EDILSON BRUNO ROMANINI

Título da Tese: EXTRAÇÃO E ESTABILIDADE DE COMPOSTOS BIOATIVOS DE SUBPRODUTOS DE UVA BRS Violeta

Orientadora: Profa. Dra. Grasiele Scaramal Madrona

Data da Defesa: 18/04/2022

RESUMO GERAL

INTRODUCTION
The hybrid cultivar BRS Violet is well adapted to southern Brazil. It is recommended for the preparation of juice, containing high levels of anthocyanins and other polyphenols, found mainly in the skin. Grape pomace is an important source of polyphenols such as phenolic acids, flavonoids, anthocyanins, proanthocyanidins and resveratrol, which are recognized for their antioxidant properties, including free radical scavenging ability, prevention of cancer, inflammation, cardiovascular disease and disorders related to aging. Therefore, the bioactive phenolic compounds recovered from grape pomace can potentially be used in the food, cosmetic and pharmaceutical areas, promoting health benefits and reducing the environmental impact. Among the current extraction technologies, ultrasound-assisted extraction (UAE) has been widely recognized as environmentally friendly, cheap, fast and efficient for phenolic extraction due to acoustic cavitation caused by the passage of the ultrasonic wave. Acoustic cavitation consists of the formation, growth and collapse of microbubbles on the solid surface, leading to corrosion and erosion, resulting in the breakdown of cell walls, allowing solvent penetration and improving mass transfer, which leads to an increase in the yield and to a decrease in the extraction time. Now, the great challenge is to apply these phenolic compounds in order to guarantee their properties after extraction from the grape, since they are unstable when exposed in the long term as they easily undergo degradation, oxidation, epimerization and polymerization. The chemical structure of polyphenols responsible for the antioxidant activity is itself targeted by those instabilities by the action of radiation or enzymes and variations in temperature, pH and oxygen. In this context, the use of substances that can transport these bioactive compounds in order to provide protection until their final destination has been described in the literature, for example, the encapsulation techniques by lyophilization, ionic gelation and spray drying are the most common. In this case, the core (phenolic compounds) is physically protected by an encapsulating material such as alginate-Ca2+, maltodextrin and xanthan gum.
AIMS
The objective of this research was to investigate the effect of ultrasound on the extraction of total phenolics and anthocyanins from grape pomace, followed by encapsulation in an alginate-Ca2+ matrix and in a mixture of maltodextrin and xanthan gum to increase the shelf life and stability of phenolics and antioxidants, aiming their application in a food matrix (gelatin).
MATERIAL AND METHODS
Initially, grape juice was extracted by steam distillation using an artisanal juice extractor. The pomace generated during the juice production was subjected to a drying process and the skin were manually separated from the rest of the residue and ground in a food processor. A high intensity ultrasonic processor was used for the experimental design of ultrasound-assisted extraction (UAE). The extraction was carried out using water as a solvent at a ratio of 1:200 (w/v, g/mL). After preliminary tests, the factors were defined. The independent variables were sonication time (X1 = 2.5, 5 and 10 min), ultrasound frequency (X2 = 20, 30 and 40%) and temperature (X3 = 25, 40 and 55 °C), and the responses were the concentration of total anthocyanins (TA) and total phenolic compounds (TP). Conventional extraction (CE) was performed for 6 min, at room temperature (25 ºC) and without ultrasound, as a control sample. After determining the best extraction conditions, samples were characterized regarding the contents of bioactive compounds (TA, TP and TF-Total Flavonoids), antioxidant activities
(DPPH, FRAP, ABTS) and quantification of individual phenolic compounds by HPLC (rutin, gallic acid, p-coumaric acid, quercetin, myricetin and cyanidin chloride). The extract was encapsulated with alginate-Ca2+ at a concentration of 2% w/v (2 g/100 mL) with stirring and heating (70 ºC ± 4°C) for complete dispersion. For capsule formation, the dispersion was dripped using the Caviar Box® kit in an aqueous solution of calcium chloride (1 g/100 mL) kept for 10 min and washed with deionized water. The capsules containing the extracts were lyophilized. The stability of the microcapsules was evaluated through the degradation of the bioactive compounds (TP and TA) during 28 days of storage in the presence or absence of light. Their concentrations were assessed in intervals of 7 days, until the end of the 28 days. In the second step of the study, encapsulation was carried out with maltodextrin and xanthan gum (99.5% and 0.5%, respectively). The encapsulating agents were added at a 1:1 (w/w) agent:extract ratio. The capsules containing the extracts were lyophilized and stored in different conditions: a chamber (BOD) at a temperature of 25 ºC, in the presence and absence of light; and at 4 ºC for 120 days. In intervals of 30 days, they were monitored for the parameter of total monomeric anthocyanins, total phenolics and color. The lyophilized extract was used as a control sample. The microcapsules, the extract and an artificial dye were applied in a colorless gelatin powder, aiming to evaluate the instrumental color. Thus, the gelatin samples were refrigerated (4 °C) for 30 days, and the color parameters were evaluated at 0, 15 and 30 days. Analyzes were performed in triplicate and submitted to analysis of variance and Tukey's test for the minimum significant difference (p < 0.05) between means. Correlation and contour surface were also calculated using the statistical software Excel and Statistic 7.0.
RESULTS AND DISCUSSION
The effect of the three independent variables in ultrasonication (frequency, temperature and time) on extraction results (total phenolic compounds and anthocyanins) was revealed by generating a contour surface. TA values ranged from 3.11 to 3.90 mg of cyanidin-3-O-glycoside/g and TP from 22.30 to 25.03 mg GAE/g. The best extraction conditions for total phenolics and anthocyanins were at 55 °C, with 40% of ultrasound amplitude and for 6 min of treatment. Regarding the color of the extracts, the conventional extraction resulted on a brighter sample. On the other hand, the ultrasound method produced a red extract, indicating a better obtention of the compounds, which correlates with the extraction of anthocyanins with UAE 1.35 higher than with the CE. The concentration and antioxidant capacity results (DPPH, ABTS and FRAP) for UAE were statistically higher when compared to the CE. The results of the three analyzes of total bioactive compound content performed (TP, TA and TF) were different comparing UAE with CE, demonstrating that the use of the ultrasound method was significantly better, on average, 11% for TP, 25% for TA and 34% for TF. Positive correlations were observed between these contents (TP, TA and TF) and DPPH (r = 0.963, 0.967 and 0.940 respectively), ABTS (r = 0.989, 0.986 and 0.997, respectively) and FRAP (r = 0.976, 0.972 and 0.990, respectively). The CE and UAE methods also resulted in significantly different concentrations of anthocyanins and of other compounds identified, demonstrating that the use of ultrasound was significantly better, on average, 29.41% for rutin and quercetin, 50% for acid p-coumaric, 23.93% for cyanidin chloride, 16.67% for myricetin and 15.79% for gallic acid. An encapsulation efficiency (EE) of 62.52% was observed. The stability assay of the extracts with alginate-Ca2+ showed that the degradation of anthocyanins and total phenolics (32.80 and 60.26%, respectively) was higher in the presence than in the absence of light (18.51 and 43.59%, respectively). When evaluating the effect of the three parameters (presence, absence of light, and temperature 4 ºC) using maltodextrin + xanthan gum as encapsulating matrix, the degradation was higher in the unprocessed extract (TP = 24.49, 21.38 and 23.07% TA = 20.61, 18.53 and 19.47%, respectively) than in the encapsulated extract (TP = 17.92, 12.06 and 9.69%; TA = 14.71, 12.78 and 12.78%, respectively). Thus, one can observe the protective effect of the capsule comparing to the unprocessed extract. There was a significant difference in the TP
content during the stability test of the encapsulated extract, with higher degradation in the presence of light. On the other hand, the TA content showed practically similar degradation under the conditions studied. Regarding the color at 120 days of storage, the encapsulated extract Cap4 (exposed to a temperature of 4 °C) presented the lowest color change (ΔE < 5), while this parameter was high (ΔE > 5) for the extracts that are independent of the storage conditions and the encapsulated extracts Cap25 (absence of light) and CapLight (presence of light). A first order degradation curve was obtained for all encapsulated samples. The half-life values of the encapsulated content (alginate-Ca2+) of total monomeric and phenolic anthocyanins decreased from 104 to 59 and from 40 to 25 days, respectively, in the presence of light, indicating a longer half-life in the absence of light. When evaluating the encapsulated product (maltodextrin + xanthan gum) versus the extract at both temperatures (25 and 4 ºC), there was a more accentuated loss in the TP content present in the unprocessed extract (t(1/2) of 321 days) compared to the capsules (984 days) 4 ºC. When evaluating the effect of light on the capsules and the extract, TA and TP content losses were similar (t(1/2)). In a general evaluation of the conditions studied, both total anthocyanin and phenolic compounds showed higher degradation in the extract, inferring that the capsule provides protection to bioactive substances. The application of the capsules and the coloring in gelatin showed little difference in total color in 30 days (ΔE = 3.35 and 3.58). During storage, on days 15 and 30, the total color of the extract changed significantly: ΔE = 8.37 and 8.85, respectively. Comparing to the dye and the capsules, the color change when the extract is used is 2.47 and 2.65 times higher, respectively. This demonstrates that the encapsulation of colored bioactive substances can be used as an alternative to artificial coloring in food products, for example, when stored for up to 15 days under refrigeration.
CONCLUSIONS
The aqueous extracts of grape skins (industry by-product) obtained by ultrasound showed higher levels of total anthocyanins and phenolics compared to conventional extraction. The increase in some specific compounds is also evident (29% for rutin and quercetin and 24% for cyanidin chloride). The extract encapsulated in alginate-Ca2+ showed less degradation when stored in the absence of light than in its presence. The use of an encapsulating matrix of maltodextrin and xanthan gum proved to be effective in maintaining the stability and color of antioxidant compounds, increasing their half-life. Therefore, it is recommended for protecting antioxidant substances that also provide color in food applications. Finally, grape pomace (BRS Violet) showed a high concentration of phenolic compounds, better extracted by ultrasound, and that can be reused, mainly by encapsulation, for future applications in food matrices.
Keywords: Ultrasound, phenolic compounds, antioxidant, stability, encapsulation.

Artigos Publicados Vinculados a Tese:

https://www.sciencedirect.com/science/article/pii/S0308814620319634