MARIA ROSA TRENTIN ZORZENON

Título da Dissertação: MICROCÁPSULAS DE EXTRATOS DE FOLHAS DE Stevia rebaudiana: CARACTERIZAÇÃO FÍSICO-QUÍMICA E FUNCIONAL

Orientador: Prof. Dr. Silvio Claudio da Costa.

Data da Defesa: 21/02/2020

INTRODUCTION
The microencapsulation process is based on the incorporation of substances in a matrix in order to preserve the physical and chemical structure of the retained compounds, protect them from degradation by environmental factors such as sunlight, oxygen, humidity, heat and pH, prevent the exposure of volatile compounds, inhibit possible adverse effects of the compounds and increase their bioavailability. Some methods are commonly used to encapsulate food ingredients, being spray drying a widely applied method due to the ease of the process and for preserving the bioactivity of hydrophilic and hydrophobic phenolic compounds. In foods, carbohydrates such as maltodextrin are used as encapsulating agents due to their low cost, high solubility and efficiency, absence of color and for not being as sweet when compared to classic sugars. Furthermore, maltodextrin proves to be efficient and stable when microencapsulating steviol glycosides, which are the major compounds of the Stevia rebaudiana plant. This plant is known worldwide for containing steviol glycosides in its leaves, with rebaudioside A and stevioside being the natural sweeteners present in greater quantities. In addition to sweeteners, several bioactive substances are present in the leaves, crude and semi-purified extracts (fractions) of Stevia, such as phenolic compounds, flavonoids, alkaloids, xanthophylls, derivatives of caffeine and chlorogenic acid, soluble oligosaccharides, free sugars, amino acids, lipids, essential oils and trace elements that are reported by the literature with positive effects in the prevention and treatment of metabolic diseases, such as diabetes. Among the extracts and fractions with these properties, there is the ethyl acetate fraction, a fraction with high antioxidant potential and content of phenolic compounds, but which has low solubility in water and therefore has limited use in food applications. In addition to this fraction, the ethanolic extract of the leaves, which would be a by-product in the process of obtaining sweeteners, also has compounds with functional effects, but is unstable and easily degraded. Thus, this dissertation, being the first study in the literature with crude and semi-purified extracts of Stevia leaves, proposes to microencapsulate the ethyl acetate fraction and the ethanolic extract in order to preserve the present compounds and increase the physicochemical characteristics and consequently the application of these products as a food supplement or additive in the food industry.
AIMS
The aim of this work was to microencapsulate extracts of Stevia leaves, ethyl acetate fraction and ethanolic extract, with possible functional effects and to evaluate their physicochemical parameters through analysis such as microencapsulation efficiency, solubility, moisture content, hygroscopicity, morphology by scanning electron microscopy, quantification of antioxidant activity and identification of bioactive compounds by ultra-high performance liquid chromatography coupled to mass spectrometer (UHPLC-MS), in order to evaluate the preservation of these compounds under different conditions of in vitro digestion and provide application as a food supplement or additive in the food industry.
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MATERIAL AND METHODS
Stevia shrubs were harvested in the period of maximum vegetative growth and dried in an air circulation oven. Then, the leaves were separated from the stems and ground in a knife mill. The milled leaves were stored for later extraction. In order to obtain the ethyl acetate fraction, the dried and ground leaves were submitted to the Soxhlet apparatus with methanol until exhaustion. After the extraction, the methanolic extract was dried in a rotary evaporator and resuspended in water. Thereafter, the aqueous phase was fractionated with organic solvents with a polarity gradient: hexane, chloroform and ethyl acetate. The ethyl acetate fraction (FA) obtained was dried in a rotary evaporator and stored for later analysis. In order to obtain the ethanolic extract, the leaves were subjected to extraction with absolute ethanol through percolation at room temperature and 14 fractions were collected and dried in a rotary evaporator. The dry ethanol extract (EE) was stored for further analysis. The FA and EE samples were microencapsulated, separately, with maltodextrin as an encapsulating agent by means of Spray Dryer, and for the EE microencapsulation, two types of maltodextrin were tested: DE 10 and DE 19. Free and microencapsulated samples were evaluated through the physicochemical characterization: microencapsulation efficiency, solubility, moisture content, hygroscopicity, water activity and structure through scanning electron microscopy. In the free and microencapsulated EE sample, colorimetric analysis was also performed. In both free and microencapsulated samples, sweeteners and phenolic compounds were quantified. These compounds were identified by HPLC and UHPLC-MS. For the free and microencapsulated EE sample, cytotoxicity and possible antidiabetic effect by inhibiting the α-amylase enzyme were analyzed. It was also assessed the bioavailability of bioactive compounds through simulation of in vitro digestion.
RESULTS AND DISCUSSION
The FA microencapsulation showed 84% of efficiency, with a significant increase in solubility from 20.77% to 81.93%. The moisture content decreased from 7.87% to 2.59% and there was an increase in hygroscopicity from 28.0 to 132.5mgH2O/g, and this behavior was also observed in EE microencapsulation, from 10.43 to 11.88% when microencapsulated with maltodextrin DE 19, which can negatively interfere with the applicability of the microcapsules. In contrast, the microencapsulation efficiency of EE was 87.70% with maltodextrin DE 10 and 76.70% with maltodextrin DE 19. The microcapsules showed greater solubility compared to the free extract, going from 62.33% to approximately 95% and without statistically significant difference for water activity. There was also no significant difference for moisture content, even with numerically significant values. All process of microencapsulation showed the formation of a uniform spherical structure with slight cracks and agglomerations. Regarding the content of bioactive compounds, the FA fraction had a high content of phenolic compounds (31.39g/100g) and antioxidant activity (94.7%), being identified by UHPLC-MS a variety of nutritionally important compounds that prove their possible functional effect in metabolic syndromes such as diabetes. For free EE, significant levels of phenolics and antioxidant activity were found, 7.21g/100g and 87.47%, respectively. Microcapsules, on the other hand, showed lower values due to the presence of maltodextrin, but with significant values of approximately 2.70g/100 and 59.20%. The content of sweeteners was 28% for the free extract and approximately 9.65%
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for the microcapsules and a series of compounds reported by the literature with various functional effects have been identified. The free and microencapsulated EE sample showed neither inhibition of the α-amylase enzyme nor cytotoxicity. Regarding the bioavailability assessment of bioactive compounds by simulation of in vitro digestion, the microencapsulation preserved these compounds from degradation with both maltodextrins used as an encapsulating agent. Microcapsules with DE 10 maltodextrin had a higher percentage of bioavailable of phenolic compounds in the gastric condition (without statistically significant difference in the other conditions) and also greater antioxidant activity in the mouth and intestinal conditions.
CONCLUSIONS
The process of microencapsulation of the FA and EE fraction with maltodextrin by Spray Dryer proved to be effective in improving the physicochemical parameters of the samples, such as solubility, allowing the increased applicability of these products in higher doses in the formulation of functional foods, in addition to preserving the significant content of phenolic compounds and antioxidant activity present. In both samples, compounds directly related to the nutritional benefits of Stevia were identified. Furthermore, microcapsules showed greater availability of bioactive compounds under different conditions of in vitro digestion, indicating that microencapsulation decreases the degradation of these compounds in the digestion process. However, additional studies are needed to evaluate possible variations, such as different spray inlet temperatures, the amount of encapsulating agent and the use of other types of encapsulating agents to optimize the microencapsulation process of Stevia leaves extracts.
Key words: Spray-dryer, maltodextrin, phenolics, antioxidant activity, UHPLC-MS.

 Artigos Publicados Vinculados a Dissertação:

https://doi.org/10.3303/CET1975052
https://doi.org/10.1111/1750-3841.15437