基于超声辅助酶解技术的大豆抗氧化肽制备、活性评价及分子机制

摘要/Abstract

摘要： 本研究以大豆分离蛋白（soy protein isolate，SPI）为原料，采用超声辅助酶解技术制备大豆蛋白抗氧化肽，超滤分离后测定其不同分子量肽段的1,1-二苯基-2-三硝基苯肼（1,1-diphenyl2-picrylhydrazyl，DPPH）自由基、2,2’-联氮双(3-乙基苯并噻唑啉-6-磺酸)（2,2’-azino-bis(3ethylbenzothiazoline-6-sulfonic acid)，ABTS）阳离子自由基、羟自由基清除能力和铁还原能力（Ferric reducing antioxidant power, FRAP），分析其氨基酸组成，鉴定并筛选出抗氧化活性较高的肽段进行分子对接，评价其抗氧化活性及分子机制。结果表明，分子量<3kDa的肽段具有最强的DPPH自由基、ABTS阳离子自由基、羟自由基清除能力，以及最高的铁还原能力（P＜0.05）。氨基酸组成结果表明，<3kDa小分子肽段中疏水性和抗氧化氨基酸含量较高，与其抗氧化活性呈正相关。液相色谱-串联质谱联用（Liquid Chromatography-Tandem Mass Spectrometry，LC-MS/MS）鉴定出1217条肽段，筛选出10条高活性肽段（活性评分＞0.90），分子对接分析表明其与Keap1蛋白结合能较低（-9.7至-7.2 kcal/mol），主要通过氢键和疏水作用发挥抗氧化活性。本研究为大豆蛋白资源的高值化利用和天然抗氧化肽的开发提供了理论和技术支持。

关键词: 大豆分离蛋白, 抗氧化肽, 抗氧化活性, 分子对接

Abstract: This study used soy protein isolate (SPI) as the raw material to prepare soybean protein-derived antioxidant peptides through ultrasound-assisted enzymatic hydrolysis. After ultrafiltration separation, the scavenging capacities of peptides with different molecular weights against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cationic radicals, hydroxyl radicals, and ferric reducing antioxidant power (FRAP) were determined. The amino acid composition was analyzed, and peptides with high antioxidant activity were identified and screened for molecular docking to evaluate their antioxidant activity and molecular mechanisms. The results showed that peptides with a molecular weight of <3 kDa exhibited the strongest DPPH radical, ABTS cationic radical, and hydroxyl radical scavenging capacities, as well as the highest FRAP value (P < 0.05). Amino acid composition analysis revealed that the <3 kDa small-molecular-weight peptides contained higher levels of hydrophobic and antioxidant amino acids, which were positively correlated with their antioxidant activity. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1,217 peptides, and 10 highly active peptides (activity score > 0.90) were screened. Molecular docking analysis indicated that these peptides had low binding energy (-9.7 to -7.2 kcal/mol) with Keap1 protein, primarily exerting antioxidant activity through hydrogen bonds and hydrophobic interactions. This study provides theoretical and technical support for the high-value utilization of soybean protein resources and the development of natural antioxidant peptides.

Key words: Soy protein isolate, Antioxidant peptides, Antioxidant activity, Molecular docking

中图分类号:

TS251.5

林晓华马欣史东红孙健. 基于超声辅助酶解技术的大豆抗氧化肽制备、活性评价及分子机制[J]. 食品科学, 2026, 47(2): 0-0.

sun jian. Preparation, Activity Evaluation and Molecular Mechanism of Soybean Antioxidant Peptides by Ultrasound-Assisted Enzymatic Hydrolysis[J]. FOOD SCIENCE, 2026, 47(2): 0-0.

参考文献

[1]朱秀清, 韩田露, 刘纪元, 等.大豆蛋白淀粉样纤维制备、功能特性及应用研究进展[J].食品科学, 2025, 46(03):296-305 [2]HUANG Shanfen, LI Yunliang, LI Chengliang, et al.Effects of ultrasound-assisted sodium bisulfite pretreatment on the preparation of cholesterol-lowering peptide precursors from soybean protein[J].International Journal of Biological Macromolecules, 2021, 183:295-304 [3]SANGIORGIO S, VIDOVI? N, BOSCHIN G, et al.Preparation,characterization and in vitro stability of a novel ACE-inhibitory peptide from soybean protein[J].Foods, 2022, 11(17):2667- [4]谢晋祥, 耿树香, 宁德鲁, 等.云南个主栽品种核桃多肽的制备及体外抗氧化活性的研究[J].中国油脂, 2025, 50(2):51-5694 [5]XU Yue, YANG Yang, MA Chunmin, et al.Characterization of the structure, antioxidant activity and hypoglycemic activity of soy (Glycine max L.) protein hydrolysates[J].Food Research International, 2023, 173:113473- [6]ZHENG Zhaojun, SI Dayong, AHMAD B, et al.A novel antioxidative peptide derived from chicken blood corpuscle hydrolysate[J].Food Research International, 2018, 106:410-419 [7]SUN Jin, ZHOU Changyu, CAO Jinxuan, et al.Purification and Characterization of Novel Antioxidative Peptides From Duck Liver Protein Hydrolysate as Well as Their Cytoprotection Against Oxidative Stress in HepG2 Cells13[J].Frontiers in Nutrition, 2022, 9:9848289-848289 [8]MAGALH?ES I S, GUIMAR?ES A D B, TRIBST A A L, et al.Ultrasound-assisted enzymatic hydrolysis of goat milk casein: Effects on hydrolysis kinetics and on the solubility and antioxidant activity of hydrolysates[J].Food Research International, 2022, 157:111310- [9]YOSHIKAWA M, FUJITA H, MATOBA N, et al.Bioactive peptides derived from food proteins preventing lifestyle‐related diseases[J].Biofactors, 2000, 12(1-4):143-6 [10]范路好.超声波辅助酶解对核桃蛋白酶解物抗氧化性的影响研究[J]., 2024, :5-57 [11]TIAN Shuangqi, MENG Fanhao, DU Ke.Physicochemical properties and structure characteristics of different molecular weight peptides from ultrasonic assisted papain hydrolysate of wheat germ albumin[J].Industrial Crops and Products, 2024, 211:118254- [12]王立, 卞欢, 吴海虹, 等.超声辅助酶解鹅肝蛋白的动力学和热力学及酶解物抗氧化性能[J].江苏农业学报, 2017, 33(06):1395-1401 [13]BRAND-WILLIAMS W, CUVELIER M E, BERSET C.Use of a free radical method to evaluate antioxidant activity[J].LWT-Food Science and Technology, 1995, 28(1):25-30 [14]JARA A M R, LIGGIERI C S, BRUNO M A.Preparation of soy protein hydrolysates with antioxidant activity by using peptidases from latex of Maclura pomifera fruits[J].Food Chemistry, 2018, 264:326-333 [15]ZHENG Xiqun, WANG Juntong, LIU Xiaolan, et al.Effect of hydrolysis time on the physicochemical and functional properties of corn glutelin by Protamex hydrolysis[J].Food Chemistry, 2015, 172:407-415 [16]SIDDIQ M, DOLAN K D, PERKINS-VEAZIE P, et al.Effect of pectinolytic and cellulytic enzymes on the physical, chemical, and antioxidant properties of blueberry (Vaccinium corymbosum L.) juice[J].Lwt, 2018, 92:127-132 [17]中华人民共和国国家标准.GB 5009.124-2016食品安全国家标准食品中氨基酸的测定[S]. 北京: 国家食品药品监督管理总局, 2016. [18]JE J Y, PARK P J, KIM S K.Antioxidant activity of a peptide isolated from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate[J].Food Research International, 2005, 38(1):45-50 [19]SARMADI B H, ISMAIL A.Antioxidative peptides from food proteins: A review[J].Peptides, 2010, 31(10):1949-1956 [20]JIMéNEZ‐RUIZ E I, CALDERóN DE LA BARCA A M, SOTELO‐MUNDO R R, et al.Partial characterization of ultrafiltrated soy protein hydrolysates with antioxidant and free radical scavenging activities[J].Journal of food science, 2013, 78(8):C1152-C1158 [21]WEN Chaoting, ZHANG Jixia, FENG Yuqin, et al.Purification and identification of novel antioxidant peptides from watermelon seed protein hydrolysates and their cytoprotective effects on H2O2-induced oxidative stress[J].Food Chemistry , 2020, 327:127059- [22]TANG Y, DEBNATH T, CHOI E-J, et al.Changes in the amino acid profiles and free radical scavenging activities of Tenebrio molitor larvae following enzymatic hydrolysis[J].PLoS One, 2018, 13(5):e0196218- [23]ALARJANI W M A, Mohammed M E A.Antioxidant activities of Saudi honey samples related to their content of short peptides[J].Scientific Reports, 2024, 14(1):24318- [24]范丽琪.基于肽组学的大豆抗氧化二肽酶解释放与高效制备[J]., 2024, : 27-28 [25]谢晓妍, 张芸溪, 王颖, 等.菌-酶协同法制备白芸豆抗氧化肽工艺优化及其抗氧化活性分析[J]., 2025, :1-17 [26]YESILTAS B, GARCíA-MORENO P J, GREGERSEN S, et al.Antioxidant peptides derived from potato, seaweed, microbial and spinach proteins: oxidative stability of 5% fish oil-in-water emulsions[J].Food Chemistry, 2022, 385:132699- [27]张婉婷, 张丽, 龚婉莹, 等.核桃碱性蛋白酶酶解物水解特性及其低苦味抗氧化肽的筛选与分子对接解析[J]., 2025, :1-14 [28]于梦怡, 刘世林, 黄志远, 等.青刺果抗氧化肽的分离纯化、结构鉴定及其分子对接解析[J].食品科学技术学报, 2024, 42(2):109-119 [29]李良煜.基于Keap1-Nrf2通路的蛋清源抗氧化小肽筛选及其作用机制研究[J]., 2017, :2-5