Grain Chemistry and Utilization Laboratory, National Small Grains and Potato Germplasm Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Aberdeen, Idaho, USA.
J Food Sci. 2022 May;87(5):2018-2033. doi: 10.1111/1750-3841.16141. Epub 2022 Apr 22.
Protease inhibitors of protein nature are rich in seeds of legume crops. There are two common types: Kunitz inhibitor, which mainly inhibits trypsin, and Bowman-Birk inhibitor, which inhibits both trypsin and chymotrypsin. Historically, trypsin inhibitor activity in legume products has been of primary interest for measurement. However, as plant proteins are increasingly used for food or feed in recent years, there is a growing interest in monitoring chymotrypsin inhibitor activity (CIA) in these products as well. Reported methods for CIA assay vary greatly and are incompletely described. No standardized or official method is available. The present study focused on developing a robust method for accurately measuring CIA, using N-benzoyl-L-tyrosine p-nitroanilide (BTpNA) as a substrate. Since BTpNA is not water soluble, a water-miscible organic solvent must be present. After investigating the effects of several factors, such as absorption spectra, organic solvent type and concentration, substrate and enzyme concentrations, inhibitor levels (which affected % chymotrypsin inhibition), the sequence of adding reagents, extractant and extraction time, and so forth, an optimized method for CIA measurement was finally developed. It features dimethylformamide as the organic solvent, the enzyme-last sequence, 5 ml total assay volume, and calculation of the inhibitor activity based on 40% chymotrypsin inhibition. The method can also be slightly modified for measuring chymotrypsin activity. The robust performance of the method was verified by measuring 11 assorted protein products, paving a way for standardization. PRACTICAL APPLICATION: With an increasing use of plant proteins, there is an urgent need to measure chymotrypsin inhibitor activity in various protein products with accuracy. After thoroughly investigating several factors, an optimized method for measuring chymotrypsin inhibitor activity in various protein products was developed. The proposed method is sensitive and robust, providing a basis for standardization. It can also be used for measuring chymotrypsin activity.
富含蛋白质的蛋白酶抑制剂存在于豆类作物的种子中。有两种常见类型：Kunitz 抑制剂，主要抑制胰蛋白酶；Bowman-Birk 抑制剂，同时抑制胰蛋白酶和糜蛋白酶。从历史上看，豆类产品中胰蛋白酶抑制剂的活性一直是主要的测量对象。然而，近年来植物蛋白越来越多地被用于食品或饲料，人们对监测这些产品中糜蛋白酶抑制剂活性（CIA）的兴趣也越来越大。报告的 CIA 测定方法差异很大，且描述不完整。没有标准化或官方方法。本研究集中于使用 N-苯甲酰-L-酪氨酸对硝基苯胺（BTpNA）作为底物，开发一种准确测量 CIA 的稳健方法。由于 BTpNA 不溶于水，因此必须存在可与水混溶的有机溶剂。在研究了几种因素的影响，如吸收光谱、有机溶剂类型和浓度、底物和酶浓度、抑制剂水平（影响糜蛋白酶抑制的百分比）、试剂添加顺序、萃取剂和萃取时间等之后，最终开发出了 CIA 测量的优化方法。该方法以二甲基甲酰胺为有机溶剂，采用酶后添加顺序，总测定体积为 5ml，并根据 40%的糜蛋白酶抑制计算抑制剂活性。该方法稍加修改即可用于测量糜蛋白酶活性。通过测量 11 种不同的蛋白质产品，验证了该方法的稳健性能，为标准化铺平了道路。实际应用：随着植物蛋白的使用越来越多，迫切需要准确测量各种蛋白质产品中的糜蛋白酶抑制剂活性。在彻底研究了几种因素之后，开发了一种用于测量各种蛋白质产品中糜蛋白酶抑制剂活性的优化方法。所提出的方法灵敏且稳健，为标准化提供了基础。它也可用于测量糜蛋白酶活性。
