【作者】 赵静；

【导师】 徐中其；

【作者基本信息】 东华大学 ， 分析化学， 2021， 硕士

【摘要】 牛奶中含有许多人类维持生命活动所必需的矿物质和蛋白质。牛奶中矿物质和乳清蛋白的含量往往直接决定其营养价值和质量并可以提供有关是否掺假,生产工艺和奶牛疾病等重要信息。随着各种分离技术的发展与完善,测定牛奶中的金属阳离子和乳清蛋白含量已成为食品领域的一个热门课题。迄今,对于牛奶中金属阳离子和乳清蛋白的分离与测定,传统的分离检测技术面临了操作程序繁琐复杂和分析成本高两大主要缺陷,而毛细管电泳技术可以提供一种高效、快速、简便和成本低的分离检测方法测定牛奶中主要的金属阳离子和乳清蛋白。因此,本研究开发了一种有效的毛细管电泳分析方法成功地同时分离和检测了牛奶中主要的金属阳离子和乳清蛋白,并对其展开了详细的讨论。首先,介绍了毛细管电泳（CE）的发展由来与基础理论,介绍了电容耦合非接触式电导检测器（C⁴D）的构造和检测原理。其次,介绍了牛奶中主要金属阳离子和乳清蛋白的对人体的作用、研究的意义以及常用的检测方法。最后,阐述了本课题研究的目的与意义。建立了一种可靠、简单的C⁴D和紫外（UV）检测器串联模式同时测定牛奶样品中主要金属阳离子(K⁺,Ca²⁺,Na⁺,Mg²⁺)的CE方法。样品预处理包括稀释、10 m M HAc酸化牛奶样品至p H 4.55和离心。开发和优化了一种1.0 M HAc（乙酸）和12 m M L-His（L-组氨酸）组成的简单BGE溶液,在10 min内可以实现金属阳离子(K⁺,Ca²⁺,Na⁺,Mg²⁺)的完全分离。样品的进样压力设置为50 mbar,优化的进样时间为5 s,优化的分离电压为+15 k V,温度为25℃,优化的C⁴D检测器的激发电压/频率为80 V/1000 k Hz,紫外检测器的检测波长设置为200 nm。在金属阳离子分析中,C⁴D检测器和UV检测器的检出限范围分别为0.05～0.10 mg/L和0.10～0.50 mg/L,两者都具有较低的定量限和良好的线性系数,四种阳离子的回归系数（R²）都优于0.995。四种目标阳离子的日内和日间分析的相对迁移时间（相对于Li⁺）的相对标准偏差（RSD%,n=5）分别为0.35～0.52%和0.47～0.69%,四种目标阳离子的日内和日间分析的峰面积的相对标准差（RSD%,n=5）分别为2.42～4.16%和4.07～5.60%。最后,获得和比较了四种主要金属阳离子在各种不同品牌和价格的牛奶样品中的含量。运用上述BGE溶液、相同的电泳条件以及相同的样品预处理程序在20 min内成功的测定了牛奶中的两种乳清蛋白（α-Lac,β-Lg）。同时,也探索了最佳的BGE浓度和最优的电泳条件。在乳清蛋白分析中,两种乳清蛋白分析均获得了较好的线性结果,相关回归系数（R²）均优于0.995。两种乳清蛋白β-Lg和α-Lac的CE分析方法的检出限分别为5 mg/L和3 mg/L。日内和日间分析的迁移时间的相对标准差（RSD%,n=5）分别为0.25～0.33%和0.42～0.45%,峰面积的相对标准差（RSD%,n=5）分别为2.88～3.22%和3.64～4.16%。最后,运用该CE方法获得了乳清蛋白β-Lg和α-Lac在各种不同热处理方式牛奶样品中的含量。此外,在巴氏杀菌奶和超瞬时杀菌（INF）牛奶分析中,该方法能实现金属阳离子和乳清蛋白通过单次进样分析同时测定。另外,建立了一种经济成本低,简单环保,快速的毛细管CE分析方法,能够有效测定牛奶中三种主要的乳清蛋白（α-Lac、β-Lg、BSA）的含量。研究了背景电解质的浓度以及缓冲液添加剂的种类及浓度对乳清蛋白分离效果的影响,确定了最佳的BGE为1.6 M HAC,20 m M L-His,0.1%HPMC,p H 2.70。样品的进样压力设置为50 mbar,优化的进样时间为5 s,优化的分离电压为+15 k V,温度为25℃,紫外检测器的检测波长设置为200 nm。三种乳清蛋白在30～1000 mg/L范围内线性关系良好,线性相关系数（R²）均大于0.994,方法的检出限的范围为5～15 mg/L,日内和日间分析的迁移时间的相对标准差（RSD%,n=5）分别为0.34～0.47%和0.44～0.61%,日内和日间分析的峰面积的相对标准差（RSD%,n=5）分别为2.67～4.28%和3.44～5.47%。该CE方法也已应用于不同热处理类型的牛奶中乳清蛋白含量测定。更多还原

【Abstract】 The most important nutrients in milk are minerals and proteins.Minerals in milk are indispensable nutrients for regulating physiological function and promoting growth and development of human beings.The amount of whey proteins in milk often directly determines its nutritional value and quality and can also provide important information about adulteration,production techniques and cow diseases.Therefore,the study of metal cations and whey proteins content in milk has become a hot topic in the field of food.At present,the traditional analysis technologies for the determination of metal ions and whey proteins in milk have two main defects:complicated operation procedures and high analysis cost.However,capillary electrophoresis technology can provide a rapid,simple and low-cost method to determine the major metal ions and whey proteins in milk.A reliable and simple CE method for simultaneous determination of major metal cations and whey proteins in milk samples is developed and it is discussed in detail in this thesis.Firstly,the thesis introduces the basic theoretical knowledge of capillary electrophoresis（CE）and capacitively coupled contactless conductivity detection（C⁴D）.Secondly,the role of metal cations and whey proteins in milk on human body,the significance of the study of metal cations and whey proteins in milk and the commonly used detection methods for determining metal cations and whey proteins in milk are introduced.Finally,the purpose and significance of this research are introduced.A reliable and simple CE method with dual C⁴D and UV detection modes for simultaneous determination of major metal cations in milk samples was developed.Sample pretreatment comprised dilution,acidification to p H 4.55 with 10 m M HAc and centrifugation.The complete separation of metal cations K⁺,Ca²⁺,Na⁺,Mg²⁺could be achieved within 10 min in a simple BGE composed of 1.0 M HAc and 12 m M L-His with p H 2.74 under+15 k V.The samples were injected for 5 s at 50 mbar pressure,the excitation voltage and excitation frequency of the C⁴D detector were 80 V and 1000 k Hz,respectively and the detection wavelength of UV detection was set at 200 nm.In cation analysis,the range of detection limit was 0.05～0.10 mg/L for C⁴D detection and 0.10～0.50 mg/L for UV detection,respectively.It is found that the working ranges of four cations have satisfactory linearity and low quantitative limits and the correlation coefficients are better than 0.995 under both detectors,which is suitable for milk sample analysis.And the relative standard deviations（RSD%,n=5）of intraday and interday analysis were 0.35～0.52%and 0.47～0.69%for relative migration time,2.42～4.16%and 4.07～5.60%for peak area,respectively.Finally,the contents of four major metal cations in milk samples of various brands and prices were obtained.Whey proteinsα-Lac,β-Lg in milk are successfully determined within 20 min by using the same BGE solution,the same electrophoretic conditions and the same sample pretreatment procedures.In whey protein analysis,satisfactory linearity were obtained for both whey proteinsα-Lac,β-Lg analysis,and correlation coefficients（R²）were both better than 0.995 and the detection limits ofβ-Lg andα-La analysis were 5 mg/L and 3 mg/L,respectively and the relative standard deviations（RSD%,n=5）of intraday and interday analysis were 0.25～0.33%and 0.42～0.45%for migration time and 2.88～3.22%and 3.64～4.16%for peak area,respectively.Finally,the content of whey proteinβ-Lg andα-Lac in milk samples of various heat treatments were obtained.Furthermore,this CE method can realize the simultaneous determination of metal cations and whey proteins by single injection analysis in the analysis of pasteurized milk and INF milk.An economical,simple and environmentally friendly,and rapid CE analysis method was developed,which can effectively determine the three major whey proteins（α-lac,β-lg,BSA）in milk.The influence of the concentration of BGE,the kinds and the concentration of buffer additive on the separation effect of whey proteins was studied,and the optimal BGE was determined to be 1.6 M HAC,20 m M L-His,0.1%HPMC,p H 2.70.The sample was injected for5 s under a pressure of 50 mbar,the operating voltage was+15 k V,the temperature was 25°C,and the detection wavelength of the UV detector was 200 nm.The three kinds of whey proteins have a good linear relationship in the range of 30～1000 mg/L,and the linear correlation coefficient（R²）is better than 0.994,and the detection limit of the method ranges from 5～15 mg/L.The relative standard deviations（RSD%,n=5）of the intra-day analysis of migration time and peak area ranged from 0.34～0.47%and 2.67～4.28%,respectively,and the relative standard deviations（RSD%,n=5）of the inter-day analysis of migration time and peak area ranged from 0.44～0.61%and3.44～5.47%,respectively.The CE method has been applied to the determination of whey protein contents in different types of heat-treated milk.更多还原