A Comprehensive Method for Determination of Residual Protein in Rebaudioside M

The sweetener Rebaudioside M (Reb M) has broad application prospects and can be produced on a large scale by enzymatic methods. However, detecting exogenous protein residues in Reb M products remains a major challenge, and methods for detecting protein residues in Reb M have rarely been reported. In this study, Reb M was dissolved in a sodium hydroxide solution to improve its solubility, and proteins were precipitated using deoxycholic acid sodium and trichloroacetic acid solutions. This precipitation method also eliminated the interference of Reb M in the detection results. After reconstituting the residual protein with water, the protein content in Reb M was measured using the bicinchoninic acid (BCA) method. The analytical method was internally validated according to the following parameters: range of linearity, limits of detection, limits of quantification, sensitivity, accuracy, and precision. The recoveries ranged from 100.90 to 109.40%, and the relative standard deviation (RSD) for precision was less than 4%. Results from 10 samples, including process products, final products, and products from different manufacturers, demonstrated that the method was suitable for the quantitative detection of residual proteins in enzyme-catalyzed Reb M, providing technical support for the quality control of Reb M.