[0070] (5) Molecular docking was performed between the absorbed soybean peptide and the StAR receptor. The protein structure of StAR (PDB ID: 3P0L) 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). The cholesterol molecular structure was obtained from the Chenmspider database (https: / / www.chemspider.com / ). The StAR receptor was molecularly docked with the substrate cholesterol, and the complex was imported into PyMol. The getbox plugin was used to determine the center coordinates of the docking box. AutoDockTools 1.5.7 was used to dehydrate and hydrogenate StAR and the peptide. The center coordinates of the StAR docking box were set as follows: x = 57.7, y = 25.7, z = -7.5. The size of the docking box was: Molecular docking was performed using AutoDockVina to obtain the binding energies of StAR and different peptides. DQTPRVF, TDLPSVVE, AENNQRNF, GEKDNVVRQ, and SRDPIYSN showed the lowest affinity energies, at -6.8, -6.8, -5.5, -5.5, and -4.7 kcal / mol, respectively. The docking results for each peptide were imported into Pymol for visualization (results are shown in Figure 1). Figure 1-2 (As shown in Table 3). The complex was then uploaded to 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 (results are shown in Table 3).

[0071] Table 3. Interaction analysis of different peptides with the StAR receptor

[0072]

[0073]

[0074] Table 3 shows that the five peptides with the lowest affinity bind to the StAR receptor mainly through hydrogen bonds, salt bridges, and π-cation interactions, with hydrophobic interactions and hydrogen bonds being the most prevalent. P1-P5 primarily form hydrophobic interactions with Asn150, Val151, Val179, Arg182, and Phe184 residues of the StAR receptor, hydrogen bonds with Asn150, Glu169, Arg182, Arg188, and Gln262 residues, and a salt bridge with His220 residue. Hydrophobic interactions and hydrogen bonds play the main roles in the binding of P1-P5 to the StAR receptor.

[0075] (6) Y1 cells were stimulated with ACTH (adrenocorticotropic hormone), and the mood-regulating peptides of this embodiment were obtained by screening cell viability, CORT content, and StAR gene expression levels:

[0076] Mouse adrenocortical tumor cells Y1 (Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences) were cultured in F12 medium (Gibco, USA) containing 10% FBS and 1% PS (penicillin-streptomycin solution) at 37°C and 5% CO2 / 95% aerosol. When the cell density reached 80%, cells were passaged using 0.25% TE (trypsin-EDTA solution, Gibco, USA) at a passage ratio of 1:2. The effects of different peptides and ACTH on Y1 cell viability were assessed using CCK-8 assay: Y1 cells were cultured at a rate of 4 × 10⁶ cells / year. 5 Cells were seeded at a density of [number] cells / mL in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. After cell attachment, the culture medium was removed, and 100 μL of different concentrations 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 ACTH solutions (0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, and 10 mg / mL) dissolved in F12 medium were added. After incubation for 10 h, the peptide solutions or ACTH solutions were removed, and the cells were washed with PBS. 100 μL of 10% CCK-8 solution (Shanghai Beyotime Biotechnology Co., Ltd.) (dissolved in F12 medium) was added, and the cells were incubated for 1.5 h. The absorbance was measured at 450 nm using a microplate reader. A blank group (no cells seeded, F12 medium added) and a control group (cells seeded, F12 medium added) were also set up. Results are shown below. Figure 3As shown, P1-P3 did not damage Y1 cells within the range of 0.125-4 mg / mL, and cell viability remained largely unchanged (P>0.05). However, P4 at 4 mg / mL significantly reduced Y1 cell viability to 87.6% ± 3.0% (P<0.05), and P5 at 4 mg / mL significantly reduced Y1 cell viability to 94.0% ± 1.8% (P<0.05). To rule out cell damage from peptides, all peptide concentrations were standardized to 2 mg / mL for subsequent experiments. Figure 3 As shown in Figure F, 0.5-5 mg / mL ACTH had no significant effect on cell viability (P>0.05), while 10 mg / mL ACTH significantly reduced Y1 cell viability to 46.4±3.8% (P<0.05). To rule out the effect of ACTH on cell damage, 5 mg / mL ACTH was selected for subsequent experiments.

[0077] CORT levels in Y1 cell supernatant were determined using ELISA: Y1 cells were inoculated at a concentration of 4 × 10⁶ cells / mL. 5 Cells were seeded at a density of [number] cells / mL in 6-well plates and incubated for 24 h. The control and ACTH groups were added to F12 medium, while the experimental groups were added to 2 mg / mL P1, P2, P3, P4, and P5, respectively. After 2 h of nutrient intervention, the control group was added to F12 medium, the ACTH group was added to 5 mg / mL ACTH, and the experimental groups were added to 2 mg / mL P1, P2, P3, P4, P5, and 5 mg / mL ACTH, respectively. All groups were incubated for 8 h. After incubation, the supernatant was collected, and CORT was measured according to the kit (QuicKey Pro, Wuhan Yilairuit Biotechnology Co., Ltd.). The results are as follows: Figure 4 As shown, compared with the control group, 5 mg / mL ACTH significantly increased CORT levels (P<0.05), which was 5 times higher than that of the control group. Compared with the ACTH group, P1-P5 all significantly decreased CORT levels (P<0.05), with CORT levels from low to high being P2 (41.2±0.9 ng / mL), P5 (41.4±1.1 ng / mL), P1 (42.9±0.7 ng / mL), P3 (44.6±0.5 ng / mL), and P4 (44.8±0.8 ng / mL). The results indicate that P2 and P5 significantly inhibited CORT production in Y1 cells, regulating the biosynthesis of steroid hormones.

[0078] Gene expression levels in Y1 cells were determined using RT-qPCR: Y1 cells were inoculated at a concentration of 4 × 10⁶ cells / cells. 5Cells were seeded at a density of [number] cells / mL in 6-well plates and incubated for 24 h. The control and ACTH groups were added to F12 medium, while the experimental groups were added to 2 mg / mL P2 and P5, respectively. After 2 h of nutrient intervention, the control group was added to F12 medium, the ACTH group was added to 5 mg / mL ACTH, and the experimental groups were added to 2 mg / mL P2, P5, and 5 mg / mL ACTH, respectively, and incubated for 8 h. After incubation, cells were lysed and collected, and total RNA was extracted using the TransZol Up Plus RNA kit (Beijing TransGen Biotech Co., Ltd.). RNA purity was assessed using agarose gel electrophoresis, following [specific procedures / methods]. The Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR kit (Beijing TransGen Biotech Co., Ltd.) instructions state that reverse transcription should be performed using a temperature gradient PCR instrument. Follow the... The Green qPCR SuperMix kit (Beijing TransGen Biotech Co., Ltd.) instructions state that it is used for quantification using a real-time quantitative PCR instrument to calculate the gene expression level of the sample. -ΔΔCt Actb was used as an internal reference gene. The results were as follows: Figure 5 As shown, ACTH stimulation significantly increased the relative mRNA expression level of the StAR gene (P<0.05), which was 1.4 times that of the control group. Compared with the ACTH group, the expression of the StAR gene in the P2 group was significantly decreased by 28.8% (P<0.05), while there was no significant change in the P5 group. The results indicate that P2 can downregulate the expression of the StAR gene, reduce the transport of cholesterol to mitochondria, decrease steroid levels, and ultimately lead to a decrease in the levels of COR and CORT.

[0079] Experimental Example 1

[0080] Following and modifying the method of Valdes-Tresanco et al., Gromacs 2023 was used to construct the initial simulation system by placing the peptide-StAR receptor complex generated through molecular docking and the StAR receptor's PDB file within a dodecahedral water box. The force field was Amber14sb, the water molecule model was TIP3P, and sodium and chloride ions were used to achieve a total charge of 0. A two-step energy minimization was performed on the initial simulation system using the steepest descent method, repeated 10,000 times. After energy optimization, 100 ps NVT and NPT simulations were performed, followed by a 100 ns formal simulation. The temperature control algorithm used was C-rescale, and the pressure control algorithm was Parrinello-Rahman. The temperature was 310 K, and the conformation was saved every 10 ps. Gromacs commands were used to analyze and obtain the simulation results. The stability of the system was assessed using root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (Rg). The results are as follows: Figure 6 As shown.

[0081] Depend on Figure 6 As shown in Figure A, during the 100 ns molecular dynamics simulations, the RMSD of both systems almost reached equilibrium, indicating that all simulated systems reached a stable state, and the dynamic simulation results are reliable. Furthermore, the StAR-P2 complex exhibited less fluctuation, suggesting that P2 binding increases the stability of the StAR receptor. Figure 6 As shown in B, the overall fluctuation trends of RMSF changes in the StAR receptor and the StAR-P2 complex are similar. After P2 binding, the local fluctuations of the StAR receptor decrease, indicating that P2 may stabilize specific residues. According to Table 3, the binding sites of P2 to the StAR receptor are distributed at positions 150, 180, 220, 240, and 250, which correspond to the peak values ​​of RMSF. Figure 6 C represents the Rg change of the StAR receptor and the StAR-P2 complex. After 75 ns, both curves stabilized, indicating that both the StAR receptor and the StAR-P2 complex reached a stable conformation. Compared with StAR, the Rg value of the StAR-P2 complex after 75 ns was higher than that of the StAR receptor. These results suggest that P2 (peptide TDLPSVVE) enhances the stability of the StAR receptor and stabilizes the residues at their respective binding sites, reducing cholesterol transport by the StAR receptor and thus leading to a decrease in steroid (CORT) production, further enhancing its mood-regulating effect.

[0082] Experimental Example 2

[0083] 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.

[0084] The results are shown in Table 4:

[0085] Table 4. TDL PLPSVVE Peptide Analysis

[0086]

[0087] Experimental results show that TDLPSVVE 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. Furthermore, TDLPSVVE peptides exhibit good processing performance and safety, making them significant for the development of mood-regulating functional foods and pharmaceuticals, as well as their applications in the food and pharmaceutical industries.

[0088] 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. A mood-regulating peptide, characterized in that, The amino acid sequence of the mood-regulating peptide is TDLPSVVE, and its molecular weight is 859 Da.

2. The method for preparing the mood-regulating peptide as described in claim 1, characterized in that, Includes the following steps: (1) Soybean peptides were obtained by enzymatic hydrolysis of soybean protein isolate using alkaline protease, papain and flavor protease, and the soybean peptides were subjected to in vitro simulated digestion to obtain soybean peptide digestion products. (2) Using the Caco-2 monolayer cell model, the digestion products of the soybean peptides were simulated for in vitro absorption to obtain absorbed soybean peptides; (3) The absorbed soybean peptides were identified to obtain the mood-regulating peptide TDLPSVVE. Step (1) specifically includes the following steps: A. The soybean peptides are mixed with simulated saliva and reacted to obtain a soybean peptide oral digestive fluid. B. Add simulated gastric juice to the soybean peptide oral digestive fluid, adjust the pH to 3.0, then add pepsin, react, and obtain soybean peptide gastric digestive fluid; C. Add simulated intestinal fluid to the soybean peptide gastric digestive fluid, adjust the pH to 7.0, then add trypsin, react, and obtain soybean peptide intestinal digestive fluid; D. The soybean peptide digest solution is heated to terminate digestion, and then freeze-dried to obtain the soybean peptide digest product; 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•6H2O6, 0.06 mL of 0.5 M (NH4)2CO3, and 0.09 mL of 6 M HCl, with dH2O replenished. 0.025 mL of 0.3 M CaCl2•2H2O2 is added separately before use. 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•6H2O, 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•2H2O is added separately before use. 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•6H2O, 0.7 mL of 6 M HCl, and dH2O is added to make up the difference. 0.04 mL of 0.3 M CaCl2•2H2O is added separately before use.

3. The preparation method according to claim 2, characterized in that, Step (3) specifically includes the following steps: I. The absorbed soybean peptides obtained in step (2) are molecularly docked with the StAR receptor, and peptides are screened based on affinity. II. A cell model was established by stimulating Y1 cells with adrenocorticotropic hormone (ACTH). The mood-regulating peptides were obtained by testing the effects of peptides screened in step A on cell viability, CORT content, and StAR gene expression.

4. The preparation method according to claim 2, characterized in that, The total amount of alkaline protease, papain, and flavor protease added is 0.8%-1.2% of soy protein isolate by mass fraction, and the mass ratio of alkaline protease, papain, and flavor protease is 1:2:2-2:3:

3.

5. The preparation method according to claim 2, characterized in that, In the preparation of soybean peptides, soybean 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 min, and the enzymatic hydrolysis temperature is 45-55 ℃. Then, papain is added, and the enzymatic hydrolysis continues for 30-35 min, and the enzymatic hydrolysis temperature is 45-55 ℃. Finally, flavor protease is added, and the enzymatic hydrolysis continues for 160-175 min, and the enzymatic hydrolysis temperature is 50-60 ℃. After the enzymatic hydrolysis is completed, the temperature is raised to above 90 ℃ and water bath is used for more than 10 min to inactivate the enzyme.

6. The preparation method according to claim 2, characterized in that, In step A, the concentration of soybean peptide in the mixed solution of soybean peptide and simulated saliva is 1-2 g / mL, the reaction temperature is 35-40 ℃, and the reaction time is 2-4 min. In step B, the pepsin is added until the final concentration of pepsin is 2000-2500 U / mL, the reaction temperature is 35-40 ℃, and the reaction time is 2-3 h; In step C, the trypsin is added until the final concentration of trypsin is 100-150 U / mL, the reaction temperature is 35-40 °C, and the reaction time is 2-3 h; In step D, digestion is terminated by a water bath at 95-100℃ for 10-15 min, and the freeze-drying temperature is -40~-80℃, with a freeze-drying time of 48-72 h. The volume ratio of the simulated saliva, simulated gastric juice, and simulated intestinal juice is 1:1:

2.

7. The preparation method according to claim 2, characterized in that, In step (2), the soybean peptide digestion product 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.

8. A mood-regulating composition comprising the mood-regulating peptide of claim 1.

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