Raman Spectroscopic Authentication of Rebaudioside M: Discriminating Natural, Fermentation-Derived, and Enzymatically...

Rebaudioside M (Reb M) is a high-value steviol glycoside responsible for the most desirable sensorial profile among stevia-derived sweeteners, owing to its intense sweetness and near absence of bitter aftertaste. However, its extremely low natural abundance in Stevia rebaudiana leaves has driven the development of alternative production strategies, including microbial fermentation and enzyme-assisted bioconversion. In this work, Raman spectroscopy is employed as a rapid, non-destructive, and label-free analytical tool to discriminate Reb M obtained from three distinct sources: (i) naturally occurring leaf extracts, (ii) fermentation-derived products, and (iii) enzymatically bioconverted products. Distinct vibrational fingerprints are identified that reflect differences in glycosylation patterns, residual steviol glycoside populations, matrix components, and process-related byproducts. The results demonstrate that Raman spectroscopy enables prompt authentication of Reb M origin and provides a powerful platform for real-time quality control. Importantly, the technique allows near-zero-cost screening, thus offering a decisive advantage over conventional chromatographic methods. These findings highlight Raman spectroscopy as a key method enabling a swift procedure for ensuring transparency, safety, and consistency in next-generation Stevia sweeteners.