Ruditapes philippinarum antioxidant peptide as well as preparation method and application thereof

Abstract

The invention discloses ruditapes philippinarum antioxidant peptide as well as a preparation method and application thereof, and relates to the technical field of food biology. Comprising at least one of the following antioxidant peptides; the amino acid sequences of the ruditapes philippinarum antioxidant peptides 1-5 are shown as SEQ ID NO.8, 9, 13, 16 and 18. The preparation method of the ruditapes philippinarum antioxidant peptide comprises the following steps: (1) enzymatic hydrolysis of ruditapes philippinarum; (2) preparing the ruditapes philippinarum antioxidant peptide; (3) purifying the ruditapes philippinarum antioxidant peptide; (4) determining the amino acid sequence of the ruditapes philippinarum antioxidant peptide; (5) identifying active sites of the ruditapes philippinarum antioxidant peptide; and (6) carrying out solid-phase synthesis on the ruditapes philippinarum antioxidant peptide. The antioxidant peptide provides a deep processing technology for comprehensive utilization of ruditapes philippinarum, and has the advantage of high-value utilization of low-value aquatic products.

Description

Technical Field

[0001] The present invention relates to the field of food biotechnology, and more particularly to antioxidant peptides from Manila clams, their preparation methods and applications. Background Art

[0002] During the metabolic process of human cells, a large amount of reactive oxygen species (ROS) will be generated. Excessive generation of ROS will cause the body to be in a state of oxidative stress. Excessive ROS will induce autophagy, trigger apoptosis, and cause irreversible tissue damage. Therefore, finding safe antioxidants to eliminate excessive free radicals in the body is one of the current research hotspots; compared with chemically synthesized antioxidants, natural antioxidants have the advantages of low toxicity and side effects and low price, and are expected to be an effective antioxidant strategy in the future, with important application values.

[0003] The activity mechanism of antioxidant peptides is closely related to signal pathways, which play a key role in regulating intracellular redox balance and protecting cells from oxidative stress damage. Myeloperoxidase (MPO) plays a crucial role in the generation of ROS in the body and can be used to evaluate whether the screened peptides have the ability to spontaneously inactivate this enzyme; Keap1-Nrf2 / ARE is the main signal pathway regulating cellular antioxidant responses, and activation of this pathway can reduce oxidative damage caused by external adverse stimuli. The structures of both proteins have been elucidated, and antioxidant peptides can be screened through receptor proteins, thus saving resource costs.

[0004] China has rich shellfish resources. The Manila clam, also known as the variegated clam, is a common low-value shellfish in the coastal areas of China. It has the characteristics of low price, but due to its small size, it is not convenient for eating and processing, so it is mostly consumed fresh, and its development and utilization rate is low. The Manila clam is rich in a large amount of protein and has a rich variety of amino acids, making it a good raw material for preparing antioxidant peptides.

[0005] Therefore, how to deeply process the protein resources of Manila clams using modern biotechnology and optimize the antioxidant peptide products through processes is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides antioxidant peptides from Manila clams, their preparation methods and applications.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The antioxidant peptides from Manila clams include at least one of the following antioxidant peptides;

[0009] The amino acid sequence of the antioxidant peptide 1 from Ruditapes philippinarum is LGGEDFDNRM, as shown in SEQ ID NO.13;

[0010] The amino acid sequence of the antioxidant peptide 2 from Ruditapes philippinarum is FDCSQFKPEE, as shown in SEQ ID NO.18;

[0011] The amino acid sequence of the antioxidant peptide 3 from Ruditapes philippinarum is KTDAMAGMGGM, as shown in SEQ ID NO.9;

[0012] The amino acid sequence of the antioxidant peptide 4 from Ruditapes philippinarum is IDPRSDCAF, as shown in SEQ ID NO.8;

[0013] The amino acid sequence of the antioxidant peptide 5 from Ruditapes philippinarum is IAQTWDGEF, as shown in SEQ ID NO.16.

[0014] The preparation method of the antioxidant peptide from Ruditapes philippinarum comprises the following steps:

[0015] (1) Enzymatic hydrolysis of Ruditapes philippinarum:

[0016] The enzymatic hydrolysis conditions are as follows: the enzymatic hydrolysis temperature is 40 - 60 °C, the enzymatic hydrolysis time is 2 - 6 h, the enzyme - substrate ratio is 0.05% - 0.6%, and the solid - liquid ratio is 1:1 - 5;

[0017] (2) Preparation of the antioxidant peptide from Ruditapes philippinarum:

[0018] The above enzymatic hydrolysate is ultrafiltered and separated using ultrafiltration membranes with molecular weight cut - off ranges of 5 kDa, 3 kDa, and 1 kDa for the enzymatic hydrolysis products, and it is divided into 4 fractions with different molecular weight ranges, and is evaluated with DPPH· free radical scavenging rate, ABTS·+ free radical scavenging rate, and Fe 2+ chelation rate as indicators;

[0019] (3) Purification of the antioxidant peptide from Ruditapes philippinarum:

[0020] Collect the fraction with the best antioxidant activity, filter it through a 0.22 - μm filter membrane, and then perform gel chromatography separation using Sephadex G - 15 as the separation medium. The eluent is ultrapure water, the flow rate is 1 mL / min, measure the antioxidant activity of the elution fractions corresponding to each absorption peak, and freeze - dry to obtain high - purity antioxidant peptide;

[0021] (4) Determination of the amino acid sequence of the antioxidant peptide from Ruditapes philippinarum:

[0022] Collect the elution fraction with the highest antioxidant activity (RPPH - 1A), and use LC - MS / MS to determine the amino acid sequence;

[0023] (5) Identification of the active sites of antioxidant peptides from Ruditapes philippinarum:

[0024] Download the Keap1 protein (PDB ID: 6QMK) and MPO protein (PDB ID: 3F9P) from NCBI, and perform molecular docking of these two receptor protein models with the screened amino acid sequences;

[0025] Molecular docking of antioxidant peptide 1 from Ruditapes philippinarum (amino acid sequence: LGGEDFDNRM) with the receptor Keap1, and the main binding sites are Arg415, Arg483, Arg380, His436, Ser602, Ala366; Molecular docking of the peptide segment with the amino acid sequence of LGGEDFDNRM of antioxidant peptide 1 from Ruditapes philippinarum with the receptor MPO, and the main binding sites are Thr502, Lys505, Asp321, LEU33, Trp32, Phe439, Lys308, Arg504;

[0026] Molecular docking of antioxidant peptide 2 from Ruditapes philippinarum (amino acid sequence: FDCSQFKPEE) with the receptor Keap1, and the main binding sites are Tyr334, Phe577, Arg483, Tyr572, Tyr525, Val418, Val465, Val512, Ile559, Leu365, Leu557, Val604; Molecular docking of antioxidant peptide 2 from Ruditapes philippinarum with the receptor MPO, and the main binding sites are Lys308, Phe439, Cys497, Lys505, Ser319, Arg504, Asp321, Lys505.

[0027] (6) Solid-phase synthesis of antioxidant peptides from Ruditapes philippinarum:

[0028] Synthesize the bioactive peptide by solid-phase synthesis and determine its in vitro antioxidant activity; its antioxidant activity is equivalent to that of Vc at the same concentration.

[0029] Furthermore, in step (1), the enzyme is any one of alkaline protease, flavor protease, and compound protease;

[0030] Furthermore, in step (3), the four components with different molecular weight ranges are: components with molecular weight < 1 kDa, 1 - 3 kDa, 3 - 5 kDa, > 5 kDa.

[0031] Furthermore, in step (3), before purifying the antioxidant peptide from Ruditapes philippinarum, first verify the in vivo antioxidant activity of the bioactive peptide through the Caenorhabditis elegans model, evaluate it with indicators such as nematode lifespan, stress resistance, and antioxidant enzyme activity, and then use this component for subsequent separation and purification.

[0032] Application of antioxidant peptides from Ruditapes philippinarum in functional foods, health products, food additives and cosmetics.

[0033] As can be seen from the above technical solutions, compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] (1) The antioxidant peptide sequence of the present invention is reported for the first time and is different from all the publicly disclosed antioxidant peptide sequences;

[0035] (2) The molecular weight of the antioxidant peptide from Ruditapes philippinarum of the present invention is 800 Da to 2100 Da, and the low-molecular-weight antioxidant peptide is more conducive to human digestion and absorption;

[0036] (3) The raw material price of Ruditapes philippinarum is low and there are few processed products. The antioxidant peptide of the present invention provides a deep processing technology for the comprehensive utilization of Ruditapes philippinarum, and has the advantage of high-value utilization of low-value aquatic products. Description of the Drawings

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative work.

[0038] Figure 1 Shows the effects of different proteases on the degree of hydrolysis and the scavenging rate of ABTS·+ free radicals;

[0039] Figure 2 Shows the effects of hydrolysis temperature on the degree of hydrolysis and the scavenging rate of ABTS·+ free radicals;

[0040] Figure 3 Shows the effects of hydrolysis time on the degree of hydrolysis and the scavenging rate of ABTS·+ free radicals;

[0041] Figure 4 Shows the effects of enzyme dosage on the degree of hydrolysis and the scavenging rate of ABTS·+ free radicals;

[0042] Figure 5 Shows the effects of solid-liquid ratio on the degree of hydrolysis and the scavenging rate of ABTS·+ free radicals;

[0043] Figure 6 Shows the interaction diagram of hydrolysis temperature and hydrolysis time;

[0044] Figure 7 Shows the interaction diagram of hydrolysis temperature and solid-liquid ratio;

[0045] Figure 8It is the interaction diagram of enzymatic hydrolysis time and solid-to-liquid ratio;

[0046] Figure 9 It is the antioxidant activity of each component after ultrafiltration separation;

[0047] Figure 10 It is the effect of antioxidant peptides on the lifespan of nematodes under different conditions; Figure 10 a is the effect of antioxidant peptides on the lifespan of nematodes; Figure 10 b is the effect of antioxidant peptides on the lifespan of nematodes under oxidative stress; Figure 10 c is the effect of antioxidant peptides on the lifespan of nematodes under heat stress;

[0048] Figure 11 It is the effect of antioxidant peptides on the ROS content and lipofuscin content of nematodes; Figure 11 a is the ROS content; Figure 11 b is the lipofuscin content;

[0049] Figure 12 It is the effect of antioxidant peptides on the CAT content, GSH-Px content, SOD content, and MDA content of nematodes; Figure 12 a is the CAT content; Figure 12 b is the GSH-Px content; Figure 12 c is the SOD content; Figure 12 d is the MDA content;

[0050] Figure 13 It is the Sephadex G-15 gel chromatogram;

[0051] Figure 14 It is the antioxidant activity of the peptide segment purified by Sephadex G-15 gel chromatography;

[0052] Figure 15 It is the first-order mass spectrum obtained by LC-MS / MS identification;

[0053] Figure 16 It is the 3D and 2D docking diagrams of LGGEDFDNRM with the active site of Keap1;

[0054] Figure 17 It is the 3D and 2D docking diagrams of LGGEDFDNRM with the active site of MPO;

[0055] Figure 18 It is the 3D and 2D docking diagrams of FDCSQFKPEE with the active site of Keap1;

[0056] Figure 19 It is the 3D and 2D docking diagrams of FDCSQFKPEE with the active site of MPO;

[0057] Figure 20For the antioxidant activity of synthetic peptides. Specific embodiments

[0058] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0059] In the following embodiments, the determination of the DPPH· radical scavenging rate is as follows:

[0060] Referring to the method of (ZHANG Q Z, TONG X H, SUI X N, et al. Antioxidant activity and protective effects of Alcalase-hydrolyzed soybean hydrolysate in human intestinal epithelial Caco-2 cells [J]. Food Research International, 2018, 111, 256-264.), the DPPH· radical scavenging rate is determined and calculated according to formula (1):

[0061]

[0062] Among them, Ac——the absorbance of the control group; As——the absorbance of the test group.

[0063] In the following embodiments, the determination of the ABTS·+ radical scavenging rate is as follows:

[0064] Referring to the method of (HU R J, XU J W, QI G Y, et al. Antioxidative hydrolysates from corn gluten meal may effectively reduce lipid oxidation and inhibit HepG2 cancer cell growth [J]. Journal of Agriculture and Food Research, 2022, 7.), the ABTS·+ radical scavenging rate is determined and calculated according to formula (2):

[0065]

[0066] Among them, Ac is the absorbance of the control group; As is the absorbance of the test group.

[0067] In the following examples, Fe 2+ The determination of the chelation rate is as follows:

[0068] Refer to the method of (ZHANG Y J, DING X J, LI M Q, et al. Preparation, characterization and in vitro stability of iron-chelating peptides from mung beans[J]. Food Chemistry, 2021, 349: 129101 - 129101.) to determine the Fe 2+ The chelation rate is determined and calculated according to formula (3):

[0069]

[0070] Among them, A1 is the absorbance of the test group; A2 is the absorbance of the control group; A0 is the absorbance of the blank group.

[0071] Example 1

[0072] A preparation method of an antioxidant peptide from Ruditapes philippinarum protein source, the preparation process flow is as follows: Ruditapes philippinarum → enzymatic hydrolysis → ultrafiltration separation to retain the fraction with a molecular weight < 1 kDa → in vitro animal experiment to verify the activity → gel chromatography separation and purification → LC-MS / MS determination of the amino acid sequence → molecular docking to determine the active site → solid-phase synthesis to determine the antioxidant property.

[0073] The preparation method of the antioxidant peptide from Ruditapes philippinarum includes the following steps:

[0074] (1) Enzymatic hydrolysis of Ruditapes philippinarum:

[0075] The enzymatic hydrolysis conditions are as follows: the enzymatic hydrolysis temperature is 40 - 60 °C, the enzymatic hydrolysis time is 2 - 6 h, the enzyme-substrate ratio is 0.05% - 0.6%, and the solid-liquid ratio is 1:1 - 5; the enzyme is any one of alkaline protease, flavor protease, and compound protease;

[0076] (2) Preparation of the antioxidant peptide from Ruditapes philippinarum:

[0077] The above enzymatic hydrolysate is ultrafiltered and separated by ultrafiltration membranes with molecular weight cut-off ranges of 5 kDa, 3 kDa, and 1 kDa to divide the enzymatic hydrolysis product into 4 components with different molecular weight ranges. The 4 components with different molecular weight ranges are: components with a molecular weight < 1 kDa, 1 - 3 kDa, 3 - 5 kDa, and > 5 kDa; with DPPH· radical scavenging rate, ABTS·+ radical scavenging rate, Fe2+ Evaluated by the chelation rate.

[0078] (3) Purification of the antioxidant peptide from Ruditapes philippinarum:

[0079] Before purifying the antioxidant peptide from Ruditapes philippinarum, first verify the in vivo antioxidant activity of this bioactive peptide through the Caenorhabditis elegans model, evaluate it using indicators such as the lifespan of nematodes, stress resistance, and antioxidant enzyme activity, and then use this fraction for subsequent separation and purification. Collect the fraction with the best antioxidant activity, filter it through a 0.22 μm filter membrane, and perform gel chromatography separation using Sephadex G-15 as the separation medium. The eluent is ultrapure water, the flow rate is 1 mL / min, measure the antioxidant activity of the eluted fractions corresponding to each absorption peak, and freeze-dry to obtain high-purity antioxidant peptide;

[0080] (4) Determination of the amino acid sequence of the antioxidant peptide from Ruditapes philippinarum:

[0081] Collect the eluted fraction with the highest antioxidant activity (RPPH-1A) and determine the amino acid sequence using LC-MS / MS;

[0082] (5) Identification of the active site of the antioxidant peptide from Ruditapes philippinarum:

[0083] Download the Keap1 protein (PDB ID: 6QMK) and MPO protein (PDB ID: 3F9P) from NCBI, and perform molecular docking of these two receptor protein models with the screened amino acid sequence;

[0084] (6) Solid-phase synthesis of the antioxidant peptide from Ruditapes philippinarum:

[0085] Synthesize the bioactive peptide by solid-phase synthesis and determine its in vitro antioxidant activity.

[0086] Single-factor optimization experiment: In the enzymatic hydrolysis process, take the degree of hydrolysis of Ruditapes philippinarum protein and the scavenging rate of ABTS·+ free radicals as the investigation indexes, and explore the effects of different enzyme types, solid-liquid ratio, enzyme dosage, hydrolysis time, and hydrolysis temperature on the extraction rate of Ruditapes philippinarum polypeptide and the free radical scavenging rate respectively; Weigh 10 g of fresh clam meat into a conical flask, add 30 g of deionized water and homogenize. After homogenization, add any one of alkaline protease, flavor protease, and compound protease at an addition amount of 0.2%, and hydrolyze at 40-60 °C for 2-6 h. Then centrifuge at 9,000 rpm for 10 min to remove the precipitate and obtain the hydrolysate.

[0087] (1) Influence of enzyme types: In order to screen the protease with higher enzymatic hydrolysis efficiency for Ruditapes philippinarum, this experiment selected alkaline protease, flavor protease, compound protease, and double enzyme (alkaline protease + compound protease) for hydrolysis; The results are as Figure 1As shown, the antioxidant capacities of the polypeptides obtained by enzymatic hydrolysis with three proteases are not the same. Among them, the enzymatic hydrolysate of compound protease has the strongest antioxidant capacity, with an ABTS·+ scavenging rate of 94.24% and a degree of hydrolysis of 45.57%. Considering comprehensively, compound protease was selected for subsequent research.

[0088] (2) Effect of hydrolysis temperature: As can be seen from Figure 2 it, the degree of hydrolysis of Ruditapes philippinarum protein shows a trend of first increasing and then decreasing with the increase of hydrolysis temperature. The degree of hydrolysis is the highest at 50 °C, and the ABTS·+ scavenging rate can reach 94.36%. It decreases significantly after 50 °C. Therefore, considering comprehensively, hydrolysis temperatures of 45 °C, 50 °C, and 55 °C were selected for the response surface experiment.

[0089] (3) Effect of hydrolysis time: As can be seen from Figure 3 it, the degree of hydrolysis of Ruditapes philippinarum protein shows a trend of first increasing and then decreasing with the increase of hydrolysis temperature. The degree of hydrolysis reaches the peak at 4 h, and then the degree of hydrolysis decreases slightly. Considering comprehensively, hydrolysis times of 3 h, 4 h, and 5 h were selected for the response surface experiment.

[0090] (4) Effect of enzyme dosage: As can be seen from Figure 4 it, with the increase of enzyme dosage, the binding probability of protein and enzyme increases, and the degree of hydrolysis of the reaction system gradually increases. When the enzyme dosage is 0.2%, the ABTS·+ scavenging activity reaches the maximum value of 96.34%. Further increasing the enzyme dosage has little effect on the free radical scavenging rate. Considering comprehensively, 0.2% was selected as the fixed experimental condition for the follow-up.

[0091] (5) Effect of solid-liquid ratio: As can be seen from Figure 5 it, the degree of hydrolysis of Ruditapes philippinarum protein shows a trend of first increasing and then decreasing with the increase of hydrolysis temperature. The ABTS·+ scavenging activity reaches the maximum value of 96.65% at a solid-liquid ratio of 1:3. Considering comprehensively, solid-liquid ratios of 1:2, 1:3, and 1:4 were selected for the response surface experiment.

[0092] Example 2

[0093] (1) Response surface optimization experiment: According to the aforementioned experimental design scheme, with the degree of hydrolysis of Ruditapes philippinarum antioxidant peptides as the response value, hydrolysis temperature (A), hydrolysis time (B), and solid-liquid ratio (C) were selected as the investigation indexes. The Box-Behnken test design of three factors and three levels was carried out using Design Expert 13.0 software, and the variance analysis of the significance test of the regression equation was carried out. The results are shown in Table 1. The model P = 0.0011 < 0.05, and the model reaches significance. The lack-of-fit item P = 0.4503 > 0.05 is not significant. Therefore, the quadratic model holds, and this model can be used to predict the optimal hydrolysis conditions. The regression coefficient R of the model 2When it is > 0.8, the model can be used to explain the data changes and the relationships between various parameters. The determination coefficient of the prediction quadratic model is 0.9471, indicating a good fit between the model and the actual situation. The optimal technological conditions for clam meat obtained by response surface optimization are temperature 54.216 °C, time 4.019 h, solid-to-liquid ratio 1:3, and degree of hydrolysis 46.815%. For ease of industrial operation, the enzymolysis conditions were set as temperature 54 °C, time 4 h, and solid-to-liquid ratio 1:3. Under these conditions, the experiment was repeated 3 times, and the degree of hydrolysis of the obtained enzymolysis solution was 46.9%, with no significant difference from the software optimization results. Therefore, it was selected as the final enzymolysis condition.

[0094] Table 1 Variance analysis of the response surface quadratic model

[0095]

[0096] Note: ** indicates extremely significant difference (P < 0.01), and * indicates significant difference (P < 0.05).

[0097] Table 2 Variance analysis results of the quadratic model

[0098]

[0099] (2) Response surface analysis of the interaction of each factor: According to the obtained regression equation, response surface plots and contour plots were made. Two of the three factors of enzymolysis temperature (A), enzymolysis time (B), and solid-to-liquid ratio (C) were fixed at the origin in turn, and the response surface plot and contour plot of the other factor were obtained. The three-dimensional response surface plot and contour plot of the interaction of each factor on the degree of hydrolysis are as Figures 6 - 8 shown, indicating that there is a certain interaction among the factors.

[0100] Example 3

[0101] (1) Preparation of antioxidant peptides from Ruditapes philippinarum: The above enzymolysis solution was ultrafiltered and separated using ultrafiltration membranes with molecular weight cut-off ranges of 5 kDa, 3 kDa, and 1 kDa. The enzymolysis products were divided into 4 components with different molecular weight ranges, namely components with molecular weight < 1 kDa, 1 - 3 kDa, 3 - 5 kDa, and > 5 kDa, denoted as RPPH-1, RPPH-2, RPPH-3, RPPH-4, and the unultrafiltered component RPPH. The DPPH· radical scavenging rate, ABTS·+ radical scavenging rate, and Fe 2+ chelating rate were used as evaluation indicators. As Figure 9 can be seen, within the concentration range of 2 - 10 mg / mL, each component showed good in vitro antioxidant activity and had a dose-dependent relationship. Among them, RPPH-1 had the strongest antioxidant ability, so RPPH-1 was selected for subsequent analysis.

[0102] (2) To determine the antioxidant effect of this bioactive peptide in vivo, RPPH-1 was dissolved in OP50 and the final concentrations of 0.5 mg / ml, 1 mg / mL, and 1.5 mg / mL were prepared, and then fed to Caenorhabditis elegans to observe the in vivo activity of this antioxidant peptide. Lifespan is the most intuitive biological indicator to evaluate the aging process of nematodes. The results are as Figure 10 shown in a. The average lifespan of nematodes in the Control group and the 1.5 mg / mL group was 13.15 ± 0.52 days and 17.22 ± 0.61 days, respectively, and the lifespan extended with the increase of the administration concentration; Figure 10 b and 10c are the lifespan curves of nematodes under oxidative stress and heat stress, respectively. The survival rate and lifespan of the treated nematodes were significantly increased, and the stress resistance was enhanced.

[0103] Lipofuscin, an oxidative by-product of lysosomal degradation, is a recognized aging marker. As Figure 11 shown in a, exposure to RPPH-1 significantly reduced the accumulation of lipofuscin in nematodes. The 1.5 mg / mL fraction could reduce the lipofuscin level to 86.38%. As Figure 11 shown in b, the accumulation of ROS showed a concentration-dependent trend with RPPH-1. The fluorescence intensity of the treated nematodes became weaker, and the ROS content was significantly reduced. RPPH-1 at 1.5 mg / mL could reduce the ROS content to 85.88%. Aging is also closely related to the redox system in nematodes. Excessive accumulation of ROS is affected by superoxide dismutase (SOD) and catalase (CAT), while malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) are common indicators reflecting the level of lipid peroxidation. Compared with the Control group, the CAT content, GSH-Px content, and SOD content in the 1.5 mg / mL treatment group increased by 45.1% (P < 0.0005), 59.05%, and 35.15% (P < 0.00005), respectively, and the MDA content decreased by 39.64% (P < 0.0005). The above results indicate that RPPH-1 has a certain activating effect on the antioxidant system.

[0104] Example 4

[0105] (1) Purification of the antioxidant peptide from Ruditapes philippinarum: The fraction RPPH-1 with the best antioxidant activity was collected, freeze-dried, filtered through a 0.22 μm filter membrane, and gel chromatography separation was carried out using Sephadex G-15 as the separation medium. The eluent was ultrapure water, and the flow rate was 1 mL / min. The gel chromatogram is as Figure 13 shown. A total of three different peak components were separated, which were labeled as RPPH-1A, RPPH-1B, and RPPH-1C in sequence. After collecting each component and freeze-drying, its antioxidant activity was measured as Figure 14As shown, RPPH-1A has the strongest antioxidant capacity: the DPPH· radical scavenging rate reaches 21.37%, the ABTS·+ radical scavenging rate reaches 55.69%, and the Fe 2+ chelating rate reaches 55.87%, which is significantly higher than other components. Therefore, RPPH-1A was selected for subsequent analysis.

[0106] (2) Amino acid sequence determination of antioxidant peptides from Ruditapes philippinarum: To further study the structure-activity relationship between the structure of RPPH-1A and its antioxidant effect, the structure of RPPH-1A was first identified. The results of its first-order mass spectrometry are shown in Figure 15 , and a total of 359 amino acid sequences of Ruditapes philippinarum were identified. For these sequences, the Peptide Ranker website was first used to predict the biological activity of the screened amino acid sequences, and the screening criterion was a predicted score > 0.5. The ToxinPred website predicted the toxicity of the amino acid sequences, the Innovagen website predicted the water solubility of the amino acid sequences, and the protein database BIO-PEP-UWM was used to search for new peptide sequences, so as to screen out active peptide segments with unknown functions. A total of 19 non-toxic, water-soluble, and bioactive peptide segments were screened for subsequent research, and the results are shown in Table 3.

[0107] Table 3 Prediction results of amino acid sequences of antioxidant peptides

[0108]

[0109]

[0110] (3) Identification of the active sites of antioxidant peptides from Ruditapes philippinarum: The structures of the selected amino acid sequences were constructed using Chemdraw 23. The Keap1 and MPO receptor protein models were imported into the Discovery Studio software, and the water molecules of the receptor proteins were removed and hydrogen atoms were added under the CHARMM force field environment. The maximum cavity volumes of the Keap1 and MPO receptor proteins were selected as the docking pockets. The docking active position coordinates of the Keap1 receptor protein were: X = 14.204, Y = 64.957, Z = -28.186. The docking active position coordinates of the MPO receptor protein were: X = -28.997, Y = 10.271, Z = -33.493. The remaining parameters were set as default values. The successfully constructed peptide segments were molecularly docked with the Keap1 and MPO receptor protein models using the Discovery Studio software, and the docking results are shown in Table 4. The lower the energy value of the CDOCKER interaction energy, the stronger the binding ability of the peptide segment to the receptor protein, and the more potential antioxidant effect the peptide segment has. Therefore, based on the molecular docking results of the peptide segments with the Keap1 and MPO receptor proteins, the amino acid sequences of LGGEDFDNRM, FDCSQFKPEE, KTDAMAGMGGM, IDPRSDCAF, and IAQTWDGEF shown in SEQ ID NO.8, 9, 13, 16, and 18 were selected for subsequent studies.

[0111] Table 4 Docking energy values of protein peptides from Ruditapes philippinarum with two receptor proteins

[0112]

[0113]

[0114] (4) Conformational analysis

[0115] To further clarify the interaction mechanism between the amino acid sequences of LGGEDFDNRM, FDCSQFKPEE, KTDAMAGMGGM, IDPRSDCAF, and IAQTWDGEF shown in SEQ ID NO.8, 9, 13, 16, and 18 and the Keap1 and MPO receptor proteins, conformational analysis was performed on the two amino acid sequences of LGGEDFDNRM and FDCSQFKPEE with the lowest docking scores in step (2).

[0116] Figure 16 Figure is the conformational analysis result of LGGEDFDNRM and the Keap1 receptor protein. As can be seen from the figure, Arg415, Arg483, Arg380, His436, Ser602, and Ala366 are the main amino acid sites for the interaction between LGGEDFDNRM and the Keap1 receptor protein, and the main force is van der Waals force.Figure 17 It is the conformational analysis result of LGGEDFDNRM and the MPO receptor protein. As can be seen from the figure, the direct binding between the two is achieved through the amino acid sites of Thr502, Lys505, Asp321, LEU33, Trp32, Phe439, Lys308, and Arg504, and the indirect binding is achieved through the amino acid sites of Val320, Ser319, Ala435, Arg438, Leu33, Val320, Gly441, Cys440, and Tyr309. The van der Waals force is the main acting force.

[0117] Figure 18 It is the conformational analysis result of FDCSQFKPEE and the Keap1 receptor protein. As can be seen from the figure, Tyr334, Phe577, Arg483, Tyr572, Tyr525, Val418, Val465, Val512, Ile559, Leu365, Leu557, and Val604 are the main amino acid sites for the interaction between FDCSQFKPEE and the Keap1 receptor protein. In addition, FDCSQFKPEE binds to the Lys308 and Lys505 amino acid sites of MPO through electrostatic interaction, and binds to the Thr501, Arg504, Phe439, Asp321, and Trp32 amino acid sites of MPO through hydrogen bond interaction. Figure 19 It is the conformational analysis result of FDCSQFKPEE and the MPO receptor protein. As can be seen from the figure, FDCSQFKPEE binds to the Asp321, Ser319, Phe439, and Lys505 amino acid sites of the MPO receptor protein through hydrogen bonds, and interacts with the Thr501, Asn317, Arg438, Tyr309, Cys440, Thr501, Leu33, and Val320 amino acid sites of the MPO receptor protein through indirect interaction.

[0118] Example 5

[0119] To further verify the antioxidant activities of LGGEDFDNRM and FDCSQFKPEE, two peptide segments of LGGEDFDNRM and FDCSQFKPEE were synthesized by solid-phase synthesis method, and their purities exceeded 99%. Their molecular weights were 1062.505 Da and 876.455 Da respectively. 5 mg of each of the antioxidant peptides of LGGEDFDNRM and FDCSQFKPEE was dissolved in 1 mL of ultrapure water to prepare antioxidant peptide solutions with a concentration of 5 mg / mL. Vitamin C with a mass concentration of 5 mg / mL was used as the control group for in vitro antioxidant determination and analysis. Each peptide segment was tested 3 times as the original data, and the measurement results are as Figure 3 shown, by Figure 20It can be seen that FDCSQFKPEE has the most obvious antioxidant effect, comparable to that of vitamin C, and LGGEDFDNRM follows next.

[0120] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Antioxidant peptide from Philippine clam, characterized in that: Includes at least one of the following antioxidant peptides; The amino acid sequence of the Philippine clam antioxidant peptide 1 is LGGEDFDNRM, as shown in SEQ ID NO.13; The amino acid sequence of the Philippine clam antioxidant peptide 2 is FDCSQFKPEE, as shown in SEQ ID NO.18; The amino acid sequence of the Philippine clam antioxidant peptide 3 is KTDAMAGMGGM, as shown in SEQ ID NO.9; The amino acid sequence of the Philippine clam antioxidant peptide 4 is IDPRSDCAF, as shown in SEQ ID NO.8; The amino acid sequence of the Philippine clam antioxidant peptide 5 is IAQTWDGEF, as shown in SEQ ID NO.

16.

2. The method for preparing the antioxidant peptide of Ruditapes philippinarum according to claim 1, characterized in that: The following steps are involved: (1) Enzymatic hydrolysis of Philippine clams: The enzymolysis conditions are as follows: an enzymolysis temperature of 40 to 60° C., an enzymolysis time of 2 to 6 hours, an enzyme-substrate ratio of 0.05% to 0.6%, and a solid-liquid ratio of 1:1 to 5; (2) Preparation of antioxidant peptides from Philippine clam: The enzymatic hydrolysate was separated by ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off range of 5 kDa, 3 kDa, and 1 kDa to divide it into four components with different molecular weight ranges. 2+ The chelation rate was used as an indicator for evaluation; (3) Purification of antioxidant peptides from Philippine clam: The fractions with the best antioxidant activity were collected, filtered through a 0.22 μm filter membrane, and separated by gel chromatography using Sephadex G-15 as the separation medium. The eluent was ultrapure water at a flow rate of 1 mL / min. The antioxidant activity of the eluted components corresponding to each absorption peak was determined, and high-purity antioxidant peptides were obtained by freeze drying. (4) Determination of amino acid sequence of antioxidant peptide from Philippine clam: The eluted fractions with the highest antioxidant activity were collected and the amino acid sequence was determined by LC-MS / MS; (5) Identification of the active site of the antioxidant peptide from the Philippine clam: Molecular docking is performed between the target receptor protein model and the amino acid sequence obtained by screening; (6) Solid phase synthesis of Philippine clam antioxidant peptides: The active peptide was synthesized by solid phase and its in vitro antioxidant activity was determined.

3. The method for preparing the antioxidant peptide of Ruditapes philippinarum according to claim 2, characterized in that: In step (1), the enzyme is any one of alkaline protease, flavor protease and composite protease.

4. The method for preparing the antioxidant peptide of Ruditapes philippinarum according to claim 2, characterized in that: In step (3), the four components with different molecular weight ranges are: components with molecular weights of <1 kDa, 1-3 kDa, 3-5 kDa, and >5 kDa.

5. The method for preparing the antioxidant peptide of Ruditapes philippinarum according to claim 2, characterized in that: In step (3), before purifying the Philippine clam antioxidant peptide, the antioxidant activity of the active peptide in vivo is first verified using a Caenorhabditis elegans model, and the nematode lifespan, stress resistance, antioxidant enzyme activity, etc. are used as indicators for evaluation, and then the component is used for subsequent separation and purification.

6. Use of the Philippine clam antioxidant peptide according to claim 1 in functional foods, health products, food additives and cosmetics.

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