Çekiç Sema Demirci, Demir Aslı, Başkan Kevser Sözgen, Tütem Esma, Apak Reşat
Faculty of Engineering,Department of Chemistry,Istanbul University,34320 Istanbul,Turkey.
J Dairy Res. 2015 May;82(2):177-84. doi: 10.1017/S0022029915000114. Epub 2015 Mar 3.
Most milk-applied antioxidant assays in literature are based on the isolation and quantification of individual antioxidative compounds, whereas total antioxidant capacity (TAC) gives a more holistic picture due to cooperative action of antioxidants. Recently, the cupric reducing antioxidant capacity (CUPRAC) method has been modified to measure the antioxidant capacities of thiol-containing proteins, where the classical ammonium acetate buffer - that may otherwise precipitate proteins- was replaced with concentrated urea buffer (able to expose embedded thiol groups of proteins to oxidative attack) adjusted to pH 7.0. Thus, antioxidant capacity of milk was investigated with two competing TAC assays, namely CUPRAC and ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid))/persulphate, because only these assays were capable of evaluating protein contribution to the observed TAC value. As milk fat caused turbidity, experiments were carried out with skim milk or defatted milk samples. To determine TAC, modified CUPRAC method was applied to whole milk, separated and redissolved protein fractions, and the remaining liquid phase after necessary operations. Both TAC methods were investigated for their dilution sensitivity and antioxidant power assessment of separate milk fractions such as casein and whey. Proteins like β-lactoglobulin and casein (but not simple thiols) exhibited enhanced CUPRAC reactivity with surfactant (SDS) addition. Addition of milk protein fractions to whole skim milk produced significant 'negative-biased' deviations (up to -26% relative standard error) from TAC absorbance additivity in the application of the ABTS method, as opposed to that of the CUPRAC method less affected by chemical deviations from Beer's law thereby producing much smaller deviations from additivity (i.e. the property of additivity is valid when the measured TAC of a mixture is equal to the sum of individual antioxidant capacities of its constituents).
文献中大多数应用于牛奶的抗氧化剂测定方法是基于对单个抗氧化化合物的分离和定量，而总抗氧化能力（TAC）由于抗氧化剂的协同作用能提供更全面的情况。最近，铜离子还原抗氧化能力（CUPRAC）方法已被改进以测量含硫醇蛋白质的抗氧化能力，其中经典的醋酸铵缓冲液（否则可能会使蛋白质沉淀）被pH值调至7.0的浓缩尿素缓冲液（能够使蛋白质中嵌入的硫醇基团暴露于氧化攻击）所取代。因此，用两种相互竞争的TAC测定方法，即CUPRAC和ABTS（2,2'-联氮双（3-乙基苯并噻唑啉-6-磺酸））/过硫酸盐，来研究牛奶的抗氧化能力，因为只有这些测定方法能够评估蛋白质对观察到的TAC值的贡献。由于乳脂肪会导致浑浊，实验使用脱脂牛奶或脱脂乳样品进行。为了测定TAC，将改进的CUPRAC方法应用于全脂牛奶、分离并重新溶解的蛋白质组分以及必要操作后的剩余液相。研究了这两种TAC方法的稀释敏感性以及对酪蛋白和乳清等单独牛奶组分的抗氧化能力评估。像β-乳球蛋白和酪蛋白这样的蛋白质（而不是简单的硫醇）在添加表面活性剂（SDS）时表现出增强的CUPRAC反应性。在应用ABTS方法时，向全脂脱脂牛奶中添加牛奶蛋白质组分产生了与TAC吸光度加和性显著的“负偏差”（相对标准误差高达-26%），而CUPRAC方法受比尔定律化学偏差的影响较小，因此产生的加和性偏差要小得多（即当混合物的实测TAC等于其成分的各个抗氧化能力之和时，加和性特性才有效）。
