摘要
采用超滤、强阴离子交换层析法分离并纯化青刺果蛋白酶解物,以1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基清除率为评价指标,筛选具有较好抗氧化活性的肽组分。通过L C-MS/MS鉴定肽序列,结合生物信息学方法分析其理化特性,并进一步利用红外光谱、分子对接技术解析其二级结构特征及与Keap1蛋白的对接位点。研究结果表明,分离得到的4个组分中F-1组分具有更好的抗氧化活性,其DPPH自由基清除率达到27.67%。选择活性更高,且分子质量小于1 kDa的肽组分,测定得到的3条肽(TALDVPPPR、TLSDAGVGGL和DLINGGKDA)的DPPH自由基清除率,其IC 50值分别为0.43、0.83、0.51 mg/mL,其中TALDVPPPR的抗氧化活性相对较高。二级结构分析结果表明,3条抗氧化肽均由α-螺旋、β-折叠和β-转角构成,其中TALDVPPPR的α-螺旋含量相对较低,使其更易与自由基结合。分子对接结果显示,TALDVPPPR、TLSDAGVGGL、DLINGGKDA以氢键和疏水作用与Keap1蛋白紧密结合,释放Nrf2进入细胞核,并激活一系列具有抗氧化和细胞保护作用的蛋白质表达,即激活Keap 1-Nrf2/ARE通路而发挥抗氧化作用。因此,TALDVPPPR、TLSDAGVGGL、DLINGGKDA可作为新型抗氧化剂,研究旨在为青刺果功能性肽的开发利用提供理论参考。
Ultrafiltration and strong anion exchange chromatography were used to isolate and purify the protease hydrolysates of Prinsepia utilis Royle.The free radical scavenging rate of 1,1-diphenyl-2-picrylhydrazyl(DPPH)was used as the evaluation index to screen the peptide components with better antioxidant activity.The peptide sequence was identified by L C-MS/MS,and its physicochemical properties were analyzed by bioinformatics methods,and the secondary structure characteristics and docking sites with Keap1 protein were further analyzed by infrared spectroscopy and molecular docking technology.The results showed that the F-1 fractions had better antioxidant activity among the four component,and its DPPH free radical scavenging rate reached 27.67%.The peptide components with higher activity and molecular weight less than 1 kDa were selected,and the IC 50 values of DPPH free radical scavenging of the three peptides(TALDVPPPR,TLSDAGVGGL,and DLINGGKDA)were 0.43,0.83,and 0.51 mg/mL,respectively,the antioxidant activity of TALDVPPPR was relatively high.Secondary structure analysis showed that the three antioxidant peptides were composed ofα-helix,β-fold andβ-turn,and the content ofα-helix in TALDVPPPR was relatively low,making it easier to bind to free radicals.Molecular docking results showed that TALDVPPPR,TLSDAGVGGL and DLINGGKDA bound to Keap1 protein by hydrogen bond and hydrophobic interaction,and released Nrf2 into the nucleus to activate the expression of a series of proteins with antioxidant and cell protective effects,that is,activate the Keap 1-Nrf2/ARE pathway to play an antioxidant role.Therefore,TALDVPPPR,TLSDAGVGGL and DLINGGKDA could be used as new antioxidants.The purpose of this study was to provide theoretical reference for the development and utilization of functional peptides from Prinsepia utilis Royle.
作者
于梦怡
刘世林
黄志远
董文明
范江平
王雪峰
YU Mengyi;LIU Shilin;HUANG Zhiyuan;DONG Wenming;FAN Jiangping;WANG Xuefeng(College of Food Science and Technology,Yunnan Agricultural University,Kunming 650201,China)
出处
《食品科学技术学报》
EI
CAS
CSCD
北大核心
2024年第2期109-119,共11页
Journal of Food Science and Technology
基金
国家自然科学基金地区科学基金项目(31960462)
云南省农业联合专项重点项目(202101BD070001-013)
云南省科协第三届青年科技人才托举工程项目(2022—2024)。