Casein anti-anxiety peptide and preparation method thereof

Abstract

The invention belongs to the technical field of anti-anxiety peptides, and particularly relates to a casein anti-anxiety peptide, a preparation method and an identification method, and the anti-anxiety peptide sequence is FFVAPFPE, VYPFPGPIPN, HLPLPL, FLPYPY and WQVL. The anti-anxiety peptide is prepared from casein through enzymolysis, and components with the molecular weight smaller than 3 kDa are obtained through an ultrafiltration tube. The anti-anxiety peptide screened by the invention has strong anti-anxiety activity, has the characteristics of high efficiency and simple preparation method, improves the utilization value of casein in food industry, and provides a theoretical basis for developing anti-anxiety functional food.

Description

Technical Field

[0001] The present invention relates to the technical field of antianxiety peptides, and specifically relates to antianxiety peptides and a preparation method and an identification method. Background Art

[0002] Anxiety disorder is a common mental disorder. According to statistics from the World Health Organization (WHO), it is listed as the ninth leading cause of health-related disability in the world. At present, studies have shown that food-derived functional peptides are closely related to brain health and are considered to be effective health products for relieving anxiety. The preparation and utilization of the above casein anti-anxiety peptides will contribute to the development of natural health functional products.

[0003] Casein is considered a promising precursor protein because it is rich in Pro and Trp and can produce peptides with anti-anxiety activity. In recent years, studies have shown that hydrolysates and peptides obtained from casein have anti-anxiety activity. Traditional methods require a long enzymatic hydrolysis time, a low degree of hydrolysis, and a limited number of repeat sequences in the protein. With the rapid development of bioinformatics, computer-assisted screening methods have been applied to the prediction and design of bioactive peptides. In addition, molecular docking is widely used in the prediction of interactions between peptide ligands and enzyme receptors. Compared with traditional methods, the combination of ultrasound and enzymatic methods and computer-assisted methods are more efficient and economically feasible in the preparation and evaluation of new bioactive peptides. Summary of the invention

[0004] The present invention provides a casein active peptide having antianxiety activity

[0005] The present invention further provides a method for preparing, separating and screening the casein peptide having antianxiety activity.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0007] The present invention provides a casein peptide with anti-anxiety activity, wherein the casein anti-anxiety peptide is selected from SEQ ID NO.1-SEQ ID NO.5; the sequence of the anti-anxiety peptide is specifically: FFVAPFPE (SEQ ID NO.1), VYPFPGPIPN (SEQ ID NO.2), HLPLPL (SEQ ID NO.3), FLPYPY (SEQ ID NO.4), and WQVL (SEQ ID NO.5).

[0008] Most preferably, the casein anxiolytic peptide is selected from FFVAPFPE, VYPFPGPIPN, HLPLPL, FLPYPY, WQVL.

[0009] The present invention also provides the preparation and separation screening methods of the above-mentioned casein anti-anxiety peptide: multi-enzyme screening, selecting the best enzyme, enzymatically extracting the polypeptide from bovine milk casein, and obtaining the component with molecular weight less than 3kDa by ultrafiltration.

[0010] The specific steps are as follows:

[0011] (1) Trypsin, alkaline protease, or chymotrypsin were used to hydrolyze casein for 6 h, and samples were taken at regular intervals to inactivate the enzymes. After inactivation, the samples were cooled to room temperature, and the supernatant was collected after centrifugation and stored at -20°C for further analysis;

[0012] (2) diluting the sample prepared in step (1) by 300 times and determining the degree of hydrolysis;

[0013] (3) diluting the sample prepared in step (1) 10-fold and measuring the GABA-T inhibitory activity;

[0014] (4) separating the supernatant obtained in step (1) by membrane separation technology, retaining component F3 with a molecular weight of less than 3 kDa, and freeze-drying casein hydrolysates of different molecular weights to obtain anti-anxiety peptides;

[0015] (5) separating and purifying the hydrolyzate with the best inhibitory activity to obtain component F3, and performing mass spectrometry identification on F3;

[0016] (6) F3 was subjected to mass spectrometry identification, and the peptide sequence was identified through computer analysis and molecular docking simulation to screen out casein peptides with potential anxiolytic activity;

[0017] (7) Anti-anxiety peptides were synthesized by solid phase HPLC and prepared into different concentrations to determine IC 50 value;

[0018] (8) The optimal hydrolase for steps (2) and (3) is alkaline protease;

[0019] (9) The experimental conditions of step (3) are as follows: the GABA-T detection method is that the mice are killed by breaking the spine, and the brain is immediately taken out and stored in liquid nitrogen until the test is measured; the brain tissue is stored in 20% glycerol, 0.10mmol / L glutathione, 0.10mmol / L PLP, 1mmol / L EDTA disodium salt, 0.13% Triton-X100, 0.01mmol / L K 2 HPO 4, pH 6.8, and ground into brain homogenate, centrifuged at 4°C 10000g for 20 min, and collected the supernatant. 180 μL of incubation medium (100 mmol / L potassium pyrophosphate, 5 mmol / L α-ketoglutaric acid, 4 mmol / L nicotinamide adenine dinucleotide (NAD), 3.50 mmol / L 2-mercaptoethanol, 0.01 mmol / L pyridoxal 5-phosphate (PLP), pH 8.6), 20 μL of tissue homogenate and 10 μL of sample solution were added to each well of a 96-well plate; the plate was pre-incubated at 37°C for 15 min, and then 50 mL of 115 mmol / L GABA was added to start the reaction, and the production of NADH within 15 min at 37°C 340 nm was measured with an enzyme reader; all experiments were repeated three times; calculated according to the following formula:

[0020]

[0021] T is the test (incubation solution, GABA, homogenate, and sample), T0 is the test blank (water, homogenate, and sample), A is the control (incubation solution, homogenate, water, GABA), and A0 is the control blank (water, brain homogenate);

[0022] (10) The ultrafiltration conditions of step (4) are as follows: the hydrolyzate is placed in an ultrafiltration centrifuge tube and centrifuged at 5400 g for 20 min to obtain three components with different molecular weights, >10 KDa, 3-10 KDa, and <3 KDa. Each component is stored at -20°C and the GABA-T inhibitory activity of each component is measured. The measurement results are shown in Figure 5 ;

[0023] (11) The mass spectrometry conditions of step (6) are as follows: Chromatographic conditions: Liquid chromatography 0.15 mm*150 mm, RP-C18, Column Technology Inc.) was balanced with 95% A solution (0.1% formic acid aqueous solution), and the sample was loaded into Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) by an automatic sampler, and then separated by a liquid chromatography column, and the liquid phase gradient was set as follows: 0-50 min, 4%-50% B solution (0.1% formic acid acetonitrile aqueous solution); 50-54 min, 50%-100% B solution; 54-60 min, 100% B solution. Mass spectrometry conditions: Mass spectrometry analysis was performed using a Q Exactive mass spectrometer (ThermoFisher), analysis time: 60 min; detection mode: positive ion; primary ion mass tolerance: 20 ppm; primary ion mass tolerance: 0.1 Da;

[0024] (12) The computer screening steps of step (6) are: Peptide Ranker (http: / / distilldeep.ucd.ie / PeptideRanker / ) predicts the biological activity of peptides, AllergenFp (https: / / ddg-pharmfac.net / AllergenFP / ) predicts the sensitization, and admetSAR3.0 predicts the absorption of peptides;

[0025] (13) The molecular docking step of step (6) is as follows: using aminohexetine as a positive control, the crystal structure of GABA-T (PDB ID: 1OHW) was obtained from the PDB database (http: / / www.rcsb.org / ). The molecular docking was performed by MOE to verify the results of the model prediction, and the intermolecular interactions were visualized by pymol. The results are shown in Figure 6 ;

[0026] (14) The casein peptides with potential anxiolytic activity screened out in step (6) are: FFVAPFPR, VYPFPGPIPN, HLPLPL, FLPYPY, WQVL;

[0027] (15) IC of FFVAPFPR, VYPFPGPIPN, HLPLPL, FLPYPY, WQVL of step (7) 50 The values ​​were 3.20mM, 4.43mM, 8.49mM, 2.79mM and 7.20mM respectively. Figure 7

[0028] Compared with the prior art, the present invention is beneficial in that:

[0029] The present invention utilizes enzymatic hydrolysis, ultrafiltration and LC-MS / MS technology to efficiently screen and identify novel anti-anxiety peptides from casein; computer analysis is used to screen bioactive peptides, and through in vitro activity verification, Tongno molecular docking reveals the action site and mechanism of the target anti-anxiety peptide, providing a rapid and efficient anti-anxiety peptide identification scheme.

[0030] The anti-anxiety peptides screened out by the present invention have strong anti-anxiety activity and are highly efficient and have a preparation method, which helps to improve the utilization value of casein in the food industry and provides some theoretical thinking for the development of functional natural anti-anxiety products. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a process flow chart of the present invention;

[0032] Figure 2 It is a graph of multi-enzyme hydrolysis degree determination;

[0033] Figure 3 This is a graph of GABA-T inhibition assay of multi-enzyme casein hydrolysate;

[0034] Figure 4 It is the ultrafiltration diagram;

[0035] Figure 5 It is the LC-MS / MS total ion current and secondary mass spectra of some peptides with MW < 3 kDa;

[0036] Figure 6 It is the docking diagram of peptides FFVAPFPE, VYPFPGPIPN, HLPLPL, FLPYPY, WQVL, positive drugs and GABA-T molecules;

[0037] Figure 7 It is the peptide FFVAPFPE, VYPFPGPIPN, HLPLPL, FLPYPY, WQVL, positive drug and GABA-T IC 50 Value graph. DETAILED DESCRIPTION

[0038] The specific embodiments are further described below in conjunction with the accompanying drawings.

[0039] A method for preparing casein anti-anxiety peptides, characterized in that the method comprises the following steps:

[0040] Example 1

[0041] (1) The controlled variable method was used, with a casein concentration of 5% and an enzyme-substrate ratio of 4800 U / g. Trypsin was selected and casein was hydrolyzed for 6 h. 5 mL of sample was taken at regular intervals and the enzyme was inactivated in a 100°C water bath for 10 min. After inactivation, the sample was cooled to room temperature and the pH was adjusted to 4.6 with glacial acetic acid. The sample was then centrifuged at 8000 g for 10 min. After centrifugation, the clear liquid was collected and stored at -20°C for further analysis.

[0042] (2) diluting the sample prepared in step (1) by 300 times and determining the degree of hydrolysis;

[0043] (3) The sample prepared in step (1) was diluted 10 times and the GABA-T inhibitory activity was measured. Figure 3 and 4 As shown, casein was hydrolyzed with trypsin, the degree of hydrolysis was 4.7%, and the GABA-T inhibitory activity was 65%.

[0044] (4) The supernatant obtained in step (1) was separated by membrane separation technology, and the components A1, A2, and A3 with molecular weight cutoffs >10 kDa, 3-10 kDa, and <3 kDa were retained. The GABA-T inhibitory activity of the three components was measured. The inhibitory activity of A1 was 55%, the inhibitory activity of A2 was 60%, and the inhibitory activity of A3 was 66%. Because A3 had the smallest molecular weight and the highest GABA-T inhibitory activity, A3 was selected for subsequent experiments;

[0045] (5) A3 was subjected to mass spectrometry identification, and the peptide sequence was identified through computer analysis and molecular docking simulation to screen out casein peptides with potential anxiolytic activity;

[0046] (6) A3 was subjected to mass spectrometry identification, and the peptide sequence was identified through computer analysis and molecular docking simulation to screen out casein peptides with potential anxiolytic activity. The results are shown in Table 1 below.

[0047] Table 1

[0048]

[0049] From the results in Table 1, it can be seen that these five peptides all have good biological activity, and the peptides with a score of more than 0.5 are considered to be potential biologically active peptides. In addition, these five peptides are non-toxic and non-allergenic.

[0050] Example 2

[0051] (1) Using the controlled variable method, casein concentration was 5%, enzyme substrate ratio was 4800U / g, chymotrypsin was selected, casein was hydrolyzed for 6h, 5mL of sample was taken at regular intervals, enzyme was inactivated in a 100℃ water bath for 10min, cooled to room temperature after enzyme inactivation, pH was adjusted to 4.6 with glacial acetic acid, and then centrifuged at 8000g for 10min. After centrifugation, the clear liquid was collected and stored at -20℃ for further analysis.

[0052] (2) diluting the sample prepared in step (1) by 300 times and determining the degree of hydrolysis;

[0053] (3) The sample prepared in step (1) was diluted 10 times and the GABA-T inhibitory activity was measured. Figure 3 and 4 As shown, casein was hydrolyzed with chymotrypsin, the degree of hydrolysis was 11%, and the GABA-T inhibitory activity was 60%.

[0054] (4) The supernatant obtained in step (1) was separated by membrane separation technology, and the components B1, B2, and B3 with molecular weight cutoffs >10 kDa, 3-10 kDa, and <3 kDa were retained. The GABA-T inhibitory activity of the three components was measured. The inhibitory activity of B1 was 43%, the inhibitory activity of B2 was 56%, and the inhibitory activity of B3 was 62%. Because B3 had the smallest molecular weight and the highest GABA-T inhibitory activity, B3 was selected for subsequent experiments;

[0055] (5) B3 was subjected to mass spectrometry identification, and the peptide sequence was identified through computer analysis and molecular docking simulation to screen out casein peptides with potential anxiolytic activity;

[0056] (6) B3 was subjected to mass spectrometry identification, and the peptide sequence was identified through computer analysis and molecular docking simulation to screen out casein peptides with potential anxiolytic activity. The results are shown in Table 2 below.

[0057] Table 2

[0058]

[0059] From the results in Table 2, it can be seen that these five peptides all have good biological activity, and the peptides with a score of more than 0.5 are considered to be potential biologically active peptides. In addition, these five peptides are non-toxic and non-allergenic.

[0060] Example 3

[0061] (1) Using the controlled variable method, the casein concentration was 5%, the enzyme-substrate ratio was 4800U / g, and alkaline protease was selected to hydrolyze casein for 6 hours. 5mL of samples were taken at regular intervals and inactivated in a 100℃ water bath for 10 minutes. After inactivation, the samples were cooled to room temperature and the pH was adjusted to 4.6 with glacial acetic acid. The samples were then centrifuged at 8000g for 10 minutes. After centrifugation, the clear liquid was collected and stored at -20℃ for further analysis.

[0062] (2) diluting the sample prepared in step (1) by 300 times and determining the degree of hydrolysis;

[0063] (3) The sample prepared in step (1) was diluted 10 times and the GABA-T inhibitory activity was measured. Figure 3 and 4 As shown, casein was hydrolyzed with alkaline protease, the degree of hydrolysis was 17%, and the GABA-T inhibitory activity was 75%;

[0064] (4) The supernatant obtained in step (1) was separated by membrane separation technology, and the fractions F1, F2, and F3 with molecular weight cutoffs of >10 kDa, 3-10 kDa, and <3 kDa were retained. The GABA-T inhibitory activity of the three fractions was determined. The results were as follows: Figure 4As shown, the inhibitory activity of F1 was 75%, the inhibitory activity of F2 was 82%, and the inhibitory activity of F3 was 86%. Because F3 had the smallest molecular weight and the highest GABA-T inhibitory activity, F3 was selected for subsequent experiments;

[0065] (5) F3 was subjected to mass spectrometry identification, and the peptide sequence was identified by computer analysis, and molecular docking simulation was used to screen out casein peptides with potential anxiolytic activity. The results are shown in Table 3 below;

[0066] Table 3

[0067]

[0068] From the results analysis in Table 3, it can be seen that these five peptides all have high biological activity, peptides with a score of more than 0.5 are considered to be potential bioactive peptides, and peptides with a score close to 1 are considered to have very high biological activity, and these five peptides are all non-toxic and non-sensitizing.

[0069] (6) Using aminohexetine as a positive control, the crystal structure of GABA-T was obtained from the PDB database (http: / / www.rcsb.org / ) (PDB ID: 1OHW). Molecular docking was performed by MOE to verify the results of the model prediction, and the intermolecular interactions were visualized by pymol. The results are shown in Figure 6 , indicating that the above five peptides can bind tightly to GABA-T through potential specific binding sites, thereby reducing the degradation of GABA and playing a role in alleviating anxiety.

[0070] (7) The above five peptides were synthesized by HPLC solid phase, and samples of different concentrations were prepared for determination of GABA-T inhibitory activity. The results are as follows: Figure 7 As shown, FFVAPFPE, VYPFPGPIPN, HLPLPL, FLPYPY, WQVL IC 50 The values ​​are 3.20mM, 4.43mM, 8.49mM, 2.79mM, and 7.20mM respectively.

Claims

1. A casein peptide with antianxiety activity, characterized in that The amino acid sequence of the casein antianxiety peptide is: FFVAPFPE, VYPFPGPIPN,HLPLPL,FLPYPY,WQVL.

2. The method for preparing the casein anti-anxiety peptide according to claim 1, characterized in that: The method comprises the following steps: (1) selecting trypsin, alkaline protease, and chymotrypsin to hydrolyze casein for 6 hours, taking samples at regular intervals and inactivating the enzymes, cooling the samples to room temperature after inactivation, taking the supernatant after centrifugation, freezing the supernatant at -20°C, and selecting alkaline protease for subsequent experiments based on comprehensive considerations by measuring the degree of hydrolysis and GABA-T inhibitory activity; (2) separating the supernatant obtained in step (1) by using a membrane separation technique, retaining a component F3 with a molecular weight of less than 3 kDa, and freeze-drying the casein hydrolysates with different molecular weights; The antianxiety peptide was obtained; (3) F3 was identified by mass spectrometry, and the amino acid sequence of the component was FFVAPFPE, VYPFPGPIPN, HLPLPL, FLPYPY, WQVL; (4) FFVAPFPE, VYPFPGPIPN, HLPLPL, FLPYPY, WQVL were synthesized by HPLC solid phase, and the concentrations were prepared into 0.625, 1.25, 2.5, 5, and 10 mg / mL, respectively, and the GABA-T inhibitory activity was determined and the curve was drawn to calculate the IC 50 value.

3. The hydrolysis method according to claim 2, characterized in that In the above (1), the enzyme is alkaline protease.

4. The method for preparing the casein anti-anxiety peptide according to claim 2, characterized in that In the above (1), the casein concentration is 5%, the enzyme-substrate ratio is 4800 U / g, and the degree of hydrolysis is 17%.

5. The method for preparing the casein anti-anxiety peptide according to claim 2, characterized in that In the above (1), the GABA-T detection method is as follows: mice are killed by spinal fracture, and the brain is immediately taken out and stored in liquid nitrogen until the test is measured; the brain tissue is ground into a buffer solution composed of 20% glycerol, 0.10mmol / L glutathione, 0.10mmol / L PLP, 1mmol / L EDTA disodium salt, 0.13% Triton-X100, 0.01mmol / L K2HPO4, pH 6.8 to prepare a brain homogenate, and centrifuge at 4°C 10000×g for 20min to collect the supernatant; 180μL incubation medium (100mmol / L potassium pyrophosphate, 5mmol / L α-ketoglutaric acid, 4mmol / L nicotinamide adenine dinucleotide (NAD), 3.50mmol / L 2-mercaptoethanol, 0.01mmol / L pyridoxal 5-phosphate (PLP), pH 8.6), 20μL tissue homogenate and 10μL sample solution; the plate was pre-incubated at 37℃ for 15min, then 50μL 115mmol / L GABA was added to start the reaction, and the NADH production within 15min at 37℃ 340nm was measured by microplate reader; all experiments were repeated three times; calculated according to the following formula: T is the test (incubation solution, GABA, homogenate and sample), T0 is the test blank (water, homogenate and sample), A is the control (incubation solution, homogenate, water, GABA), and A0 is the control blank (water, homogenate).

6. The method for preparing the casein anti-anxiety peptide according to claim 2, characterized in that In the above (2), casein is hydrolyzed to prepare polypeptide hydrolysate, and a component with a molecular weight of less than 3 kDa is obtained by ultrafiltration.

7. The 5 anti-anxiety peptides according to claim 2, characterized in that In (4): FFVAPFPE, VYPFPGPIPN, HLPLPL, FLPYPY, WQVL, IC 50 They are 3.20mM, 4.43mM, 8.49mM, 2.79mM and 7.20mM respectively.

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