基于特种乳差异蛋白组分析建立乳间掺伪检测方法的研究

With China's rapid economic development, the production and consumption of dairy products have been ranked in the world, the safety of dairy products have gained much national attention. After the public of two laws "People's Republic of China Food Safety" and the new "dairy safety standards", China's dairy industry and market get further regulation; but with implementing of the sustainable development strategy of China's agriculture, further strengthen of the characteristics aquaculture, dairy raw materials are becoming more diverse - cattle, sheep, horses , donkeys, camel milk, etc. The appearance of these features dairy also raised new dairy safety issues. For example, how to prevent adding relatively inexpensive milk adulteration in features dairy. In this project, proteomics methods will be used for comparative analysis of protein type and content of cattle, horses, donkeys, camels and so on, other milk between the comparative analysis to determine significant differences between lactoprotein and their separation, purification and identification. Using acetate cellulose membrane electrophoresis, non-denaturing PAGE electrophoresis, SDS-PAGE electrophoresis and RP-HPLC technique to detect the incorporation of another protein in milk ingredients, verify the difference between the milk protein detection sensitivity and reliability; establish ELISA technical to test milk adulteration, comprehensive evaluation of various detection methods, optimize pre-treatment methods of the dairy, and eventually, establish a rapid quantification for all types of dairy milk proteins to detect adulteration between the best solution. Improve the dairy detection technology; protect the development of features dairy industry.

随着我国经济的高速发展，乳品生产及消费均已位居世界前列，其安全问题备受关注，《中华人民共和国食品安全法》和新《乳品安全国家标准》颁布后，我国乳业及市场进一步规范；但随着各地特色养殖经济进一步加强，乳品原料趋于多样化，牛、羊、马、驴、驼等特色乳品的出现对乳品安全提出了新课题，如何防范特色乳品中添加相对便宜乳类的掺伪行径。本项目将通过蛋白组学的研究方法对牛、马、驴、驼等各乳间的蛋白种类和含量进行对比分析，确定乳间差异显著蛋白，并对其分离、纯化、鉴定。通过醋酸纤维膜电泳、非变性PAGE电泳、SDS-PAGE电泳和RP-HPLC技术检测各乳中彼此掺入的蛋白成分，验证各乳间差异蛋白质检测中的灵敏度和可靠性；建立Elisa检测乳间掺伪技术方法，对各检测技术进行综合评价，优化各乳品的前处理方法，最终建立适合各类乳品的快速检测乳间蛋白掺伪的最佳方案；完善国家乳品检测技术，为特色乳业发展保驾护航。

本项目建立了SDS-PAGE、Native-PAGE、间接ELISA、HPLC和双重PCR等方法用于检测新疆特种乳中掺伪牛乳，并探讨热处理对方法检出限的影响，结论如下：.1. 利用SDS-PAGE以β-乳球蛋白为标志物检测驼乳中牛乳的掺伪，以αs1-酪蛋白为标志物检测驴乳中牛乳的掺伪，牛乳检出限在10~20%之间。.2. 利用Native-PAGE，以乳清蛋白3种为标志物检测特种乳中掺伪牛乳，牛乳检出限在5~30%之间；以酪蛋白为标志物牛乳检出限在10~50%之间。.3. 采用RP-HPLC分析乳样品发现：以β-乳球蛋白为检测标志物，检测掺入驼乳的牛乳，鲜驼乳掺伪最低可检测至45.55 mg/mL，巴氏驼乳掺伪为63.15 mg/mL，高温灭菌驼乳掺伪为84.11mg/mL。依据α-乳白蛋白为检测标志物，鲜驼乳掺伪最低检测限为12.45mg/mL，巴氏驼乳掺伪为0.46mg/mL，高温灭菌驼乳掺伪为12.07mg/mL。.4.以线粒体基因序列为靶基因，设计特异性引物，建立的双重PCR方法,驼、马、驴和山羊乳乳样中牛乳掺伪的检出限在鲜乳中分别为0.1%，0.1%，0.5%和0.1%，在巴氏消毒乳中分别为0.2%，0.2%，2%和1%，在高温灭菌乳中分别为0.5%，0.5%，2%和2%，在冷冻干燥乳中分别为0.1%，0.1%，2 %和0.2%；由于热处理对样品DNA的破坏，导致检出灵敏度降低。.5. 采用驼乳α-乳白蛋白，牛乳β-乳球蛋白制备多克隆，建立检测掺伪乳的ELISA方法，最低检出限均为5%，掺伪乳量在掺入0-50%范围内具有良好的线性关系。.结果表明，项目建立的双重PCR，SDS-PAGE、Native-PAGE、间接ELISA和HPLC方法适用于原料乳及不同加工方式处理（巴氏消毒、高温灭菌和冷冻干燥）新疆特种乳乳制品中牛乳掺伪的检测，其中不同的热处理会对DNA和蛋白质会产生一定的破坏，但程度不同，造成灵敏度有所降低。其中双重PCR方法灵敏度高，可用于准确定性检测；SDS-PAGE、Native-PAGE灵敏度较低，但设备简单、价格低廉、操作方便、耗时短；间接ELISA方法灵敏度高，速度快，有望开发成试剂盒，用于原料乳快速检测；HPLC法准确度高，但是时间长、成本高可用于成品检测。