Orientadora: Profa. Dra. Grasiele Scaramal Madrona

Data da Defesa:09/03/2021


Camu-camu (Myrciaria dubia) is a reddish-colored fruit found typically in the Amazon region. Uvaia (Eugenia pyriformis Cambess), also known as Uvalha, Uvaia do mato, is yellow to orange in color. Both fruits belong to the Myrtaceae family and have acidic flavors. These fruits have aroused the interest of industries (pharmaceutical, nutraceutical, food), due to the presence of vitamin C and other substances beneficial to health. Factors like these contribute to the development of extraction, identification and quantification of these compounds. Currently, the use of clean technologies that improve the extracts yield, reduce the extraction time and are low cost has been sought. Associated with clean solvents such as water, it is possible to avoid toxicity and reduce the environmental impact, in addition to being considered potentially green and ecofriendly. In this context, among several extraction techniques, it can be highlight ultrasound-assisted extraction (UAE) for facilitating mass transfer and reducing the size of fragments through the cavitation effect, in addition, it has greater efficiency and reduced energy when compared to conventional techniques. The reverse osmosis (RO) membrane concentration is a simple operation process, economically viable, and stands out for performing the separation / concentration of the compounds at room temperature, allowing the thermolabile compounds to be processed without modification or functional properties losses. There are few reports in the literature that evaluate the association of green technologies (ultrasound and reverse osmosis) for the extraction of bioactive compounds from camu-camu and uvaia, and there are few studies that point out alternatives for the reuse of uvaia residues. AIMS The objective of the study was to evaluate the best conditions for aqueous extraction of bioactive compounds from the camu-camu and Uvaia fruit (pulp and residue) using clean technologies, extraction assisted by ultrasound followed by reverse osmosis concentration, as well as characterizing the process steps and all obtained products. MATERIAL AND METHODS First, an experimental project was used to obtain the aqueous extract of the fruits (1: 4 w / v), camu-camu and uvaia (pulp and residue), by means of ultrasound-assisted extraction (UAE) to evaluate different variables, being they, time (X1 = 2.5, 5 and 10 min), amplitude (X2 = 20, 30 and 40%) and temperature (X3 = 40, 50 and 60 °C). After obtaining the best extraction conditions, the samples were concentrated by reverse osmosis (R25a, 500 Da, polyamide and and 5 bar area 3 ft2), and the flow rate was monitored. The process flow and the volumetric concentration factor were calculated. At the end of the sequential process, the products obtained (control sample, sample obtained after extraction and concentrated sample), were characterized according to the contents of phenolic compounds, antioxidant activity (DPPH, FRAP, ABTS), total flavonoids, anthocyanins (for camu-camu ) and carotenoids (uvaia), quantification of compounds (vitamin C, myricetin, cyanidin-3-glucoside, p-cumaric acid, rutin, gallic acid, quercetin, chlorogenic acid and ellagic acid) using HPLC-DAD UV / vis, and identification of compounds using UHPLC-MS / MS. All analyzes were subject to analysis of variance and Tukey test (p <0.05) using the Sisvar 5.6 statistical program, and the standard curves for the antioxidant tests were plotted using the GraphPad Prism 5 program.
RESULTS AND DISCUSSION It was verified that the Ultrasound Assisted Extraction (UAE) process, applied to obtain extracts rich in bioactive compounds of camu-camu and uvaia, generated satisfactory results, when followed by Reverse Osmosis membrane (OR) in order to concentration of the compounds. In the case of camu-camu, the best region for compounds extracting was 5 min, 60 °C and 30% amplitude according to the experimental design with the response to total phenolics compounds (TPC) and vitamin C. During the sample concentration, the permeate flow was measured as a function of the operating time for the reverse osmosis process at intervals of 3 minutes to 48 minutes, and varied from 15.0 L / (h.m2) at the beginning of the process to 1.8 L / (h.m2), when the solids content reached 4.1 ° Brix. The reduction in permeate flow occurs because the membrane used is very dense and almost all soluble solids are retained as the process time increases. Fouling of the membrane impairs its useful life and performance, with low fouling being best. In the present study, the level of membrane fouling was relatively low (19%), and the concentration factor was 4.1 times that the initial volume (feed). The final concentrated sample (CC) of camu-camu showed the following results of phenolic compounds (25.798 mg GAE / g fw) and total anthocyanins (66.169 mg of cyanidin-3-glucoside / 100 g total, being 3.2 and 6.5 times higher respectively than initial sample (CS). For the antioxidant analyzes the best result was obtained by the FRAP method (528.667 mmol TE / g) for final sample (CC). A total of twenty bioactive compounds were identified by UHPLC-Q – TOF-MS/MS, such as Cyanidin-3-O-glucoside, ellagic acid and Alnusiin for the first time detected in camu-camu. The quantification of some compounds was performed using HPLC-DAD / UV vis, and vitamin C was highlighted with 7.0 times higher in the final concentrated sample (52.01 mg /g) in relation to CS, followed by gallic acid (97.298 mg / 100 g), rutin (9.783 mg / 100 g) and Cyanidin-3-glucoside (2.783 mg / 100 g) The same processes mentioned above were applied to pulp and residues (peel and seed) of uvaia. Best region of extraction for both was similar: 40 ° C temperature, 40% amplitude, with a difference only for the extraction time, residue (2.5 min) and for the pulp (10 min). For the residue concentration, the fouling found was slightly higher (39%) than for the pulp (31%), probably due to the presence of more solids in the sample. The concentration factor was 7.02 times higher than feed for residue sample, and 4.4 times for pulp of uvaia. The samples final concentration, of residue (CF) and pulp (CP), increased by 6.2 times (332.225 mg GAE / 100 g) and 3.7 times (189.542 mg GAE / 100 g), respectively, for total phenolic content, in relation to the initial extract (RS). For flavonoids an increase of 7.8 (1300.179 mg QE / 100 g) and 6.5 (207.870 mg QE / 100 g) with respect to the initial sample. For both concentrated final samples (CF and CP) the best results for antioxidant were obtained by the FRAP method (136.761 and 39.396 mmol TE / g). Fifteen and fourteen compounds were identified in the residue and pulp samples by UHPLC-Q –TOF-MS / MS, respectively. Quantification by HPLC-DAD / UV vis, allowed to highlight compounds such as: vitamin C (4.420 and 93.367 mg / 100g), galic acid (8.119 and 3.181 mg / 100 g), rutin (2.243 and 1.034 mg / 100g) and ellagic acid (9.407 and 1.876 mg / 100 g), for the final samples (CF and CP). The combination of the two techniques (UAE and OR) applied to camu-camu and uvaia (residue and pulp), showed great potential for recovery of bioactive compounds and can be applied in food, nutraceutical and cosmetic matrices, bringing their benefits to consumers.

CONCLUSIONS It can be concluded that a combination of aqueous extraction assisted by ultrasound followed by the reverse osmosis concentration, were efficient for the bioactive compounds extraction, with emphasis on being clean and viable techniques. At the end of the processes, products with higher levels of phenolic compounds, total flavonoids, antioxidant activity were obtained, when compared to each other (control sample, after extraction and concentrated sample), both for camu-camu and for the different parts analyzed for uvaia. The uvaia residue (seed and peel) used as raw material, was highly promising for the bioactive compounds recovery. The final commercial fruit concentrate can be considered functional and viable for use by the food, nutraceutical and cosmetic industries.

Keywords: Myrtaceae, functional foods, antioxidant activity, phenolic compounds, HPLC-DAD/UV-vis, UHPLC-QTOF-MS/MS.

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