A Modified BCA Method for Determination of Residual Protein in Enzymatic Biosynthetic 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 quantitation, sensitivity, accuracy, and precision. The optimized method was then successfully applied to the determination of protein residues in Reb A, Reb D, and Reb M. The recoveries ranged from 90.60% to 109.40%, and the intraday and interday relative standard deviation (RSD) for precision was both less than 5%. 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.