该文以生物信息学为理论基础,使用计算机模拟酶解藏羊血红蛋白,利用在线活性评分、活性预测和分子对接技术对藏羊血红蛋白抗氧化肽进行筛选,并通过DPPH自由基、ABTS阳离子自由基清除率实验对其抗氧化性进行验证。使用UniProt数据库筛选藏羊血红蛋白的氨基酸序列,对其潜在的生物活性进行评价,应用ExPASy Peptide Cutter在线工具对筛选的肽链进行酶解,应用Peptide Ranker对生物活性预测评分,利用在线工具Innovagen、ToxinPred、iBitter-SCM、A11erTOP和AdmetSAR对其溶解性、毒性、苦味、致敏性和ADMET进行筛选,使用Keap1受体进行分子对接,预测其活性,最后利用DPPH自由基、ABTS阳离子自由基清除率实验验证筛选得到的藏羊血红蛋白抗氧化肽。结果表明,5条藏羊血红蛋白链含有20种氨基酸,经酶解共得到258种多肽(485条),通过生物活性评分系统筛选出110种评分大于0.5的多肽(194条),经过活性预测,得到18种溶解性好、无毒、无苦味且非过敏原的多肽,对肠道吸收较好的10条多肽进行分子对接,将分子对接评分前3位的多肽KRM、FPL、FL进行体外抗氧化活性实验,结果显示,FPL和KRM的自由基清除能力均超过了阳性对照,其中KRM对DPPH自由基和ABTS阳离子自由基清除能力超过了阳性对照,IC50分别为(0.125 0±0.012 3)和(0.339 1±0.031 4) mg/mL,FPL对DPPH自由基和ABTS阳离子自由基清除能力也超过了阳性对照,IC50分别为(0.256 5±0.014 1)和(0.374 6±0.047 1) mg/mL。FL对DPPH自由基和ABTS阳离子自由基清除能力分别达到了阳性对照的88.90%和94.40%,IC50分别为(1.106 1±0.279)和(1.601±0.296) mg/mL,均表现出较强的抗氧化活性。该方法与传统酶解实验相比,能快速、经济地筛选抗氧化肽,为藏羊血液资源的高值化利用与开发提供研究基础。
Based on the theoretical basis of bioinformatics, this paper used the computer to simulate the enzymatic digestion of Tibetan sheep hemoglobin, using online activity scoring, activity prediction, and molecular docking techniques to screen their antioxidant peptides and verify their antioxidant properties by DPPH free radical and ABTS cationic free radical scavenging rate experiments.The amino acid sequences of Tibetan sheep hemoglobin were screened using the UniProt database to evaluate its potential bioactivities, ExPASy Peptide Cutter online tool was applied to enzymatically digest the screened peptide chains, Peptide Ranker was applied to score the bioactivity prediction, and the online tools Innovagen, ToxinPred, iBitter-SCM, A11erTOP, and AdmetSAR for solubility, toxicity, bitterness, sensitization and ADMET, molecular docking using Keap1 receptor for activity prediction, and finally validation of the screened antioxidant peptides of Tibetan sheep hemoglobin using DPPH free radical and ABTS cationic free radical scavenging rate assay.Results showed that five Tibetan sheep hemoglobin chains contained 20 amino acids, and a total of 258 kinds of peptide (485 peptides) were obtained by enzymatic digestion, and 110 kinds of peptide (194 peptides) with ratings greater than 0.5 were screened out by the bioactivity scoring system, and 18 kinds of peptide with good solubility, non-toxicity, non-bitterness, and non-allergenicity were obtained through the prediction of their activities, and the molecular docking was carried out for the 10 peptides that had a better intestinal absorption, and the top three peptides with molecular docking scores, KRM, FPL, and FL, were subjected to in vitro antioxidant activity assay, and the results showed that the free radical scavenging ability of FPL and KRM exceeded the positive control, with KRM exceeding the positive control for DPPH radicals and ABTS cationic radicals with IC50 of (0.125 0±0.012 3) and (0.339 1±0.031 4) mg/mL, respectively, and FPL exceeding the positive control for DPPH radicals and ABTS cationic radicals with IC50 of (0.256 5±0.014 1) and (0.374 6±0.047 1) mg/mL, respectively. and the scavenging ability of FL for DPPH radicals and ABTS cation radicals reached 88.90% and 94.40% of that of the positive control, with IC50 of (1.106 1±0.279) and (1.601±0.031 4) mg/mL, respectively, both of which showed strong antioxidant activity. This method could screen antioxidant peptides quickly and economically compared with the traditional enzyme digestion experiments, and provide a research basis for the high-value utilization and development of Tibetan sheep blood resources.
[1] 张强, 李伟华.抗氧化肽的研究现状[J].食品与发酵工业, 2021, 47(2):298-304.
ZHANG Q, LI W H.Research progress of antioxidant peptides[J].Food and Fermentation Industries, 2021, 47(2):298-304.
[2] BENEDÉ S, MOLINA E.Chicken egg proteins and derived peptides with antioxidant properties[J].Foods, 2020, 9(6):735.
[3] RIZZELLO C G, TAGLIAZUCCHI D, BABINI E, et al.Bioactive peptides from vegetable food matrices:Research trends and novel biotechnologies for synthesis and recovery[J].Journal of Functional Foods, 2016, 27:549-569.
[4] 刘聪, 尹乐斌, 罗鑫阳, 等.响应面法优化辣椒籽抗氧化肽的酶解制备工艺[J].粮食与油脂, 2024, 37(2):111-115.
LIU C, YIN L B, LUO X Y, et al.Optimization of enzymatic hydrolysis of antioxidant peptides from Capsicum annuum seeds by response surface method[J].Cereals & Oils, 2024, 37(2):111-115.
[5] ZHU B Y, HE H, HOU T.A comprehensive review of corn protein-derived bioactive peptides:Production, characterization, bioactivities, and transport pathways[J].Comprehensive Reviews in Food Science and Food Safety, 2019, 18(1):329-345.
[6] 陈昶宇, 陈博睿, 赵卿宇, 等.小米多肽酶解工艺及其抗氧化和ACE抑制活性[J].中国食品学报, 2023, 23(12):125-133.
CHEN C Y, CHEN B R, ZHAO Q Y, et al.Enzymatic hydrolysis process of millet polypeptides and its antioxidant and ACE inhibitory activities[J].Journal of Chinese Institute of Food Science and Technology, 2023, 23(12):125-133.
[7] YANG J, HUANG J C, ZHU Z S, et al.Investigation of optimal conditions for production of antioxidant peptides from duck blood plasma:Response surface methodology[J].Poultry Science, 2020, 99(12):7159-7168.
[8] ZHENG Z J, SI D Y, AHMAD B, et al.A novel antioxidative peptide derived from chicken blood corpuscle hydrolysate[J].Food Research International, 2018, 106:410-419.
[9] 周成伟, 李芮洋, 李景敏, 等.羊血的开发和利用[J].科技视界, 2019(13):81-82.
ZHOU C W, LI R Y, LI J M, et al.Development and utilization of sheep blood[J].Science & Technology Vision, 2019(13):81-82.
[10] 韩晴, 王维婷, 王守经, 等.羊血亚铁血红素的酶法制备及抗氧化活性研究[J].食品科技, 2023, 48(3):147-154.
HAN Q, WANG W T, WANG S J, et al.Enzymatic preparation and antioxidant activity of ferroheme from sheep blood[J].Food Science and Technology, 2023, 48(3):147-154.
[11] SOUSA R S, MINERVINO A H H, OLIVEIRA F L C, et al.Impact of blood storage duration on hematologic, blood gas, biochemical, and oxidative stress variables in sheep undergoing allogeneic blood transfusions[J].Veterinary Clinical Pathology, 2020, 49(4):545-556.
[12] LEÓN MADRAZO A, SEGURA CAMPOS M R.In silico prediction of peptide variants from Chia (S.hispanica L.) with antimicrobial, antibiofilm, and antioxidant potential[J].Computational Biology and Chemistry, 2022, 98:107695.
[13] OLSEN T H, YESILTAS B, MARIN F I, et al.AnOxPePred:Using deep learning for the prediction of antioxidative properties of peptides[J].Scientific Reports, 2020, 10(1):21471.
[14] MINNEE H, RACK J G M, VAN DER MAREL G A, et al.Solid-phase synthesis and biological evaluation of peptides ADP-ribosylated at histidine[J].Angewandte Chemie (International Ed), 2024, 63(4):e202313317.
[15] MINKIEWICZ P, IWANIAK A, DAREWICZ M.BIOPEP-UWM database of bioactive peptides:Current opportunities[J].International Journal of Molecular Sciences, 2019, 20(23):5978.
[16] CHEN M D, PAN D D, ZHOU T Q, et al.Novel umami peptide IPIPATKT with dual dipeptidyl peptidase-IV and angiotensin I-converting enzyme inhibitory activities[J].Journal of Agricultural and Food Chemistry, 2021, 69(19):5463-5470.
[17] HU J, ZHOU X Y, CAO Y K, et al.Bioinformatics-based analyses and B/Th cell epitope prediction of mugwort pollen allergen art v 1 protein[J].Journal of Visualized Experiments, 2023:202.
[18] PURCELL D, PACKER M A, HAYES M.Identification of bioactive peptides from a Laminaria digitata protein hydrolysate using in silico and in vitro methods to identify angiotensin-1-converting enzyme (ACE-1) inhibitory peptides[J].Marine Drugs, 2023, 21(2):90.
[19] MARCET I, DELGADO J, DÍAZ N, et al.Peptides recovery from egg yolk lipovitellins by ultrafiltration and their in silico bioactivity analysis[J].Food Chemistry, 2022, 379:132145.
[20] NINGRUM A, WARDANI D W, VANIDIA N, et al.In silico approach of glycinin and conglycinin chains of soybean by-product (okara) using papain and bromelain[J].Molecules, 2022, 27(20):6855.
[21] DESALE V J, MALI S N, THORAT B R, et al.Synthesis, admetSAR predictions, DPPH radical scavenging activity, and potent anti-mycobacterial studies of hydrazones of substituted 4-(anilino methyl) benzohydrazides (Part 2)[J].Current Computer-Aided Drug Design, 2021, 17(4):493-503.
[22] PARK Y R, PARK C I, SOH Y.Antioxidant and anti-inflammatory effects of NCW peptide from clam worm (Marphysa sanguinea)[J].Journal of Microbiology and Biotechnology, 2020, 30(9):1387-1394.
[23] ZAKY A A, ABD EL-ATY A M, MA A J, et al.An overview on antioxidant peptides from rice bran proteins:Extraction, identification, and applications[J].Critical Reviews in Food Science and Nutrition, 2022, 62(5):1350-1362.
[24] ZHU L J, MA M H, AHN D U, et al.Hatched eggshell membrane can be a novel source of antioxidant hydrolysates to protect against H2O2-induced oxidative stress in human chondrocytes[J].Antioxidants, 2022, 11(12):2428.
