Optimization of bioactive compounds extraction from olive pomace by microwave hydro diffusion and gravity

Abstract

There are few studies on the extraction of bioactive compounds from olive pomace by the microwave method of hydro diffusion and gravity (MHG). Therefore, this study extracted bioactive compounds from pomace by MHG using a central rotational compound (DCCR). The statistical model showed a significant difference for the variable potency (W) in the extractions of phenolic compounds, showing linear behavior. Simple linear regression analysis suggested the use of 100W of power for the extraction of phenolic compounds in olive pomace. There were no significant differences in flavonoid extractions and antioxidant capacity of the extracts, at different potencies (W) and times (min), in the olive pomace extracts.

Alternate abstract:

Existem poucos estudos sobre a extração de compostos bioativos do bagaço de azeitona pelo método de micro-ondas de hidrodifusão e gravidade (MHG). Portanto, este estudo teve como objetivo extrair compostos bioativos do bagaço por MHG utilizando um composto rotacional central (DCCR). O modelo estatístico apresentou diferença significativa para a variável potência (W) nas extrações de compostos fenólicos, apresentando comportamento linear. A análise de regressão linear simples sugere a utilização de 100W de potência para a extração de compostos fenólicos em bagaço de azeitona. Não houve diferenças significativas nas extrações de flavonóides e na capacidade antioxidante dos extratos, em diferentes potências (W) e tempos (min), nos extratos de bagaço de azeitona.

INTRODUCTION

Among the methods commonly used for bioactive compound extraction and the growing search for new green technologies, the technique of microwave extraction by hydro diffusion and gravity (MHG) has gained prominence. It is a sustainable method that performs the extraction of phenolic compounds without the addition of solvents or by the presence of water (EKEZIE et al., 2017).

MHG has other advantages, such as high extraction speed, low energy cost, low levels of contaminants present in the extracts, and a high degree of extract purity, in addition to preserving the matrix and enabling its reuse. The extraction process also allows sample fractions to be collected at different periods, making it possible to identify the physicochemical properties of each constituent present (BITTAR et al., 2013; EKEZIE et al., 2017).

The reuse of different agro-industrial by-products is widely studied to add value to products already on the market and presents a fundamental approach from an economic and environmental point of view (GULLÓN et al., 2018a). For example, in the processing of olives, the by-product called biomass or olive pomace makes up a much higher percentage than obtaining the olive oil itself, as a large part of the constituents remain in the olive pomace after processing (RUIZ et al., 2017a).

One of the great uses of olive pomace is as organic compost for cultivating olive trees as a source of nutrients. It is also applied for the production of animal feed, extraction of organic compounds (pectin, antioxidant, and enzymes), residual oil, pesticides, and in the participation of other products such as alcohol and energy production (CABRERA et al., 2010; MEDEIROS, 2016; NEGRO et al., 2017).

Olive pomace has interesting characteristics due to the presence of bioactive compounds since during extraction, a large part of these compounds remains in the pomace, making the reuse of this by-product promising since these bioactive compounds are linked to beneficial properties to the health of the consumer (FRANKEL et al., 2013). However, further studies are needed to optimize the extraction and characterization of bioactive compounds from olive pomace.

Therefore, this study extracted bioactive compounds from olive pomace by the microwave method by hydro diffusion and gravity (MHG) and to analyze the antioxidant activity of the extracts.

MATERIALS AND METHODS

Olive pomace samples.

The samples were olive pomace of the Arbsana variety donated by Aroeira Colony (Formigueiro, RS, Brazil). They came from the processing of olives harvested in March 2019. Portions of 1000 g of bagasse with an initial moisture content of 72.35% were placed in plastic bags and stored at -18 ºC until extraction. The samples were thawed at room temperature for approximately 24 hours before undergoing MHG extractions.

Obtaining the extract by microwave hydro diffusion and gravity (MHG)

The extracts obtained by MHG were according to the methodology described by KHAN et al. (2016), 200g of fresh olive pomace were placed in the glass holder of the equipment, which consists of a filter mesh and polytetrafluoroethylene cap. (PTFE). They were submitted to powers of 40, 100, 250, 400, and 462 Watts and times of 3, 10, 15, 20, and 22 minutes. Extraction method followed the statistical model applying a central rotational composite design (DCCR), with two independent variables (2), with different solvent powers and times, containing three central points and four axial, resulting in 11 experiments.The extracts were centrifuged (Centrifuda Serilab MTD III Plus) at 1085 G for 10 minutes, filtered, then placed in amber flasks, and stored in a freezer (-18 ºC) until the time of analysis.

Preliminary studies aimed to analyze the matrix’s behavior since the extraction of bioactive compounds from olive pomace by MHG is still little explored. Initially, high powers were tested, with variations between 200 to 600W, commonly used in the extraction of bioactive compounds in studies related to other food matrices, noting that olive pomace behaved differently from the other matrices, obtaining higher concentrations of phenolic compounds at lower powers. Therefore, the statistical design was developed over the optimal range of extraction, which was between 100 to 250W.

Analysis of total phenolic compounds

The total phenolic compounds were determined by the methodology of SINGLETON et al. (1999) with modifications by ROESLER et al. (2007). Gallic acid was used as a standard for the calibration curve, which presented concentrations from 10 to 500 mg.L^-1 and R2 = 0.9953. Results were expressed as gallic acid/g of dry olive pomace (mg GAE g^-1).

Total flavonoids

The methodology used to evaluate the concentration of total flavonoids was that of ZHISHEN et al. (1999). Quercetin standard solutions with concentrations from 25 to 200 mg.L^-1 were used in the calibration curve that presented R² = 0.9672. Results were expressed as mg quercetin equivalent/g dried olive pomace (mg EQ g^-1).

Oxygen radical absorption capacity/ORAC

Antioxidant capacity was measured by the oxygen radical absorption capacity (ORAC) method (OU et al., 2001). The method seeks to estimate the scavenging capacity of antioxidant compounds present in the extract against peroxyl radicals induced by 2,2’ - azobis (2-amidinopropane) dihydrochloride (AAPH). The antioxidant capacity of the extract is measured by monitoring the target molecules using fluorescein as a marker. The non-fluorescein product of fluorescein is monitored by spectrometry, using excitation wavelengths of 485 nm and emission wavelengths of 528 nm. The antioxidant capacity was expressed in µmol Trolox equivalent per gram of dry olive pomace (µmol Trolox/g).

RESULTS AND DISCUSSION

Potency and temperature did not influence (P < 0.05) the flavonoid content and antioxidant capacity extracted from olive pomace. Despite this, potency influenced (P < 0.05) the concentration of phenolics, showing a linear behavior (Table 1). It is suggested that 100W was the best power for phenolic compounds, as shown by the maximum point in the linear equation (Figure 1 and Table 2).

Studies carried out by BRITO (2016) using microwaves (Miero-Chef 2535, Moulinex, France) led to the conclusion that the necessary effect in the extraction of phenolic compounds from olive pomace would not be pressure but heat. The reason is that when the same power of 850W is applied for different seconds (10, 20, and 30), it is obtained higher concentrations of phenolic compounds in 30 seconds, where the process reaches a maximum temperature of 76 ºC and concentrations of 9.9 mg EAG/g of dry bagasse.

The extraction process by MHG is different from conventional microwaves. According to TURK et al. (2017), the extraction process has two cycles, latency, which is characterized by the period in which electromagnetic energy is converted into thermal energy, and the extraction phase, where this process of thermal energy continues, and the food matrix cell breaks, extracting the water contained in it and consequently the bioactive compounds. However, for olive pomace, this latency process lasted around the initial 5 minutes in the extraction process by MHG, reaching an internal temperature in the sample of 40 ºC. After 5 minutes, the extraction process began, where the optimal extraction range was from 10 to 15 minutes, reaching a maximum temperature of up to 94 ºC in extractions using 100W power.

ALUPULUI et al. (2012), showed in their studies that the longer the extraction time by MHG, the higher the process temperature becomes, promoting the heating of the matrix, which may be favorable for some bioactive compounds, facilitating the extraction.

TABARAKI et al. (2012) report that temperature also positively influences the extraction of flavonoids, as heat makes the cell wall more permeable, increasing the solubility of compounds and the diffusion coefficient of the solvent. The type of solvent also plays a crucial role in the extraction of bioactive compounds (GULLÓN et al., 2018a; MOKRANI & MADANI, 2016). Considering that the extraction with MHG is carried out in a polar medium due the presence of the sample’s own water (in situ water) which acts as a solvent (VINATORU et al., 2017), it is estimated that, because some flavonoids are less polar, they were recovered over a long period extraction, since the longer the extraction time, the higher the temperature.

Studies carried out by GULLÓN et al. (2018a), on the extraction of flavonoids from olive pomace by the conventional method using different solvents revealed that the extraction of flavonoids with pure water at 55 ºC for 90 minutes was the least effective solvent, presenting values close to 15 mg EQ / g of dry olive pomace. These values are higher than those found in this study, ranging from 0.27 to 1.16 mg EQ / g of dry olive pomace. Phenolic compounds and flavonoids are good indicators of antioxidant properties in food matrices (MENESES et al., 2013; BALLESTEROS et al., 2014; MOKRANI & MADANI, 2016). However, the flavonoids and antioxidant activity results had no significant difference in the potency and times used in the olive pomace extracts.

CONCLUSION

The study showed significant results, suggesting that 100W was the best potency for the extraction, presenting linear behavior, in the concentration of phenolic compounds 8,35 mg GAE g^-1. For flavonoids and antioxidant activity, there was no significant difference in the different potencies and in the times evaluated in the extractions of these compounds in olive pomace.

ACKNOWLEDGMENTS

The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeioamento de Pessoal de Nível Superior (CAPES), and the Universidade Federal de Santa Maria (UFSM).

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