Anti-fatigue peptide as well as screening method and application thereof

Abstract

The invention relates to an anti-fatigue peptide. The amino acid sequence of the anti-fatigue peptide is Asp-Asp-Trp-Glu-Asn-Trp-Ala-Lys. The amino acid sequence of the anti-fatigue peptide is shown in the description. The anti-fatigue peptide provided by the invention has good cell DPPH free radical scavenging ability (IC50 is 0.60 mg / mL) and mitochondrial energy homeostasis regulation effect, and has good anti-oxidation and anti-fatigue effects. The anti-fatigue peptide can improve the activity of antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT), in cells, reduce the release of lactic dehydrogenase (LDH), and reduce the level of malondialdehyde (MDA); the damage of a mitochondrial network structure is improved, the ATP level of cells is improved, and the cell oxidative damage induced by H2O2 can be protected. The anti-fatigue peptide developed by the invention is good in safety and effectiveness, overcomes the defects of drug dependence, cytotoxicity and the like of the existing anti-fatigue agent, and can be widely applied to the industries of foods, medicines, health-care products and the like.

Description

Technical Field

[0001] The invention relates to the field of biology and food technology, and in particular to an anti-fatigue peptide and a screening method and application thereof. Background Art

[0002] Reactive oxygen species (ROS) are byproducts of standard cellular metabolism and may also originate from environmental factors such as stress, pollution, and industrial chemicals. Excessive free radicals may cause oxidative stress, triggering diseases such as fatigue. An oxidative environment can alter the body's metabolism, leading to changes in energy homeostasis. Cells constantly maintain cellular energy homeostasis by adjusting the production of adenosine triphosphate (ATP) according to metabolic demand. The involvement of mitochondria is essential to meet this energy demand. In addition to being the biological engine of eukaryotic cells, mitochondria are also the main producers and targets of ROS within the organelle. Excessive ROS generation can damage mitochondrial DNA (mtDNA) and other key components, disrupt mitochondrial function, and exacerbate mitochondrial ROS production, ultimately leading to tissue damage and decreased physical function, causing fatigue. In addition, oxidative stress induces the opening of important mitochondrial channels, triggering mitochondrial-related apoptotic pathways, leading to cell death. Reducing oxidative stress and mitochondrial dysfunction represents a new therapeutic approach to relieve antioxidant and anti-fatigue effects in vivo. Currently, many synthetic anti-fatigue agents cannot be used long-term due to other potential side effects such as drug dependence and cytotoxicity. Therefore, searching for natural anti-fatigue peptides, especially bioactive peptides from food-derived proteins to replace traditional anti-fatigue agents has become a new strategy to solve the above problems.

[0003] Sea bass (Lateolabrax japonicus) is a marine fish with high economic value, which is favored by consumers for its rich nutritional value. The development and preparation of anti-fatigue peptides using whole sea bass not only improves the comprehensive utilization of aquatic product resources, conforms to the concept of green, coordinated and sustainable development, but also lays the foundation for the development and utilization of anti-fatigue peptide-based functional foods or drugs, and provides new perspectives and inspirations for the application of bioactive peptides. At present, the process of preparing anti-fatigue peptides using whole sea bass has not been reported so far. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an anti-fatigue peptide and a screening method and application thereof.

[0005] The technical solution adopted by the present invention is as follows:

[0006] In one aspect, the present invention provides an anti-fatigue peptide, wherein the amino acid sequence of the anti-fatigue peptide is Asp-Asp-Trp-Glu-Asn-Trp-Ala-Lys.

[0007] The anti-fatigue peptide proposed in the present invention has good cell DPPH free radical scavenging ability (IC 50 0.60mg / mL) and mitochondrial energy homeostasis regulation, and good antioxidant and anti-fatigue effects. The anti-fatigue peptide can increase the activity of intracellular antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT), reduce the release of lactate dehydrogenase (LDH), and reduce the level of malondialdehyde (MDA); improve the damage of mitochondrial network structure and increase the level of cell ATP, and can protect H2O2-induced cell oxidative damage. The anti-fatigue peptide developed by the present invention has good safety and effectiveness, overcomes the shortcomings of existing anti-fatigue agents such as drug dependence and cytotoxicity, and can be widely used in industries such as food, medicine and health products.

[0008] Preferably, the molecular weight of the anti-fatigue peptide is 1063.19 Da.

[0009] On the other hand, the present invention provides a method for screening anti-fatigue peptides as described in any of the above technical solutions, comprising the following steps: using whole seabass as raw material, performing proteolysis and separation and purification on it, collecting components with the highest free radical scavenging activity, sequencing them using nano-liquid chromatography-tandem mass spectrometry, and then obtaining anti-fatigue peptides through computer-assisted screening.

[0010] The present invention uses whole seabass as a raw material to carry out controllable enzymatic hydrolysis preparation of anti-fatigue peptides, has the advantages of being simple and efficient, being able to track the activity of enzymatic hydrolysis products to achieve directional enzyme cutting, and the like, and is conducive to full utilization of marine proteins; at the same time, the present invention screens the anti-fatigue peptides through computer-assisted screening, has the advantages of simple operation, low production cost, high accuracy, and the like, and is conducive to large-scale production and application of the anti-fatigue peptides.

[0011] Preferably, the enzymatic hydrolysis method is: mixing sea bass and ultrapure water in a certain solid-liquid ratio to prepare a solution, adding papain for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, inactivating the enzyme at high temperature, and collecting the supernatant by centrifugation to obtain sea bass enzymatic hydrolyzate.

[0012] Preferably, the material-liquid ratio is 1:7 w / v;

[0013] The enzymatic hydrolysis conditions are as follows: the pH of the solution is 7.0, the enzymatic hydrolysis temperature is 50° C., the enzymatic hydrolysis time is 9 hours, and the enzyme-substrate ratio is 4.3% w / w.

[0014] Preferably, the separation and purification method is: using Sephadex G-25 gel column chromatography to separate and purify the seabass hydrolysate obtained by the protein hydrolysis, the eluent is deionized water, the sample volume is 5 mL, the flow rate is 0.5 mL / min, the detection wavelength is 214 nm, the graded components are collected, and the DPPH free radical scavenging activity of each component is determined, and the component with the highest DPPH free radical scavenging activity is freeze-dried.

[0015] Preferably, the computer-assisted screening method is: firstly screen peptide sequences with activity>0.5 through the Peptide Ranker database, and then further screen non-toxic and edible peptide sequences with hydrophilicity>0 through the ToxinPred database to obtain anti-fatigue peptides.

[0016] In another aspect, the present invention provides an anti-fatigue peptide according to any one of the above technical solutions or an anti-fatigue peptide obtained by the screening method of anti-fatigue peptides according to any one of the technical solutions in the field of preparing anti-fatigue drugs, health products or foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The elution diagram of Sephadex G-25 gel column chromatography in Example 1 (A) and the DPPH free radical scavenging activity result diagram of each component (B), different letters indicate significant differences (p<0.05);

[0018] Figure 2 is the sequence model of the anti-fatigue peptide of Example 1;

[0019] Figure 3 This is a graph showing the DPPH free radical scavenging activity results of the anti-fatigue peptide of Example 1;

[0020] Figure 4 The effect of the anti-fatigue peptide of Example 1 on the survival rate of Caco-2 cells induced by H2O2, different letters indicate significant differences (p<0.05);

[0021] Figure 5 The protective effect of the anti-fatigue peptide of Example 1 on the oxidative stress of Caco-2 cells induced by H2O2, wherein (A) is LDH leakage; (B) is SOD activity; (C) is CAT activity; (D) is the effect on MDA content, and different letters indicate significant differences (p<0.05);

[0022] Figure 6 The effect of the anti-fatigue peptide of Example 1 on the ultrastructure of mitochondria in Caco-2 cells induced by H2O2;

[0023] Figure 7The effect of the anti-fatigue peptide of Example 1 on the ATP level of Caco-2 cells induced by H2O2. Different letters indicate significant differences (p<0.05). DETAILED DESCRIPTION

[0024] In order to better understand the above technical solution, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0025] An embodiment of the present invention provides an anti-fatigue peptide, wherein the amino acid sequence of the anti-fatigue peptide is Asp-Asp-Trp-Glu-Asn-Trp-Ala-Lys.

[0026] In a preferred embodiment of the present invention, the molecular weight of the anti-fatigue peptide is 1063.19 Da.

[0027] The screening is carried out by the following method, which includes the following steps: using whole sea bass as raw material, performing proteolysis and separation and purification on it, collecting components with the highest free radical scavenging activity, sequencing using nano-liquid chromatography-tandem mass spectrometry, and then obtaining anti-fatigue peptides through computer-assisted screening.

[0028] In a preferred embodiment of the present invention, the enzymatic hydrolysis method is: mixing sea bass with ultrapure water at a certain solid-liquid ratio to prepare a solution, adding papain for enzymatic hydrolysis, inactivating the enzyme at high temperature after the enzymatic hydrolysis, and collecting the supernatant by centrifugation to obtain sea bass enzymatic hydrolyzate. In an embodiment of the present invention, the whole sea bass is crushed before enzymatic hydrolysis.

[0029] In a preferred embodiment of the present invention, the solid-liquid ratio is 1:7 w / v;

[0030] The conditions of enzymatic hydrolysis were as follows: pH of the solution was 7.0, the enzymatic hydrolysis temperature was 50° C., the enzymatic hydrolysis time was 9 h, and the enzyme-substrate ratio was 4.3% w / w.

[0031] In a preferred embodiment of the present invention, the separation and purification method is: Sephadex G-25 gel column chromatography is used to separate and purify the seabass hydrolysate obtained by protein hydrolysis, the eluent is deionized water, the sample volume is 5 mL, the flow rate is 0.5 mL / min, the detection wavelength is 214 nm, the graded components are collected, and the DPPH free radical scavenging activity of each component is determined, and the component with the highest DPPH free radical scavenging activity is freeze-dried.

[0032] In a preferred embodiment of the present invention, the computer-assisted screening method is: firstly, the peptide sequences with activity>0.5 are screened through the PeptideRanker database, and then the peptide sequences with non-toxic edible properties and hydrophilicity>0 are further screened through the ToxinPred database to obtain anti-fatigue peptides. In an embodiment of the present invention, the screened peptide sequences are modeled through the PEP-FOLD 3.5 polypeptide structure prediction modeling program in the RPBS structural biology database, the number of simulations is selected as 100, the model ranking is based on sOPEP, and the modeling result with the highest score is screened.

[0033] The anti-fatigue peptide of any one of the above technical solutions or the anti-fatigue peptide obtained by the screening method of any one of the technical solutions can be used in the field of preparing anti-fatigue medicines, health products or foods.

[0034] The technical solution of the present invention is further described and illustrated below based on specific embodiments.

[0035] Example 1

[0036] In the present embodiment, the anti-fatigue peptide having an amino acid sequence of Asp-Asp-Trp-Glu-Asn-Trp-Ala-Lys (single-letter abbreviation DDWENWAK) is named DK-8 for illustration.

[0037] A method for screening anti-fatigue peptides comprises the following steps:

[0038] (1) Enzyme hydrolysis: The whole seabass was used as the raw material and was pulverized. 4 g of the seabass pulverized material was weighed and mixed with 28 mL of ultrapure water to prepare a solution. The pH of the solution was adjusted to 7.0. 0.172 g of papain was added and the solution was subjected to enzymatic hydrolysis at 50°C for 9 h. After the enzymatic hydrolysis was completed, the solution was boiled in a boiling water bath for 10 min to inactivate the enzyme. The solution was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected to obtain a seabass enzymatic hydrolysate. The solution was freeze-dried to obtain a seabass enzymatic hydrolysate.

[0039] (2) Separation and purification: 500 mg of the above sea bass hydrolysate was added with 5 mL of deionized water, stirred until completely dissolved, centrifuged at 10,000 rpm for 10 min, the supernatant was collected, filtered through a 0.22 μm filter membrane, and separated and purified by Sephadex G-25 gel column chromatography. The eluent was deionized water, the sample volume was 5 mL, the flow rate was 0.5 mL / min, the detection wavelength was 214 nm, the fractionated components were collected, and the elution curve corresponding to the absorption peak of each component was determined (see Figure 1 (A)) and DPPH free radical scavenging activity (see Figure 1 (B)), the fraction (F3) with the highest DPPH radical scavenging activity was freeze-dried.

[0040] (3) Sequencing: The amino acid sequence of the component with the highest DPPH free radical scavenging activity was identified using nano-liquid chromatography-tandem mass spectrometry.

[0041] (4) Computer-assisted screening: The identified sequences were subjected to computer-assisted screening. Peptide sequences with activity > 0.5 were first screened through the PeptideRanker database, and then non-toxic, edible, and hydrophilic > 0 peptide sequences were further screened through the ToxinPred database to obtain an anti-fatigue peptide with an amino acid sequence of Asp-Asp-Trp-Glu-Asn-Trp-Ala-Lys and a molecular weight of 1063.19 Da.

[0042] (5) Peptide sequence modeling: The peptide sequences obtained by screening were modeled using the PEP-FOLD 3.5 peptide structure prediction modeling program in the RPBS structural biology database. The number of simulations was selected as 100, and the model ranking was based on sOPEP. The modeling results with the highest scores were selected (see Figure 2 ).

[0043] The anti-fatigue peptide obtained in Example 1 was evaluated for its DPPH free radical scavenging ability and anti-fatigue activity using the DPPH method and H2O2-induced cell model, and the results are recorded in Figures 3 to 7 .

[0044] 1. Effect of DPPH free radical scavenging activity of anti-fatigue peptides

[0045] The determination method is as follows:

[0046] Take 1mL of anti-fatigue peptide solution of different concentrations and 1mL of DPPH solution (0.1mM, prepared with 95% ethanol) and mix them thoroughly as the sample group; use 1mL of 95% ethanol solution instead of DPPH solution as the sample reference group; use 1mL of DPPH solution and 1mL of 95% ethanol solution as the blank group. Stand at room temperature and avoid light for 30min, measure the absorbance at 517nm, and calculate the DPPH free radical scavenging activity of the sample according to the following formula (1):

[0047]

[0048] Where: A i is the absorbance value of the sample group, A j is the absorbance value of the sample reference group, and A0 is the absorbance value of the blank group.

[0049] from Figure 3 It can be seen that the IC value of the anti-fatigue peptide of the present invention for DPPH free radical scavenging rate is 50 It is 0.60 mg / mL, which has a good ability to scavenge DPPH free radicals.

[0050] 2. Effects of anti-fatigue peptides on H2O2-induced oxidative stress model in Caco-2 cells

[0051] The determination method is as follows:

[0052] (I) Effects of anti-fatigue peptides on the survival rate of Caco-2 cells induced by H2O2

[0053] The Caco-2 cells were rapidly revived in a 37°C constant temperature water tank, washed and centrifuged several times, and expanded and cultured in a DMEM medium containing 20% ​​fetal bovine serum, 1% penicillin-streptomycin mixture and 1% non-essential amino acids for later use.

[0054] The density of Caco-2 cells was set to 4 × 10 4 / mL. 100μL was inoculated in each well of a 96-well plate and cultured for 24h. For the model group (MC), it was first treated with 800μMH2O2 for 4h, the supernatant was removed, and fresh culture medium was added for 24h. After the sample group was treated with H2O2 and the supernatant was removed, different concentrations of anti-fatigue peptide solutions and fresh culture medium were added and cultured for 24h. The cells in the blank group (BC) were cultured normally with PBS instead of H2O2 and anti-fatigue peptide solution. 20μL MTT solution (5mg / mL) was added to the 96-well plate, the supernatant was discarded after incubation for 4h, 150μL DMSO solution was added and shaken for 10min, the absorbance was measured at 570nm, and the cell viability was calculated as shown in the following formula (2):

[0055]

[0056] from Figure 4 It can be seen that the anti-fatigue peptide of the present invention has a certain restorative effect on the decrease in the survival rate of Caco-2 cells induced by H2O2.

[0057] (II) Effects of anti-fatigue peptides on LDH, SOD, CAT and MDA levels in Caco-2 cells induced by H2O2

[0058] The Caco-2 cell density was set to 3 × 10 5 / mL. Then, 2mL of cell suspension was inoculated in a 6-well plate, cultured for 24h, and treated and cultured according to the experimental grouping in (I). The cell supernatant was collected and the LDH level was detected using a kit. The cells were collected and treated with an ultrasonic cell disruptor. The levels of SOD, CAT and MDA were then detected using a kit.

[0059] Depend on Figure 5 It can be seen that the anti-fatigue peptide of the present invention protects Caco-2 cells induced by H2O2 by increasing the activities of SOD and CAT, reducing the release of LDH, and lowering the level of MDA.

[0060] (III) Effects of anti-fatigue peptides on the ultrastructure of mitochondria in Caco-2 cells induced by H2O2

[0061] The Caco-2 cell density was set to 3 × 10 5 Cells were collected after the same treatment as in (a), and then resuspended and fixed in electron microscopy fixative at 4°C for 4 hours. The cells were pre-embedded in agar, and then fixed in 1% osmium acid prepared in 0.1M PBS (pH=7.4) at room temperature for 2 hours in the dark. After dehydration at room temperature, the cells were infiltrated and embedded. After the resin block was polymerized, it was sliced ​​and stained using an ultrathin microtome, dried overnight at room temperature, and observed under a transmission electron microscope.

[0062] pass Figure 6 It can be seen that the anti-fatigue peptide of the present invention can improve the damage of mitochondrial network structure, and its state is significantly improved.

[0063] (IV) Effects of anti-fatigue peptides on ATP levels in Caco-2 cells induced by H2O2

[0064] The Caco-2 cell density was set to 3 × 10 5 After the cells were treated as in (a), they were collected and the ATP value of the cells was determined using an ATP assay kit according to the manufacturer's instructions.

[0065] Figure 7 The results show that the anti-fatigue peptide of the present invention can effectively increase the cellular ATP level.

[0066] In summary, the anti-fatigue peptide of the present invention has good anti-fatigue activity against H2O2-induced cellular oxidative stress.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-fatigue peptide, characterized in that The amino acid sequence of the anti-fatigue peptide is Asp-Asp-Trp-Glu-Asn-Trp-Ala-Lys.

2. The anti-fatigue peptide according to claim 1, characterized in that The molecular weight of the anti-fatigue peptide is 1063.19 Da.

3. A method for screening anti-fatigue peptides as claimed in claim 1, comprising the following steps: The whole sea bass was used as raw material, and it was enzymatically hydrolyzed, separated and purified. The components with the highest DPPH free radical scavenging activity were collected, sequenced by nano-liquid chromatography-tandem mass spectrometry, and then anti-fatigue peptides were obtained through computer-assisted screening.

4. The method for screening anti-fatigue peptides according to claim 3, characterized in that: The enzymatic hydrolysis method comprises the following steps: mixing sea bass and ultrapure water in a certain material-liquid ratio to prepare a solution, adding papain to perform enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, inactivating the enzyme at high temperature, and collecting the supernatant by centrifugation to obtain sea bass enzymatic hydrolyzate.

5. The method for screening anti-fatigue peptides according to claim 4, characterized in that: The material-liquid ratio is 1:7 w / v; The enzymatic hydrolysis conditions are as follows: the pH of the solution is 7.0, the enzymatic hydrolysis temperature is 50° C., the enzymatic hydrolysis time is 9 hours, and the enzyme-substrate ratio is 4.3% w / w.

6. The method for screening anti-fatigue peptides according to claim 3, characterized in that: The separation and purification method comprises: using Sephadex G-25 gel column chromatography to separate and purify the seabass enzymatic hydrolysate obtained by protein hydrolysis, the eluent is deionized water, the sample volume is 5 mL, the flow rate is 0.5 mL / min, the detection wavelength is 214 nm, the graded components are collected, and the DPPH free radical scavenging activity of each component is determined, and the component with the highest DPPH free radical scavenging activity is freeze-dried.

7. The method for screening anti-fatigue peptides according to claim 3, characterized in that: The computer-assisted screening method is: firstly screening peptide sequences with activity>0.5 through the Peptide Ranker database, and then further screening non-toxic edible peptide sequences with hydrophilicity>0 through the ToxinPred database to obtain anti-fatigue peptides.

8. Use of the anti-fatigue peptide according to any one of claims 1 to 2 or the anti-fatigue peptide obtained by the screening method of any one of claims 3 to 7 in the preparation of anti-fatigue medicines, health products or foods.

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