猪骨源抗氧化肽分离纯化、结构鉴定及抗氧化作用机制  

Isolation,Purification,Structural Identification and Action Mechanism of Antioxidant Peptides Derived from Porcine Bones

作　　者：陈露露 雷阳[1] 郝阳雪 吕扬勇[1] 魏闪 赵风光 胡元森[1,3] CHEN Lulu;LEI Yang;HAO Yangxue;LÜYangyong;WEI Shan;ZHAO Fengguang;HU Yuansen(School of Biological Engineering,Henan University of Technology,Zhengzhou 450001,China;Henan Yuxin Sugar Alcohol Co.Ltd.,Anyang 456171,China;Food Laboratory of Zhongyuan,Luohe 462300,China)

机构地区：[1]河南工业大学生物工程学院,河南郑州450001 [2]河南豫鑫糖醇有限公司,河南安阳456171 [3]中原食品实验室,河南漯河462300

出　　处：《肉类研究》2025年第3期8-17,共10页Meat Research

基　　金：河南省自然科学基金青年科学基金项目(242300421556);河南工业大学高层次人才科研启动基金项目(2024BS003);横向技术开发项目(062H2023SW062)。

摘　　要：以猪骨粉为原料制备猪骨源抗氧化肽,通过单因素试验和响应面试验优化猪骨抗氧化肽酶解工艺参数,利用超滤、葡聚糖凝胶色谱分离纯化,采用液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)结合PeptideRanker数据库鉴定并筛选抗氧化活性较高的肽段,借助分子对接技术分析该肽段与Kelch样ECH相关蛋白1(Kelch-like ECH-associated protein 1,Keap1)结合方式。结果表明:酶解最优工艺参数为酶解pH 6.8、温度50℃、风味蛋白酶添加量6000 U/g、酶解时间4 h、底物质量浓度4 g/100 mL;酶解后的粗肽液经超滤和Sephadex G-25逐级分离得到3个组分(F-1、F-2、F-3),其中F-2组分抗氧化活性最高,2,2'-联氮-双(3-乙基苯并噻唑啉-6-磺酸)阳离子自由基和1,1-二苯基-2-三硝基苯肼自由基清除率分别达(94.57±1.08)%和(79.08±0.61)%;经LC-MS/MS鉴定,F-2组分含有232个肽段,经PeptideRanker预测得到的3条肽段分别为GPVGAAGP、APGPVGP、QPGPAGPG,其疏水性氨基酸占比在80%以上,说明可能具有较高的抗氧化活性;3条肽段以氢键和疏水相互作用与Keap1结合,释放核转录因子E2相关因子2(nuclear factor erythroid 2 related factor 2,Nrf2)进入细胞核,激活Keap1-Nrf2/抗氧化反应元件通路,从而发挥抗氧化作用。因此,GPVGAAGP、APGPVGP、QPGPAGPG可作为新型抗氧化剂,本研究旨在为猪骨源抗氧化肽的开发利用提供理论参考。The enzymatic preparation of antioxidant peptides from porcine bone meal was optimized by the combined use of single factor experiments and response surface methodology.The antioxidant peptides were separated and purified by successive ultrafiltration and Sephadex G-25 column chromatography,and the peptides with high antioxidant activity were selected and identified using liquid chromatography-tandem mass spectrometry(LC-MS/MS)combined with PeptideRanker.The binding of the peptides to the protein Kelch-like ECH-associated protein 1(Keap1)was analyzed by molecular docking technology.The results showed that the optimal process parameters were as follows:pH 6.8,50℃,6000 U/g flavourzyme,4 h hydrolysis,and 4 g/100 mL substrate concentration.Among three fractions(F-1,F-2,and F-3)obtained from successive ultrafiltration and Sephadex G-25 column chromatography,F-2 had the highest antioxidant activity,which scavenged(94.57±1.08)%of 2,2’-azino-bis(3-ethylbenzothiazoliline-6-sulfonic acid)(ABTS)cationic radical and(79.08±0.61)%of 1,1-diphenyl-2-picrylhydrazyl(DPPH)radical.LC-MS/MS identified 232 peptides from F-2,and three peptides,GPVGAAGP,APGPVGP and QPGPAGPG,were predicted by PeptideRanker,hydrophobic amino acids accounting for more than 80%of their total amino acids,indicating that they may have high antioxidant activity.The three peptides bound to Keap1 protein by hydrogen bonding and hydrophobic interaction,causing the release of nuclear factor erythroid 2 related factor 2(Nrf2)into the nucleus and the activation of the Keap1-Nrf2/antioxidant response element pathway to exert antioxidant effects.Therefore,GPVGAAGP,APGPVGP and QPGPAGPG can be used as new antioxidants,and the findings from this research provide a theoretical basis for the development and utilization of porcine bone-derived antioxidant peptides.

关 键 词：猪骨 酶解 抗氧化肽 分子对接 

分 类 号：TS251.94[轻工技术与工程—农产品加工及贮藏工程]