Dual-Enzyme Biocatalytic Synthesis of Novel Rebaudioside M Analogs with Enhanced Aqueous Solubility, Physicochemical Stability, and Sensory Profiles

Rebaudioside M (RM), a steviol glycoside derived from Stevia rebaudiana Bertoni, has emerged as a promising next-generation zero-calorie sweetener. However, its low natural abundance and poor aqueous solubility limit its practical application in beverage formulations. To address these limitations, two novel RM analogs, RMLC1 and RMLC2, were synthesized from stevioside (ST) through a two-step biocatalytic strategy. ST was first α-glucosylated by Leuconostoc kimchii dextransucrase to generate the α-glucosylated intermediate RD2, which was subsequently β-glucosylated by UGT76G1 to produce RMLC1 and RMLC2, with more than 95% conversion of RD2. RMLC1 and RMLC2 were stable at temperatures up to 120 °C and across a pH range of 2–10. RMLC1 and RMLC2 exhibited 139- and 127-fold higher aqueous solubility than RM, respectively, due to α-glucosylation-induced changes in molecular conformation. In a commercial low-pH beverage, both analogs showed approximately 3-fold greater storage stability than RM. In vitro digestion assays demonstrated that both analogs released less than half the glucose generated from enzymatically modified stevia. Electronic tongue analysis suggested that RMLC1 exhibited a balanced, blend-friendly taste profile, while RMLC2 displayed a sweetness-forward profile with minimal bitterness.