Stevioside (ST) and rebaudioside A (Reb A) are 250 times sweeter than sucrose, but their bitterness and low water solubility limit their application. Rebaudioside Aα (Reb Aα) was synthesized using dextransucrase. Reb Aα exhibited 80-fold greater water solubility and lower thermal mass loss than Reb A and had melting point >180 °C. Reb Aα was stable within pH range 1.4–10 and exhibited excellent storage stability in aqueous solution. In vitro digestion revealed that Reb Aα was hydrolyzed into ST and glucose. In vivo, Reb Aα administration did not affect body weight gain, glucose tolerance, energy homeostasis, or liver histology. Also, in the Reb Aα group, inflammatory crown-like structures were not observed in white adipose tissue and uncoupling protein 1 was significantly upregulated in brown adipose tissue. Gut microbiota analysis revealed no significant differences in microbial composition between the control and Reb A or Reb Aα groups.

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.