Antioxidant peptide derived from lactoferrin and application thereof

Abstract

The invention belongs to the field of biological medicine, and discloses an antioxidant peptide derived from lactoferrin and application thereof. The amino acid sequence of the antioxidative peptide derived from lactoferrin is shown as SEQ ID NO: 55, and the antioxidative peptide has an antioxidative effect and can be applied to preparation of antioxidative products, for example, the antioxidative peptide is used as an active ingredient in food, cosmetics or health care products.

Description

[0001] This application is a divisional application of the invention patent application with the original application number 2023102303563, the invention name is "Antioxidant peptides derived from lactoferrin and their applications", and the application date is March 10, 2023. Technical Field

[0002] The present invention belongs to the field of biomedicine, and particularly relates to an antioxidant peptide derived from lactoferrin and applications thereof. Background Art

[0003] With the development of free radical biology and medicine, the study of free radicals and antioxidants has attracted increasing attention. Reactive oxygen species are byproducts of physiological metabolism in human cells, including hydroxyl radicals, superoxide anion radicals, singlet oxygen, and hydrogen peroxide, which are promptly cleared by the cellular antioxidant system (Guo Y, Zhang T, Jiang B, et al. The effects of an antioxidative pentapeptide derived from chickpea protein hydrolysate on oxidative stress in Caco-2 and HT-29 cell lines [J]. Journal of Functional Foods, 2014, 7: 719-726.). In some cases, excessive accumulation of free radicals can trigger oxidative stress in the body, causing oxidative damage to intracellular biomacromolecules such as proteins, lipids, and DNA, thereby accelerating aging and triggering a variety of diseases such as neurodegenerative diseases, atherosclerosis, chronic inflammation, and cancer (García-Nebot MJ, Recio I, Hernández-Ledesma B. Antioxidant activity and protective effects of peptide lunasin against oxidative stress in intestinal Caco-2 cells[J]. Food and Chemical Toxicology, 2014, 65: 155-161.). Studies have shown that in vitro intake of antioxidants can scavenge excessive free radicals in the body, reduce the level of oxidative stress in the body, and play a role in preventing or even treating certain diseases (He R, Ju X, Yuan J, et al. Antioxidant activities of rapeseed peptides produced by solid state fermentation[J]. Food Research International, 2012, 49(1): 432-438.). Oxidation is also one of the important reasons for food spoilage, reduced nutritional value and even the production of harmful substances. Therefore, antioxidants are widely demanded in the fields of health products, food, medicine and cosmetics.Because long-term intake of synthetic antioxidants poses potential hazards to human health, natural food-derived antioxidants are becoming increasingly popular (Liu C, Ren D, Li J, et al. Cytoprotective effect and purification of novel antioxidant peptides from hazelnut (C. heterophylla Fisch) protein hydrolysates [J]. Journal of Functional Foods, 2018, 42: 203-215.). Among natural antioxidants, antioxidant peptides derived from plant and animal food proteins have attracted widespread attention due to their low molecular weight, high activity, easy absorption, colorlessness, odorlessness, and no side effects (Mahgoub S, Alagawany M, Nader M, et al. Recent development in bioactive peptides from plant and animal products and their impact on the human health [J]. Food Reviews International, 2021: 1-26.). There are many types of antioxidant peptides, with different molecular sizes and solubility, which provides more options for their development and utilization.

[0004] There are mainly the following ways to obtain antioxidant peptides: first, directly screening from animal and plant cell lysates; second, using animal and plant proteins as raw materials, selecting appropriate enzymes for hydrolysis, using ultrafiltration, chromatography and other methods to separate and purify polypeptides of a certain molecular weight, and measuring the antioxidant activity of the corresponding separated components; after the sequence of the antioxidant peptide is clarified, a large amount of antioxidant peptides can be obtained through separation and purification or synthesis technology; third, using bioinformatics methods to analyze proteins of known sequences in animals and plants, predict highly active polypeptide fragments, and synthesize them through chemical synthesis methods to verify their activity (Current Status of Research on Antioxidant Peptides).

[0005] Lactoferrin (LF) is a non-heme iron-binding glycoprotein secreted by mammalian mucosal epithelial cells (Mayeur S, Spahis S, Pouliot Y, et al. Lactoferrin, a pleiotropic protein in health and disease[J]. Antioxidants&redox signaling, 2016, 24(14): 813-836.), with a molecular weight of approximately 80 kDa. It is widely present in tears, saliva, sweat, gastrointestinal fluid, bovine milk and human colostrum, and is considered to be one of the basic elements of the human anti-infection defense system (Park JH, Park GT, Cho IH, et al. An antimicrobial protein, lactoferrin exists in the sweat: proteomic analysis of sweat[J]. Experimental Dermatology, 2011, 20(4): 369-371. & Masson P, Heremans JF, Prignot J. Immunohistochemical localization of the iron-binding protein lactoferrin in human bronchial glands[J].Experientia,1965,21(10):604-605.). It is reported that LF has biological functions such as antibacterial, antiviral, antiparasitic, anticancer, antioxidant, antiallergic, anti-inflammatory and immunomodulatory functions (Zhang Enpeng. Design of bovine lactoferrin peptide-derived peptides and their expression in Pichia pastoris[D].Jiangsu University,2019.&Fan F,Shi P,Liu M,et al.Lactoferrin preserves bone homeostasis by regulating the RANKL / RANK / OPG pathway of osteoimmunology[J].Food&Function,2018,9(5):2653-2660.).Due to the benefits of LF, it is often added to a variety of products such as infant formula, health products, cosmetics, pet care supplements, beverages, fermented milk, chewing gum and toothpaste. It helps regulate iron absorption and protect the gastrointestinal tract of newborn infants from infection (Brock J H. Lactoferrin in human milk: its role in iron absorption and protection against enteric infection in the newborn infant [J]. Archives of Disease in Childhood, 1980, 55 (6): 417.), and is used to enhance the body's immunity (Wang B, Timilsena YP, Blanch E, et al. Lactoferrin: Structure, function, denaturation and digestion [J]. Critical Reviews in Food Science and Nutrition, 2019, 59 (4): 580-596.). At the same time, many have been shown to have activities that exceed those of the entire protein and have multiple active functions. For example, LF polypeptide has angiotensin-converting enzyme inhibitory activity, antioxidant and anti-inflammatory activities (Ruiz-Giménez P, Salom JB, Marcos JF, et al. Antihypertensive effect of a bovine lactoferrin pepsin hydrolysate: Identification of novel active peptides [J]. Food Chemistry, 2012, 131 (1): 266-273. & Gu Y, Wu J. Bovine lactoferrin-derived ACE inhibitory tripeptide LRP also shows antioxidative and anti-inflammatory activities in endothelial cells [J]. Journal of Functional Foods, 2016, 25: 375-384.).Although studies have shown that the degradation products of lactoferrin retain biological activities such as antioxidant activity, the preparation of active peptides using enzymatic hydrolysis methods is complex, time-consuming, costly, and has uneven product composition. The quality of different batches is difficult to control, making it difficult to conduct in-depth research on its mechanism of action (Zhang Qiang, Li Weihua. Research status of antioxidant peptides [J]. Food and Fermentation Industries, 2021, 47(2): 298-304.). Therefore, it is necessary to use bioinformatics analysis to supplement the traditional methods of studying antioxidant peptides. However, computer simulation-based screening of antioxidant peptides, especially antioxidant peptides derived from lactoferrin, has not yet been reported. Summary of the Invention

[0006] The present invention uses bioinformatics methods to design a lactoferrin peptide library based on overlapping peptide libraries. AnOxPePred-1.0 is used to predict and screen for highly active antioxidant peptides, which can be used as additives for food, cosmetics, or health products. Traditional protein separation and purification methods for bioactive peptides are abandoned, and bioinformatics methods are used instead, thereby significantly saving experimental time and money costs. Given the lack of effective discovery methods for antioxidant peptides using traditional methods, the present invention relates to a simple and effective method for high-throughput screening of antioxidant polypeptides, offering unique advantages. The screened antioxidant peptides of the present invention are derived from lactoferrin sequences, achieving the goals of good biocompatibility, environmental friendliness, and safety.

[0007] In order to achieve the above object, the technical solution provided by the present invention is:

[0008] The present invention provides an antioxidant peptide derived from lactoferrin, the amino acid sequence of which is shown in any one of SEQ ID NOs: 1 to 99.

[0009] Preferably, its amino acid sequence is shown in any one of SEQ ID NO:1-2, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO:23-26, SEQ ID NO.34, SEQ ID NO.37, SEQ ID NO.40, SEQ ID NO.43, SEQ ID NO.48, SEQ ID NO.50, SEQ ID NO:53-57, SEQ ID NO.61, SEQ ID NO.64-65, SEQ ID NO.68, SEQ ID NO:71-73, SEQ ID NO.75, SEQ ID NO.77, SEQ ID NO.79, SEQ ID NO:85-88, SEQ ID NO:92-94, SEQ ID NO.96 and SEQ ID NO:98-99.

[0010] More preferably, the amino acid sequence is as shown in SEQ ID NO.1, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.55, SEQ ID NO.61, SEQ ID NO.65, SEQ ID NO.68, SEQ ID NO.73, SEQ ID NO.79 or SEQ ID NO.98. Further preferably, the amino acid sequence is as shown in any one of SEQ ID NO.24, SEQ ID NO.55 and SEQ ID NO.73.

[0011] The present invention also provides a nucleic acid encoding the antioxidant peptide, an expression vector containing the nucleic acid, and a recombinant cell containing the expression vector.

[0012] The present invention also provides the use of the antioxidant peptide or its encoding nucleic acid in the preparation of antioxidant products.

[0013] Preferably, the product is food, cosmetics or health products, and the antioxidant peptide is used as an active ingredient or additive.

[0014] The present invention innovatively employs overlapping peptide libraries to design a lactoferrin peptide library, achieving full sequence construction of a lactoferrin peptide library, saving time and cost, and improving the accuracy of hydrolyzate sequence composition, thereby demonstrating originality in the field of bioinformatics bioactive peptides. Furthermore, the invention innovatively employs the AnOxPePred-1.0 server to predict and screen lactoferrin antioxidant peptides with high activity, thereby achieving a high-throughput screening method for lactoferrin antioxidant peptides, saving capital and manpower, and improving efficiency, thereby demonstrating originality in the field of bioactive peptides. The present invention is characterized in that a specific amino acid sequence containing cysteine ​​is present, and these innovative peptides are capable of effectively scavenging free radicals to enhance antioxidant activity, and are natural antioxidants with good safety.

[0015] Specifically, the innovative peptide provided by the present invention has excellent antioxidant activity after testing. Under the assay conditions of the present invention, its DPPH free radical scavenging ability and ABTS free radical scavenging ability are close to or better than those of glutathione and carnosine, respectively. DETAILED DESCRIPTION

[0016] Unless otherwise specified, the reagents and instruments used in the following examples are all commercially available products.

[0017] Example 1 Design of lactoferrin peptide library and rapid screening method based on overlapping peptide library

[0018] 1. Establishment of Lactoferrin Peptide Library

[0019] (1) Obtain the amino acid sequence of lactoferrin in the UniProt protein database;

[0020] (2) The above amino acid sequence was designed using the overlapping peptide library method to obtain 1398 overlapping peptide segments with a length of 11, and adjacent peptide segments overlapped by 10 amino acids.

[0021] 2. Bioinformatics prediction of antioxidant activity sequences

[0022] (1) The 1398 peptide sequences were grouped into 28 groups of 50 peptides each, and submitted to the AnOxPePred-1.0 server (AnOxPePred-1.0-Services-DTU Health Tech). The peptide length range was set to 2-30 amino acids. The peptide model was used to obtain the free radical scavenging (FRS) or chelation (CHEL) scores of the predicted peptides.

[0023] (2) Based on the FRS score threshold ≥0.45 predicted in step (1), 179 peptide sequences were screened;

[0024] (3) connecting all x consecutive peptide sequences with overlapping partial amino acid sequences in step (2) into a long peptide chain consisting of y amino acid residues in length, wherein the C-terminus of the nth amino acid sequence is connected to the non-overlapping sequence of the n+x-1th amino acid sequence, to obtain 53 peptide sequences;

[0025] (4) The peptide sequence obtained in step (3) was submitted to the AnOxPePred-1.0 server according to step (1) to obtain the FRS score of the predicted peptide. The results are shown in Table 1.

[0026] Table 1. FRS scores of 53 peptides

[0027]

[0028]

[0029] (5) Based on the new sequences SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.10 obtained in step (4), the N-terminal amino acid residues were added to obtain the sequences SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28 and SEQ ID NO.29, respectively. The sequences were submitted to the AnOxPePred-1.0 server according to step (1) to obtain the FRS scores of the predicted peptides. The results are shown in Table 2;

[0030] Table 2. FRS scores of four peptides

[0031] SEQ ID NO. Peptide sequence Length FRS score 54 CVPNSKEKYYGYTGAFRCL 19 0.60702 55 CACSSREPYFGYSGAFKCLQ 20 0.58787 56 CLAKLGGRPTYEEYLGTEYVTA 22 0.56280 57 ADALNLDGGYIYTAGKCG 18 0.53777

[0032] (6) Based on the CHEL score threshold ≥ 0.25 predicted in step (1), 97 peptide sequences were screened;

[0033] (7) The peptide sequence obtained in step (5) was connected using the peptide chain method in step (3) to obtain 42 peptide sequences;

[0034] (8) The peptide sequence obtained in step (7) was submitted to the AnOxPePred-1.0 server according to step (1) to obtain the CHEL score of the predicted peptide. The results are shown in Table 3.

[0035] Table 3. CHEL scores of 42 peptides

[0036] SEQ ID NO. Peptide sequence Length CHEL score 58 SRSFQLFGSPPGQRDLLF 18 0.3046 59 TTVFENLPEKADRD 14 0.29661 60 VKQVLLHQQALFG 13 0.29169 61 CSTSPLLEACAFL 13 0.29070 62 QRDLLFKDSAL 11 0.27729 63 RSDRAAHVKQVLL 13 0.27319 64 FDEFFSQSCAPG 12 0.26668 65 IDRQAYPNLCQL 12 0.26610 66 TKNLLFNDNTE 11 0.26273 67 PALLSLGALGL 11 0.25982 68 CHLAVAPNHAVV 12 0.25873 69 KKGSNFQLDQLQG 13 0.25581 70 TKESPQTHYYA 11 0.25520 71 AFKECHLAQVPSH 13 0.25462 72 DKCVPNSKEKY 11 0.25383 73 FSQSCAPGADPKS 13 0.25370 74 SWTESLEPLQGAVAK 15 0.25314 75 YELLCLNNSRA 11 0.24621 76 KDSALGFLRIPS 12 0.24478 77 QSSDPDPNCVDRP 13 0.30134 78 FQLFGSPSGQKDLL 14 0.30037 79 CSTSPLLEACEFL 13 0.29067 80 RLKQVLLHQQAKFG 14 0.28647 81 TLRPFLNWTGPP 12 0.26766 82 GKDKSPKFQLF 11 0.26655 83 FEDLSDEAERD 11 0.26533 84 DAIWNLLRQAQ 11 0.26458 85 IKRDSPIQCIQAI 13 0.26190 86 CLFQSETKNLL 11 0.25615 87 ELLCPDNTRKP 11 0.25582 88 FSQSCAPGSDP 11 0.25566 89 PMGLLFNQTGS 11 0.25483 90 LVLLFLGALGL 11 0.25374 91 TGPPEPIEAAV 11 0.25279 92 FRCLAENAGDV 11 0.25211 93 DKGQFPNLCRL 11 0.25208 94 GDEQGENKCVP 11 0.25188 95 IWNLLRQAQEK 11 0.25110 96 KCAFSSQEPYF 11 0.25098 97 KGGSFQLNELQGLK 14 0.24319 98 GSDCPDKFCLFQ 12 0.24260 99 EARSCHLAMAPNHAVV 16 0.21622

[0037] Example 2 Verification of the activity of antioxidant peptides

[0038] 1. Synthetic Antioxidant Peptides

[0039] According to the predicted and screened lactoferrin antioxidant peptide sequence in Example 1, it was synthesized by GenScript Biotech Co., Ltd.

[0040] 2. Activity Verification of Antioxidant Peptides

[0041] (1) DPPH free radical scavenging ability

[0042] Preparation of relevant reagents:

[0043] 1) DPPH solution: 0.2 mM (dissolved in anhydrous ethanol).

[0044] 2) Peptide solution: Custom-made and synthesized from GenScript Biotech Co., Ltd., lyophilized powder was dissolved in pure water.

[0045] Experimental plan:

[0046] Sample#1

[0047] 100 μL of each peptide solution of varying concentrations was added to 100 μL of a 0.2 mM DPPH ethanol solution. The mixture was shaken and incubated in the dark at room temperature for 30 minutes. The absorbance at 517 nm (At) was measured. Simultaneously, the absorbance at 517 nm (Ar) of 100 μL of the peptide solution plus 100 μL of the ethanol solution was measured, and the absorbance at 517 nm (A0) of 100 μL of the DPPH solution plus 100 μL of the ethanol solution was measured. Three replicates were performed for each assay. The results are shown in Tables 4 and 5.

[0048] DPPH free radical scavenging rate (%) = [1-(As -A b ) / A c ]×100

[0049] A c : Absorbance of 100 μL anhydrous ethanol + 100 μL DPPH solution; A s : absorbance of 100 μL sample solution + 100 μL DPPH solution; A b : Absorbance value of 100 μL sample solution + 100 μL anhydrous ethanol.

[0050] Experimental results:

[0051] The DPPH free radical is a purple, stable nitrogen-centered free radical, and antioxidants can reduce it to a yellow compound. This phenomenon occurs because the DPPH free radical can accept an electron or hydrogen atom to form a stable diamagnetic molecule. In this study, 99 peptides and GSH were collectively tested for DPPH free radical scavenging activity to comprehensively evaluate their antioxidant activity. The results are shown in Table 4. For the 99 lactoferrin antioxidant peptides, at a concentration of 0.5 mg / mL, their DPPH free radical scavenging rates ranged from 18.69% to 94.64%. It was also found that peptides containing Cys amino acid residues exhibited relatively high DPPH free radical scavenging activity, particularly the peptide containing three Cys amino acid residues (SEQ ID NO. 55). For 41 polypeptides containing Cys amino acid residues, SEQ ID NO: 1-2, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO: 23-26, SEQ ID NO.34, SEQ ID NO.37, SEQ ID NO.40, SEQ ID NO.43, SEQ ID NO.48, SEQ ID NO.50, SEQ ID NO: 53-57, SEQ ID NO.61, SEQ ID NO: 64-65, SEQ ID NO.68, SEQ ID NO.71-73, SEQ ID NO.75, SEQ ID NO.77, SEQ ID NO.79, SEQ ID NO: 85-88, SEQ ID NO: 92-94, SEQ ID NO.96 and SEQ ID NO.98-99, their DPPH free radical scavenging activity IC 50The results showed that the number of Cys amino acid residues not only had a high influence on the DPPH free radical scavenging rate, but also the position of the Cys amino acid residue had a great influence on the DPPH free radical scavenging activity. In general, among the 41 polypeptides above, when the Cys amino acid residue was at the N-terminus of the peptide, such as SEQ ID NO.25, SEQ ID NO.48, SEQ ID NO.56, SEQ ID NO.68 and SEQ ID NO.86 containing one Cys amino acid residue, and SEQ ID NO.2, SEQ ID NO.54, SEQ ID NO.61 and SEQ ID NO.79 containing two Cys residues, had relatively high DPPH free radical scavenging activity. Structurally, their strong antioxidant activity may be related to the Cys residue at the N-terminus of the peptide chain. Cys is a sulfur-containing amino acid with a highly reactive side chain sulfhydryl group (-SH), exhibiting weak acidity (pK = 8.4) and prone to proton loss. Therefore, Cys readily couples with electrons from DPPH. In contrast, peptides containing Cys residues at the C-terminus and within the sequence exhibit relatively low DPPH radical scavenging activity. Furthermore, the DPPH radical scavenging activity of GSH was 0.032 mg / mL. Clearly, GSH possesses very high DPPH radical scavenging activity. The DPPH free radical scavenging activities of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.48, SEQ ID NO.55, SEQ ID NO.61, SEQ ID NO.68, and SEQ ID NO.79 were comparable to those of GSH, and the activities of SEQ ID NO.2, SEQ ID NO.48, SEQ ID NO.55, and SEQ ID NO.86 were even higher than those of GSH, with no statistically significant differences. It can be clearly seen that the above polypeptides and GSH all share a common Cys amino acid residue, and the presence of a thiol group on the Cys amino acid residue allows for direct reaction with free radicals (Jiang H, Tong T, Sun J, et al. Purification and characterization of antioxidant peptides from round scad (Decapterus maruadsi) muscle protein hydrolysate [J]. Food Chemistry, 2014, 154: 158-163.), resulting in these polypeptides possessing strong DPPH free radical scavenging activity. In addition, peptides without Cys amino acid residues, such as the remaining 58 peptides, showed low DPPH radical scavenging activity.Therefore, not only the amino acid residue composition, such as the Cys amino acid residue, but also the position of the active amino acid residue, such as the C-terminus or N-terminus, has a great influence on the DPPH free radical scavenging activity of the polypeptide.

[0052] Table 4. Scavenging ability of 0.5 mg / mL peptide on DPPH free radicals

[0053]

[0054]

[0055] Table 5. IC values ​​of DPPH free radical scavenging ability of peptides containing Cys amino acid residues and glutathione 50 value

[0056] Sample <![CDATA[IC 50 Value (mg / mL)]]> Sample <![CDATA[IC 50 Value (mg / mL)]]> Glutathione 0.032 SEQ ID NO.57 0.098 SEQ ID NO.1 0.039 SEQ ID NO.61 0.039 SEQ ID NO.2 0.029 SEQ ID NO.64 0.145 SEQ ID NO.8 0.041 ​ 0.146 ​ 0.147 ​ 0.040 ​ 0.135 ​ 0.156 ​ 0.152 ​ 0.150 ​ 0.088 ​ 0.124 ​ 0.062 ​ 0.175 ​ 0.046 ​ 0.091 ​ 0.090 ​ 0.040 ​ 0.088 ​ 0.092 ​ 0.092 ​ 0.029 ​ 0.087 ​ 0.078 ​ 0.090 ​ 0.080 ​ 0.030 SEQ ID NO.92 0.079 SEQ ID NO.50 0.080 SEQ ID NO.93 0.101 SEQ ID NO.53 0.094 SEQ ID NO.94 0.110 SEQ ID NO.54 0.044 SEQ ID NO.96 0.121 SEQ ID NO.55 0.027 SEQ ID NO.98 0.041 SEQ ID NO.56 0.096 SEQ ID NO.99 0.087

[0057] (2) ABTS free radical scavenging ability

[0058] Preparation of relevant reagents:

[0059] 1) ABTS solution: 7 mM, weigh 19.2 mg of ABTS and dissolve in 5 mL of pure water.

[0060] 2) Potassium persulfate solution: 140 mM, weigh 189 mg of potassium persulfate and dissolve in 5 mL of pure water.

[0061] 3) ABTS stock solution: Mix 5 mL of ABTS solution and 5 mL of potassium persulfate solution and let it stand overnight at room temperature in the dark for 16 h to form the ABTS stock solution.

[0062] 4) Peptide solution: Custom-made and synthesized from GenScript Biotech Co., Ltd., lyophilized powder was dissolved in pure water.

[0063] Experimental plan:

[0064] Sample#1

[0065] 1) Dilute the ABTS stock solution with pure water to prepare a working solution with an absorbance of 0.7 ± 0.02 at 734 nm.

[0066] 2) Pipette 100 μL of peptide solution of different concentrations, add 100 μL of ABTS working solution, shake for 1-2 minutes, place at 37°C for 10 minutes, and measure the absorbance value A at 734 nm. s , with 100 μL ABTS working solution + 100 μL pure water) as blank absorbance A c The absorbance of 100 μL of peptide solution + 100 μL of pure water mixed evenly is A bThe same assay was designed with three replicates. The results are shown in Tables 6 and 7.

[0067] ABTS free radical scavenging rate (%) = [1-(A s -A b ) / A c ]×100

[0068] A s : absorbance of 100 μL sample solution added with 100 μL ABTS solution; A b : absorbance of 100 μL sample solution added to 100 μL distilled water; A c : Absorbance of 100 μL ABTS solution added to 100 μL distilled water.

[0069] Experimental results:

[0070] In the reaction system, ABTS will be oxidized to generate ABTS free radicals, which are stable and water-soluble free radicals with a blue-green color and a maximum absorbance value at 734nm. The reaction of ABTS free radicals with antioxidants can make the characteristic color of the former fade, thereby reducing the absorbance value. Under the same conditions, the antioxidant activity of the biological sample is proportional to the degree of decrease in the absorbance value of the reaction solution. The ABTS free radical scavenging rates of lactoferrin peptides with different sequences are shown in Table 6. At a concentration of 0.5 mg / mL, their ABTS free radical scavenging rates are 8.66% to 99.96%. It was also found that peptides containing Cys amino acid residues have relatively high ABTS free radical scavenging activity. For the 41 peptides containing Cys amino acid residues, their ABTS free radical scavenging rates are 80.99% to 99.96%. Among them, their IC 50 The IC values ​​of the ABTS free radical scavenging activity of the 41 peptides containing Cys amino acid residues are 0.020~0.067mg / mL. Meanwhile, the ABTS free radical scavenging activity of the positive control carnosine is 1.244mg / mL. 50The values ​​are significantly lower than those of carnosine. Studies have shown that the reaction rate of ABTS free radicals with amino acids is mainly determined by whether the amino acid side chain has unstable hydrogen atoms, and is related to the pH of the solution and the concentration of the sample. Under the same reaction conditions, the activity of several amino acids in scavenging free radicals is ranked as follows: Cys>Trp>Tyr>His (Aliaga C, Lissi E A.Reactions of the radical cation derived from 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS·+)with aminoacids.Kinetics and mechanism[J].Canadian Journal of Chemistry,2000,78(8):1052-1059.). Consistent with the above research conclusions, lactoferrin polypeptides with strong ABTS free radical scavenging ability all contain Cys amino acids that are prone to lose hydrogen atoms in the reaction. This shows that the ABTS free radical scavenging activity of lactoferrin polypeptides is related to the type of its amino acid residues, and different types of amino acid residues have different activities in free radical reactions.

[0071] Table 6. Scavenging ability of 0.5 mg / mL peptide on ABTS free radicals

[0072]

[0073]

[0074] Table 7. IC values ​​of ABTS free radical scavenging abilities of peptides containing Cys amino acid residues and carnosine 50 value

[0075] Sample <![CDATA[IC 50 Value (mg / mL)]]> Sample <![CDATA[IC 50 Value (mg / mL)<!-- 13 --> ]]> Carnosine 1.244 SEQ ID NO.57 0.052 SEQ ID NO.1 0.040 SEQ ID NO.61 0.027 SEQ ID NO.2 0.045 SEQ ID NO.64 0.060 SEQ ID NO.8 0.041 SEQ ID NO.65 0.038 SEQ ID NO.10 0.061 SEQ ID NO.68 0.039 SEQ ID NO.15 0.058 SEQ ID NO.71 0.057 SEQ ID NO.17 0.058 SEQ ID NO.72 0.060 SEQ ID NO.23 0.032 SEQ ID NO.73 0.027 SEQ ID NO.24 0.020 SEQ ID NO.75 0.067 SEQ ID NO.25 0.046 SEQ ID NO.77 0.056 SEQ ID NO.26 0.044 SEQ ID NO.79 0.029 SEQ ID NO.34 0.050 SEQ ID NO.85 0.058 SEQ ID NO.37 0.056 SEQ ID NO.86 0.055 SEQ ID NO.40 0.056 SEQ ID NO.87 0.058 SEQ ID NO.43 0.043 SEQ ID NO.88 0.060 SEQ ID NO.48 0.056 SEQ ID NO.92 0.055 SEQ ID NO.50 0.042 SEQ ID NO.93 0.048 ​ 0.059 ​ 0.049 ​ 0.043 ​ 0.060 ​ 0.026 ​ 0.030 ​ 0.058 ​ 0.059 .

Claims

1. An antioxidant peptide derived from lactoferrin, characterized in that Its amino acid sequence is shown in SEQ ID NO:

55.

2. A nucleic acid encoding the antioxidant peptide according to claim 1.

3. An expression vector containing the nucleic acid according to claim 2.

4. A recombinant cell containing the expression vector according to claim 3.

5. Use of the antioxidant peptide or the nucleic acid encoding the peptide according to claim 1 in the preparation of an antioxidant product.

6. The use according to claim 5, characterized in that The product is a food or a cosmetic.

7. The use according to claim 6, characterized in that The food is a health product.

8. The use according to claim 6 or 7, characterized in that The antioxidant peptide is used as an active ingredient or additive.

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