Orientador: Prof. Dr. Silvio Claudio da Costa

Data da Defesa: 27/09/2021


Steviol glycosides are compounds with sweetening power extracted from the Stevia rebaudiana plant, a shrub of the Asteraceae family native to South America. These sweeteners have a sweetening power that can be up to 450 times greater than the sweetening power of sucrose and among them are stevioside, steviolbioside, rebaudiosides A, B, C, D, E, F and dulcoside. Among these, rebaudioside A is the one with the best sensory profile. To overcome some problems in plant cultivation, NEPRON, Nucleus of Research in Natural Products at the State University of Maringá, selected an elite plant called Stevia UEM-13, which has rebaudioside A as the major glycoside. This variety is multiplied by seeds, has a better rooting level and a lower level of plant mortality. To obtain the sweeteners, extraction processes are necessary, which can be conventional or unconventional. In general, researchers seek the highest rate of glycoside recovery using the lowest possible solvent:leaf ratio. These extraction processes commonly generate crude extracts with high levels of soluble solids and suspended materials, making it impossible to directly use these products without a purification process. In an attempt to obtain crude extracts with better sensory profiles and facilitate clarification and purification, pretreatments performed with different solvents are used in order to selectively extract characteristic Stevia compounds that have a bitter aftertaste such as some antioxidant compounds, jhanol, austroinulin and others labdanic alcohols and at the same time keep the sweeteners in the leaves so that they can be later extracted.
This work aimed to study the extraction of sweeteners from untreated leaves (UL) and leaves pretreated with ethanol (PL) from the Stevia UEM-13 variety, using the semicontinuous percolation method with water as solvent, in order to obtain a crude extract that can be used directly in the preparation of teas or coffees or that has the potential to achieve purity above 70 % by using a simple microfiltration step.
Stevia rebaudiana plants were harvested at their vegetative growth peak and dried in a forced air circulation oven at 60 ºC until the moisture content was lower than 10 %. Then, stems and leaves were separated and crushed in a knife mill. The leaves were stored in a polyethylene bag protected from the light to be later analyzed and extracted. For the ethanolic pretreatment, the leaves were packed with absolute ethanol in a column. This same solvent eluted through the column and 14 fractions of 300 ml were collected. These fractions were dried and stored for other purposes while the leaves were placed in a stainless steel tray for drying and then stored away from the light for further analysis. The quantification of steviol glycosides from leaves in natura, leaves pretreated with ethanol and from the extracts obtained from glycoside extraction was performed by HPLC. The extraction of steviol glycosides was performed with untreated leaves and leaves pretreated with ethanol by semicontinuous percolation, using water as a solvent at a ratio of 1:40 (w/v). For this purpose, 200 ml of boiling deionized water were percolated through 5 g of leaves that were placed on an analytical paper filter. This process was repeated 4 more times totaling 1000 ml of solvent percolated and the filtrates were collected and stored separately. The extracts obtained in each cycle had their volume and total solids content determined. Color and turbidity analyses were performed by measuring the absorbance at 420 and 670 nm. The contents of soluble and insoluble impurities were estimated through calculations that correlate the absorbance with these contents.
Three hundred grams of Stevia rebaudiana leaves of the Stevia UEM-13 variety were pretreated. For this, 5.1 L of absolute ethanol were used and after drying the leaves, a mass of 280.14 g was obtained, resulting in a yield of 93.38 %, values compatible with those in the literature. The content of total glycosides for UL was 13.6 % and 13.1 % for PL, which shows the efficiency of the ethanolic pretreatment in selectively extracting undesirable substances leaving practically unaltered the sweetener content in the pretreated leaves. Both untreated leaves and those pretreated with ethanol kept rebaudioside A as the major sweetener in their composition, with a ratio of 1.58 and 1.52, respectively. The extraction by percolation allowed the recovery of 91.64 % of the glycosides for UL and 93.80 % for PL, leaving the bagasse with a glycoside content of 0.18 % and 0.28 % respectively. In the first extraction cycle, the purity content of the extract obtained was 25.68 % for UL and 24.14 % for PL, with a glycoside recovery of, respectively, 49.73 % and 42.33 %. In the second extraction cycle, there was an increase in the purity content of the extracts obtained, with 35.21 % for UL and 38.03 % for PL, with a recovery of glycosides present in the leaves of, 34.29 % and 37.82 %, respectively. In the third extraction cycle, the purity content of the extract obtained was 44.54 % for UL and 45.96 % for PL, with a sweetener recovery of 6.16 % and 10.31 %, respectively. Absorption values at 420 nm (color) ranged from 1.4933 to 0.0543 from the first to the fifth cycle for UL and from 1.2858 to 0.0696 for PL, causing a color reduction of 96.36 % and 94.59 %, respectively. At 670 nm (turbidity), the values ranged from 0.2653 to 0.0096 for UL and from 0.1999 to 0.0105 for PL, which generates a reduction in turbidity of 96.38 % and 94.75 %, respectively. With the pretreated leaves, 903.33 ml of extract were obtained and 623.2 mg of sweeteners were recovered. With the untreated leaves, 877.33 ml of extract were obtained, recovering 614.4 mg of sweeteners. It is concluded that from 5 g of UL, it was obtained enough sweeteners to sweeten 934.8 ml of coffee, while from PL it was obtained enough sweeteners to sweeten 921.6 ml of coffee. Five grams of untreated leaves and leaves pretreated with ethanol were mixed with 80 g of coffee powder and preliminary sensory tests unequivocally demonstrated that the coffee prepared with PL did not have an unpleasant, herbaceous taste as presented in the coffee prepared with UL. It is estimated that the removal of insoluble solids by microfiltration present in the lower purity extract has the potential to raise the level of purity of the UL extract from 25.68 % to 81.12 % and of the PL extract from 24.14 % to 87.26 %. Regarding the effect of extraction cycles on the levels of stevioside, rebaudioside C, rebaudioside A and RebA/Stev ratio, it was observed that the increase in extractive cycles was accompanied by a reduction in the RebA/Stev ratio. For UL and PL, 124.63 and 122.88 mg of total glycosides per gram of leaves were recovered respectively. Regarding the recovery of rebaudioside A, the major glycoside, the values obtained were 66.53 mg/g for UL and 65.03 mg/g for PL.
The method of semicontinuous percolation using boiling water as solvent allowed steviol glycosides recovery levels higher than 90 % from both untreated leaves and leaves pretreated with ethanol, resulting in bagasse with very low glycoside contents. Extracts with lower purity can be easily purified by using microfilters, resulting in extracts with purity above 70 %. It is estimated that the removal of insoluble solids by microfiltration from the lowest purity cycle can result in an increase in the purity level of the UL extract from 25.68 % to 81.12 % and of the PL extract from 24.14 % to 87.26 %. The extracts generated from 5 g of leaves had enough sweeteners to prepare approximately one liter of coffee, with both UL and PL. Preliminary sensory tests indicated that extracts obtained from PL did not have the same pronounced and unpleasant herbaceous taste as the extracts obtained from the untreated leaves, which should allow the PL extracts to be used without any additional clarification or purification steps in the formulation of juices and beverages, in total or partial replacement of sucrose or other synthetic sweeteners.
Keywords: clarification, extraction, rebaudioside, steviol glycosides.

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