CASSANDRA MEIRELES TERRES

Título da Dissertação: OBTENÇÃO DE PRODUTOS TRANSGLICOSILADOS DO ESTEVIOSÍDEO E AVALIAÇÃO DE EFEITOS BIOLÓGICOS E SENSORIAIS

Orientador: Prof. Dr. Sílvio Cláudio da Costa

Data da Defesa: 19/04/2013

RESUMO GERAL

 INTRODUCTION: Stevioside is one of the major glycosides in Stevia rebaudiana (Bert.) Bertoni leaves and is a by-product from the industrial production of stevia sweeteners due to its bitter aftertaste relative to rebaudiosides A and D. The processes for obtaining rebaudioside A generate large amounts of stevioside with a low capacity for insertion in the sweetener market. Large amounts of stevioside are even produced from selected varieties of Stevia with high rebaudioside A contents, such as Stevia UEM-320. One strategy for improving the sensory profile of stevioside is enzymatic modification (or the transglycosylation process). With this technique, the oligosaccharides that are anchored in carbons 13 and 19 are altered, which permits the formation of products that maintain their sweetening power with an improved sensory profile. In this case, CGTase from diverse groups of microorganisms were used for the intermolecular transglycosylation of stevioside.
OBJECTIVES: The objective of this study was to test the CGTase commercial enzyme (Toruzyme® - Novozymes®) for the enzymatic treatment of stevioside that is generated during the purification of rebaudioside A in the NEPRON Pilot Unit. This test was conducted to determine the conversion rates of the glycosides and to identify new glycosides. Moreover, this study sought to assess the effects of enzymatically modified products on the blood glucose levels in normal rats and to evaluate their sensory properties.
MATERIALS AND METHODS: The stevioside was obtained at the NEPRON Pilot Unit by using the methodology described by Dacome (2003). Transglycosylation experiments were performed according to the methodology described by Abelyan et al. (2004), and chromatographic analyses were conducted according to Dacome (2005). Identification of the stevioside and transglycosylated products was performed by direct insertion LC-MS with the direct insertion method. To determine the effects of the transglycosylated products on blood glucose, the recommendations of Krishnamurti and Steffes 2001; the Diabetes control group, 1998; and UKPDS, 1998) were followed. Sensory analysis was performed with 50 untrained participants by following the 9-point hedonic scale. The resulting data were submitted for an analysis of variance (ANOVA) test followed by Tukey’s test (p<0.05%).
RESULTS AND DISCUSSION: The natural sweetener stevioside (5.8 g) was obtained from Stevia rebaudiana (Bert.) Bertoni leaves with a purity of 90%. The leaves were enzymatically treated with commercial CGTase (Toruzyme) with stevioside concentrations of 0.68 to 2.75 U/g and 5.8 g of maltodextrin for 24 h. The mean stevioside concentration decreased by a rate of 52.55% with a maximum decrease of 58%. When the incubation kinetics were measured, increased incubation times resulted in the conversion of up to 70% of the stevioside. The enzymatically treated product was analyzed by HPLC/IR and HPLC/UV (210 nm). Four additional signals were detected in addition to the signal that corresponded to stevioside. LC-MS confirmed the presence of stevioside ([M+Na] 827.4) and steviol glycosides with one, two or three attached glucose molecules (m/z 989.4, 1151.5 and 1313.4). The venous infusion of 5.6 mM of the enzymatically modified product did not significantly affect the plasma glucose concentrations in normal rats. In addition, the enzymatically treated product did not interfere with glucose tolerance. The sensory analysis revealed that the enzymatically treated product had a better aftertaste with a mean value of 6.92 (relative to 4.52) for stevioside. In addition, this product was preferred by 86% of the testers.
CONCLUSIONS: The commercial enzyme Toruzyme (CGTase), with a stevioside concentration of 0.68 to 2.75 U/g, promoted the transglycosylation of stevioside. In this case, stevioside derivatives with one, two or three attached glucose molecules were identified in the transglycosylated product. The transglycosylated product did not cause any significant effects when added at a concentration of 5.6 mM regarding the plasma glucose levels in normal rats. In addition, the transglycosylation process generated a mix of enzymatically modified products with less of a bitter aftertaste that were preferred over stevioside by the testers.
KEY WORDS: stevioside, stevia, Toruzyme, transglycosylation

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