Emotion regulating peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of protein, and particularly relates to an emotion regulating peptide as well as a preparation method and application thereof. The amino acid sequence of the emotion regulating peptide is TDLPSVVE, and the molecular weight of the emotion regulating peptide is 859Da. The emotion regulating peptide can be spontaneously combined with a StAR receptor, the stability of the StAR receptor is enhanced, the level of CORT (corticosterone) generated by Y1 cells is reduced, and therefore the emotion regulating effect is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of proteins and relates to a mood regulating peptide and a preparation method and application thereof. Background Art

[0002] In modern society, the fast-paced lifestyle and intense competitive pressures have generally led to an increase in negative emotions such as mood swings, depression, and anger. Emotional regulation has become crucial, not only affecting individual mental health but also directly impacting interpersonal relationships and work efficiency. As an emerging tool for mood regulation, research on food-derived peptides has primarily focused on anti-agitation and antidepressant peptides, which are primarily isolated and purified from raw materials such as milk, fish, eggs, and soybeans. However, research on mood-regulating peptides is relatively limited, and exploration of their preparation processes and mechanisms of action is insufficient.

[0003] As a key stress hormone, corticosterone plays an important role in mood regulation. Excessive secretion of corticosterone may lead to emotional problems such as depression. Long-term high corticosterone levels are closely related to the occurrence of emotional and mental health problems. StAR protein is a membrane-bound protein that is mainly responsible for promoting the transport of cholesterol to the steroid hormone synthesis pathway and plays a key role in the synthesis and secretion of corticosterone. By inhibiting the expression of StAR, the transport of cholesterol to mitochondria can be reduced, the level of steroids can be reduced, and then the level of corticosterone can be reduced, thereby achieving mood regulation. Therefore, exploring peptides that can inhibit the expression of StAR receptors may provide a new method for mood regulation.

[0004] Currently, mood regulation primarily relies on medication and psychotherapy. However, exploring new approaches to mood regulation through nutritional intervention has become a research hotspot in academia. Food-derived peptides, due to their diverse bioactivities, are considered to have potential applications in the field of mood regulation. Despite this, research on mood-regulating peptides is relatively limited, and their preparation and mechanisms of action remain underdeveloped. Summary of the Invention

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

[0006] The purpose of the present invention and the solution to its technical problems can be achieved through the following technical solutions.

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

[0008] In another aspect, the present invention provides a method for preparing the mood regulating peptide, comprising the following steps:

[0009] (1) using pepsin and trypsin to simulate in vitro digestion of soybean peptides to obtain soybean peptide digestion products; the soybean peptides are obtained by enzymatically hydrolyzing soybean protein isolate using alkaline protease, papain and flavor protease;

[0010] (2) using a Caco-2 monolayer cell model to simulate in vitro absorption of the soybean peptide digestion product to obtain absorbed soybean peptide;

[0011] (3) The absorbable soybean peptide is identified to obtain the mood regulating peptide TDLPSVVE.

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

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

[0014] Beneficial effects:

[0015] The mood regulating peptide of the present 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 the present invention has low molecular weight, good water solubility, strong stability, is non-toxic and non-allergenic, and is conducive to maintaining biological activity during processing and digestion. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The diagram shows the molecular docking visualization of the P1 and P2 peptide segments with the StAR receptor; (A) is the P1 peptide segment, and (B) is the P2 peptide segment;

[0018] Figure 2 The following are visualization diagrams of the molecular docking of P3, P4 and P5 peptides with StAR receptors; (C) is the P3 peptide, (D) is the P4 peptide, and (E) is the P5 peptide;

[0019] Figure 3 This is the analysis result of the effects of different peptides and ACTH on Y1 cell viability;

[0020] Figure 4This is the analysis result of the effect of different peptides on ACTH-induced CORT production in Y1 cells;

[0021] Figure 5 This is the result analysis diagram of the effects of different peptides on ACTH-induced StAR gene expression in Y1 cells;

[0022] Figure 6 The molecular dynamics simulation results of StAR and StAR-P2 complex; (A) is the RMSD fluctuation test result diagram, (B) is the RMSF change test result diagram; (C) is the Rg change test result diagram. DETAILED DESCRIPTION

[0023] 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.

[0024] Due to their diverse biological activities, food-derived peptides are considered to have potential applications in the field of mood regulation. Despite this, research on these peptides is relatively limited. The present inventors developed the present invention by processing soybean peptides to obtain these peptides.

[0025] In one aspect, the present invention provides a mood-regulating peptide having an amino acid sequence of TDLPSVVE and a molecular weight of 859 Da.

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

[0027] In another aspect, 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 soy 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 peptide was identified to obtain the mood regulating peptide TDLPSVVE of the present invention.

[0031] In an embodiment of the present invention, the mood-regulating peptides are screened when first isolated using a method comprising the following steps: I. molecular docking the absorbed soybean peptides separated in step (2) with the StAR receptor, and screening peptide segments based on affinity; II. using adrenocorticotropic hormone to stimulate Y1 cells to establish a cell model, and screening the mood-regulating peptides by testing the effects of the peptide segments screened in step A on cell viability, CORT content, and StAR gene expression.

[0032] 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).

[0033] In the preparation process of the soybean peptide of the present invention, the soy protein isolate is prepared into a 9%-10% (e.g., 9% or 10%) protein solution, the pH value is adjusted to 8.5, alkaline protease is first added, the enzymatic hydrolysis time is 30-35 min (e.g., 30 min, 31 min, 32 min, 33 min, 34 min or 35 min), the enzymatic hydrolysis temperature is 45-55 ° C (e.g., 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 min (e.g., 30 min, 31 min, 32 min, 33 min, 34 min or 35 min). 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).

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

[0035] A. Mix the soybean peptide with simulated saliva and react to obtain the soybean peptide oral digestive fluid.

[0036] B. adding simulated gastric fluid to the soybean peptide oral digestive fluid, adjusting the pH to 3.0, and then adding pepsin to react to obtain soybean peptide gastric digestive fluid;

[0037] C. adding simulated intestinal fluid to the soybean peptide gastric digestion fluid, adjusting the pH to 7.0, and then adding trypsin to react to obtain soybean peptide intestinal digestion fluid;

[0038] D. heating the soybean peptide digestion solution to terminate digestion, and freeze-drying to obtain the soybean peptide digestion product;

[0039] 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;

[0040] 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;

[0041] 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.

[0042] The reason why CaCl2(H2O)2 is added to the above-mentioned simulated liquid is that: 1) the ionic strength of calcium ions in gastrointestinal fluid is simulated; 2) calcium ions in the solution can ensure the stability and 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.

[0043] In an embodiment of the preparation method of the present invention, in step A, 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 B, pepsin is added to a final concentration of 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 h (e.g., 2 h, 2.3 h, 2.5 h, 2.8 h, or 3 h). If the amount of pepsin added is too high, excessive enzymatic hydrolysis of the peptide will result and the actual digestive 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 digestive process of the human body cannot be simulated. In step C, 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, it will result in The added amount may cause excessive enzymatic hydrolysis of the peptide and fail to simulate the actual digestion process of the human body; if the added amount is too low, the enzymatic hydrolysis of the peptide will be incomplete and the actual digestion process of the human body may not 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 solution is placed in a water bath at 95-100°C (for example, 95°C, 96°C, 97°C, 98°C, 99°C or 100°C) for 10-15 minutes (for example, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes or 15 minutes) to terminate the digestion.

[0044] In an embodiment of the preparation method of the present invention, in step D, 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).

[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 subjected to in vitro simulated absorption using a Caco-2 monolayer cell model, and the soybean peptide digestion products are separated into absorbed soybean peptides and unabsorbed soybean peptides. In a specific embodiment, the Caco-2 cells are cultured for 21 days to reduce their resistance to 400-600 Ω / cm. 2 When 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 a specific embodiment, the resistance value of the Caco-2 monolayer cell can be 410, 420, 430, 440, 450, 480, 490, 500, 550, 560, 570, 580, 590, 600Ω / 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 use of the mood-regulating peptide of the present invention in the preparation of a mood-regulating drug.

[0049] 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.

[0050] Example 1

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

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

[0053] (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 temperature of 50°C. Papain was added again and the enzymatic hydrolysis was continued for 30 minutes at a temperature of 50°C. Finally, flavor protease was added and the enzymatic hydrolysis was continued for 175 minutes at a temperature of 60°C. The enzymes were 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.

[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 fluid and simulated intestinal fluid

[0056] 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

[0057] (3) In vitro simulated gastrointestinal digestion of soybean peptides was performed with reference 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, 10 g of soybean peptide was weighed, SSF solution was added to 10 mL, and the mixture was reacted at 37°C for 2 min. In the gastric digestion stage, SGF was added to 20 mL of the oral digestive fluid and the pH was adjusted to 3.0. Pepsin was added to make its activity in the gastric digestive fluid reach 2000 U / mL, and the mixture was incubated at 37°C for 2 h. In the intestinal digestion stage, SIF was added to 40 mL of the gastric digestive fluid and the pH was adjusted to 7.0. Trypsin was added to make its activity in the intestinal digestive fluid reach 100 U / mL, and the mixture was incubated at 37°C for 2 h. After digestion was completed, the digestive fluid was placed in boiling water and heated for 10 min to terminate digestion. Finally, the digestion solution was placed at -40°C and vacuum freeze-dried for 48 hours to obtain digested soybean peptides.

[0058] (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 × 105 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 2 The digested soybean peptide transport experiment was carried out at the same time. 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 30 minutes of equilibrium. 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 for peptide identification.

[0059] 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.

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

[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: number of peaks to match.

[0066] The higher the XCorr value, the better the matching quality.

[0067] Table 2 Identification results of absorbed soybean peptide segments

[0068]

[0069]

[0070] (5) The absorbed soybean peptide was molecularly docked with 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-FO LD4). The cholesterol molecular structure was obtained from the Chenmspider database (https: / / www.chemspider.com / ). The StAR receptor was molecularly docked with the substrate cholesterol. The complex was imported into PyMol and the getbox plug-in was used to determine the center coordinates of the docking box. AutoDockTools 1.5.7 was used to dehydrate and hydrogenate the StAR and peptide segments. The center coordinates of the StAR docking box were set as follows: x = 57.7, y = 25.7, z = -7.5, and the size of the docking box was: Molecular docking was performed using AutoDockVina to obtain the binding energies between StAR and different peptides. Among them, the affinity of DQTPRVF, TDLPSVVE, AENNQRNF, GEKDNVVRQ, and SRDPIYSN was relatively low, at -6.8, -6.8, -5.5, -5.5, and -4.7 kcal / mol, respectively. The docking results of each peptide were imported into Pymol for visualization (the results are shown in Figure 2). 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 about the interaction between the receptor and the peptide (the results are shown in Table 3).

[0071] Table 3 Analysis of the interaction between different peptides and StAR receptors

[0072]

[0073]

[0074] Table 3 shows that the five peptides with the lowest affinity bind to the StAR receptor primarily through hydrogen bonding, salt bridges, and π-cation interactions, with the majority being hydrophobic interactions and hydrogen bonding. 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 bonding play a major role in the binding of P1-P5 to the StAR receptor.

[0075] (6) ACTH (adrenocorticotropic hormone) was used to stimulate Y1 cells, and the mood-regulating peptides of this example were screened by detecting cell viability, CORT content, and StAR gene expression:

[0076] Mouse adrenocortical tumor Y1 cells (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 in 5% CO2 / 95% air. When the cell density reached 80%, 0.25% TE (trypsin-EDTA solution, Gibco, USA) was used for subculture at a subculture ratio of 1:2. The effects of different peptides and ACTH on Y1 cell viability were evaluated by CCK-8: Y1 cells were cultured at 4×10 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 the cells adhered, 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 ACTH solution (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 hours, the different peptide solutions or ACTH solutions were removed and washed with PBS. 100 μL of 10% CCK-8 solution (Shanghai Biyuntian Biotechnology Co., Ltd.) (dissolved in F12 medium) was added and incubated for 1.5 hours, and the absorbance was measured at 450 nm using a microplate reader. At the same time, a blank group (no cells were seeded, and F12 medium was added) and a control group (cells were seeded, and F12 medium was added) were set up. The results are shown in Figure 2. Figure 3As shown, P1-P3 had no damage to Y1 cells in the range of 0.125-4 mg / mL, and cell viability did not change significantly (P>0.05), while 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). In order to exclude the damage of peptides to cells, all peptide concentrations were unified to 2 mg / mL for subsequent experiments. Figure 3 F showed that 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 exclude the effect of ACTH on cell damage, 5 mg / mL ACTH was selected for subsequent experiments.

[0077] The CORT level in Y1 cell supernatant was determined by ELISA: Y1 cells were plated at 4 × 10 5 Cells were seeded at a density of 10 cells / mL in a 6-well plate and incubated in an incubator for 24 hours. F12 medium was added to the control group and the ACTH group, and 2 mg / mL P1, P2, P3, P4 and P5 were added to the experimental group. After 2 hours of nutritional intervention, F12 medium was added to the control group, 5 mg / mL ACTH was added to the ACTH group, and 2 mg / mL P1, P2, P3, P4, P5 and 5 mg / mL ACTH were added to the experimental group and incubated for 8 hours. After the incubation, the supernatant was collected and CORT was determined according to the instructions of the kit (QuicKey Pro, Wuhan Elaruite Biotechnology Co., Ltd.). The results are shown in the figure. Figure 4 As shown in the results, compared with the blank group, 5 mg / mL ACTH significantly increased the CORT content (P<0.05), which was 5 times that of the blank group. Compared with the ACTH group, P1-P5 all significantly reduced the CORT content (P<0.05). The CORT levels from low to high were 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 can significantly inhibit the production of CORT in Y1 cells and regulate the biosynthesis of steroid hormones.

[0078] The gene expression levels of Y1 cells were determined by RT-qPCR. Y1 cells were cultured at 4 × 10 5The cells were seeded at a density of 10 cells / mL in a 6-well plate and incubated in an incubator for 24 hours. F12 medium was added to the control group and the ACTH group, and 2 mg / mL P2 and P5 were added to the experimental group. After 2 hours of nutritional intervention, F12 medium was added to the control group, 5 mg / mL ACTH was added to the ACTH group, and 2 mg / mL P2, P5 and 5 mg / mL ACTH were added to the experimental group and incubated for 8 hours. After the incubation, the cells were lysed and collected, and the total RNA of the cells was extracted using the TransZol Up Plus RNA kit (Beijing Quanshijin Biotechnology Co., Ltd.). The purity of the RNA was evaluated by agarose gel electrophoresis, and the Reverse transcription was performed using a temperature gradient PCR instrument according to the instructions of the Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Kit (Beijing Quanshijin Biotechnology Co., Ltd.). Green qPCR SuperMix Kit (Beijing Quanshijin Biotechnology Co., Ltd.) was used for quantitative analysis using a real-time fluorescence quantitative PCR instrument to calculate the gene expression level of the sample. -ΔΔCt , Actb was used as the internal reference gene. Figure 5 As shown, ACTH stimulation significantly increased the relative mRNA expression of the StAR gene (P < 0.05), reaching 1.4 times that of the blank group. Compared with the ACTH group, StAR gene expression in the P2 group decreased significantly by 28.8% (P < 0.05), while there was no significant change in the P5 group. These results suggest that P2 can downregulate StAR gene expression, reduce cholesterol transport into mitochondria, and decrease steroid levels, ultimately leading to decreased COR and CORT levels.

[0079] Experimental Example 1

[0080] Using the modified method of Valdes-Tresanco et al., Gromacs 2023 was used to place the peptide-StAR receptor complex generated by molecular docking and the StAR receptor PDB file as the initial structure within a dodecahedral water box. The force field used was Amber14sb, the water molecule model was TIP3P, and sodium and chloride ions were used to minimize the total charge of the system to zero, thus constructing the initial simulation system. A two-step energy minimization was performed on the initial simulation system using the steepest descent method for 10,000 iterations. After energy optimization, NVT and NPT simulations were performed for 100 ps, respectively. Following the NPT simulation, a 100 ns full simulation was performed. C-rescale was used for temperature control, and parrinello-rahman for pressure control. The temperature was set to 310 K, and conformations were saved every 10 ps. Simulation results were analyzed using 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). Figure 6 shown.

[0081] Depend on Figure 6 A shows that during the 100ns molecular dynamics simulation, the RMSD of the two systems almost reached equilibrium, indicating that all simulation systems reached a stable state and the dynamics simulation results were reliable. In addition, the fluctuation of the StAR-P2 complex was smaller, indicating that the binding of P2 increased the stability of the StAR receptor. Figure 6 As shown in Figure 3, the overall fluctuation trends of the RMSF changes of the StAR receptor and the StAR-P2 complex are similar. P2 binding leads to a decrease in local fluctuations in the StAR receptor, suggesting that P2 may stabilize specific residues. Combined with Table 3, the binding sites of P2 for the StAR receptor are distributed at positions 150, 180, 220, 240, and 250, corresponding to the peaks of the RMSF. Figure 6 C represents the Rg changes of the StAR receptor and the StAR-P2 complex. After 75 ns, both curves stabilize, indicating that both the StAR receptor and the StAR-P2 complex have reached a stable conformation. Compared to StAR, the StAR-P2 complex has a higher Rg value after 75 ns than the StAR receptor. These results suggest that P2 (peptide TDLPSVVE) enhances StAR receptor stability and simultaneously stabilizes residues within its binding sites, reducing cholesterol transport by the StAR receptor and, consequently, reducing steroid production (CORT), further contributing to its mood-regulating effects.

[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 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.

[0084] The results are shown in Table 4:

[0085] Table 4 Analysis of TDLPSVVE peptide segments

[0086]

[0087] Experimental results demonstrate that the TDLPSVVE peptide exhibits low molecular weight, good water solubility, strong stability, and is non-toxic and non-allergenic, maintaining its biological activity during processing and digestion. Furthermore, the peptide exhibits excellent processability and safety, making it of great significance for the development of mood-modulating functional foods and pharmaceuticals, as well as their application in the food and pharmaceutical industries.

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

2. The method for preparing the mood regulating peptide according to claim 1, wherein: The following steps are involved: (1) enzymatically hydrolyzing soy protein isolate with alkaline protease, papain, and flavor protease to obtain soy peptides, and subjecting the soy peptides to in vitro simulated digestion to obtain soy peptide digestion products; (2) using a Caco-2 monolayer cell model to simulate in vitro absorption of the soybean peptide digestion product to obtain absorbed soybean peptide; (3) The absorbed soybean peptide is identified to obtain the mood regulating peptide TDLPSVVE.

3. The preparation method according to claim 2, wherein When the mood regulating peptide is first isolated, it is screened using a method comprising the following steps: I. performing molecular docking of the absorbed soybean peptide separated in step (2) with the StAR receptor, and screening the peptide segments based on affinity; II. Using adrenocorticotropic hormone to stimulate Y1 cells to establish a cell model, the mood-regulating peptide was screened by testing the effects of the peptides screened in step A on cell viability, CORT content, and StAR gene expression.

4. The preparation method according to claim 2, wherein The added amount of the total protease of alkaline protease, papain and flavor protease is 0.8%-1.2% of the soy protein isolate in terms of mass fraction, and the mass ratio of the 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 soybean peptide preparation process, 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 minutes at a temperature of 45-55°C; then papain is added again, and the enzymatic hydrolysis is continued for 30-35 minutes at a temperature of 45-55°C; finally, flavor protease is added, and the enzymatic hydrolysis is continued for 160-175 minutes at a temperature of 50-60°C; after the enzymatic hydrolysis is completed, the solution is heated to above 90°C and bathed in water for more than 10 minutes to inactivate the enzyme.

6. The preparation method according to claim 2, wherein step (1) specifically comprises the following steps: A. mixing the soybean peptide with simulated saliva, reacting to obtain soybean peptide oral digestive fluid, B. adding simulated gastric fluid to the soybean peptide oral digestive fluid, adjusting the pH to 3.0, and then adding pepsin to react to obtain soybean peptide gastric digestive fluid; C. adding simulated intestinal fluid to the soybean peptide gastric digestion fluid, adjusting the pH to 7.0, and then adding trypsin to react to obtain soybean peptide intestinal digestion fluid; D. heating the soybean peptide digestion solution to terminate digestion, and freeze-drying to obtain the soybean peptide digestion product; 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 5, wherein In step A, in the mixed solution of the soybean peptide and the simulated saliva, the concentration of the soybean peptide is 1-2 g / mL, the reaction temperature is 35-40° C., and the reaction time is 2-4 min; In step B, 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 h; In step C, 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; In step D, the digestion is terminated by placing in a 95-100°C water bath for 10-15 minutes, the freeze-drying temperature is -40 to -80°C, and the freeze-drying time is 48-72 hours; The volume ratio of the simulated saliva, simulated gastric fluid and simulated intestinal fluid is 1:1:

2.

8. 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.

9. A mood regulating composition comprising the mood regulating peptide according to claim 1.

10. Use of the mood-regulating peptide according to claim 1 in the preparation of a mood-regulating drug.

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