Active peptide and brain peptide enzymolysis extract for improving memory as well as preparation method and application of active peptide and brain peptide enzymolysis extract

Abstract

The invention belongs to the technical field of active peptides, and particularly relates to an active peptide capable of improving memory, a brain peptide enzymolysis extract and a preparation method and application of the brain peptide enzymolysis extract. The amino acid sequence of the active peptide is shown as SEQ ID NO.1 Ala-Ala-Val-Pro, and the active peptide can improve sleep and improve memory; on the other hand, the invention provides a preparation method of the brain peptide enzymolysis extract. The preparation process comprises the steps of raw material pretreatment and grinding, microwave treatment and centrifugal separation, protein precipitation and enzymolysis, microwave-assisted enzymolysis and purification and nanofiltration membrane filtration. Pepsase is added in the microwave treatment, and the temperature is not more than 50 DEG C in the enzymolysis process, so that the small molecule peptide keeps relatively high activity; furthermore, carbon dioxide gas is introduced in the microwave treatment for aeration, and the aeration pressure and the pH value are optimized, so that the activity and the yield of the brain peptide enzymolysis extract are further improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of active peptides. In particular, the present invention relates to an active peptide for improving memory, a brain peptide enzymatic hydrolysis extract, and a preparation method and application thereof. Background Art

[0002] Active peptides are small molecular compounds formed by amino acids connected through peptide bonds, and have the characteristics of small molecular weight, easy absorption, strong biological activity, etc. In recent years, the application of brain peptides in the field of brain rehabilitation has attracted much attention, mainly reflected in the following aspects:

[0003] Neuroprotective effect: Active peptides can cross the blood-brain barrier and directly act on nerve cells, inhibit neuroinflammation, reduce oxidative stress, and thus protect neurons from damage.

[0004] Promote nerve regeneration: Certain active peptides can stimulate the proliferation and differentiation of neural stem cells, and promote the repair and regeneration of damaged nerve tissues.

[0005] Improve cognitive function: Active peptides improve memory, learning and cognitive functions by regulating the release and metabolism of neurotransmitters, and have potential therapeutic effects on neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0006] Anti-fatigue and anti-stress: Active peptides can regulate the energy metabolism in the brain, relieve brain fatigue, and enhance the anti-stress ability of the body.

[0007] Due to the multiple effects of brain peptides in brain rehabilitation, their preparation and application have become research hotspots. However, the extraction and preparation process of brain peptides has an important impact on their activity and efficacy, especially the temperature control during the enzymatic hydrolysis process is crucial.

[0008] Brain peptide enzymatic hydrolysis extract is a polypeptide mixture extracted from animal brain tissues (such as pig brain) through enzymatic hydrolysis technology, and brain peptide is an important component in the brain peptide enzymatic hydrolysis extract.

[0009] Temperature is a key parameter in the enzymatic hydrolysis process. Too high enzymatic hydrolysis temperature may cause the enzyme protein to denature, resulting in a significant decrease in enzyme activity; the polypeptide chain breaks or amino acid residues are oxidized, destroying the active structure of the polypeptide; side reactions increase.

[0010] Too high or too low enzymatic hydrolysis temperature will affect the enzyme activity and the yield of polypeptides, and thus affect the quality and efficacy of the final product. Therefore, developing a low-temperature and high-efficiency enzymatic hydrolysis technology to prepare small molecule peptides, and selecting small molecule brain peptides with high activity has become a new research trend. Summary of the Invention

[0011] To solve the problem that the temperature is too high during the preparation of existing small molecule brain peptides, resulting in low activity and yield of the obtained small molecule peptides, the present application provides an active peptide, a brain peptide enzymatic hydrolysate, and its preparation method and application.

[0012] In the first aspect, the present application provides an active peptide:

[0013] An active peptide, whose amino acid sequence is SEQ ID NO.1: Ala-Ala-Val-Pro.

[0014] In the second aspect, the present application provides a nucleic acid molecule:

[0015] A nucleic acid molecule that encodes the amino acid sequence Ala-Ala-Val-Pro, or is reverse complementary to the nucleotide sequence encoding the active peptide with the amino acid sequence Ala-Ala-Val-Pro.

[0016] In the third aspect, the present application provides a recombinant vector:

[0017] A recombinant vector containing the nucleic acid molecule of the present application and capable of expressing the active peptide of the present application.

[0018] In the fourth aspect, the present application provides a host cell:

[0019] A host cell, whose genome incorporates the nucleic acid molecule of the present application and is capable of expressing the active peptide of the present application.

[0020] In the fifth aspect, the present application provides a drug for improving sleep and treating memory-related diseases:

[0021] A drug for improving sleep and treating memory-related diseases, comprising the active peptide of the present application and pharmaceutically acceptable excipients, and the excipients include one or more of diluents, preservatives, buffers, disintegrants, antioxidants, suspending agents, and coloring agents.

[0022] In the sixth aspect, the present application provides a preparation method of a brain peptide enzymatic hydrolysate:

[0023] A preparation method of a brain peptide enzymatic hydrolysate containing the active peptide of the present application, and its preparation method includes the following preparation process:

[0024] 1) Raw material pretreatment and grinding: Grind the animal brain into a slurry to obtain a homogenate;

[0025] 2) Microwave treatment and centrifugal separation: After microwave-treating the homogenate, centrifuge to remove lipids and phospholipids. The temperature in the microwave treatment is 30 - 55 °C, and the treatment time is 15 - 35 minutes;

[0026] 3) Protein precipitation and enzymatic hydrolysis, and trypsin is used in the enzymatic hydrolysis;

[0027] 4) Microwave-assisted enzymatic hydrolysis and purification. In the purification, chitosan solution after acidification is used to remove macromolecular impurities, and then microfiltration membrane filtration is carried out.

[0028] 5) Nanofiltration membrane filtration.

[0029] Furthermore, in the microwave treatment, the temperature is 30 - 50 °C, and pepsin accounting for 5 - 7 wt% of the mass of the homogenate is added, and the pH value is adjusted to 1.5 - 3.5.

[0030] Furthermore, in the microwave treatment, carbon dioxide gas is introduced for aeration, the pressure is 0.6 - 2 MPa, and the pH value is adjusted to 2 - 2.5.

[0031] In the seventh aspect, the present application provides a brain peptide enzymatic hydrolysis extract, which is obtained by using the preparation method of the brain peptide enzymatic hydrolysis extract described in the present application and contains the active peptide described in the present application.

[0032] In the eighth aspect, the present application provides an application in a drug:

[0033] The active peptide described in the present application, or the nucleic acid molecule described in the present application, or the recombinant vector described in the present application, or the host cell described in the present application, or the brain peptide enzymatic hydrolysis extract described in the present application is used in the preparation of a drug for treating related diseases such as enhancing memory and promoting sleep.

[0034] Beneficial effects: 1. The present application preferably selects an active peptide, and its amino acid sequence is as SEQ ID NO.1: Ala - Ala - Val - Pro. This active peptide has high activity and good therapeutic effects in improving sleep and enhancing memory.

[0035] 2. The present application preferably selects a preparation method of a brain peptide enzymatic hydrolysis extract. Its preparation process includes raw material pretreatment and grinding, microwave treatment and centrifugal separation, protein precipitation and enzymatic hydrolysis, microwave-assisted enzymatic hydrolysis and purification, and nanofiltration membrane filtration; and pepsin is added in the microwave treatment, and the temperature during the enzymatic hydrolysis process is not higher than 50 °C, so that the obtained brain peptide enzymatic hydrolysis extract and the separated small molecule peptides have high activity and yield.

[0036] 3. Furthermore, carbon dioxide gas is introduced for aeration in the microwave treatment, and the pressure and pH value are preferably selected, further improving the activity and yield of the brain peptide enzymatic hydrolysis extract and small molecule peptides. Description of the Drawings

[0037] Figure 1 : Mass spectrometry diagram of the small molecule brain peptide enzymatic hydrolysate obtained by the preparation method of Example 1 of the present application. Detailed Embodiments

[0038] The present invention will be further described in detail below in conjunction with specific embodiments, but the implementation manners of the present invention are not limited to the scope shown in the embodiments. These embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, after reading the content of the present invention, those skilled in the art can make various modifications to the present invention, and these equivalent changes also fall within the scope defined by the appended claims of the present invention.

[0039] Related terms

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0041] "Small molecule peptide" is a biochemical substance between amino acids and proteins. It has a smaller molecular weight than proteins and a larger molecular weight than amino acids. Small molecule peptides have a simple structure and a small molecular weight, can be quickly absorbed through the small intestinal mucosa without further digestion and without consuming energy, and have the characteristic of 100% absorption. Small molecule peptides can directly enter cells through the skin barrier, blood-brain barrier, placental barrier, and gastrointestinal mucosal barrier. Therefore, the absorption, transformation, and utilization of small molecule peptides are efficient and complete.

[0042] "Active peptide" specifically refers to peptide molecules with specific biological activities, and its function is determined by its amino acid sequence and structure. The active peptides involved in the present invention have a relatively small molecular weight and belong to small molecule peptides.

[0043] "Vector" refers to a nucleic acid vehicle into which polynucleotides can be inserted. When the vector can enable the protein encoded by the inserted polynucleotide to be expressed, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic element carried by it can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as lambda phages or M13 phages, and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40).

[0044] "Host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as Drosophila S2 cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0045] The small molecule peptides screened and constructed in the present invention can improve sleep and enhance memory.

[0046] The nucleic acid molecule of the embodiment of the present invention encodes the small molecule peptide as described above, i.e., the coding strand, or is reverse complementary to the nucleotide sequence encoding the small molecule peptide as described above, i.e., the antisense strand.

[0047] It can be understood that due to the degeneracy of codons, nucleic acid sequences capable of expressing the same small molecule peptide have multiple forms. Those skilled in the relevant art can determine the nucleic acid sequence through the codon table and further optimize the codons to improve the expression efficiency, etc.

[0048] The recombinant vector of the embodiment of the present invention contains the nucleotide sequence of the nucleic acid molecule as described above.

[0049] Optionally, the recombinant vector is constructed based on a prokaryotic cell expression vector or a eukaryotic cell expression vector. Prokaryotic cell expression vectors such as pBAD vector, pCAl-n / pCAl-pelB vector, pPOW3.0 expression vector, etc., and eukaryotic cell expression vectors such as pCMVp-NEO-BAN vector, pEGFP expression vector, pEGFP-Actin expression vector, pSV2 expression vector, CMV4 expression vector, etc. However, the type of the vector is not limited to this and can be selected according to specific needs.

[0050] It can be understood that the vector may also contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, etc. and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0051] The host cell of the embodiment of the present invention has the nucleic acid molecule as described above incorporated into its genome.

[0052] It can be understood that the small molecule peptide, nucleic acid molecule, recombinant vector or host cell as described above can all be applied to the preparation of products for treating diseases related to improving sleep and enhancing memory.

[0053] The preparation method of the small molecule peptide of the embodiment of the present invention can be to artificially synthesize the small molecule peptide or to obtain the small molecule peptide by gene expression using the above host cell.

[0054] The medicament for improving sleep and treating memory-related diseases according to the embodiments of the present invention comprises the above-mentioned small molecule peptides and pharmaceutically acceptable excipients.

[0055] In a specific example, the excipients include one or more of diluents, preservatives, buffers, disintegrants, antioxidants, suspending agents, colorants, and excipients.

[0056] In a specific example, the diluent is selected from one or more of polyethylene glycol, propylene glycol, vegetable oil, and mineral oil. In a specific example, the preservative is selected from one or more of sorbic acid, methyl sorbate, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, benzyl p-hydroxybenzoate, sodium methyl p-hydroxybenzoate, benzoic acid, and benzyl alcohol. In a specific example, the buffer is selected from one or more of sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium citrate, sodium tartrate, and sodium acetate. In a specific example, the disintegrant is selected from one or more of cross-linked carboxymethylcellulose sodium, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, or low-substituted hydroxypropylcellulose. In a specific example, the antioxidant is selected from one or more of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, dibutylhydroxytoluene, glycine, inositol, ascorbic acid, sodium ascorbate, lecithin, malic acid, hydroquinone, citric acid, succinic acid, and sodium metabisulfite. In a specific example, the suspending agent is selected from one or more of beeswax, ethyl hydroxyethyl cellulose, chitin, chitosan, methyl cellulose, carboxymethyl cellulose, agar, hydroxypropyl methyl cellulose, and xanthan gum. In a specific example, the colorant is selected from one or more of carbon black, iron black, iron brown, iron red, and titanium dioxide. In a specific example, the excipient is selected from one or more of mannitol, glucose, lactose, dextran, dextrorotatory dextran, and sodium chloride.

[0057] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial sources.

[0058] Example 1, a preparation method of a brain peptide hydrolysate, comprising the following steps:

[0059] 1) Pretreatment and grinding of raw materials:

[0060] Take out fresh pig brains or thawed frozen pig brains, wash them with pure water, and remove fascia and blood vessels; after mixing the pig brains with pure water at a ratio of 1:1.5, first coarsely grind (100 mesh), then finely grind (150 mesh), cycle for 5 minutes per batch, then add 1.3 times the amount of pure water and continue to homogenize for 5 minutes, mix evenly, and then adjust the pH value to 9.5 with potassium hydroxide solution to obtain a homogenate.

[0061] 2) Microwave treatment and centrifugal separation:

[0062] The homogenate is placed in a microwave tank, and pepsin (5000 u / g, 6 wt% based on the mass of the homogenate) is added. The pH value is adjusted to 2.5 using a 1% acetic acid (CH3COOH) solution. Under the condition of a temperature of 40 °C, microwave treatment (microwave frequency 3300 MHz, power 25 KW) is carried out and carbon dioxide is introduced (pressure 1 MPa) for aeration treatment for 20 minutes, and then incubated for 15 minutes.

[0063] A carbon dioxide pressure of 1 MPa means that the carbon dioxide gas transported into the microwave tank is 1 MPa; in other embodiments, the treatment time can be any time within 10 - 30 minutes, and in other embodiments, the incubation time can be any time within 10 - 30 minutes.

[0064] Then the homogenate is separated into protein and lipid at 38 °C using a disc high-speed centrifuge:

[0065] First centrifugation: The rotation speed is 9000 r / min, and 42 wt% of the light liquid (containing lipid) is discharged.

[0066] Second centrifugation: The rotation speed is 12000 r / min, the temperature is not lower than 38 °C, and the light liquid (phospholipid solution) is discharged; the concentrated liquids from the two centrifugations are mixed to obtain a mixed concentrated liquid.

[0067] 3) Protein precipitation and enzymatic hydrolysis:

[0068] S1 Protein precipitation: The mixed concentrated liquid obtained in step 1) is adjusted to a pH value of 5.0 with dilute hydrochloric acid to precipitate and layer the protein, and the liquid is removed using a 250-mesh filter cloth centrifuge, and the filter residue (brain separated protein) is collected.

[0069] S2 Enzymatic hydrolysis: The filter residue is dissolved in 2 times of pure water to obtain a 5 wt% solution, and the pH value is adjusted to 9.0; then trypsin (5000 u / g, 4.5 wt% based on the mass of the separated protein) is added, and homogenization and circulation are carried out; when the pH value drops to 7.5, alkali is added to adjust it to 8.6, and the circulation continues for 3 minutes.

[0070] 4) Microwave-assisted enzymatic hydrolysis and purification:

[0071] S1 Microwave-assisted enzymatic hydrolysis: Detect the pH value of the slurry obtained in step 3), if it is less than 7.0, add alkali to adjust it to 8.2; carry out microwave-assisted enzymatic hydrolysis, incubate for 15 min, and inactivate the enzyme.

[0072] Precipitation of S2 impurities: Drain the inactivated enzyme solution. When the temperature of the enzyme solution drops below 40 °C, add a mixed solution formed by dissolving 1.5% (w / v) chitosan in 15% (w / v) acetic acid solution under stirring conditions, adjust the pH value to 4.8 to precipitate the unhydrolyzed brain proteins, phospholipids and other macromolecular impurities, and then filter by centrifugation with a 200-mesh filter cloth to remove the precipitate.

[0073] Purification of S3 polypeptide: Filter the centrifugate with a 0.25-μm hollow fiber membrane, and filter the filtrate with an ultrafiltration membrane that can intercept a molecular weight of 10,000 Da. The permeate is the porcine brain polypeptide solution. If only polypeptides are prepared, the solution is concentrated under reduced pressure and freeze-dried to obtain polypeptide powder.

[0074] 5) Preparation of small molecule peptides:

[0075] Filter the porcine brain polypeptide solution with a nanofiltration membrane (intercepting molecular weight 1200 Da), concentrate the filtrate by reverse osmosis membrane, freeze the concentrated solution and dry it to obtain a small molecule brain peptide hydrolysate. The mass spectrum is as shown in Figure 1 ; Figure 1 peaks at m / z 115.0866, 116.0607, 215.39 appear, and the amino acid fragment Ala-Ala-Val-Pro exists in the hydrolysate.

[0076] Example 2, A preparation method of a brain peptide hydrolysate extract, which is different from Example 1 in that in step 2) of microwave treatment and centrifugal separation, carbon dioxide gas (pressure 2.5 MPa) is introduced for aeration, and the pH value is adjusted to 3.5.

[0077] Example 3, A preparation method of a brain peptide hydrolysate extract, which is different from Example 1 in that in step 2) of microwave treatment and centrifugal separation, carbon dioxide gas (pressure 0.4 MPa) is introduced for aeration, and the pH value is adjusted to 2.5.

[0078] Example 4, A preparation method of a brain peptide hydrolysate extract, which is different from Example 1 in that in step 2) of microwave treatment and centrifugal separation, no carbon dioxide gas is introduced for aeration.

[0079] Example 5, A preparation method of a brain peptide hydrolysate extract, which is different from Example 1 in that in step 2) of microwave treatment and centrifugal separation, no carbon dioxide gas is introduced for aeration and pepsin is not used; the specific operation steps are to place the homogenate in a microwave tank, perform microwave treatment at 60 °C (microwave frequency 3300 MHz, power 25 KW) for 20 minutes, and keep warm for 15 minutes.

[0080] LC-MS / MS separation and identification

[0081] The amino acid sequence and molecular weight of small molecule peptide components were determined by LC-MS / MS. Before the sample was loaded onto the machine, reduction alkylation and desalting treatments were first carried out.

[0082] The pre-column of the capillary liquid chromatography column in LC-MS / MS was an Acclaim PepMapRPLC C18 analytical column (5 μm) with a size of 300 μm × 5 mm; the analytical column was an Acclaim PepMap RPLC C18 analytical column (1.9 μm) with a size of 150 μm × 150 mm; mobile phase A was a 0.1% (v / v) formic acid solution and a 2% (v / v) acetonitrile solution, and mobile phase B was a 0.1% (v / v) formic acid solution and an 80% (v / v) acetonitrile solution. The flow rate was 600 nL / min, and the analysis time for each component was 60 min.

[0083] The MS and MS / MS parameters were as follows:

[0084] (1) MS parameters: The resolution was 70000; the maximum injection time was 40 ms; the scanning range was 300 - 1400 m / z.

[0085] (2) MS / MS parameters: The resolution was 175000; the maximum injection time was 60 ms; the scanning range was 300 - 1400 m / z;

[0086] Top N = 20; NCE / steeped NCE = 27.

[0087] The original MS / MS file was analyzed by Mascot software according to the sample type and searched against the Uniprot database Sus scrofa (Pig) to determine the amino acid sequence of the peptide.

[0088] After the R4 component was retrieved by mass spectrometry data, PSM FDR ≤ 0.01 and Protein FDR ≤ 0.01 were used as the screening criteria for peptide segment, site and protein identification respectively, and 110 peptide segments were obtained, and the peptide content was tested by LC-MS / MS.

[0089] Peptide segment screening, synthesis and verification

[0090] 1) Prediction of polypeptide activity

[0091] Peptide segments were screened according to the peptide segment matching score (Score), peptide segment matching error probability (PEP) and peptide segment peak intensity value (Intensity), and peptide segments with zero scores and intensities and too high matching error rates were screened out. The domestic PeptideRanker was used to evaluate the possibility of peptide segments having biological activity and to rank them. Among the 115 peptides, 51 peptide segments had a score exceeding 0.5, and 10 peptide segments had a score greater than 0.9.

[0092] The bioactivities of 70 short peptides with a PeptideRanker score greater than 0.5 were evaluated by searching the BIOPEP database, and the toxicity and PI values of 51 short peptides were evaluated by Toxin Pred (toxicity prediction software). Most short peptides potentially have or possess antihypertensive (ACE inhibition, renin inhibition, and vasoactive substance release activities), hypoglycemic (DPP-IV inhibition, glucose absorption stimulation activity, and α-glucosidase inhibition activity), pain control (DPP-Ⅲ inhibition activity), and AChE inhibition activities, with antioxidant activity being in a small part. The evaluation found that none of the 70 short peptides were toxic, and the PI values mostly ranged from 5 to 7, with a few ranging from 8 to 10. 18 polypeptides were screened through the above conditions for molecular docking.

[0093] 2) Molecular docking

[0094] Ligand processing: The molecular structural formula of the peptide segment was drawn using Chem3D 19.0 software, optimized by the MM2 force field, and saved as a pdb file. Using AutoDock software (molecular docking software), the pdb file was imported and hydrogenated, and saved as a pdbqt ligand file.

[0095] Receptor processing: The crystal structures of Fyn (2DQ7) and Keap 1 (6SP1) were downloaded from the PDB database. The redundant chloride ions and water molecules were removed using PyMOL software and saved as a pdb file. The receptors 2DQ7 and 6SP1 were dehydrated and hydrogenated using AutoDock Tools software and saved as pdbqt receptor files for standby.

[0096] Molecular docking: The vina function of AutoDock Tools software was used for molecular docking to simulate the interaction between small peptide molecules and large molecule proteins. The docking center coordinates of 2DQ7 were set to (-3.031, -9.266, 39.033) (x, y, z) in sequence, and the box size was 50×51×58. The docking center coordinates of 6SP1 were set to (-26.315, 20.346, -16.173) (x, y, z), and the box size was 51×51×49. Other parameters were taken as default values. Then, the amino acids and polypeptide ligands were docked with the 2DQ7 crystal structure one by one.

[0097] Results: The above-screened 21 peptide segments were respectively subjected to Vina simulation docking with 2DQ7 and 6SP1. According to the Vina scores, 5 small molecule peptides were screened, and the specific properties are shown in Table 1.

[0098] Table 1 Verification results of the relevant properties of small molecule peptides

[0099]

[0100] As can be seen from the data in Table 1, in the preparation method of the brain peptide enzymatic hydrolysate, a lower temperature is adopted in the microwave treatment step, and the combination of microwave treatment and carbon dioxide gas aeration results in a higher content and higher activity in the prepared brain peptide enzymatic hydrolysate; the possible reasons for achieving beneficial effects are as follows: at a lower temperature, the slurry or enzymatic hydrolysate better maintains the activity of peptide segments; microwave treatment and carbon dioxide gas aeration improve the pepsin enzymatic hydrolysis efficiency; further carbon dioxide gas aeration reduces the oxygen content in the slurry, which is beneficial for the peptide segments in the slurry to maintain activity in subsequent processing, thereby enhancing the activity and yield of the brain peptide enzymatic hydrolysate.

[0101] Optimizing the carbon dioxide gas aeration pressure and pH value further improves the enzymatic hydrolysis efficiency.

[0102] 3) Synthesis of small molecule peptides and detection of the effects of improving sleep and enhancing memory:

[0103] Synthesis: Five small molecule peptides, namely WVFYF, PRVAP, VAF, APF, and AAVP, were all synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0104] Animal experiments:

[0105] Experiment 1: Experiment on promoting sleep effect:

[0106] Experimental animals: Kunming mice, half male and half female, with a body weight of 18 - 22 g, randomly divided into 14 groups, with 7 mice in each group. The production license number is: SCXK(Zhe)2022 - 0005. The model control group was gavaged with the same volume of normal saline; the example group was gavaged with 0.6 g of small molecule peptide / kg. Continuous gavage administration was carried out for 10 days, once a day.

[0107] On the 10th day after gavage administration, continue normal feeding for 1 day, and gavage administration once. 75 minutes after the last administration, intraperitoneal injection of pentobarbital sodium at 40 mg / kg (for the example group and the control group). Record the sleep onset time and wake-up time of the mice.

[0108] Sleep onset time: Taking the disappearance of the righting reflex of the mouse as the sleep onset sign, it is the time from the injection of pentobarbital sodium to the appearance of the sleep onset sign;

[0109] Wake-up time: Taking the recovery of the righting reflex of the mouse as the wake-up sign, it is the time from sleep onset to wake-up; Sleep time = Wake-up time - Sleep onset time, and the experimental results are shown in Table 2.

[0110] Experiment 2: Experiment on brain strengthening and intelligence enhancing effect:

[0111] Experimental animals: SPF-grade SD rats, 8 weeks old, half male and half female, adaptively fed for 1 week.

[0112] Grouping of experimental animals: The rats were randomly divided into 9 groups, with 7 rats in each group. The model control group was intragastrically administered the same volume of normal saline; the example group was intragastrically administered the corresponding small molecule peptide at a dose of 0.6 g / kg. Continuous intragastric administration was carried out for 14 days, once a day.

[0113] Experimental method: Morris water maze method. The diameter of the Morris water maze was 1.65 m, the inner wall and bottom were all black, and the water maze area was divided into four quadrants. A black platform with a diameter of 12 cm was placed 2 cm below the water surface in the center of the second quadrant. After each water change in the water maze experiment, a certain amount of black dye was added to make the water black and opaque, and the water temperature was maintained at 20 ± 2 °C during the experiment. During the 14th day of drug administration, the place navigation experiment was carried out. During the place navigation experiment, each rat was trained 4 times a day, with an interval of 10 min. During training, a random entry point was selected, and the rat was placed into the water facing the pool wall. The 4 trainings were carried out from 4 different entry points. The time required for each rat to find the underwater platform starting from 4 different points, that is, the latency to escape to the platform (escaping latency), was recorded. The animal was allowed to find the platform within 60 s and stay on the platform for 15 s after finding it. The average value of the latencies at the 4 entry points on the 15th day of drug administration was used as the result of directional swimming; the latency results of the water maze test for each group of rats are shown in Table 2.

[0114] During the water maze test, the reference objects such as the lights around the water maze remained unchanged to avoid environmental interference.

[0115] Table 2. List of the results of the sleep-promoting effect experiment and the brain-strengthening and intelligence-enhancing effect experiment for the rats in the example group and the control group

[0116]

[0117] Compared with the model control group for each group: # P ≤ 0.05, ## P ≤ 0.01.

[0118] Without departing from the scope of the present invention, the present invention can well obtain further advantages and improvements. Although the present invention has been shown and described in a manner that is considered to be the most practical and preferred embodiment, it should be recognized that departures from the present invention can be made within the scope of the present invention. The present invention is not limited to the details disclosed herein, but should be given the full scope of the claims to include any and all equivalent devices and equipment. Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is well known, nor should it constitute a part of the common general knowledge in the art.

Claims

1. A bioactive peptide, characterized in that, Its amino acid sequence is as shown in SEQ ID NO.1: Ala-Ala-Val-Pro.

2. A nucleic acid molecule, characterized in that, It encodes the active peptide described in claim 1, or is reverse complementary to the nucleotide sequence encoding the active peptide described in claim 1.

3. A recombinant vector, characterized in that, It contains the nucleic acid molecule described in claim 2 and is capable of expressing the active peptide described in claim 1.

4. A host cell, characterized in that, Its genome is doped with the nucleic acid molecule described in claim 2 and is capable of expressing the active peptide described in claim 1.

5. A drug for improving sleep and treating diseases related to memory improvement, characterized in that, It includes the active peptide described in claim 1, and a pharmaceutically acceptable excipient, which includes one or more of a diluent, a preservative, a buffer, a disintegrant, an antioxidant, a suspending agent, and a coloring agent.

6. A preparation method of a brain peptide enzymatic hydrolysate extract, characterized in that, Its preparation method includes the following preparation process: 1) Raw material pretreatment and grinding: Grind the animal brain into a slurry to obtain a homogenate; 2) Microwave treatment and centrifugal separation: After microwave treatment of the homogenate, centrifuge to remove lipids and phospholipids. In the microwave treatment, the temperature is 30-60 °C and the treatment time is 15-35 minutes; 3) Protein precipitation and enzymatic hydrolysis: Trypsin is used in the enzymatic hydrolysis; 4) Microwave-assisted enzymatic hydrolysis and purification. In the purification, an acidified chitosan solution is used to remove macromolecular impurities, and then microfiltration membrane filtration is used; 5) Nanofiltration membrane filtration.

7. The preparation method of a brain peptide enzymatic hydrolysate extract according to claim 6, characterized in that, In the microwave treatment, the temperature is 30-50 °C, and 5-7 wt% of pepsin based on the mass of the homogenate is added, and the pH value is adjusted to 1.5-3.

5.

8. A method for preparing a brain peptide enzymatic hydrolysate extract according to claim 7, characterized in that, In the microwave treatment, carbon dioxide gas is introduced for aeration, the pressure is 0.6-2 MPa, and the pH value is adjusted to 2-2.

5.

9. The brain peptide enzymatic hydrolysis extract obtained by the preparation method according to any one of claims 6-8 contains the active peptide described in claim 1.

10. The use of the active peptide described in claim 1, or the nucleic acid molecule described in claim 2, or the recombinant vector described in claim 3, or the host cell described in claim 4, or the brain peptide enzymatic hydrolysis extract described in claim 9 in the preparation of a drug for improving sleep and treating memory-related diseases.

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