Department of Food and Drug, University of Parma, parco area delle scienze 17/A, Parma 43124, Italy.
J Agric Food Chem. 2023 Jun 7;71(22):8648-8655. doi: 10.1021/acs.jafc.3c01173. Epub 2023 May 23.
The greater awareness of consumers regarding the sustainability of food chains has shifted part of the consumption from animal protein sources to vegetable sources. Among these, of relevance both for human food use and for animal feed, is soy. However, its high protein content is unfortunately accompanied by the presence of antinutritional factors, including Kunitz's trypsin inhibitor (KTI). Now there are few analytical methods available for its direct quantification, as the inhibitory activity against trypsin is generically measured, which however can be given by many other molecules and undergo numerous interferences. Therefore, in this work, a direct label-free liquid chromatography-mass spectrometry (LC-MS) method for the identification and quantification of trypsin Kunitz inhibitor KTI3 in soybean and derivative products has been developed. The method is based on the identification and quantification of a marker peptide, specific for the protein of interest. Quantification is achieved with an external calibration curve in the matrix, and the limit of detection and the limit of quantification of the method are 0.75 and 2.51 μg/g, respectively. The results of the LC-MS method were also compared with trypsin inhibition measured spectrophotometrically, highlighting the complementarity of these two different pieces of information.
消费者对食物链可持续性的认识不断提高，导致部分动物蛋白来源的消费转向植物蛋白来源。在这些来源中，大豆不仅对人类食品有重要意义，对动物饲料也同样重要。然而，大豆的高蛋白含量却不幸伴随着抗营养因子的存在，其中包括 Kunitz 胰蛋白酶抑制剂（KTI）。目前，针对其直接定量的分析方法非常有限，因为通常只能测量其对胰蛋白酶的抑制活性，但实际上许多其他分子也具有抑制活性，而且还会受到许多干扰因素的影响。因此，本研究开发了一种用于鉴定和定量大豆及其衍生产品中胰蛋白酶 Kunitz 抑制剂 KTI3 的直接无标记液相色谱-质谱（LC-MS）方法。该方法基于对特定目标蛋白的标记肽进行鉴定和定量。通过在基质中进行外部校准曲线定量，该方法的检测限和定量限分别为 0.75 和 2.51μg/g。LC-MS 方法的结果还与分光光度法测量的胰蛋白酶抑制活性进行了比较，突出了这两种不同信息的互补性。
