DJÉSSICA TATIANE RASPE

Título da Tese: Métodos não convencionais de extração de Glicosídeos e Compostos Bioativos da Stevia (Stevia rebaudiana Bertoni)

Orientador: Prof. Dr. Silvio Claudio da Costa

Data da defesa: 31/05/2022

RESUMO:

INTRODUCTION AND AIMS:
Stevia rebaudiana (Bertoni) is a perennial herbaceous shrub that has benefits due to its antioxidant, antimicrobial, antifungal, antitumor and antidiabetic properties. This plant has a sweetish flavor in its leaves, characteristic of compounds called steviol glycosides (SG), among which stevioside (Stv) and rebaudioside A (Reb A) stand out mainly, and rebaudioside C (Reb C) and dulcoside A (Dul A), as well as traces of rebaudiosides B, D, E and F. The presence of this matrix in the scenario of compounds with food and phytotherapic relevance and its possible attributions are widely discussed in the literature, mainly because their particularity is to provide up to 450 times more sweetening power than sucrose, not generating energy accumulation or any change to the glycemic index. However, there are still few reports involving obtaining its compounds through techniques that envision higher yields with the application of technology, linked to the precepts of green chemistry. With a view to contributing to the improvement of the matrix and techniques investigations, bringing unprecedented findings of the application of strategies still little explored in the literature, the main objectives of the four articles that make up this thesis were: 1) To present and discuss recent experimental reports, bringing together strategies that have not yet been explored, focused on obtaining compounds from Stevia rebaudiana leaves through unconventional techniques on the rise, such as ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and extractions under pressurized conditions, by pressurized liquid (PLE), subcritical water (SWE) and supercritical fluid (SFE). 2) Investigate the UAE of compounds from Stevia rebaudiana leaves, evaluating the influence of ultrasound power intensity on leaves submitted and not submitted to ethanolic pretreatment, and through the application of an experimental design, examine the effect of experimental variables (temperature, solvent/leaf ratio and percentage of ethanol in the extracting solvent), under the mass yield (YM) and sweeteners (YS), SG content, as well as its active compounds, represented by the content of total phenolic compounds (TPC) and antioxidant activity (AA) of pre-treated leaves. 3) Evaluate the efficiency of PLE by obtaining SG from leaves of Stevia rebaudiana submitted and not submitted to ethanolic pretreatment, investigating the effect of experimental variables (solvent/leaf ratio and percentage of ethanol in the extracting solvent) on YM it was. 4) Investigate the extraction of compounds from pre-treated Stevia rebaudiana leaves under pressurized conditions, through the effect of experimental variables (percentage of ethanol in the extracting solvent, static time, pressure and temperature) on YM, TPC and AA, and through of the condition that resulted in the maximization of the response variables, to determine the content of active compounds (TPC, total flavonoid content - TFC and AA) and SG, aiming to compare the extracts obtained with those resulting from the extraction by Soxhlet and UAE.
METHODS:
1) For the development of this survey, articles and patents were consulted in journals from the main databases (Science Direct, Scielo, Web of Science, Scopus, Springer and Wiley, and SciFinder, respectively). For the compilation of data, only scientific articles and patents published in the last 12 years (2009–2021) were considered, limiting themselves to data referring to i) extraction of compounds from Stevia rebaudiana leaves, ii) data referring to compounds obtained in extraction and iii) data referring to the types of unconventional extraction. The selection and screening process to form the basis of the writing was carried out independently, summarized in the analysis of a total of 35 articles and 09 patents. 2) In this study, the intensity of ultrasound power of 0, 50 and 100% (0, 83 and 165 W, respectively) was evaluated in the extraction of compounds from Stevia leaves with and without ethanolic pretreatment, in YM, YS, SG, TPC and AA, the latter represented by the concentration of extract capable of reducing the DPPH radical by 50% (EC50). A Box-Behnken experimental design with three levels, three variables and five repetitions of the central point was applied to examine the effect of temperature (35, 50 and 65 °C), solvent/leaf ratio (5, 10 and 15 mL g−1) and percentage of ethanol in the extracting solvent (10, 40 and 70%, v/v), aiming to reach the conditions that maximize the YM and YS, within the tested experimental range. Subsequently, these extracts were characterized in terms of composition, and the correlation between the operational conditions and the evaluated responses was used in the analysis of principal components. 3) To evaluate the PLE efficiency in obtaining the YM and YS, the variables temperature, pressure and time were kept fixed at 120 ºC, 100 bar and 60 min, respectively. An experimental apparatus operated in semi-continuous mode was used to conduct the evaluation of the effect of the solvent/leaf ratio (30 to 90 mL g-1) and ethanol concentration in the extracting solvent (100 and 70%, v/v) in the extraction of leaf compounds with and without ethanolic pretreatment. 4) In this study, the effect of the percentage of ethanol in the extracting solvent (40 and 70%, v/v), static time (10, 20 and 30 min) and pressure (50 and 100 bar) were investigated in the YM, TPC and AA of pre-treated Stevia leaf compounds. The conditions that maximized the response variables were then investigated in the extraction kinetics (100, 125 and 150 °C). Subsequently, a comparison was made between the extract obtained by PLE resulting from the maximization of the response variables, Soxhlet (50 mL g-1 for 8 hours) and UAE (15 mL g-1 for 3 cycles of 10 min), in relation to the YM, YS, SG, and active compounds (TPC, TFC and AA).
MAIN RESULTS AND DISCUSSION:
1) The reports leveraged through unconventional extraction methods, contemplate promising technological criteria in the world market demand for natural compounds, where Stevia rebaudiana presents itself as a matrix with efficiency and profitability in the recovery of its sweeteners and active compounds. They allow quick and easy operation, through the use of renewable solvents under operating conditions that do not affect the quality of the extract obtained. Among these processes, techniques involving ultrasound acoustic cavitations, non-ionizing microwave irradiation, compressed fluids in pressurized liquid and subcritical water extraction, as well as supercritical fluid extraction, are highlighted on the rise. In addition, there were no reports of the application of these techniques linked to strategies that could improve the process and the sensory characteristics of the extract obtained, as is the case of the application of ethanolic pre-treatment. 2) The application of maximum power (165 W) of the ultrasound cavitations provided the attainment of higher YM and YS, and extracts with higher contents of TPC and AA. The increase in the solvent/leaf ratio and the percentage of ethanol in the extracting solvent provided the highest values of YM and YS, while the temperature promoted an increase only in the YM. Maximum values of YM (44.7 wt%) and YS (88.5 wt%) were obtained using solvent with 40% ethanol, 50 °C and 15 mL g-1 (solvent/sheet). The extract obtained had ~26.0 wt% SG, corresponding to 9.5, 4.1 and 12.0 wt% of Stv, Reb C and Reb A, respectively. The analysis of the principal components indicated a high correlation of the variables percentage of ethanol in the extracting solvent and solvent/leaf ratio in obtaining extracts with higher levels of active compounds (TPC and AA), in addition to demonstrating that there is no correlation in obtaining sweeteners and compounds active. 3) Under pressurized conditions, the results showed greater extraction of SG when pre-treated leaves were used. Increasing the solvent/leaf ratio from 30 mL g-1 to 90 mL g-1 did not favor the extraction of sweeteners, demonstrating that an excess of solvent in the medium does not result in higher yields. These results made it possible to preliminarily evaluate the effects of this technique on the matrix, gathering data and information for further investigations. 4) The increase in the percentage of ethanol in the extractor solvent favored the YM, TPC and AA, while the increase in static time and pressure (>10 min and >50 bar) did not affect the extraction of compounds under the conditions evaluated. The temperature provided an increase in the YM and the highest levels of active compounds were obtained after 30 min of the process. The YM was similar between the extraction techniques evaluated, with PLE (125°C) providing greater recovery of TPC and AA, and Soxhlet favoring the extraction of SG and TPC. The composition of the extract obtained by PLE was ~26.0 wt% of SG, corresponding to 9.5, 3.9 and 12.68 wt% of Stv, Reb C and Reb A, respectively, representing 87.8% of the total obtained by Soxhlet. The active potential (TPC, TFC and AA) of the PLE extract was 3.6 and 11.0% higher than that of Soxhlet and UAE, respectively.
CONCLUSIONS:
1) The UAE, MAE, PLE, SWE and SFE techniques address environmental and food safety issues by allowing the use of renewable solvents, in addition to being aligned with the development of alternative processing routes, contemplating sustainability concepts. With a view to contributing to the improvement of these techniques and adding information to future research related to the matrix, by 2) evaluating the UAE, efficiency in obtaining an extract with high yield, TPC and AA content, and great potential for use as a food additive, it was verified. In terms of the feasibility of the process, the use of an unconventional technique of rapid processing, together with a binary mixture between ethanol and water as an extracting solvent, stands out, which provides reduced production costs compared to using only ethanol and easier solvent separation when compared to using water. 3), 4) When investigating PLE, higher YM and active compounds (TPC and AA) was observed, through the use of less energy and solvent, which combined with operational strategies such as the pre-treated matrix and the application of a binary mixture of ethanol and water as a solvent, allowed the conduction of a process in less time, with ease of separation of the extracting solvent and, at the same time, potential reduction of operational costs. Therefore, obtaining compounds from this matrix through the unconventional methods proposed here, in addition to being a viable and adequate alternative for improving the solubility and availability of analytes, has been shown to provide a reduction in operating conditions, enabling the profitable use of GRAS (generally recognized as safe) solvents in these processes. that fit with merit in the precepts of green chemistry, generating an extract with high phytotherapic and food use potential. Although a possible implementation of these processes on a pilot scale presents itself as a relevant alternative, as it allows for fewer inconveniences when compared to conventional processes, the reduction in consumption of inputs and the feasibility of reusing the solvent in the process still need to be improved in their investigation. Combined or sequential processes could be applied as alternatives to this problem, but their exploitation has not yet been reported, as well as the consequences of exposing this matrix to extreme conditions of some of the mentioned technologies, mainly in relation to the possible degradation of the target compounds. These gaps and the lack of information about these techniques are the main challenges in expanding the use of these technologies, emerging as perspectives for future work.
KEY-WORS: Active compounds; Antioxidant activity; Green solvents; Natural sweeteners; Phenolic compounds; Sweeteners; Technology application.

Publicação vinculada a Tese:

Artigo 1. RASPE, D. T.; SILVA, C.; COSTA, S. C. Compounds from Stevia rebaudiana Bertoni leaves: An overview of non-conventional extraction methods and challenge. Food Bioscience, v. 46, 101593, 2022. https://doi.org/10.1016/j.fbio.2022.101593
Artigo 2. RASPE, D. T.; CIOTTA, S. R.; ZORZENON, M. R. T.; DACOME, A. S.; SILVA, C.; MILANI, P. G.; COSTA, S. C. Ultrasound-assisted extraction of compounds from Stevia leaf pretreated with ethanol. Industrial Crops and Products, v. 172, 114035, 2021. https://doi.org/10.1016/j.indcrop.2021.114035
Artigo 3. RASPE, D. T.; CIOTTA, S. R.; MELLO, B. R. F.; MILANI, P. G.; SILVA, C.; COSTA, S. C. Pressurized liquid extraction of steviol glycosides from Stevia rebaudiana leaves. Chemical Engineering Transactions, v. 87, p. 301-306, 2021. https://doi.org/10.3303/CET2187051
Artigo 4. RASPE, D. T.; SILVA, C.; COSTA, S. C. Pressurized liquid extraction of compounds from Stevia leaf: Evaluation of process variables and extract characterization. (submetido)

Cabe ressaltar que, contemplando parte desta pesquisa, o trabalho intitulado “Efeito de diferentes métodos de extração no rendimento e obtenção de compostos bioativos de folhas de Stevia”, recebeu menção honrosa por mérito científico no V Congresso Internacional de Ciência, Tecnologia e Inovação da Universidade Paranaense, no ano de 2021.