大鲵肝脏金属硫蛋白提取工艺优化及生物活性分析  

Extraction Process Optimization and Bioactivity Analysis of Andrias davidianus Liver Metallothionein

作　　者：郝林英 骆建林 随坤宇 王明波 黄青青 华雪 叶婷 王开卓 商文聪 孟永禄 李灿 HAO Linying;LUO Jianlin;SUI Kunyu;WANG Mingbo;HUANG Qingqing;HUA Xue;YE Ting;WANG Kaizhuo;SHANG Wencong;MENG Yonglu;LI Can(College of Biological and Environmental Engineering,Guiyang University/Guizhou Provincial Key Laboratory of Rare Animals and Economic Insects in Mountainous Areas/Guizhou Provincial Engineering Research Center for Biological Resources Protection and Efficient Utilization of the Mountainous Region/Guiyang Agricultural(Ecological Fishery)Industry Technology Innovation Center,Guiyang 550005,China)

机构地区：[1]贵阳学院生物与环境工程学院/贵州省山地珍稀动物与经济昆虫重点实验室/贵州省山地生物资源保护和高效利用工程研究中心/贵阳市农业(生态渔业)产业技术创新中心,贵州贵阳550005

出　　处：《食品工业科技》2024年第22期188-199,共12页Science and Technology of Food Industry

基　　金：贵州省高层次创新人才“百”层次人才(黔科合平台人才(2016)4020号);贵州省贵阳学院院士工作站(20195605);贵州省科技计划项目(黔科合基础-ZK[2021]一般251);贵阳学院博士科研启动项目(GYU-KY-[2024]);贵州省大学生创新创业训练项目(2022年);贵阳学院硕士研究生科研基金项目(GYU-YJS[2021]-62)。

摘　　要：为提高大鲵加工副产物利用价值,探索两栖动物金属硫蛋白结构、活性和功能,本研究采用匀浆离心法从大鲵肝脏提取金属硫蛋白(AdMT),通过单因素和正交试验优化提取工艺,利用离子交换层析分离纯化,以自由基清除能力检测抗氧化活性,乳酸脱氢酶释放率测试细胞毒性,脂多糖诱导RAW264.7细胞炎症模型评价抗炎活性,CCK-8法分析细胞增殖能力和免疫原性,流式细胞术检测细胞周期,划痕实验测试细胞迁移能力,CdCl_(2)诱导L929和HaCaT细胞中毒模型评价重金属解毒能力。结果表明:最佳提取工艺为液料比5:1、提取液浓度0.02 mol/L、提取温度40℃,提取时间1 h,提取含量为0.362 mg/g。AdMT对ABTS^(+)自由基的清除率达到95.01%,对L929和HaCaT细胞乳酸脱氢酶释放率及RAW264.7细胞存活率均无显著影响。AdMT处理48 h后,L929和HaCaT细胞存活率分别达到192.63%和207.92%,细胞增殖指数分别上升至0.56和0.53。同时,细胞迁移能力随着AdMT浓度增加不断增强,划痕愈合率分别为63.38%和76.23%。此外,AdMT能显著(P<0.05,P<0.01,P<0.001)降低细胞炎症模型中炎症因子的表达水平,将镉中毒细胞存活率恢复至对照组水平。研究结果为大鲵金属硫蛋白的进一步开发和大鲵副产物加工利用提供了参考。In this study,the by-products processed from Andrias davidianus were evaluated,and the structure,activity,and functionality of the metallothionein from this amphibian species(AdMT)were investigated.AdMT was extracted from the liver using a centrifugal homogenization method,with the extraction process optimized by means of single-factor and orthogonal experiments.Subsequently,the protein was purified using ion-exchange chromatography.The antioxidative activity of AdMT was assessed by measuring its free radical-scavenging ability,and its cytotoxicity was evaluated using the lactate dehydrogenase release assay.The anti-inflammatory activity of the protein was determined by RAW264.7 cell model of lipopolysaccharide-induced inflammation,and its immunogenicity and effect on cell proliferation were analyzed using the CCK-8 assay.Its effects on the cell cycle and cell migration ability were assessed using flow cytometric and scratch assays,respectively.Its heavy metal detoxification capacity was evaluated using L929 and HaCaT cell models of CdCl_(2)-induced toxicity.Results showed that,the optimal AdMT extraction conditions were determined to be as follows:Liquid-to-material ratio of 5:1,extraction solution concentration of 0.02 mol/L,extraction temperature of 40℃,and extraction time of 1 h,yielding 0.362 mg/g of the protein.AdMT exhibited a 95.01%scavenging rate against ABTS^(+)free radicals and had no significant impact on the lactate dehydrogenase release rates of L929 and HaCaT cells or on the viability of RAW264.7 cells.After 48 h of AdMT treatment,the viability of L929 and HaCaT cells improved to 192.63%and 207.92%,respectively,and their cell proliferation indices increased to 0.56 and 0.53,respectively.Additionally,the migration ability of the L929 and HaCaT cells increased with increasing AdMT concentration,showing scratch healing rates were 63.38%and 76.23%,respectively.In the cell inflammation model,AdMT significantly(P<0.05,P<0.01,P<0.001)reduced the expression levels of inflammatory factors.Furthermore,the

关 键 词：大鲵 金属硫蛋白 抗氧化 抗炎 细胞增殖 细胞迁移 重金属解毒能力 

分 类 号：TS254.9[轻工技术与工程—水产品加工及贮藏工程]