Badekow Svenja, Treblin Mascha, Spöttel Jenny, Rohn Sascha
University of Hamburg, Hamburg School of Food Science, Institute of Food Chemistry, Grindelallee 117, D-20146 Hamburg, Germany.
University of Hamburg, Hamburg School of Food Science, Institute of Food Chemistry, Grindelallee 117, D-20146 Hamburg, Germany; Technische Universität Berlin, Institute of Food Technology and Food Chemistry, Department of Food Chemistry and Analysis, TIB 4/3-1, Gustav-Meyer-Allee 25, 13355 Berlin, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Sep 1;1181:122937. doi: 10.1016/j.jchromb.2021.122937. Epub 2021 Sep 15.
In complex food matrices, non-directed reactions between food proteins and secondary plant metabolites (SPM) are conceivable. In this study, the interaction between the bioactive metabolite from garden cress (Lepidium sativum) and selected Brassicaceae - benzyl isothiocyanate (BITC) - and the dairy protein α-lactalbumin (α-LA) was investigated. It was focused on monitoring the proteolytic degradation behaviour of unmodified and BITC-modified α-LA with two-dimensional high-performance thin-layer chromatography (2D-HPTLC). The two-dimensional approach of HPTLC offers high resolution in the separation of complex peptide mixtures and might enable differentiation of protein modifications. Based on the specific peptide patterns of native and modified peptides, conclusions can be drawn about differences in protein/peptide polarity, location of a modification, and digestibility. The aim was to characterize tryptically hydrolyzed unmodified and BITC-modified peptides using the 2D method and to investigate the influence of BITC modification of α-LA on polarity and digestibility. To determine the repeatability of peptide separation by 2D-HPTLC, the unmodified and BITC-modified protein hydrolyzates were separated six times. The absolute standard deviations between the retardation factors of the individual peptide spots varied between 0.52 and 4.79 mm for the x-coordinates and between 0.41 and 6.47 mm for the y-coordinates for all three samples. Here, the mean relative standard deviations ranged from 5.80 to 10.4% for the x-coordinates and from 5.91 to 18.3% for the y-coordinates. The results of the tryptic hydrolysis indicated that, depending on the concentration of BITC used, the modification sterically hinders the cleavage sites for the enzyme, resulting in a reduced digestibility. Covalent binding of the hydrophobic BITC altered the digestibility and polarity of the protein, leading to a difference in peptide patterns between the unmodified and modified α-LA. It was concluded that the reaction was undirected, resulting in a mixture of unmodified and modified peptides, and that elongated modified peptides were formed by BITC blocking of trypsin cleavage sites.
在复杂的食物基质中，食物蛋白与次生植物代谢产物（SPM）之间可能会发生非定向反应。在本研究中，对来自水田芥（独行菜）的生物活性代谢产物与选定的十字花科植物——苄基异硫氰酸酯（BITC）——和乳蛋白α-乳白蛋白（α-LA）之间的相互作用进行了研究。研究重点是通过二维高效薄层色谱法（2D-HPTLC）监测未修饰和BITC修饰的α-LA的蛋白水解降解行为。HPTLC的二维方法在分离复杂肽混合物方面具有高分辨率，并且可能有助于区分蛋白质修饰。基于天然肽和修饰肽的特定肽谱，可以得出关于蛋白质/肽极性、修饰位置和消化率差异的结论。目的是使用二维方法表征经胰蛋白酶水解的未修饰和BITC修饰的肽，并研究α-LA的BITC修饰对极性和消化率的影响。为了确定2D-HPTLC分离肽的重复性，将未修饰和BITC修饰的蛋白质水解产物分离了六次。对于所有三个样品，各个肽斑点的比移值在x坐标上的绝对标准偏差在0.52至4.79毫米之间，在y坐标上的绝对标准偏差在0.41至6.47毫米之间。此处，x坐标的平均相对标准偏差在5.80%至10.4%之间，y坐标的平均相对标准偏差在5.91%至18.3%之间。胰蛋白酶水解的结果表明，根据所用BITC的浓度，修饰在空间上阻碍了酶的切割位点，导致消化率降低。疏水性BITC的共价结合改变了蛋白质的消化率和极性，导致未修饰和修饰的α-LA之间的肽谱出现差异。得出的结论是，该反应是非定向的，导致未修饰和修饰肽的混合物，并且BITC对胰蛋白酶切割位点的阻断形成了延长的修饰肽。
