Orientador: Prof. Dr. Silvio Cláudio da Costa

Data da Defesa: 19/06/2020


INTRODUCTION: The leaves, stems and flowers of the Stevia rebaudiana Bertoni (Family – Asteraceae) contains steviol glycosides with sweetness power ranging between 300 and 450 times greater than sucrose, acting at the limit of the sweet taste perception. Among the steviol glycosides, the most remarkable are stevioside and rebaudiosides-A and C classified according to its sweet sensorial profile. The stevioside and some phytochemical components found in S. Rebaudiana leaves have as characteristic residual bitter taste in its composition, limiting its use in the food industry. Sweetener extractions studies have been carried out with the intention of minimizing or retarding the characteristic residual flavour in the stevia leaf in natura. The development of new formulations of non-sweetener-based products, which are endowed of characteristic and taste close to sucrose is a challenge for the food industry due to the constant concern of people about the health risks of high sucrose intake, such as diabetes, obesity and cardiovascular disease. The aim of this work was to perform ethanolic pretreatment of stevia leaves and to characterize to use them in formulations of cereal bars. The food formulation (cereal bar) developed was approved by judges who performed the sensory analysis. In view of the approved formulation, the animal supplement was developed to evaluate its antidiabetic effects. OBJECTIVE: The aim of this study was to perform the ethanol pretreatment of stevia leaves of the seminal variety UEM-13. To develop cereal bars sweetened with stevia leaves pretreated with ethanol, aiming to replace in these products the use of sucrose and synthetic dietary sweeteners and to develop an animal supplementation to evaluate its anti-diabetic activity.

MATERIALS: The stevia leaves of the seminal variety UEM-13, cultivated at NEPRON (Núcleo de Produtos Naturais da Universidade Estadual de Maringá) were harvested at the stage of maximum vegetative growth (55-60 days). The solvents and reagents used were analytical grade (P.A) with purity >99.5%. Ethanolic pretreatment: The ethanolic treatment of stevia samples was performed using ion exchange chromatography, according to the methodology described by Formigoni et al. (2018). Total glycosides contents (CLAE), phenolic compounds (Folin), total flavonoids, antioxidant capacity (DPPH), centesimal and physicochemical characterization of the ethanolic pretreatment of stevia leaves were performed. Development of cereal bars: The formulations were prepared at Laboratory of Vegetal Products (UEM – Universidade Estadual de Maringá, campus of Umuarama-PR), and the ingredients were purchase from local markets. The formulations of the cereal bars were expressed as ingredients percentage: oat flake (18%), granola without added sugar (29%), dehydrated apricot (9%), dehydrated cranberry (9%), stevia leaves (1.8%), acacia gum (8.2%) and water (25%). The binder solution (acacia gum, water, and stevia leaves macerated) was heated to 105 ºC by 3 min. After this time, the dry ingredients were slowly incorporated into this solution still hot, under continuous agitation in a cooking pot. The following formulations were prepared: F1 containing pre-treated stevia leaves and F2 containing stevia leaves in natura. The samples were packed using PVC film and stored for further analysis.

Microbiological analyses: Microbiological analyses were performed at Laboratory of Food Microbiology (UEM – Universidade Estadual de Maringá, Campus of Umuarama – PR), according to the methodology proposed by Silva et al. (2001), and the recommendations from RDC n0 12/2001 of ANVISA (Brasil, 2001) for cereals. Sensorial evaluations: The sensory evaluations were performed at Sensorial Analysis Laboratories (DTC/UEM), after the project

approval by Ethics Committee of UEM (CAAE 18718013.3.0000.0104). In order to verify the acceptability and preference of the panelists, a 9-point hedonic scale was adopted (1 = extremely disagreeable) (9 = extremely liked). The evaluated attributes were the general appearance, aroma, flavour and texture. Moreover, the intent to purchase the product according to the scale (5 = definitely buy; 1 = definitely not buy) has been verified. The characterization of the sensory team, regarding the consumption of diet products, was also evaluated. The sensory panel consisted of 100 untrained individuals of both gender and ages 15 to 55 years. Physiological properties evaluations: In order to perform antidiabetic activity assays, four experimental groups were established: 1) group of non-diabetic animals (ND) – this group did not receive any kind of treatment (n=5); 2) group of diabetic control animals (CD) – this group received clean food (n=5); 3) group of diabetic animals treated with food supplemented with cereal bars sweetened with ethanol pre-treated stevia leafs

(DSTV) (n=10); 4) group of diabetic animals treated with food supplemented with sucrosesweetened cereal bars (DSAC) (n=10). The groups were monitored along 30 days. Fasting blood glucose, fed state and body weight were assessed weekly. After treatments, plasma and serum glucose, total cholesterol, triglyceride and fructosamine rates were evaluated.

RESULTS AND DISCUSSION: Among the main results obtained from leaves pretreated with ethanol and without treatment, in relation, the levels of total glycosides, phenolic compounds, total flavonoids, antioxidant capacity, centesimal and physicochemical characterization. It can be seen that the stevia leaves pretreated with ethanol slightly decrease the total glycoside content. However, without changing the sensory characteristics of the sweet taste, in addition to slightly decreasing the contents of phytochemicals and proximate composition, with the exception of total fibers. Two formulations of cereal bars were developed with the same base, sweetened with stevia leaves without treatment and pretreated with ethanol. The results of the microbiological analyzes of Salmonella, total yeast and mold count, Bacillus cereus and coliforms at 45 ° C were lower than the reference values, which means that the samples are within the standards established by Brazilian legislation, resolution

RDC nº 12 , of January 2, 2001, which allow cereal bars to be used in sensory analysis. The sensory evaluation of these products showed that, among regular consumers of cereal bars, the formulation sweetened with leaves pretreated with ethanol found more than twice the acceptance in relation to the formulation sweetened with the leaf without treatment. According to the results obtained in the antidiabetic evaluation, the groups of diabetic animals that received the diet supplemented with cereal bars sweetened with stevia leaves pretreated with ethanol and sucrose, presented significantly higher food consumption, in relation to the group non-diabetic and control animals. Despite the higher food intake of these two groups of diabetic rats, they showed body weight gain similar to the group of diabetic control animals. It was found that the diabetic animals that received the diet supplemented with cereal bars sweetened with stevia leaf pretreated with ethanol showed significantly lower values than the diabetic group that received the diet supplemented with cereal bars sweetened with sucrose.

CONCLUSIONS: The ethanolic pretreatment carried out on stevia leaves resulted in an excellent extract, with a taste close to the sweet taste and minimized the residual taste characteristic of sweeteners. Cereal bars were successfully developed, within microbiological quality criteria and the sensory evaluation of these products showed that, among regular consumers of cereal bars, the formulation sweetened with pre-treated leaves obtained twice as much acceptance in relation to the formulation sweetened with untreated leaf. This demonstrates that the use of stevia leaves pretreated with ethanol can be a viable alternative as a natural sweetener for the development of food products. According to the physiological tests, diabetic animals fed with cereal bars sweetened with stevia leaves pretreated with ethanol had lower levels of liver fat when compared to diabetic control animals. A diet supplemented with cereal bars sweetened with stevia leaves pretreated with ethanol can contribute significantly to prevent degenerative diseases, cardiovascular and metabolic

diseases, such as diabetes mellitus.

Keywords: Stevia rebaudiana; cereal bar; natural sweetener; diabetes; mouse; liver fat.

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