Orientador: Prof. Dr. Silvio Claudio da Costa

Data da Defesa: 13/09/2020


INTRODUCTION. Stevia rebaudiana is a plant with relatively recent commercial exploitation. Since the discovery of its sweetening power reported and documented by Bertoni in the mid-1900s, its leaves and methods of recovering steviol glycosides, sweetening compounds, have been extensively studied. In recent years, special attention has turned to bioactive compounds and physiological benefits that plant extracts can contain. Also, little is known about the other parts of the plant, such as stems, branches and flowers, considered as residues of the productive chair, since the research published in the literature focuses on the leaves. Studies show that in addition to steviol glycosides, other components of the plant can be recovered and used for various purposes, such as food preservation, pathogen control, food fortification for nutraceutical purposes, among others. AIMS. The aim of this study was, within the production chain of stevia rebaudiana, to obtain extracts with high antioxidant potential adding value to the by-products, and also to identify the components present in the extracts that add value to both, as well as to verify their bioactivity and propose possible applications. MATERIAL AND METHODS. A purification process of steviol glycoside extracts was investigated after each step (extraction, ultra, micro and nanofiltration, ion exchange, adsorption). The residue obtained after the nanofiltration step was dried on a rotary evaporator and analyzed for phenolic compounds, flavonoids and antioxidant activity and for identification of compounds in Ultra High Efficiency Liquid Chromatography coupled with Mass Spectrometry - UHPLC-MS / MS-Qtof compiling the mass fragments with search in online databases like Massbank, Humam Metabolome Database and Respect for Phytochemicals. In addition, stevia stems previously separated from the leaves and ground were extracted with methanol. In order to separate the present compounds, the dry methanolic extract was hydrated (1: 3 w / v) and extracted in solvents with different polarities (hexane, chloroform, ethyl acetate and isobutanol). For the characterization of the stems, the methanolic extract was analyzed for antioxidant activity, phenolic compounds, in vitro digestion, cytotoxicity, and identification of compounds through UHPLC-MS/MS-Qtof. Afterwards, the fractionated extracts obtained from the different solvents and dried on a rotary evaporator were also investigated for antioxidant activity, phenolic compounds, identification of compounds by UHPLC-MS/MS-Qtof, Gas Chromatography coupled to Mass Spectrometry - GC/MS and antifungal activity in four species (Aspergillus flavus, Penicilliume citrinum, Rhizopus stolonifer and Botrytis cinerea). RESULTS AND DISCUSSION. It can be seen that Membrane Separation Processes (MSP), applied to obtain steviol glycosides, generate a permeate in the nanofiltration stage, discarded as residue, with a high concentration of phenolic compounds (58g EAG / 100 g of extract) and presence 3% steviol glycosides. Flavonoids, terpenoids such as sterebin and austroinulin were identified in the extract, in addition to steviol glycosides, thus characterizing the fraction as a high added value residue. This study also demonstrates for the first time that stems and leaves have a very different steviol glycoside composition. The rebaudioside A, which is the main glycoside present in the leaves of Stevia UEM-13, is not present in the STM. Significant differences in the composition of labdanic diterpenes indicate that, for the purpose of obtaining high quality sensory Stevia extracts, it may be advantageous to process Stevia leaves without stems, as they are rich in labdanic diterpenes, which are extremely bitter and difficult to remove during the process of separating and refining Stevia extracts. The crude methanolic extract after drying, was rehydrated and fractionated, resulting in the hexane (HF), chloroform (CF), ethyl acetate (EAF) and isobutanol (IF) fractions. The HF obtained is rich in hydrocarbons and presented the best MIC and MFC results, among the tested fractions, against different strains. CF, also rich in hydrocarbons, inhibited the growth of all tested microorganisms and had a fungicidal action on Rhizopus stolonifer. The ethyl acetate fraction (EAF) showed a composition rich in phenolic compounds of different classes, with minimal inhibitory action for all studied species; however, antifungal action was detected only against Botrytis cinerea. In IF, terpenoids, quinic acid, caffeic and derivatives were recovered. Among the four fractions studied, IF, had the worst performance, with MIC and MFC only for Botrytis cinerea. The fractionation of stevia stem extract proved to be a good alternative for separation and concentration of classes of compounds with potential antifungal applications; Botrytis cinerea being the microorganism most sensitive to all fractions. HF rushed the most relevant results. Such fungi are widely found in food and represent a major post-harvest problem, especially in cereals and fruit and vegetables. Finally, a wide analysis of the chemical composition of the different stem fractions was performed, obtaining unprecedented results, according to the best of our knowledge. Integral stem and its fractions, demonstrated great potential to be used as a source of antioxidant compounds and products to control harmful pathogens in the production, conservation and safety of cereals and hotifruits CONCLUSIONS. It can therefore be concluded that during industrial processing to obtain high purity steviol glycosides, there is the possibility of recovering residues rich in phenolic compounds and with high value. Furthermore, it was found that stevia stem is potential source rich in a wide variety of compounds and that the fractionation technique is suggested as an alternative to obtain purified fractions with antioxidant and antifungal activity in food spoilage microorganisms, drawing attention from the academic community for the utilization of this part of the plant and prompting further studies to apply these extracts in food preservation. Key words: Stevia rebaudiana, stems, phenolic compounds, chromatography, in vitro digestion, antifungal activity, cytotoxicity


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