Cantharellus cibarius antioxidant peptide as well as preparation method and application thereof
By preparing crude extracts of chanterelles polypeptides and screening using bioinformatics tools, combining molecular docking technology to determine the binding sites of antioxidant peptides, the chanterelles antioxidant peptides with high antioxidant activity were successfully screened, solving the problem of lack of effective screening methods in the existing technology, and achieving efficient screening and identification of chanterelles antioxidant peptides.
Patent Information
- Application Number
- CN202510118547.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-24
- Publication Date
- 2025-06-03
AI Technical Summary
There is a lack of a method for screening antioxidant peptides of chanterelle bacteria in the prior art, resulting in the extraction of chanterelle proteins and the development and utilization of antioxidant peptides.
By preparing the crude extract of chanterelles polypeptide, polypeptide sequence analysis was performed using LC-MS/MS, and screening was combined with bioinformatics tools such as Peptide Ranker, Innovagen, AllerTOPv.2.0 and ToxinPred, peptides with antioxidant activity were screened, and their binding sites to Keap1 receptor were determined through molecular docking technology.
Chanterelle antioxidant peptides with high antioxidant activity, such as LPRW, GFDF and WDFK, were successfully screened. These peptides showed significant antioxidant activity in in vitro and cellular experiments, with potential applications in the development of drugs, foods and skin care products.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of food biotechnology, and particularly relates to a cantharellus cibarius antioxidant peptide, a preparation method thereof, and an application thereof. Background Art
[0002] Antioxidant peptides refer to those short-chain or long-chain peptide substances with activity derived from organisms or produced by protein hydrolysis, which can neutralize free radicals and prevent lipid peroxidation reactions. Such peptides are highly favored due to their multiple functions such as nutrition, blood pressure reduction, and immune enhancement, and due to their characteristics of "natural, safe, non-toxic, and high nutritional value", they have been widely used in the food industry and have become one of the most popular natural ingredients in functional foods. Currently, developing both nutritious and safe antioxidant peptides using edible mushroom resources is a hot topic in the field of polypeptide research.
[0003] Screening active peptides using bioinformatics tools is a method based on known amino acid sequences. It predicts the potential biological activities, safety, and bioavailability of peptide segments through database queries and software analysis, and then synthesizes the target peptide segments and verifies their related properties. This method can significantly save time and experimental costs, but there is currently no report on screening cantharellus cibarius antioxidant peptides using bioinformatics tools.
[0004] There are many methods for evaluating the activity of antioxidant peptides, among which the DPPH method and the ABTS method are the most widely used (according to the GB / T39100-2020 standard). The Nrf2-Keap1 signaling pathway is a key pathway for cells to respond to oxidative stress. Blocking the interaction between Nrf2 and Keap1 can activate this pathway, improve the expression of antioxidant-related proteins, and reduce oxidative damage caused by external adverse stimuli to the body. In recent years, homology modeling and molecular docking techniques have been widely used to reveal the binding sites of antioxidant peptides to the Keap1 receptor, providing an effective way for the selection and discovery of antioxidant peptides.
[0005] Cantharellus cibarius (scientific name: Cantharellus cibarius Fr.) is a fungus of the genus Cantharellus in the family Cantharellaceae, also known as egg yolk mushroom, yellow mushroom, apricot mushroom, etc. Cantharellus cibarius has medicinal value, can clear the eyes and benefit the stomach and intestines. It can treat skin roughness or dryness caused by vitamin A, keratomalacia, xerophthalmia, and night blindness, and can also treat diseases caused by certain respiratory and digestive tract infections. Developing both nutritious and safe antioxidant peptides using edible mushroom resources is a hot topic in the field of polypeptide research. Currently, there are few reports on the extraction of Cantharellus cibarius proteins and further development and utilization for preparing antioxidant peptides. Summary of the Invention
[0006] The purpose of the present invention is to provide a cantharellus cibarius antioxidant peptide, a preparation method thereof, and an application thereof.
[0007] The present invention is implemented in the following manner: A cantharellus cibarius antioxidant peptide, the amino acid sequence of the antioxidant peptide being at least one of LPRW, GFDF, and WDFK.
[0008] A method for preparing a cantharellus cibarius antioxidant peptide, the method comprising the following steps: S1: Prepare a crude extract of cantharellus cibarius polypeptide S11: Raw material pretreatment: The cantharellus cibarius fruiting body is crushed and passed through a 100-mesh sieve to obtain cantharellus cibarius powder. Weigh the cantharellus cibarius powder, add ultrapure water and homogenize; add 25 - 35 mL of ultrapure water per gram of cantharellus cibarius powder; S12: Enzymolysis: Add cellulase to the homogenate obtained in step S11, the addition amount of cellulase being 0.8% - 1.2% of the mass of the cantharellus cibarius powder. Adjust the pH to 4.8 - 5.2, and enzymolyze at 55°C for 2.5 - 3.5 h. Then boil at 100°C to inactivate the enzyme for 8 - 12 min. After cooling, add alkaline protease, the enzyme addition amount being 0.28% - 0.32% of the mass of the cantharellus cibarius powder in step S11. Adjust the pH to 8.8 - 9.2, and enzymolyze at 50°C for 2.5 - 3.5 h. Then boil at 100°C to inactivate the enzyme for 8 - 12 min. Cool to room temperature, and then centrifuge at 3500 - 4500 r / min for 15 - 25 min. Take the supernatant to obtain the cantharellus cibarius alkaline protease enzymolysis solution; S13: Purification: Use a 3KDa ultrafiltration tube to separate the cantharellus cibarius alkaline protease enzymolysis solution in step S12 by membrane separation technology. The crude extract with a molecular weight cut-off < 3KDa is the crude extract of cantharellus cibarius antioxidant peptide; S14: Measure the antioxidant activity of the cantharellus cibarius polypeptide crude extract obtained in step S13, including the measurement of DPPH, ABTS radical scavenging rate and total reducing power; S2: Polypeptide composition analysis Perform polypeptide sequence analysis on the cantharellus cibarius polypeptide crude extract obtained in step S13 using LC-MS / MS. A total of 1174 sequences are obtained from the cantharellus cibarius polypeptide crude extract through peptide spectrum identification; S3: Screen antioxidant peptides using bioinformatics tools S31: Use the peptide activity prediction tool Peptide Ranker to predict the activity of the sequences identified by mass spectrometry in S2. Select the sequences with scores exceeding 0.5 as having potential activity for subsequent screening; S32: Predict the water solubility of the peptide through the Innovagen tool, predict the potential allergenicity of the peptide through the AllerTOPv.2.0 tool, and predict the potential toxicity of the peptide through the ToxinPred tool. Sequences without potential allergenicity and toxicity are subjected to subsequent molecular docking, synthesis and verification; S33: The receptor protein Keap1 is used to collect the structures of key target proteins through the PDB database. The target is optimized by removing water molecules and small molecule ligands using PyMOL software, and then hydrogenation and charge processing are carried out using AutoDock Tools and saved as the pdbqt format. Using the processed Keap1 protein as the receptor and the sequences screened in S32 as the ligands, molecular docking is performed using AutoDock Tools software, the binding energy is calculated, and peptides with lower docking energy to Keap1 are screened to obtain three antioxidant peptides, whose amino acid sequences are LPRW, GFDF, and WDFK respectively.
[0009] Step S11: Raw material pretreatment: The fruiting bodies of Cantharellus cibarius are crushed and passed through a 100-mesh sieve to obtain Cantharellus cibarius powder. Weigh the Cantharellus cibarius powder, add ultrapure water and homogenize; add 30 mL of ultrapure water per gram of Cantharellus cibarius powder. Step S12: Enzymatic hydrolysis: Add cellulase to the homogenate obtained in Step S11. The addition amount of cellulase is 1% of the mass of Cantharellus cibarius powder, adjust the pH to 5, and carry out enzymatic hydrolysis at 55 °C for 3 h. Then boil to inactivate the enzyme at 100 °C for 10 min. After cooling, add alkaline protease, and the enzyme addition amount is 0.3% of the mass of Cantharellus cibarius powder in Step S11. Adjust the pH to 9 and carry out enzymatic hydrolysis at 50 °C for 3 h. Then boil to inactivate the enzyme at 100 °C for 10 min. Cool to room temperature, and then centrifuge at 4000 r / min for 20 min to take the supernatant.
[0010] Application of an antioxidant peptide from Cantharellus cibarius in the preparation of drugs, foods, and skin care products for scavenging free radicals.
[0011] Beneficial effects: In the present invention, polypeptides are extracted and purified from Cantharellus cibarius, and multiple antioxidant peptides with good antioxidant ability are discovered, including one or more of LPRW, GFDF, and WDFK. The antioxidant peptide from Cantharellus cibarius in the present invention has high antioxidant activity and can be used as a natural antioxidant peptide, having potential application prospects in the fields of preparing drugs, foods, and skin care products for scavenging free radicals. Brief description of the drawings
[0012] Figure 1 It is the LC-MS / MS analysis mass spectrum of the alkaline protease hydrolysate (<3 KDa) of Cantharellus cibarius.
[0013] Figure 2 It is the molecular docking result of the antioxidant peptide LPRW and the Keap1 receptor protein.
[0014] Figure 3 It is the molecular docking result of the antioxidant peptide GFDF and the Keap1 receptor protein.
[0015] Figure 4It is the molecular docking result of the antioxidant peptide WDFK and the Keap1 receptor protein.
[0016] Figure 5 It is the effect of three antioxidant peptides at different concentrations on the viability of Caco-2 cells.
[0017] Figure 6 It is different concentrations of H 2 O 2 The effect on the degree of oxidative damage of Caco-2 cells.
[0018] Figure 7 It is the effect of three antioxidant peptides at different concentrations on the viability of Caco-2 cells after H 2 O 2 induced damage to Caco-2 cells.
[0019] Figure 8 It is the effect of three antioxidant peptides at different concentrations on the content of MDA in Caco-2 cells after H 2 O 2 induced damage to Caco-2 cells.
[0020] Figure 9 It is the effect of three antioxidant peptides at different concentrations on the activity of SOD enzyme in Caco-2 cells after H 2 O 2 induced damage to Caco-2 cells.
[0021] Figure 10 It is the effect of three antioxidant peptides at different concentrations on the activity of CAT enzyme in Caco-2 cells after H 2 O 2 induced damage to Caco-2 cells. Specific embodiments
[0022] The principles and features of the present invention are described below in conjunction with embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.
[0023] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.
[0024] In a specific embodiment, Cantharellus cibarius was purchased from Yunnan Chuxiang E-commerce Co., Ltd. In a specific embodiment, cellulase and alkaline protease were purchased from Henan Wanbang Chemical Technology Co., Ltd. In a specific embodiment, the detection method of antioxidant activity is as follows: The method for detecting the ability to scavenge DPPH free radicals refers to the first method (DPPH method) for determining the antioxidant activity of polypeptides in GB / T 39100-2020; the method for detecting the ability to scavenge ABTS free radicals refers to the second method (ABTS method) for determining the antioxidant activity of polypeptides in GB / T 39100-2020.
[0025] Example 1 I. Preparation of crude extract of Cantharellus cibarius polypeptides (1) Raw material pretreatment: The fruiting bodies of Cantharellus cibarius were crushed and passed through a 100-mesh sieve to obtain Cantharellus cibarius powder. Weigh the Cantharellus cibarius powder, add ultrapure water and homogenize; add 30 mL of ultrapure water per gram of Cantharellus cibarius powder.
[0026] (2) Enzymolysis: Add 1% (w / w) cellulase (enzyme activity 400,000 U / g) to the homogenate obtained in (1), that is, the addition amount of cellulase accounts for 1% of the mass of Cantharellus cibarius powder in step (1). Adjust the pH to 5 and enzymolyze at 55 °C for 3 h to dissolve the cell wall and release Cantharellus cibarius protein. Then boil at 100 °C for 10 min to inactivate the enzyme. After cooling, add 0.3% (w / w) alkaline protease (enzyme activity 20,000 U / g), that is, the addition amount of alkaline protease accounts for 0.3% of the mass of Cantharellus cibarius powder in step (1). Adjust the pH to 9 and enzymolyze at 50 °C for 3 h. Then boil at 100 °C for 10 min to inactivate the enzyme. Cool to room temperature, and then centrifuge at 4000 r / min for 20 min. After taking the supernatant, the alkaline protease enzymolysis solution of Cantharellus cibarius is obtained.
[0027] (3) Purification: Use a 3KDa ultrafiltration tube to separate the alkaline protease enzymolysis solution of Cantharellus cibarius in (2) by membrane separation technology. The crude extract with a molecular weight cut-off < 3KDa is the crude extract of Cantharellus cibarius antioxidant peptides; the membrane separation technology used in this example is ultrafiltration; (4) Determine the antioxidant activity of the crude extract of Cantharellus cibarius polypeptides (< 3KDa) obtained in (2), including the determination of DPPH and ABTS free radical scavenging rates and total reducing power. The specific method is as follows: ① Determination of the ability to scavenge DPPH free radicals Take the crude extract of Cantharellus cibarius polypeptides (< 3KDa) as the sample solution. Take 2 mL of the sample solution and mix it with 2 mL of 0.1 mmol / L DPPH absolute ethanol solution to form a mixed solution. After standing in the dark at room temperature for 20 min, detect the absorbance of the mixed solution at 517 nm. Use a mixed solution of 2 mL of ultrapure water and 2 mL of absolute ethanol as the blank control. Calculate the ability of the crude polypeptide extract to scavenge DPPH according to the following formula.
[0028] DPPH free radical scavenging rate (%) = Where: A 0is the light absorption value of the mixed solution of 2 mL of ultrapure water and 2 mL of 0.1 mmol / L DPPH at 517 nm; A 1 is the light absorption value of the mixed solution of 2 mL of sample solution and 2 mL of 0.1 mmol / L DPPH at 517 nm; A 2 is the light absorption value of the mixed solution of 2 mL of sample solution and 2 mL of absolute ethanol at 517 nm.
[0029] The results showed that the crude extract of Cantharellus cibarius polypeptides (<3 kDa) had a DPPH radical scavenging rate of 42.59%.
[0030] ② Determination of the scavenging ability of ABTS radicals Mix 1 mmol / L ABTS+ and 2.45 mmol / L potassium persulfate (final concentration), and let it stand overnight in the dark at room temperature to obtain stable ABTS + solution. Dilute the generated ABTS + solution with absolute ethanol to make its absorbance at 30 °C and 734 nm wavelength be 0.7 ± 0.02, that is, obtain the ABTS + working solution. Take 100 μL of the sample solution of the crude extract of Cantharellus cibarius polypeptides (<3 kDa) and add it to a test tube, then add 3.9 mL of ABTS + working solution. Use absolute ethanol to replace the sample solution in the blank tube, and use absolute ethanol to replace the ABTS + working solution in the control tube. Place it in the dark at room temperature for 6 min, and measure its absorbance at a wavelength of 734 nm. Each sample is operated in parallel 3 times, and the calculation formula is: ABTS radical scavenging rate (%) = In the formula: A 0 is the light absorption value of the mixed solution of 2 mL of absolute ethanol and 2 mL of ABTS + at 734 nm; A 1 is the light absorption value of the mixed solution of 2 mL of sample solution and 2 mL of ABTS + at 734 nm; A 2 is the light absorption value of the mixed solution of 2 mL of sample solution and 2 mL of absolute ethanol at 734 nm.
[0031] The results showed that the crude extract of Cantharellus cibarius polypeptides (<3 kDa) had an ABTS radical scavenging rate of 75.14%.
[0032] ③ Determination of total reducing power Take 2 mL of the crude extract of Cantharellus polypeptides (<3 kDa) sample solution, 2 mL of phosphate buffer (pH 6.6, 0.2 mol / L), and 2 mL of 1% potassium ferricyanide solution, mix them evenly, let stand at 50 °C for 20 min, add 2 mL of 10% trifluoroacetic acid to terminate the reaction. After centrifuging at 3000 r for 10 min, take 2 mL of the supernatant and mix it with 2 mL of ultrapure water and 0.4 mL of 0.1% FeCl 3 solution. After mixing evenly, let stand at 50 °C for 10 min, and measure the absorbance of the mixed solution at 700 nm. Use ultrapure water as the blank control.
[0033] The results showed that the total reducing power of the crude extract of Cantharellus polypeptides (<3 kDa) was 0.746.
[0034] II. Polypeptide Composition Analysis LC-MS / MS Mass Spectrometry Sequencing: Use LC-MS / MS to analyze the polypeptide sequence of the crude extract of Cantharellus polypeptides (<3 kDa) obtained in Step 1. A total of 1174 sequences were obtained from the crude extract of Cantharellus polypeptides (<3 kDa) through peptide spectrum identification.
[0035] III. Screening of Antioxidant Peptides by Bioinformatics Tools (1) Use the peptide activity prediction tool Peptide Ranker to predict the activity of the sequences identified by mass spectrometry in Step 2. Select the sequences with scores exceeding 0.5 as having potential activity for subsequent screening.
[0036] (2) Predict the water solubility of the peptide through the Innovagen tool, predict the potential allergenicity of the peptide through the AllerTOP v.2.0 tool, and predict the potential toxicity of the peptide through the ToxinPred tool. Sequences without potential allergenicity and toxicity are subjected to subsequent molecular docking, synthesis, and verification.
[0037] (3) The receptor protein Keap1 (PDB ID: 2FLU) was used to collect the protein structures of key targets in the PDB database. The PyMOL software was used to optimize the targets by removing water molecules and small molecule ligands, and the AutoDock Tools was used for hydrogenation and charge processing and then saved as the pdbqt format. Using the processed Keap1 protein as the receptor and the sequences screened in step three-(2) as the ligands, molecular docking was performed with the AutoDock Tools software to calculate the binding energy and output the result file. Finally, the PyMOL and DiscoveryStudio software were used for force analysis and result visualization from three-dimensional and two-dimensional angles. The docking energy is generally negative. The lower the binding ability, the more stable the ligand binds to the receptor. Use PyMOL for its visualization analysis (the lower the binding energy, the better the binding), and screen the peptides with lower docking energy to Keap1 to obtain three antioxidant peptides, and their amino acid sequences are LPRW, namely: Leu-Pro-Arg-Trp (SEQ ID NO. 1); GFDF, namely: Gly-Phe-Asp-Phe (SEQ ID NO. 2); WDFK, namely: Trp-Asp-Phe-Lys (SEQ ID NO. 3).
[0038] The amino acid sequences and corresponding docking energies of each antioxidant peptide are shown in Table 1. The predicted scores of antioxidant activity, water solubility, potential allergenicity, toxicity and other properties of each antioxidant peptide are shown in Table 2.
[0039] Table 1 Amino acid sequences and docking energies of antioxidant peptides Table 2 Prediction of antioxidant peptide properties The molecular docking results of the amino acid sequence LPRW with the Keap1 receptor are shown in Figure 2 ; the molecular docking results of the amino acid sequence GFDF with the Keap1 receptor are shown in Figure 3 ; the molecular docking results of the amino acid sequence WDFK with the Keap1 receptor are shown in Figure 4 .
[0040] As Figures 2-4As shown in the figure, from the binding modes and binding sites of three antioxidant peptides and the receptor protein Keap1, it can be seen that the binding of antioxidant peptides to Keap1 protein mainly occurs through hydrogen bond interactions and hydrophobic interactions with receptor amino acids. Among them, LPRW forms three hydrogen bonds with ARG326 and VAL608 of Keap1 protein, one carbon-hydrogen bond with VAL608, and two hydrophobic interactions with GLY419 and ALA607; GFDF has two hydrogen bonds with ARG336 of Keap1 protein, one carbon-hydrogen bond with PRO384, and hydrophobic interactions with LEU74, AER383, and PHE83; WDFK has seven hydrogen bonds with ASN517, ASN469, VAL467, ARG326, GLN563, GLY564, CYS513, and VAL561 of Keap1 protein and one carbon-hydrogen bond interaction with LEU468, and forms two hydrophobic interactions with CYS513 and VAL561.
[0041] IV. Identification of Intracellular Antioxidant Activity of Antioxidant Peptides According to the amino acid sequences of the three antioxidant peptides screened in Step 3, Shanghai Hongtai Biotechnology Co., Ltd. was commissioned to chemically synthesize the peptide segments, and the synthesized amino acid sequences were LPRW, GFDF, and WDFK.
[0042] Determine the effects of three chemically synthesized antioxidant peptides (amino acid sequences are LPRW, GFDF, and WDFK) on the contents of antioxidant enzymes (SOD, CAT) and MDA in Caco-2 cells. The experimental method is as follows: ① Caco-2 Cell Culture Human colon cancer cells (Caco-2 cells) were cultured in DMEM supplemented with 1% penicillin-streptomycin and 20% fetal bovine serum (FBS), and cultured at 37°C in 5% CO 2 2. The medium was changed every 2 days, and cells with 80-90% confluence were passaged with 0.25% trypsin-EDTA. All in vitro experiments used Caco-2 cells with passage numbers of 25-34.
[0043] ② Determination of the Viability of Caco-2 Cells by Antioxidant Peptides The CCK-8 kit was used to determine cell viability. Caco-2 cells (1×10 5 cells / well) were seeded on 96-well plates and cultured for 24 h, and then cultured with different concentrations of synthetic antioxidant peptides (0.005, 0.05, 0.15, 0.03, 0.5 mg / mL) dissolved in DMEM. The control group was cells growing normally without treatment. After 24 h, 100 μL of CCK-8 solution was added to each well and allowed to stand for 2 h. Finally, the absorbance (450 nm) was recorded with an enzyme-linked immunosorbent assay (ELISA) reader. Calculate cell viability.
[0044] ③H 2 O 2 Establishment of an oxidative stress model in Caco-2 cells Caco-2 cells (1×10 5 cells / well) were seeded onto 96-well plates and incubated for 24 hours. Then, they were stimulated with H 2 O 2 (200, 400, 600, 800, and 1000 μM) for 1, 2, 3, 4, 5, 6, and 7 hours. As described previously, cell viability was determined using CCK-8.
[0045] ④ Protective effect of antioxidant peptides on H 2 O 2 -induced damaged Caco-2 cells: an experimental study By establishing an H 2 O 2 -induced (1000 μM) oxidative stress model, the protective effect of Cantharellus cibarius antioxidant peptides on H 2 O 2 -induced damaged Caco-2 cells was investigated. Briefly, Caco-2 cells (1×10 5 cells / well) were cultured for 24 h and then pretreated with three synthetic antioxidant peptides at different concentrations (0.15, 0.3, 0.5 mg / mL) for 24 h. Finally, 1000 μM H 2 O 2 was added to the 96-well plates and the cells were incubated in an incubator for 2 h. Then, cell viability was determined using the CCK-8 method.
[0046] ⑤ Determination of SOD, CAT, and MDA in H 2 O 2 -induced damaged Caco-2 cells Caco-2 cells were seeded onto 6-well plates (2×10 5 cells / well) and cultured for 24 h. The treatment group was added with 2.0 mL of fresh medium containing the sample (0.5 mg / mL), with GSH as the positive control. The blank group and the H 2 O 2 -damaged group were added with fresh medium without the sample. After 24 h of treatment, 2.0 mL of fresh medium containing H 2 O 2 was added to the sample treatment group, the positive control group, and the H 2 O 2 -damaged group. After continuous culture for 5 h, the cells were collected and lysed, and the intracellular antioxidant enzyme activities were detected using SOD, CAT, and MDA assay kits.
[0047] Result analysis, as Figures 7-10As shown, the results of CCK-8 analysis confirmed that none of the three peptides showed cytotoxic effects on Caco-2 cells. Through cell experiments, it was found that H 2 O 2 The optimal conditions for inducing oxidative damage in Caco-2 cells were: 1000 μM H 2 O 2 induced for 5 h (at this time, the cell viability was 51.55%). The MDA contents in the treatment groups of the three peptides (LPRW, GFDF, WDFK) (concentration 0.5 mg / mL) were 1.38 ± 0.40 nmol / mg prot, 1.48 ± 0.25 nmol / mg prot, and 1.77 ± 0.30 nmol / mg prot respectively. Compared with the H 2 O 2 damage model group (2.18 ± 0.31 nmol / mg prot), they were significantly reduced by 32.09%, 18.93%, and 36.59% respectively, and the effect of WDFK on the intracellular MDA content (1.16 ± 0.09 nmol / mg prot) was comparable to that of the positive control GSH. Compared with the H 2 O 2 damage model group, all three peptides could significantly increase the activities of SOD and CAT. The results showed that the three antioxidant peptides had significant antioxidant activities.
[0048] Example 2 S1: Preparation of crude extract of Cantharellus cibarius polypeptides S11: Raw material pretreatment: The fruiting bodies of Cantharellus cibarius were crushed and passed through an 80-mesh sieve to obtain Cantharellus cibarius powder. Weigh the Cantharellus cibarius powder and add ultrapure water and homogenize; add 25 mL of ultrapure water per gram of Cantharellus cibarius powder; S12: Enzymolysis: Add cellulase to the homogenate obtained in step S11. The addition amount of cellulase is 0.8% of the mass of Cantharellus cibarius powder. Adjust the pH to 4.8 and enzymolyze at 55 °C for 2.5 h, then boil at 100 °C to inactivate the enzyme for 8 min. After cooling, add alkaline protease. The enzyme addition amount is 0.28% of the mass of Cantharellus cibarius powder in step S11. Adjust the pH to 8.8 and enzymolyze at 50 °C for 2.5 h, then boil at 100 °C to inactivate the enzyme for 8 min. Cool to room temperature, and then centrifuge at 3500 r / min for 25 min. Take the supernatant to obtain the alkaline protease enzymolysis solution of Cantharellus cibarius; S13: Purification: Use a 3 KDa ultrafiltration tube to separate the alkaline protease enzymolysis solution of Cantharellus cibarius in step S12 by membrane separation technology. The crude extract with a molecular weight cut-off < 3 KDa is the crude extract of Cantharellus cibarius antioxidant peptides; S14: Determine the antioxidant activity of the crude extract of Cantharellus cibarius polypeptides obtained in step S13, including the determination of DPPH, ABTS free radical scavenging rates, and total reducing power; Example 3 S1: Preparation of crude extract of Cantharellus cibarius polypeptide S11: Raw material pretreatment: The fruiting bodies of Cantharellus cibarius are crushed and passed through a 120-mesh sieve to obtain Cantharellus cibarius powder. Weigh the Cantharellus cibarius powder, add ultrapure water and homogenize it; add 35 mL of ultrapure water per gram of Cantharellus cibarius powder; S12: Enzymolysis: Add cellulase to the homogenate obtained in step S11. The addition amount of cellulase is 1.2% of the mass of Cantharellus cibarius powder. Adjust the pH to 5.2 and enzymolyze at 55 °C for 3.5 h, then boil at 100 °C to inactivate the enzyme for 12 min. After cooling, add alkaline protease. The enzyme addition amount is 0.32% of the mass of Cantharellus cibarius powder in step S11. Adjust the pH to 9.2 and enzymolyze at 50 °C for 3.5 h, then boil at 100 °C to inactivate the enzyme for 12 min. Cool to room temperature, and then centrifuge at 4500 r / min for 25 min. Take the supernatant to obtain the Cantharellus cibarius alkaline protease enzymolysis solution; S13: Purification: Use a 3KDa ultrafiltration tube to separate the Cantharellus cibarius alkaline protease enzymolysis solution in step S12 by membrane separation technology. The crude extract with a molecular weight cut-off < 3KDa is the crude extract of Cantharellus cibarius antioxidant peptide; S14: Determine the antioxidant activity of the crude extract of Cantharellus cibarius polypeptide obtained in step S13, including the determination of DPPH, ABTS free radical scavenging rate and total reducing power; The present invention efficiently screens and identifies novel antioxidant peptides from Cantharellus cibarius by using alkaline protease enzymolysis, ultrafiltration and LC-MS / MS techniques; screens bioactive peptides by computer analysis and verifies them through in vitro experiments and cell experiments; reveals the action sites and mechanisms of target antioxidant peptides through molecular simulation; determines the antioxidant effect of antioxidant peptides in cells through cell experiments. That is, by verifying in the laboratory and simulating the main action targets of antioxidant peptides through molecular docking, it is expected to achieve rapid and efficient identification of antioxidant peptides.
[0049] The antioxidant peptides screened by the present invention have strong antioxidant activity, and have the characteristics of high efficiency, simple preparation method and good repeatability. The Cantharellus cibarius antioxidant peptides described in the present invention have high antioxidant activity and can be used as natural antioxidant peptides, and have potential application prospects in the fields of preparing drugs, foods and skin care products for scavenging free radicals, providing an efficient way for the development of Cantharellus cibarius functional polypeptide products.
[0050] The present invention verifies the activities of the three antioxidant peptides through cell experiments. The three antioxidant peptides are all composed of 4 amino acids, and the peptide segments are relatively short, which is of great significance for the development of novel antioxidant products.
[0051] The present invention studies the interaction between Cantharellus cibarius antioxidant peptides and Keap1 receptor protein, and explores its antioxidant mechanism.
[0052] The above are only the preferred embodiments of the present invention. It should be noted that for those skilled in the art, without departing from the overall concept of the present invention, several changes and improvements can still be made, and these should also be regarded as the protection scope of the present invention.
Claims
1. A chanterelle antioxidant peptide, characterized in that The amino acid sequence of the antioxidant peptide is at least one of LPRW, GFDF and WDFK.
2. A method for preparing the chanterelle antioxidant peptide as claimed in claim 1, characterized in that: The method comprises the following steps: S1: Preparation of crude extract of chanterelle polypeptides S11: Raw material pretreatment: Chanterelle fruiting bodies were crushed through a 80 to 120 mesh sieve to obtain chanterelle powder, weighed chanterelle powder, added ultrapure water and homogenized; per gram of chanterelle powder add 25 to 35mL ultrapure water; S12: enzymolysis: add cellulase to the homogenate obtained in step S11, the addition amount of cellulase is 0.8%~1.2% of the mass of chanterelle powder, adjust pH4.8~5.2, enzymolysis at 55°C for 2.5~3.5h, then boil and deactivate enzyme at 100°C for 8~12min, after cooling, add alkaline protease, the enzyme addition amount is 0.28%~0.32% of the mass of chanterelle powder in step S11, adjust pH8.8~9.2, enzymolysis at 50°C for 2.5~3.5h, then boil and deactivate enzyme at 100°C for 8~12min, cool to room temperature, centrifuge at 3500~4500r / min for 15~25min, and obtain chanterelle alkaline protease enzymolysis liquid after taking supernatant; S13: Purification: using a 3KDa ultrafiltration tube to separate the chanterelle alkaline protease hydrolyzate in step S12 by membrane separation technology, and the crude extract with a molecular weight cutoff of less than 3KDa is the crude extract of chanterelle antioxidant peptides; S14: performing antioxidant activity assay on the crude extract of chanterelle polypeptide obtained in step S13, including assays of DPPH, ABTS free radical scavenging rates and total reducing power; S2: Peptide composition analysis The crude extract of chanterelle polypeptide obtained in step S13 was subjected to polypeptide sequence analysis using LC-MS / MS, and a total of 1174 sequences were obtained from the crude extract of chanterelle polypeptide through peptide spectrum identification; S3: Bioinformatics tools for screening antioxidant peptides S31: Use the peptide activity prediction tool Peptide Ranker to predict the activity of the sequences identified by mass spectrometry in S2, and select sequences with a score greater than 0.5 as having potential activity for subsequent screening; S32: The water solubility of peptides was predicted by the Innovagen tool, the potential allergenicity of peptides was predicted by the AllerTOPv.2.0 tool, and the potential toxicity of peptides was predicted by the ToxinPred tool. Sequences without potential allergenicity and toxicity were subsequently docked, synthesized, and verified; S33: The receptor protein Keap1 was used to collect key target protein structures through the PDB database. The targets were optimized by removing water molecules and small molecule ligands using PyMOL software, and hydrogenation and charge treatment were performed using AutoDock Tools and saved as pdbqt format. The treated Keap1 protein was used as the receptor, and the sequence screened out in S32 was used as the ligand. Molecular docking was performed using AutoDock Tools software to calculate the binding energy, and peptides with lower Keap1 docking energy were screened to obtain three antioxidant peptides, whose amino acid sequences were LPRW, GFDF and WDFK, respectively.
3. The method for preparing the chanterelle antioxidant peptide as claimed in claim 2, wherein: The step S11: raw material pretreatment: the chanterelle fruiting bodies are crushed and passed through a 100-mesh sieve to obtain chanterelle powder, the chanterelle powder is weighed, ultrapure water is added and homogenized; 30 mL of ultrapure water is added per gram of chanterelle powder; The step S12: enzymolysis: adding cellulase to the homogenate obtained in step S11, the amount of cellulase added is 1% of the mass of the chanterelle powder, adjusting the pH to 5, enzymolysis at 55° C. for 3 hours, then boiling at 100° C. for 10 minutes to inactivate the enzyme, after cooling, adding alkaline protease, the amount of enzyme added is 0.3% of the mass of the chanterelle powder in step S11, adjusting the pH to 9, enzymolysis at 50° C. for 3 hours, then boiling at 100° C. for 10 minutes to inactivate the enzyme, cooling to room temperature, then centrifuging at 4000 r / min for 20 minutes, and taking the supernatant.
4. A use of the chanterelle antioxidant peptide as claimed in claim 1, characterized in that: Application of chanterelle antioxidant peptides in the preparation of free radical scavenging drugs, foods and skin care products.