乳及乳制品中乳铁蛋白含量的快速SDS-PAGE荧光检测方法  

Rapid SDS-PAGE Fluorescent Detection Method for Lactoferrin Content in Milk and Dairy Products

作　　者：李汉芳 黄珍 路梦凡 孙娜娜 徐丹[1] 徐秦峰 LI Hanfang;HUANG Zhen;LU Mengfan;SUN Nana;XU Dan;XU Qinfeng(School of Food Science and Engineering,Shaanxi University of Science&Technology,Xi’an 710021,China)

机构地区：[1]陕西科技大学食品科学与工程学院,陕西西安710021

出　　处：《现代食品科技》2024年第4期296-302,共7页Modern Food Science and Technology

基　　金：陕西省教育厅项目(22JC008);陕西省科技厅项目(2021NY-174)。

摘　　要：该研究采用新型的Chromeo P503(P503)染料替代考马斯亮蓝染色方法,以克服脱色染色步骤繁琐、耗时长(≥5 h)的不足,建立一种乳及乳制品中乳铁蛋白的快速简便十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis,SDS-PAGE)检测方法。P503染料标记蛋白产生强烈的红色荧光发射,在30 min内一步操作即可完成,并且进行脱铁处理后消除乳铁蛋白铁饱和度对于荧光信号的干扰,保证定量的准确性。根据凝胶成像图中78 ku蛋白条带是否存在及其灰度值的大小,实现乳铁蛋白的定性与定量检测。在优化的实验条件下,线性范围为15~75μg/mL,检出限为5.11μg/mL,加标回收率在75.79%~108.09%之间,表明该方法具有较高的灵敏度和准确性。SDS-PAGE和P503结合无需进行肝素亲和柱前处理,便可直接应用于乳及乳制品中乳铁蛋白快速简便的准确定量检测,P503染料标记还可以用于其它乳蛋白的SDS-PAGE快速低成本定量检测,从而为乳品质量控制和营养评价及加工工艺研究提供有力工具。A novel dye(Chromeo P503,P503)was used as an alternative to Coomassie brilliant blue to address the drawbacks associated with the laborious and time consuming(≥5 h)staining and destaining processes.This facilitated the establishment of a rapid and simple sodium dodecyl sulfonate-polyacrylamide gel electrophoresis(SDS-PAGE)method for the detection of lactoferrin in milk and dairy products.P503 dye-labeled protein emits strong red fluorescence,which could be completed in one step within 30 min.The interference of lactoferrin iron saturation on fluorescence signal was eliminated after apolactoferrin preparation to ensure the accuracy of quantification.Qualitative and quantitative detection of lactoferrin was achieved based on the presence of a 78 ku protein band and its gray value in the gel image.Under optimized experimental conditions,the linear range was 15~75μg/mL,and the limit of detection was 5.11μg/mL,with a recovery of 75.79%~108.09%,indicating that this method has high sensitivity and accuracy.The combination of the SDS-PAGE and P503 methods enabled the rapid,simple,and accurate detection of lactoferrin in milk and dairy products,eliminating the need pretreatment of the heparin affinity column.P503 dye labeling can facilitate rapid and low-cost quantitative detection of other milk proteins using SDS-PAGE,providing a valuable tool for dairy quality control,nutrition evaluation,and processing technology research.

关 键 词：乳铁蛋白 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 Chromeo P503 快速标记 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程] O657.3[轻工技术与工程—食品科学与工程]