A bovine lung enzymatic hydrolysate, antioxidant active peptide, and preparation method and application

Abstract

The application belongs to the technical field of active peptide extraction and identification, and particularly relates to a bovine lung enzymatic hydrolysate, an antioxidant active peptide, and a preparation method and application thereof. The bovine lung enzymatic hydrolysate uses bovine lung as an extraction raw material, extracts natural active peptides by means of enzymolysis, collects the enzymolysis extract, and identifies the same by mass spectrometry. In combination with bioinformatics analysis and experimental characterization, 12 new antioxidant peptide sequences are obtained, and the antioxidant effect thereof is verified, so that the bovine lung enzymatic hydrolysate can be applied to natural antioxidant products.

Description

Technical Field

[0001] This invention belongs to the field of active peptide extraction and identification technology, specifically relating to a bovine lung enzymatic hydrolysate, an antioxidant active peptide, its preparation method, and its application. Background Technology

[0002] Bioactive peptides are low-molecular-weight polymers with special physiological regulatory functions. They are important active ingredients in certain medicines or functional foods. Compared with proteins, bioactive peptides have advantages such as small molecular weight, high stability, and easy absorption.

[0003] Oxidation of biomolecules is a ubiquitous reaction in living organisms, leading to the release of a large number of free radicals. These free radicals are highly reactive and disrupt the body's electron balance. Excessive free radicals can cause many diseases. Studies show that when there are excessive oxygen free radicals in the body, they can cause a series of oxidative damages. This is especially true when the body is aging, ill, or fatigued, as the balance of free radicals can be disrupted, causing a series of oxidative damages. In severe cases, this can even trigger or aggravate physiological disorders and pathological changes in cells, tissues, and even the entire body, leading to various diseases such as diabetes, Alzheimer's disease, heart disease, and cancer. Antioxidants neutralize free radicals and inhibit oxidative diffusion, thus scavenging excess ROS. Appropriate supplementation with antioxidants can reduce the adverse effects of oxidative stress on the body. However, while traditional synthetic antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ), possess good antioxidant functions, they also have certain drawbacks, including kidney damage, liver toxicity, teratogenicity, and carcinogenicity. In contrast, natural antioxidants extracted from organisms not only have better antioxidant effects but also have the advantages of wide availability and no toxic side effects. Therefore, natural bioactive peptides with antioxidant properties can be used as nutritional supplements and natural antioxidants in oxidative stress treatment.

[0004] Bovine lung, a byproduct of cattle production, contains a large amount of protein and is theoretically a high-quality source of antioxidant peptides. The field anticipates that enzymatic hydrolysis of bovine lung can degrade proteins into various bioactive peptides. Furthermore, by employing peptidomics combined with database screening, the antioxidant peptide sequences can be obtained from the bioactive peptide fragments obtained through enzymatic hydrolysis, and their application in production could significantly improve the economic efficiency of bovine byproducts. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a naturally extracted bovine lung hydrolysate, which has good antioxidant properties and the advantages of being widely available and having no side effects.

[0006] The second objective of this invention is to provide a method for preparing the above-mentioned bovine lung enzymatic hydrolysate and its application.

[0007] A third objective of this invention is to provide an antioxidant active peptide, which has good antioxidant properties.

[0008] To solve the above-mentioned technical problems, the present invention provides a method for preparing bovine lung enzymatic hydrolysate, comprising the following steps:

[0009] (1) Take raw beef lung, add water and make into a paste to obtain beef lung paste;

[0010] (2) Add protease to the bovine lung slurry for enzymatic hydrolysis.

[0011] Specifically, in the method for preparing the bovine lung enzymatic hydrolysate, the protease includes at least one of a complex protease, a neutral protease, an alkaline protease, a flavor protease, and papain.

[0012] Preferably, the protease includes papain.

[0013] Specifically, in the method for preparing the bovine lung enzymatic hydrolysate, step (1) includes:

[0014] The ratio of the bovine lung raw material to water is 5-25 wt%, preferably 10-20 wt%, and more preferably 15 wt%; and / or,

[0015] The pH value of the bovine lung slurry is adjusted to 5-9, preferably 6-9, more preferably 7.26-8.16, and even more preferably 7.7.

[0016] Specifically, in the method for preparing the bovine lung enzymatic hydrolysate, step (2) includes:

[0017] The protease is added at a concentration of 1000-5000 U / g based on the bovine lung; preferably 3000-5000 U / g, more preferably 3124.64-3730.11 U / g, and even more preferably 3400 U / g; and / or,

[0018] The temperature for the enzymatic hydrolysis step is 40-60℃, preferably 45-55℃, and more preferably 50℃; and / or,

[0019] The enzymatic hydrolysis time for the enzymatic hydrolysis step is 2-6 hours, preferably 3-5 hours, more preferably 3.21-4.55 hours, and even more preferably 3.9 hours.

[0020] Specifically, the method for preparing bovine lung enzymatic hydrolysate further includes a step of purifying the crude product obtained from the enzymatic hydrolysis step;

[0021] Preferably, the purification step includes ultrafiltration;

[0022] Preferably, the method includes the step of obtaining peptide components with molecular weights of <3kDa, 3-10 kDa and >10 kDa respectively by ultrafiltration.

[0023] Specifically, in the method for preparing the bovine lung enzymatic hydrolysate, the purification step includes purifying peptide components with a molecular weight of less than 3 kDa using gel chromatography.

[0024] Preferably, the gel chromatography method uses cross-linked dextran gel;

[0025] Preferably, the cross-linked dextran gel is Sephadex;

[0026] Preferably, the Sephadex is Sephadex G-25.

[0027] Gel chromatography was used for analysis. The sample concentration was 15-25 mg / ml, the sample volume was 3-7 ml, the flow rate was 1-2 ml / min, and one tube was collected every 1-3 min. The absorbance was measured at 220 nm and an absorbance curve was plotted. The eluent collected from tubes 14-16 was designated as BLP-1, and the eluent collected from tubes 24-26 was designated as BLP-2. Preferably, peptide fractions with a molecular weight less than 3 kDa were loaded onto the gel chromatography column at a concentration of 20 mg / ml, a sample volume of 5 ml, and a flow rate of 1.5 ml / min. One tube was collected every 2 min, and the absorbance was measured at 220 nm and an absorbance curve was plotted. The eluent collected from tubes 14-16 was designated as BLP-1, and the eluent collected from tubes 24-26 was designated as BLP-2.

[0028] And / or, the gel chromatography column has dimensions of 1.6 cm × 50 cm.

[0029] As used herein, the bovine lung hydrolysate contains bovine lung peptide (BLP), which can also be referred to as crude bioactive peptide extract BLP.

[0030] The present invention also discloses a bovine lung enzymatic hydrolysate prepared by the method described above;

[0031] Preferably, the bovine lung hydrolysate comprises at least one of the active peptides having the following sequence structure:

[0032] KF6: KPFPFF (see SEQ ID NO.1);

[0033] MP6: MWPPLP (see SEQ ID NO.2);

[0034] FG6: FYGWPG (see SEQ ID NO.3);

[0035] DW6: DGGGWW (see SEQ ID NO.4);

[0036] FP7: FGPPPPP (see SEQ ID NO.5);

[0037] WG6: WGPPGG (see SEQ ID NO. 6);

[0038] FW7: FFSPGVW (see SEQ ID NO.7);

[0039] PG16: PPPGPPPPPGPPPPPG (see SEQ ID NO. 8);

[0040] DGG6: DGGAWW (see SEQ ID NO.9);

[0041] GL8: GWNIPMGL (see SEQ ID NO.10);

[0042] GP9: GPPPAPPLP (see SEQ ID NO.11);

[0043] MF6: MIKPFF (see SEQ ID NO.12).

[0044] The present invention also discloses an antioxidant active peptide, wherein the antioxidant active peptide comprises at least one of the active peptides having the following sequence structure:

[0045] KF6: KPFPFF (see SEQ ID NO.1);

[0046] MP6: MWPPLP (see SEQ ID NO.2);

[0047] FG6: FYGWPG (see SEQ ID NO.3);

[0048] DW6: DGGGWW (see SEQ ID NO.4);

[0049] FP7: FGPPPPP (see SEQ ID NO.5);

[0050] WG6: WGPPGG (see SEQ ID NO. 6);

[0051] FW7: FFSPGVW (see SEQ ID NO.7);

[0052] PG16: PPPGPPPPPGPPPPPG (see SEQ ID NO. 8);

[0053] DGG6: DGGAWW (see SEQ ID NO.9);

[0054] GL8: GWNIPMGL (see SEQ ID NO.10);

[0055] GP9: GPPPAPPLP (see SEQ ID NO.11);

[0056] MF6: MIKPFF (see SEQ ID NO.12).

[0057] The present invention also discloses the use of the bovine lung enzymatic hydrolysate or the antioxidant active peptide in the preparation of antioxidants;

[0058] Preferably, the antioxidant includes food antioxidants, pharmaceutical antioxidants, or health product antioxidants.

[0059] The present invention also discloses the use of the bovine lung enzymatic hydrolysate or the antioxidant active peptide in the preparation of drugs;

[0060] The drug has the effect of improving liver morphology or protecting hepatocyte function, or the drug is used to prevent or treat alcoholic liver injury.

[0061] The method for preparing bovine lung enzymatic hydrolysate of the present invention uses bovine lung as the extraction raw material and extracts natural active peptides by enzymatic hydrolysis using protease. The bovine lung enzymatic hydrolysate has good antioxidant properties and can be used in the application of natural antioxidant products.

[0062] The present invention describes a method for preparing bovine lung enzymatic hydrolysate. Through screening of hydrolytic enzymes and optimization of single hydrolysis factors, an extraction process for the hydrolysate with strong antioxidant activity has been established. The preparation method preferably uses papain as the hydrolytic enzyme, adjusting the ratio of bovine lung raw material to water to 5-25 wt%, the pH of the bovine lung slurry to 5-9, and the concentration of the protease based on the bovine lung to 1000-5000 U / g. The method also controls the temperature of the enzymatic hydrolysis step to 40-60℃ and the hydrolysis time to 2-6 hours. This method yields an enzymatic hydrolysate with good antioxidant properties.

[0063] The method for preparing bovine lung enzymatic hydrolysate according to the present invention further utilizes response surface methodology to simulate and predict the process parameters of the preparation method. The optimal enzyme concentration is 3800 U / g, pH value is 7.20, enzymatic hydrolysis time is 3.2 h, material-to-liquid ratio is 15wt%, and temperature is 50℃, which can obtain a better enzymatic hydrolysis process.

[0064] The antioxidant active peptides of this invention were obtained by mass spectrometry identification and separation of bovine lung enzymatic hydrolysate, combined with bioinformatics analysis and experimental characterization, resulting in 12 active peptides with novel antioxidant peptide sequences. Their antioxidant effects were verified and they can be used in the application of natural antioxidant products.

[0065] The antioxidant peptides described in this invention, after further screening and identification, are preferably the antioxidant peptides FYGWPG, GPPPAPPLP, and WGPPGG protease, which have extremely strong stability and superior application performance.

[0066] The bovine lung enzymatic hydrolysate and antioxidant active peptides described in this invention not only have significant free radical scavenging activity, but also play a therapeutic role in alcoholic liver injury by improving liver morphology, protecting hepatocyte function, reducing inflammation and enhancing antioxidant capacity. Attached Figure Description

[0067] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0068] Figure 1 The extraction process of crude bovine lung enzymatic hydrolysate as described in this invention;

[0069] Figure 2 The results of single-factor screening of proteases in Example 1 are shown; where E1-E5 represent complex protease, alkaline protein powder, flavor protease, papain, and neutral protease, respectively; the different letters a, b, and c in the figure represent statistically significant differences, p < 0.05.

[0070] Figure 3 The results are the single-factor optimization results for the preparation of bovine lung enzymatic hydrolysate in Example 2; where A: experimental results for optimizing enzyme concentration; B: experimental results for optimizing enzymatic hydrolysis time; C: experimental results for optimizing material / liquid ratio; D: experimental results for optimizing enzymatic hydrolysis temperature; E: experimental results for optimizing pH.

[0071] Figure 4 The image shows the response surface plots of enzyme concentration and pH to DPPH free radical scavenging rate in Example 3; the left image represents a three-dimensional response surface plot, and the right image represents a two-dimensional contour plot.

[0072] Figure 5 This is an example of the effect of enzyme concentration and hydrolysis time on DPPH free radical scavenging rate in Example 3; the left image represents a three-dimensional response surface plot and the right image represents a two-dimensional contour plot.

[0073] Figure 6This is an example of the effect of pH and enzymatic hydrolysis time on the DPPH free radical scavenging rate in Example 3; the left image represents a three-dimensional response surface plot and the right image represents a two-dimensional contour plot.

[0074] Figure 7 The image shows the response surface plots of enzyme concentration and pH to ABTS free radical scavenging rate in Example 3; the left image represents a three-dimensional response surface plot, and the right image represents a two-dimensional contour plot.

[0075] Figure 8 This shows the effect of enzyme concentration and hydrolysis time on the ABTS free radical scavenging rate in Example 3; the left image represents a three-dimensional response surface plot, and the right image represents a two-dimensional contour plot.

[0076] Figure 9 This is an example of the effect of pH and enzymatic hydrolysis time on the ABTS free radical scavenging rate in Example 3; the left image represents a three-dimensional response surface plot and the right image represents a two-dimensional contour plot.

[0077] Figure 10 The results of the separation and purification of BLP in Example 10 are shown below; where A represents the DPPH and ABTS scavenging rates of BLP with different molecular weights; B represents the product spectrum of <3 kDa BLP after further purification by Sephadex G-25 gel chromatography; and C represents the DPPH and ABTS scavenging rates of BLP-1 and BLP-2 obtained after purification.

[0078] Figure 11 The results of UV and FTIR measurements of the purified BLP-1 component in Example 11 are shown; where A is the UV spectrum of BLP-1 and B is the FTIR spectrum of BLP-1.

[0079] Figure 12 The results of the biosafety assessment of BLP-1 in Example 13 are shown; where A: cytotoxicity assay; B: hemolytic activity assay; and PC is a positive control.

[0080] Figure 13 The figures show the in vitro antioxidant activity test results of BLP-1 in Example 14; where A: DPPH scavenging rate; B: ABTS scavenging rate; C: hydroxyl radical scavenging rate; D: superoxide anion scavenging rate; This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. This indicates that p < 0.0001;

[0081] Figure 14The protective effect of BLP-1 on H2O2-induced HepG2 cells in Example 15 is illustrated. A represents the effect of BLP-1 on the survival rate of H2O2-induced HepG2 cells; B and C represent the effects of BLP-1 on the levels of SOD (B), CAT (C), GSH-Px (D), and MDA (E) in H2O2-induced HepG2 cells. The different letters in a, b, c, and d in the figure indicate statistically significant differences, with p < 0.05.

[0082] Figure 15 The therapeutic effect of BLP-1 on alcoholic liver disease in mice in Example 16 is shown; where A: liver index; B: ALT level; C: AST level; D: TNF-α level; E: IL-6 level; F: SOD level; G: CAT level; H: GSH-Px level; I: MDA level;

[0083] Figure 16 The results of BLP-1 mass spectrometry identification and sequence screening in Example 17 are shown below; where A: peptide sequence identification by LC-MS / MS; B: active peptide screening results; C: PeptideRanker score statistics.

[0084] Figure 17 The results of molecular docking between the antioxidant peptide and the recombinant Keap1 protein in Example 19 are shown; where A: FG6; B: GP9; C: WG6;

[0085] Figure 18 The results show the cytotoxicity and antioxidant capacity of the synthesized antioxidant peptides in Example 21; where A: cytotoxicity; B: DPPH of the peptide and IC50 of ABTS. 50 Value result. Detailed Implementation

[0086] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0087] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0088] In this invention:

[0089] The Chinese name for DPPH is 2,2-diphenyl-1-picrylhydrazine;

[0090] The Chinese name for ABTS is 2,2'-adiazon-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt;

[0091] The bovine lungs used in the following examples are Simmental cattle lungs, purchased from Changchun Kainongtou Livestock Development Co., Ltd.

[0092] Example 1

[0093] As attached Figure 1 The extraction process shown involves repeatedly washing bovine lungs as raw material, then adding water and stirring to prepare a bovine lung slurry. Water is added at a material-to-liquid ratio of 10 wt%, and the mixture is then stirred with different proteases (based on a bovine lung concentration of 3000 U / g). The mixture is then hydrolyzed for 3 hours at its optimal temperature (50°C for the complex protease, 45°C for the neutral protease, 45°C for the alkaline protease, 50°C for the flavor protease, and 55°C for the papain). The hydrolysate is collected, centrifuged, and the supernatant is collected. The supernatant is the desired bovine lung hydrolysate, which contains bovine lung peptide (BLP), and can also be referred to as crude bioactive peptide extract BLP. In this embodiment, DPPH and ABTS scavenging rates are used as evaluation indicators to detect its antioxidant activity. The supernatant is then freeze-dried for later use.

[0094] Following the extraction steps described above, bovine lung was enzymatically hydrolyzed using compound protease, neutral protease, alkaline protease, flavor protease, and papain, respectively.

[0095] In this embodiment, DPPH and ABTS clearance rates were used as screening indicators. Five proteases were used to enzymatically hydrolyze bovine lung, and the resulting bovine lung hydrolysates were then subjected to DPPH and ABTS clearance rate testing (using a DPPH and ABTS extraction kit purchased from Nanjing Jiancheng Biological Products Institute). The DPPH and ABTS clearance rate test results of the bovine lung hydrolysates obtained from the enzymatic hydrolysis of the five proteases are shown below. Figure 2 As shown in the figure. The results indicate that papain has the best enzymatic hydrolysis effect and the strongest scavenging rate of DPPH and ABTS free radicals. Therefore, papain was used in subsequent experiments.

[0096] Example 2

[0097] This embodiment optimizes the single-factor conditions of the enzymatic hydrolysis process of bovine lung with papain, based on Example 1.

[0098] In this embodiment, the enzyme concentration (1000, 2000, 3000, 4000, 5000 U / g), enzymatic hydrolysis time (2, 3, 4, 5, 6 h), material-to-liquid ratio (5%, 10%, 15%, 20%, 25% (w / v), enzymatic hydrolysis temperature (40, 45, 50, 55, 60℃), and pH (5, 6, 7, 8, 9) during enzymatic hydrolysis were optimized.

[0099] This embodiment uses papain to enzymatically hydrolyze bovine lung. Based on this, five parameters were optimized during the hydrolysis process: enzyme concentration, hydrolysis time, material-to-liquid ratio, hydrolysis temperature, and pH. DPPH and ABTS free radical scavenging capacity were used as detection indicators, and measurements were performed according to the kit instructions. The optimization results are shown below. Figure 3 AE (Chinese AE)

[0100] The results showed that the best enzymatic hydrolysis effect could be obtained when the enzyme concentration was 4000 U / g, the hydrolysis time was 4 h, the material-to-liquid ratio was 15%, the hydrolysis temperature was 50 °C, and the pH was 7.

[0101] Example 3

[0102] Based on Example 2 above, this embodiment further selects three parameters—enzyme concentration, pH, and hydrolysis time—and uses Design Export software to design a three-level, three-factor BBD response test to further optimize the BLP extraction conditions and determine the optimal extraction conditions. The specific optimization method is shown in Table 1 below. Table 2 shows the 17 experimental groups and their corresponding DPPH and ABTS clearance rates generated by Design Export.

[0103] Table 1. Response Surface Design Factors and Levels

[0104]

[0105] Table 2. Response surface methodology and results of DPPH and ABTS clearance rates

[0106]

[0107] This embodiment further utilizes Design-Expert software to perform multivariate regression fitting on the data, obtaining regression model equations Y1 and Y2 for DPPH and ABTS; wherein,

[0108] Y1=87.27-0.7338A-0.8650B-0.5162C+0.9325AB+0.7150AC-0.1225BC-3.04A 2 -2.42B 2 -1.63C 2;

[0109] Y2 = 80.66 - 0.3850A - 0.7900B - 0.6650C + 0.4125AB + 0.7775AC - 0.1825BC - 2.96A 2 - 1.91B 2 - 2.65C 2 。

[0110] The following Table 3 and Table 4 represent the analysis of variance of the regression models for DPPH and ABTS respectively.

[0111] Table 3 Analysis of Variance of DPPH Regression Model

[0112]

[0113] Table 4 Analysis of Variance of ABTS Regression Model

[0114]

[0115] It can be seen that the P - values of both models are ≤ 0.0001, indicating that the two regression models fit extremely significantly. The lack - of - fit terms of the two models P are 0.0965 and 0.3017 respectively, both greater than 0.05, proving that the lack - of - fit terms of the models are not significant. The R 2 values of the DPPH and ABTS regression models are 0.9829 and 0.9734, and the R adj 2 values are 0.9563 and 0.9392 respectively, further proving the reliability of the models, high goodness of fit, and the two models can be used for prediction and analysis of the enzymatic hydrolysis process of bovine lungs.

[0116] In the above DPPH regression model, according to P the values, in the linear terms, A and B have extremely significant effects on the test results (P < 0.01), and the linear term C has a significant effect on the test results (0.01 < P < 0.05). In the interaction terms, AB is extremely significant, AC is significant, and BC is not significant. In the quadratic terms, A 2 and B 2 are super - significant (< 0.0001), and C 2 is extremely significant. The magnitude of the F - value indicates the influence of different factors on the test results. Therefore, the order of the influence of each factor on the DPPH scavenging rate is B (pH) > A (enzyme concentration) > C (enzymatic hydrolysis time). Similarly, in the ABTS regression model, in the linear terms, B is extremely significant, C is significant, and A is not significant; in the interaction terms, AC is significant, AB and BC are not significant; in the quadratic terms, A 2 and B 2 are super - significant, and C 2The effect of each factor on ABTS clearance rate was highly significant. The order of influence was B (pH) > C (enzymatic hydrolysis time) > A (enzyme concentration).

[0117] Furthermore, three-dimensional response surfaces and contour plots can more intuitively represent the strength of interactions between factors. The steepness of the response surface is positively correlated with the influence of each factor on the DPPH and ABTS free radical scavenging rates; a steep response surface indicates a greater influence of the factor on the response value, while a flat response surface indicates the opposite. In the contour plots, the closer the contour lines are to ellipses, the more significant the interaction between the analyzed factors. The results are shown in the attached figure. Figures 4-9 Therefore, for Figures 4-9 Analysis revealed that the effects of various factors on DPPH and ABTS clearance rates were consistent with the results of the analyses in Tables 3 and 4.

[0118] In summary, in this embodiment, under the DPPH model, with an enzyme concentration of 3730.11 U / g, a pH of 8.16, and a hydrolysis time of 4.55 h, the DPPH free radical scavenging rate reached a maximum of 86.12%. Under the ABTS model, with an enzyme concentration of 3124.64 U / g, a pH of 7.26, and a hydrolysis time of 3.21 h, the ABTS free radical scavenging rate reached a maximum of 77.81%.

[0119] Furthermore, taking into account both the optimal extraction conditions of the two models and the operability of the experiment, the extraction conditions were adjusted as follows: enzyme concentration of 3400 U / g, pH value of 7.70, and enzymatic hydrolysis time of 3.9 h.

[0120] Using the optimized conditions described above (enzyme concentration of 3400 U / g, pH 7.70, and hydrolysis time of 3.9 h), the specific method was as follows: water was added at a material-to-liquid ratio of 15 wt% and mixed to obtain bovine lung slurry, which was then adjusted to pH 7.7. Papain was added to the bovine lung slurry at a concentration of 3400 U / g based on the bovine lung content, and the temperature of the bovine lung slurry was adjusted to 50℃ for enzymatic extraction for 3.9 h. Three replicate experiments were conducted for verification, yielding an average DPPH radical scavenging rate of 89.17% and an ABTS radical scavenging rate of 82.78%, with minimal error compared to the predicted values.

[0121] Example 4

[0122] Bovine lungs were thoroughly cleaned and mixed with water at a material-to-liquid ratio of 15 wt% to obtain bovine lung slurry. The pH of the bovine lung slurry was adjusted to 8. Papain was added to the bovine lung slurry at a concentration of 4000 U / g based on the bovine lung. The temperature of the bovine lung slurry was adjusted to 50℃ for enzymatic extraction for 4 hours. The enzymatic hydrolysate was collected, centrifuged, and the supernatant was collected. The supernatant was the desired bovine lung enzymatic hydrolysate, denoted as crude bioactive peptide extract (BLP).

[0123] Example 5

[0124] Bovine lungs were thoroughly cleaned and mixed with water at a material-to-liquid ratio of 10 wt% to obtain bovine lung slurry. The pH of the bovine lung slurry was adjusted to 7.0. Papain was added to the bovine lung slurry at a concentration of 3000 U / g based on the bovine lung content. The temperature of the bovine lung slurry was adjusted to 45℃ for enzymatic hydrolysis and extraction for 5 hours. The hydrolysate was collected, centrifuged, and the supernatant was collected. The supernatant was the desired bovine lung hydrolysate, denoted as crude bioactive peptide extract (BLP).

[0125] Example 6

[0126] Bovine lungs were thoroughly cleaned and mixed with water at a material-to-liquid ratio of 20 wt% to obtain bovine lung slurry. The pH of the bovine lung slurry was adjusted to 6. Alkaline protease was added to the bovine lung slurry at a concentration of 5000 U / g based on the bovine lung. The temperature of the bovine lung slurry was adjusted to 55℃ for enzymatic hydrolysis and extraction for 3 hours. The hydrolysate was collected, centrifuged, and the supernatant was collected. The supernatant was the desired bovine lung hydrolysate, denoted as crude bioactive peptide extract (BLP).

[0127] Example 7

[0128] Bovine lungs were thoroughly cleaned and mixed with water at a material-to-liquid ratio of 12 wt% to obtain bovine lung slurry. The pH of the bovine lung slurry was adjusted to 7.5. Neutral protease was added to the bovine lung slurry at a concentration of 3500 U / g based on the bovine lung. The temperature of the bovine lung slurry was adjusted to 50°C for enzymatic hydrolysis and extraction for 4 hours. The hydrolysate was collected, centrifuged, and the supernatant was collected. The supernatant was the desired bovine lung hydrolysate, denoted as crude bioactive peptide extract (BLP).

[0129] Example 8

[0130] Bovine lungs were thoroughly cleaned and mixed with water at a material-to-liquid ratio of 18 wt% to obtain bovine lung slurry. The pH of the bovine lung slurry was adjusted to 7.2. Flavor protease was added to the bovine lung slurry at a concentration of 4500 U / g based on the bovine lung. The temperature of the bovine lung slurry was adjusted to 50°C for enzymatic extraction for 4 hours. The enzymatic hydrolysate was collected, centrifuged, and the supernatant was collected. The supernatant was the desired bovine lung enzymatic hydrolysate, denoted as crude bioactive peptide extract (BLP).

[0131] Example 9

[0132] Bovine lungs were thoroughly cleaned and mixed with water at a material-to-liquid ratio of 15 wt% to obtain bovine lung slurry. The pH of the bovine lung slurry was adjusted to 6.5. A complex protease was added to the bovine lung slurry at a concentration of 4000 U / g based on the bovine lung. The temperature of the bovine lung slurry was adjusted to 50°C for enzymatic hydrolysis and extraction for 4 hours. The hydrolysate was collected, centrifuged, and the supernatant was collected. The supernatant was the desired bovine lung hydrolysate, denoted as crude bioactive peptide extract (BLP).

[0133] Example 10

[0134] This embodiment follows the optimal extraction conditions described in Example 3 above (material-to-liquid ratio 15wt%, enzyme concentration 3400 U / g, pH 7.70, hydrolysis temperature 50℃, hydrolysis time 3.9 h), and further follows... Figure 1 The steps shown describe the extraction of crude bioactive peptide BLP from bovine lung, followed by separation and purification using ultrafiltration and Sephadex G-25 gel chromatography to obtain the optimal antioxidant components.

[0135] In this embodiment, the DPPH and ABTS scavenging rates were further determined using a DPPH and ABTS extraction kit purchased from Nanjing Jiancheng Biological Products Institute, and this was used as an evaluation index to optimize the extraction method of bovine lung antioxidant peptides.

[0136] This embodiment uses conventional ultrafiltration technology to separate the crude bioactive peptide extract (BLP) into three fractions: <3kDa, 3-10 kDa, and >10kDa. (See attached image) Figure 10 The results, based on DPPH and ABTS scavenging rates, show that the fraction <3 kDa exhibits the strongest antioxidant capacity (e.g., ...). Figure 10 (A). Subsequently, <3 kDa was further purified using Sephadex G-25 gel chromatography.

[0137] The specific steps for further purification of <3 kDa bioactive peptides using Sephadex G-25 gel chromatography are as follows: Sephadex G-25 was swelled in deionized water for 3 hours at room temperature. After swelling, a 40 cm³ column was packed. The collected <3 kDa bioactive peptide crude extract (BLP) was prepared as a sample solution at a concentration of 20 mg / mL and filtered through a 0.22 μm filter. The prepared BLP sample solution was loaded onto a Sephadex G-25 column (1.6 cm × 50 cm) at a flow rate of 1.5 mL / min. The loading concentration was 20 mg / mL, the loading volume was 5 mL, the flow rate was 1.5 min / mL, and one tube was collected every 2 min. The absorbance was detected at 220 nm and an absorbance curve was plotted. Two peaks, BLP-1 and BLP-2, appeared (e.g., ...). Figure 10 (B) The eluent collected from tubes 14-16 was BLP-1, and the eluent collected from tubes 24-26 was BLP-2. Component BLP-1 exhibited stronger antioxidant activity than component BLP-2 (e.g., ...). Figure 10 (C). Therefore, in this embodiment, the optimal antioxidant component BLP-1 was selected, freeze-dried, and stored for further testing.

[0138] Example 11

[0139] In this embodiment, the BLP-1 component further purified in Example 10 above was used for UV and FTIR measurements.

[0140] In this embodiment, the ultraviolet spectrum of BLP-1 powder was measured using a UV-Vis-NIR spectrophotometer, with a scanning range of 200-800 nm. ARP powder and potassium bromide powder (1:100) were mixed and ground, and then the sample was analyzed using an IS5 FTIR spectrometer (Thermo Fisher, Waltham), with a scanning range of 500-4000 cm⁻¹. -1 .

[0141] Figure 11 Image A shows the reflectance variation of component BLP-1 in the wavelength range of 200-800 nm. Notably, an absorption peak is observed at 220 nm, which is typically due to n-π* transitions in peptides at this wavelength. This electronic transition characteristic of peptide bonds is a typical optical feature of peptides.

[0142] Figure 11 B in the middle is BLP-1 at wavenumbers of 4000-500 cm⁻¹ Infrared absorption spectrum within the range. At approximately 3379.22 cm⁻¹. The strong absorption peak at approximately 1634.36 cm⁻¹ is attributed to the stretching vibration of the N-H bond in the polypeptide molecule, indicating the presence of an amino group in the polypeptide. The intensity and position of this peak provide strong evidence for the presence of an amino group in the polypeptide structure. The absorption peak at 1409.97 cm⁻¹ corresponds to the stretching vibration of the C=O bond (amide I band), a typical characteristic absorption peak of peptide bonds, reflecting the structural features of the polypeptide backbone. The nearby absorption peak (amide II band) is caused by the coupling of N-H bending vibration and CN stretching vibration, further confirming the presence of peptide bonds. Additionally, at 1032.56 cm⁻¹... The absorption peak at that point may be related to the amide III band.

[0143] Example 12

[0144] This embodiment is based on the determination of molecular weight distribution of the purified component BLP-1.

[0145] In this embodiment, high-performance liquid chromatography (HPLC) was used to analyze the molecular weight distribution of BLP-1. The mobile phase consisted of acetonitrile / water / trifluoroacetic acid (40 / 60 / 0.1, v / v / v), the detection wavelength was 220 nm, the injection volume was 10 µL, and the flow rate was 0.5 mL / min. A linear standard curve of log MW versus retention time was established using cytochrome C (12384 Da), bacitracin (1422 Da), Gly-Gly-Try-Arg (451 Da), and Gly-Gly-Gly (12384 Da), as shown below:

[0146] y = -0.2574x + 7.391, R 2 =0.995, and the results are shown in Table 5 below.

[0147] Table 5. Molecular weight distribution of BLP

[0148]

[0149] As shown in Table 5, in the tested bioactive peptide BLP-1, molecules with a molecular weight >10 kDa accounted for only 0.39%, indicating that the enzymatic hydrolysis effect of the extraction method of the present invention is relatively sufficient. At the same time, the molecular weight of the enzymatic hydrolysis products is mainly concentrated below 1 kDa, accounting for 91.03%, which is consistent with the conclusion that antioxidant peptides with a molecular weight less than 1 kDa have higher antioxidant activity, and also proves the antioxidant advantage of the bioactive peptide extracted in this application.

[0150] Example 13

[0151] This embodiment evaluates the biosafety of BLP-1, using hemolytic activity and cytotoxicity as detection indicators.

[0152] Cytotoxicity: HaCaT cells (1×10⁻⁶) 4 Cells were cultured in 96-well plates (number of cells / well) for 24 hours. The experimental groups were then treated with DMEM medium containing different concentrations of BLP-1 (100, 200, 400, 600, 800, and 1000 μg / mL) for another 24 hours. A control group was also included, treated with DMEM medium without BLP-1. The medium was then aspirated, cells were washed with PBS, and cell viability was assessed using the CCK8 assay. Cell viability (%) = A s / A c ×100%. A s and A c These represent the experimental group and the control group at OD levels, respectively. 450 nm Absorbance.

[0153] Hemolytic activity: Collected mouse blood cells were washed three times with PBS and then resuspended in PBS to achieve a final concentration of 1% (v / v) red blood cells. 50 μL of the red blood cell suspension was then incubated with 50 μL of BLP-1 (1, 2, 4, 8, 16 mg / mL) at 37°C for 1 hour, and absorbance was measured at 570 nm. PBS and 1% Triton-X-100 served as the positive and negative controls, respectively. Hemolytic activity (%) = (A-A0) / (A1-A0) × 100%. A, A0, and A1 represent the experimental group, negative control group, and positive control group at OD values, respectively. 570 nm Absorbance.

[0154] The results are attached. Figure 12 As shown, when the concentration of BLP-1 was between 100-1000 μg, no cytotoxicity was observed compared to the control group. Figure 12 (A). In the hemolysis test, even when the BLP-1 concentration reached 16 mg / ml, hemolysis did not occur. Figure 12 (B). All the above results indicate that BLP-1, as an antioxidant peptide, has good biocompatibility.

[0155] Example 14

[0156] This embodiment measures the in vitro antioxidant index of BLP-1.

[0157] The antioxidant activity of BLP-1 at different concentrations was detected using DPPH, ABTS, superoxide anion, and hydroxyl radical assay kits, respectively. Glutathione (GSH) was used as a control peptide to further demonstrate the antioxidant activity of BLP-1.

[0158] To evaluate the in vitro antioxidant activity of BLP-1, this study assessed its ability to scavenge four different free radicals at different concentrations, with GSH used as a control for comparison. The results are shown in the appendix. Figure 13 As shown.

[0159] It is evident that the scavenging effect of BLP-1 on the four free radicals increases in a dose-dependent manner, reaching 89.10 ± 2.01%, 93.52 ± 1.50%, 95.15 ± 0.71%, and 74.12 ± 3.27% for DPPH radicals, ABTS radicals, hydroxyl radicals, and superoxide anion radicals, respectively. Notably, BLP-1 exhibits the same antioxidant capacity as GSH at high concentrations. These results indicate that BLP-1 possesses significant free radical scavenging activity and has potential applications in anti-aging health products and cosmetics.

[0160] Example 15

[0161] This embodiment examines the protective effect of BLP-1 on oxidatively damaged HepG2 cells.

[0162] HepG2 cells were induced with different concentrations of H2O2 (600, 650, 700, 750, 800, 850 μmol / L) to determine the H2O2 concentration that caused 50% cell lethality. In the experimental group, HepG2 cells were induced with 1.0 × 10⁻⁶ μmol / L. 4 After adding a median-lethal dose of H2O2 to HepG2 cells, DMEM containing BLP-1 at final concentrations of 100, 200, 400, 600, 800, and 1000 μg / ml was added to verify its protective effect on oxidative stress in HepG2 cells. The model group consisted of HepG2 cells with added median-lethal H2O2 and DMEM without BLP-1; the control group consisted of HepG2 cells without added H2O2. Simultaneously, cell viability, antioxidant enzyme (SOD, CAT, GSH-Px), and MDA levels in oxidatively stressed cells were measured during the BLP-1 (100, 200, 400, 600 μg / ml) protection process. The results are shown in the attached figure. Figure 14 . Figure 14The cell viability (%) in Figure A, SOD level (%) in Figure B, CAT level (%) in Figure C, GSH-Px level (%) in Figure D, and MDA level (%) in Figure E were all calculated using the method A(A1) / A0×100%. A, A1, and A0 represent the values ​​of the experimental group, model group, and control group at the corresponding absorbance, respectively. The absorbances corresponding to cell viability, SOD, CAT, GSH-Px, and MDA were 450 nm, 450 nm, 240 nm, 412 nm, and 532 nm, respectively.

[0163] It is evident that in H2O2-induced oxidative stress-induced HepG2 cells, the survival rate of oxidatively damaged HepG2 cells significantly increased with the addition of BLP-1, demonstrating the therapeutic effect of BLP-1 in addressing oxidative damage. Figure 14 (A). Simultaneously, by measuring the activities of antioxidant enzymes (SOD, MDA, CAT) in HepG2 and the level of MDA, the end product of lipid peroxidation, it was found that BLP-1 can reduce oxidative stress-induced cell damage by increasing antioxidant enzyme activity and decreasing lipid peroxidation. Figure 14 (BE).

[0164] Example 16

[0165] This embodiment examines the protective effect of BLP-1 on the liver of mice with alcoholic liver injury.

[0166] The protective effect of BLP-1 against alcoholic liver injury was verified using a mouse model of acute alcoholic liver injury. Thirty male Kunming mice aged 6-8 weeks were randomly divided into 5 groups (control group, model group, and experimental groups: 300 mg / kg, 600 mg / kg, and 1200 mg / kg). Throughout the experiment, each mouse was administered 0.12 mL / 10g via gavage. The control group received saline via gavage; the model group received 53% ethanol via gavage; and the 300 mg / kg, 600 mg / kg, and 1200 mg / kg groups received 53% ethanol via gavage at BLP-1 concentrations of 25, 50, and 100 mg / mL, respectively. Mice in each group underwent gavage for 8 consecutive days. One hour after gavage on day 8, all mice except the control group (which received saline) received 53% ethanol via gavage. Twelve hours after fasting and withholding water, the mice were weighed, blood was collected from their eyeballs, and they were euthanized and their livers were removed.

[0167] After sample collection, to observe the protective effect of BLP-1 on the liver, the liver index (liver weight / body weight * 100%) of mice in each group was calculated. Simultaneously, serum inflammatory factors (IL-6 and TNF-α) and transaminases (AST and ALT), as well as liver antioxidant enzyme activities (SOD, CAT, and GSH-Px) and MDA levels were measured. The calculation methods for IL-6 (%), TNF-α (%), AST (%), and ALT (%) were all A(A1) / A0 × 100%. A, A1, and A0 represent the values ​​of the experimental group, model group, and control group at the corresponding absorbances. The corresponding absorbances were 450 nm, 450 nm, 510 nm, and 505 nm, respectively. The calculation methods for antioxidant enzyme activities and MDA are as shown above.

[0168] In addition, pathological changes in the liver were observed using hematoxylin-eosin (HE) staining, and the results are attached. Figure 15 Medium AI.

[0169] It is evident that in the alcoholic liver injury experiment, compared with the control group, the model group showed an increased liver weight ratio, significantly elevated levels of AST, ALT, TNF-α, IL-6, and MDA, while the activities of SOD, CAT, and GSH-Px decreased, indicating that alcohol induces pathological changes in the liver, hepatocellular damage, inflammatory response, and oxidative stress. Treatment with different concentrations (300, 600, 1200 mg / kg) of peptide BLP-1 resulted in a decrease in liver weight ratio, reduced AST and ALT levels, inhibited TNF-α and IL-6 release, increased SOD, CAT, and GSH-Px activities, and decreased MDA production. Figure 15 This suggests that BLP-1 may play a potential therapeutic role in alcoholic liver injury by improving liver morphology, protecting hepatocyte function, reducing inflammation, and enhancing antioxidant capacity.

[0170] Example 17

[0171] In this embodiment, BLP-1 was identified by mass spectrometry and its amino acid sequence was screened.

[0172] In this embodiment, LC-MS / MS was used to identify BLP-1 in order to obtain the peptide sequences contained in BLP-1.

[0173] During the identification process, the column temperature of the VANQUISH NEO nano-scale liquid chromatography system was maintained at 55°C. Mobile phase A consisted of water and 0.1% formic acid, while mobile phase B consisted of 0.1% formic acid and 80% acetonitrile. Gradient elution was used, and the specific elution procedure was as follows:

[0174] 0-1.8 min, A:B (volume ratio) 96.0%→95.5%:4.0%→4.5%, flow rate 0.50 μL / min;

[0175] 1.8-2.0 min, A:B (volume ratio) 95.5%→95.0%:4.5%→5.0%, flow rate 0.50 μL / min;

[0176] 2.0-41.0 min, A:B (volume ratio) was 95.0%→80.0%:5.0%→20.0%, and the flow rate was 0.30 μL / min;

[0177] 41.0-57.0 min, A:B (volume ratio) was 80.0%→65.0%:20.0%→35.0%, and the flow rate was 0.30 μL / min;

[0178] 57.0-57.5 min, A:B (volume ratio) 65.0%→45.0%:35.0%→55.0%, flow rate 0.30 μL / min;

[0179] 57.0-58.0 min, A:B (volume ratio) was 45.0%→1.0%:55.0%→99.0%, and the flow rate was 0.50 μL / min.

[0180] In this embodiment, 6200 peptide sequences were identified from BLP-1 by LC-MS / MS (as shown in the attached figure). Figure 16 (A)

[0181] This embodiment further uses PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ) to predict the probability of obtaining bioactive peptide sequences. A peptide sequence with a PeptideRanker score > 0.5 is considered bioactive. To increase the probability of obtaining bioactive peptides, peptides with a PeptideRanker score > 0.95 are selected and their bioactivity is further evaluated using the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php). Subsequently, ADP3 (https: / / aps.unmc.edu / AP / ) and ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / ) are used to predict and screen the average hydrophilicity coefficient (GRAVY) and toxicity of the peptides.

[0182] This experiment further used the PeptideRanker database (https: / / distilldeep.ucd.ie / PeptideRanker / ) to rank and screen the bioactivity of the aforementioned 6200 peptide sequences.

[0183] Generally, peptides with a PeptideRanker score >0.5 are considered potentially bioactive peptides. In this embodiment, a total of 3783 potentially bioactive peptides were obtained under this standard. Figure 16 (B) To further narrow down the screening scope and increase the probability of successful screening of bioactive peptides, 25 peptides with scores >0.95 were selected for further bioactivity analysis. Figure 16 (C)

[0184] This experiment used the BIOPEP-UWM database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ) for bioactivity analysis, and obtained 12 active antioxidant peptides, which were identified as: KPFPFF, MWPPLP, FYGWPG, DGGGWW, FGPPPPP, WGPPGG, FFSPGVW, PPPGPPPPPGPPPPPPG, DGGAWW, GWNIPMGL, GPPPAPPLP, and MIKPFF.

[0185] The predicted physicochemical properties of the antioxidant peptides obtained in this embodiment are shown in Table 6. Generally, GRAVY < 0 indicates good water solubility. KF6, FW7, GL8, and MF6 have GRAVY > 0, indicating poor solubility, and are therefore not used for further screening. Furthermore, FP7 and PG16 were predicted to have some toxicity and were also excluded from subsequent screening.

[0186] Table 6. Identification of the physicochemical properties of antioxidant peptides

[0187]

[0188] Example 18

[0189] This embodiment focuses on molecular docking of the aforementioned antioxidant peptides with DPPH and ABTS.

[0190] The screened antioxidant peptides were molecularly docked with DPPH and ABTS to evaluate their antioxidant activity. The structures of the DPPH radical (CID: 2735032) and ABTS radical (CID: 5360881) were obtained from the PubChem database. Peptide modeling was performed using an online platform (https: / / cloud.yinfotek.com) prior to docking. AutoDock Vina software was used for docking, with DPPH and ABTS as ligands and the peptides as acceptors. The docking results were then analyzed using PyMOL 2.3.0 and Discovery Studio 2019.

[0191] DPPH and ABTS are two stable free radicals that can be used to evaluate the antioxidant capacity of antioxidants. In this example, six screened antioxidant peptides were molecularly docked with DPPH and ABTS, respectively, to analyze their binding ability to these free radicals. The results are shown in Table 7 below.

[0192] Table 7. Molecular docking results of peptides with DPPH and ABTS

[0193]

[0194] As can be seen, the binding energies of the six further screened antioxidant peptides with DPPH range from -3.4 to -4.3 kcal / mol, and with ABTS range from -3.4 to -4.3 kcal / mol (see Table 7 below). A binding energy less than 0 indicates that the ligand and receptor can bind spontaneously. Therefore, all antioxidant peptides can spontaneously bind to both DPPH and ABTS. Based on the combined binding affinity of antioxidant peptides to DPPH / ABTS, the antioxidant capacity of the peptides is ranked as follows: GP9 > FG6 > WG6 > DGG6 > DW6 > MP6.

[0195] Example 19

[0196] In this embodiment, the above-mentioned antioxidant peptides were molecularly docked with the Keap1 recombinant protein.

[0197] Antioxidant peptides were docked with Keap1 to investigate their potential antioxidant activity via the Nrf2 / Keap1 pathway. The crystal structure of Keap1 (PDB ID: 2FLU) was obtained from the protein database (PDB, https: / / www.rcsb.org / ). The binding sites of Keap1 are x:5, y:9, z:2. Docking and analysis were performed according to the steps in Example 12 above.

[0198] Maintaining redox balance in the body is a complex process requiring the synergistic function of multiple pathways. Among them, the Nrf2 / Keap1 signaling pathway is crucial for maintaining cellular homeostasis and responding to oxidative stress. Therefore, this embodiment selected FG6, GP9, and WG6, which have strong binding affinity to DPPH / ABTS, to conduct molecular docking studies with Keap1. Figure 17 (Central AC). The docking binding energy is less than -7 kcal / mol, indicating a strong binding affinity between the ligand and receptor. Therefore, all three antioxidant peptides exhibit strong binding ability to Keap1. Notably, WG6 (-10.2 kcal / mol) shows the strongest docking interaction. Therefore, FG6, GP9, and WG6 may influence the interaction between Keap1 and Nrf2 by binding to Keap1, leading to the dissociation and release of Nrf2, thereby affecting the expression of downstream genes and mitigating oxidative stress-induced damage.

[0199] Example 20

[0200] This embodiment predicts digestive enzyme sites based on GP9, WG6, and FG6.

[0201] The stability of the selected peptides against digestive enzymes (trypsin, pepsin) was predicted using an online website (https: / / www.novopro.cn / tools / ), and the results are shown in Table 8 below.

[0202] Table 8 Prediction of antioxidant peptide enzymatic hydrolysis sites

[0203]

[0204] It is evident that the predicted digestive enzyme sites of FG6, GP9, and WG6 reveal that, except for FG6 which has three pepsin digestion sites, GP9 and WG6 lack trypsin / pepsin digestion sites, exhibit good stability, and have the potential to become oral drug peptides.

[0205] Example 21

[0206] In this embodiment, polypeptides FG6, GP9, and WG6 (synthesized by Nanjing Jietai Biotechnology Co., Ltd.) were further synthesized using solid-phase synthesis based on the identified amino acid sequences, and their cytotoxicity was tested according to the method in Example 13.

[0207] The results are attached. Figure 18 As shown in Figure A, cell viability consistently exceeded 90% across concentrations ranging from 100 to 1000 μg / mL. This finding indicates that these three antioxidant peptides did not exhibit significant cytotoxicity.

[0208] This experimental example further tested the antioxidant activity of the synthesized peptides FG6, GP9, and WG6 according to the method in Example 14, and measured the IC50 values ​​of GP9, FG6, and WG6 for scavenging DPPH and ABTS, respectively. 50 The results are attached. Figure 18 As shown in Figure B, the half-inhibitory concentrations (WICs) for DPPH scavenging by peptides GP9, FG6, and WG6 were 3.37 ± 0.15, 2.73 ± 0.09, and 5.23 ± 0.15 mg / mL, respectively. Furthermore, the WICs for ABTS were recorded as 4.47 ± 0.31, 3.53 ± 0.31, and 7.60 ± 0.30 mg / mL.

[0209] In summary, the antioxidant active peptides of this invention utilize bovine lung as the extraction raw material and employ enzymatic hydrolysis for the extraction of natural active peptides. Through screening of hydrolytic enzymes and optimization of single-factor hydrolysis, an extraction process for bovine lung enzymatic hydrolysate with strong antioxidant activity was established. The antioxidant active peptides of this invention were collected and identified by mass spectrometry, combined with bioinformatics analysis and experimental characterization, yielding 12 new antioxidant peptide sequences. This not only verified their antioxidant effects but also further screened and synthesized antioxidant peptides FYGWPG, GPPPAPPLP, and WGPPGG, verifying their extremely strong stability against proteases, making them suitable for applications in natural antioxidant products.

[0210] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An antioxidant active peptide, characterized in that, The antioxidant active peptide is at least one of the following active peptides with the sequence structure as shown below: FG6: the amino acid sequence is as shown in SEQ ID NO. 3; WG6: the amino acid sequence is as shown in SEQ ID NO.

6.

2. A method for preparing a bovine lung enzymatic hydrolysate containing the antioxidant active peptide according to claim 1, characterized in that, The method comprises the following steps: (1) taking bovine lung raw material to prepare slurry by adding water to obtain bovine lung slurry; (2) adding papain to the bovine lung slurry for enzymatic hydrolysis; The method further comprises a step of purifying the crude product obtained in the enzymatic hydrolysis step; The purification step comprises ultrafiltration; the purification step comprises a step of obtaining peptide components with molecular weights < 3 kDa, 3-10 kDa and > 10 kDa, respectively, by ultrafiltration; The purification step comprises a step of purifying the peptide component with a molecular weight less than 3 kDa by gel chromatography; The gel chromatography uses cross-linked dextran gel; The gel chromatography is used for chromatography, the loading concentration is 15-25 mg / ml, the loading amount is 3-7 ml, the flow rate is 1-2 ml / min, 1-3 min is collected per tube, the absorbance curve is detected at 220 nm, and the eluate collected from the 14th-16th tube is BLP-1, and the eluate collected from the 24th-26th tube is BLP-2; And / or, the size of the gel chromatography column is 1.6 cm x 50 cm.

3. The method of claim 2, wherein the bovine lung proteolyzate is prepared by the steps of: In the step (1): The ratio of the bovine lung raw material to water is 5-25 wt%; and / or, The pH value of the bovine lung slurry is adjusted to 5-9.

4. The method of claim 3, wherein the bovine lung proteolyzate is prepared by the steps of: In the step (2): The concentration of the added protease based on the bovine lung is 1000-5000 U / g; and / or, The temperature of the enzymatic hydrolysis step is 40-60℃; and / or, The enzymatic hydrolysis time of the enzymatic hydrolysis step is 2-6 h.

5. Use of the bovine lung enzymatic hydrolysate containing the antioxidant active peptide of claim 1, the amino acid sequence as shown in SEQ ID NO. 11 or prepared by the method of any one of claims 2-4 for preparing an antioxidant.

6. Use according to claim 5, characterized in that, The antioxidant comprises a food antioxidant or a pharmaceutical antioxidant.

7. Use according to claim 6, characterized in that, The food antioxidant comprises a health product antioxidant.

8. Use of the bovine lung enzymatic hydrolysate containing the antioxidant active peptide of claim 1 prepared by the method of any one of claims 2-4 for preparing a medicine; The medicine is used for preventing or treating alcoholic fatty liver.

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