Rebaudioside A (RA) is a low-calorie sweetener extracted from stevia and is considered an ideal substitute for sucrose.However, its potential applications in the food industry are limited due to the unpleasant aftertaste.Moderate enzymatic glycosylation of RA has been identified as an efficient approach to mitigate its aftertaste.Cyclodextrin glucosyltransferase (CGTase) is currently the predominant enzyme utilized for catalyzing RA glycosylation.Nevertheless, a major drawback of CGTase lies in the complexity and diversity of its reaction products, particularly with regard to the high proportion of long-chain glycosylated products, which severely impact the taste and quality of the resulting products.In this study, various CGTases from different sources were explored for RA glycosylation reaction.Ultimately, α-CGTase from Paenibacillus macerans was selected due to its exceptional conversion and significant specificity for short-chain products.After extensive investigation of enzymatic properties and optimization of reaction conditions, this study successfully determined the optimal reaction condition, which was employing a phosphate buffer (pH 6.0), using soluble starch as the glycosyl donor, and maintaining a substrate mass ratio of RA∶soluble starch=1∶1.With an enzyme concentration of 25 μg/mL and a reaction time of 12 h at 40 ℃, the maximum conversion of RA was achieved at 74.8%.The proportion of short-chain products (RA-nG, n≤2) in the total products increased to 86.5%, representing a 10% enhancement compared to the pre-optimized conditions.Therefore, this study effectively improves the specificity of short-chain glycosylated RA, thereby demonstrating its significant potential for industrial applications of steviol glycosides.
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