Maceration and liquid–liquid extractions of phenolic compounds and antioxidants from Algerian olive oil mill wastewater

Olive oil mill wastewater (OMW) is a major waste stream generated in olive oil industry. It is highly polluted due to phenolic compounds. The present study focused on the physicochemical properties of OMW as well as the quantitative and qualitative effects of two extraction methods of phenolic compounds which were liquid–liquid and maceration methods. Spectrophotometry and high-performance liquid chromatography-electrospray ionization–mass spectrometry (HPLC–ESI–MS) were adopted to quantify the phytochemical contents and the phenolic compounds. The extract obtained by the maceration method showed the highest yields of total polyphenol, flavonoid, and tannin contents. The LC–MS results revealed the presence of 16 phenolic compounds in the macerated, and only 12 phenolic compounds were found in the extract of the second method. Quinic acid was identified as the most abundant compound. Moreover, the macerated extracts possessed the highest antioxidant potential as evidenced by their strong ferric reducing antioxidant power (FRAP) and their 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical scavenging activities. The phytochemical contents, as well as the antioxidant potentials of OMW after extraction using maceration, were significantly greater than using liquid–liquid method. Therefore, maceration seemed to be the most effective method for extracting phenolic compounds from OMW. The OMW constitute a rich source of natural phenolic compounds that could be used as a potential source of natural antioxidants.


Introduction
The olive oil industry is an important sector of the economy, concentrated mainly in the Mediterranean countries (Haydari et al. 2022;De Matteis et al. 2021). In addition to the solid wastes that are called pomace, it generates very large amounts of liquid discharges and wastewater in olive oil presses. It consists of the water found in the fruit (olive) and the water added during olive oil extraction processes named olive oil mill wastewater (OMW) (Trigui et al. 2022;Gueboudji et al. 2021b). This industry is increasing annually with the cumulative demand for olive oil consumption, due to its very beneficial effect on health with its therapeutic, diet, and nutritional properties. Consequently, this industrial sector leads to an increase in solid and liquid wastes production that are a source of environmental pollution, especially in Mediterranean countries. The olive oil mill wastewaters are dark acidic liquids, with high conductivity, that contain interesting compounds including sugars, proteins, dietary fibers, and phenolics (Rocha et al. 2022;Haddad et al. 2017). Moreover, the soil damage is due to the slow degradation of these phenolic compounds leading to contamination and changes in soil chemical properties ). This situation is exacerbated by the seasonality of olive oil production and the large quantities of vegetable water produced (Koutrotsios and Zervakis 2014).
Phenolic compounds are products of the secondary metabolism of plants, widely distributed to many phenolic groups and include about 9000 different known structures (Wan et al. 2021), ranging from simple phenolic molecules with low molecular weight like phenolic acids to highpolymerized compounds such as tannins. The phenolics possessed potential beneficial effects on health such as the prevention of ROS (reactive oxygen species)-related diseases such as aging, cancer, and chronic diseases (Khan et al. 2020). Phenolic compounds have several biological properties and are used in many industrial fields because of their antioxidant activity including pharmaceutical, food, and cosmetic industries (Gueboudji et al. 2021d;Soberón et al. 2019).
Some factors would influence the phenolic compound's composition of OMW such as the olive varieties, climatic conditions, olive oil extraction process, and ripening degree of the fruit (Prazeres et al. 2021). However, the selective recovery of phenolic substances from OMW represents a valid approach for the reduction of their environmental toxicity and an opportunity to obtain high added-value molecules (Papaoikonomou et al. 2021;De Bruno et al. 2018). Many recovery studies of OMW polyphenols have been carried out on a small scale, and various techniques are used individually or in combination (Martins et al. 2021). These techniques mainly include solvent extraction, adsorption, membrane separation, supercritical fluid extractions, ultrasound treatment, and chromatographic processes (Soberón et al. 2019). Phenol recovery processes generally involve a condensation step before performing the sequential extraction with organic solvents such as methanol, ethanol, or hydro-alcoholic solutions. These processes aim to recover either a particular phenol in pure form or a mixture of phenolics in the form of a crude product (Alonso-Riaño et al. 2020).
It is crucial to evaluate the most suitable and reliable technique for the recovery of phenolics to maximize the extraction yield. Therefore, the main purpose of the current report was to investigate and compare the phenolic contents and the antioxidant potentials of OMW extracts obtained from maceration and liquid-liquid extraction methods.

Wastewater
Olive mill wastewater samples were collected from a modern olive oil cold extraction unit from the region of Baghai-Khenchela (eastern Algeria) during the 2021-2020 season.

Physicochemical characteristics of OMW
Electrical conductivity (EC) and pH of OMW were directly measured using a conductivity meter and pH meter. The dry matter (DM) was determined after sample drying at 105 °C. Fatty matter (FM) was determined by the chloroform/methanol method as described by Aissam (2003). Chemical oxygen demand (COD) and biological oxygen demand (BOD 5 ) were evaluated, respectively by the potassium dichromate and the respirometric methods (Gueboudji et al. 2022b;Rodier et al. 1984).

Liquid-liquid extraction
The liquid-liquid extraction method was carried out using ethyl acetate as described by De Marco et al. (2007). Briefly, a sample of 20 mL of OMW was acidified to (pH 2) with HCl and washed with hexane (30 mL). The resulting solution was mixed vigorously and centrifuged at 3000 t/min for 5 min. After that, 20 mL of ethyl acetate was added, and the homogenate was shaken vigorously for 15 min and then centrifuged at 3200 t/min for 10 min in (4 °C). The extraction was repeated four times and the obtained phases of ethyl acetate were then combined. The dissolved water was removed with sodium sulfate anhydrous, and the solvent was evaporated under vacuum in a rotary evaporator at 40 °C. The dry residues were dissolved in 6 mL of methanol and stored at − 18 °C. The extraction process was carried out in triplicate.

Extraction by maceration
Samples of OMW were firstly oven dried and then one gram (1 g) of the resulting powder was mixed with 10 mL of pure methanol. The mixture was vortexed for 15 min, let macerate overnight at 4 °C, and filtered through filter paper. The resulting macerate was then collected, dissolved in methanol, vortexed for 15 min, and left to macerate for 1 h. The obtained macerates were combined and filtered through sodium sulfate, and the solvent was evaporated at 40 °C in a rotating evaporator under a vacuum. The dry residue was stored in 6 mL of methanol at − 18 °C. The extraction was performed in triplicate.

Determination of TPC
The total phenolic content (TPC) of each extract was determined following the Folin-Ciocalteu method (Müller et al. 2010) and expressed as grams of gallic acid equivalents per 100 g of dry matter of initial OMW (g GAE/100 g DM) through the calibration curve of gallic acid. The calibration curves ranged 0-200 mg/mL (y = 0.0048 × + 0.0027, R 2 = 0.9982).

Determination of TFC
The total flavonoid content of each extract was assessed as described by . The total flavonoid content (TFC) was calculated through the calibration curve of quercetin (y = 0.0034 × + 0.0311, R 2 = 0.9991). The range of concentrations used in calibration curves was (0-25 mg/ mL). The results were expressed as gram quercetin equivalents per 100 g of dry matter of initial OMW (g QE/100 g DM).

Determination of TTC
The quantification of the total tannin content was performed according to the method described by Hagerman (2002) and expressed as grams of catechin equivalent per 100 g of dry matter of initial OMW (g CE/100 g DM).

LC-MS analysis of phenolic compounds
The identification of individual phenolic compounds in OMW extracts was determined by LC-MS, according to the methodology described in Mahmoudi et al. (2021a, b). The analysis of phenolic compounds was performed on a Shimadzu UFLC XR system (Kyoto, Japan), equipped with a SIL-20AXR auto-sampler, a CTO-20 AC column oven, a LC-20ADXR binary pump, and a quadrupole 2020 detector system. This instrument was equipped with an Inertsil ODS-4 C18 3 µm column (L150 × 3.0 mm i.d.). The column temperature was set at 40 °C, and the injection volume was 20 µL with a flow rate of 0.5 mL/min. Water 95% + MeOh 5% + acetic acid 0.2%, and CAN 50% + H 2 O 50% + acetic acid 0.2% were used as mobile phases A and B, respectively. The analysis was performed using a linear gradient programmed as follows: 0.01-14 min, from 10 to 20% B; 14-27 min, 0 from 20 to 55% B; 27-37 min, from 55 to 100% B; 37-45 min, 100% B; 45-50 min 10% B. Dissolving line temperature was 275 °C, nebulizing gas flow 1.50 L/ min; the drying gas was set at 15.00 L/min and temperature of heat block was 450 °C. LC-ESI ( −) MS mass spectra [M-H] were acquired using Lab Solutions software. The identification and the quantification of obtained pics were determined by comparison with the relative retention times and UV spectra with those of standard phenolic compounds as detailed in Mahmoudi et al. (2021b).

DPPH free radical-scavenging activity
The antioxidant activity of OMW extracts was evaluated using the free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity (Mahmoudi et al. 2020). The results were given as 50% inhibition concentration (IC 50 ) and compared with the antioxidant standards (butylated hydroxytoluene BHT, ascorbic acid and trolox). To assess this activity, 0.5 mL of extract at different concentrations was mixed with 0.5 mL of a solution of DPPH (0.2 mM in methanol). After vigorous shaking, the mixture was left to stand at room temperature for 30 min, and the absorbance was read at 517 nm.

ABTS + free radical scavenging activity
The ABTS scavenging activity was determined as described by Kalimuthu et al. (2022). The ABTS solution was prepared by mixing ABTS (7 mM) with potassium persulfate, and the mixture was incubated in the dark before use. The prepared solution was diluted in methanol to an absorbance of 0.7 ± 0.02 at 734 nm. After adding 25 µL of sample extracts or standard to 2 mL of the diluted ABTS solution, the absorbance was read for 5 min. The results were given as 50% inhibition concentration (IC 50 ) and compared with the antioxidant standards (butylated hydroxytoluene BHT, ascorbic acid and trolox).

FRAP
The ferric reducing antioxidant power (FRAP) activity was evaluated according to Kocak et al. (2016). A volume of 2 mL of FRAP reagent was added to 0.3 mL of the extract samples and adjusted to a final volume of 10 mL with ultrapure water. The resulting solution was allowed to stand at room temperature for 5 min and then centrifuged for 10 min at 10,000 rpm. Absorbance was measured at 593 nm, and the results were given as 50% inhibition concentration (IC 50 ) and compared with the antioxidant standards (BHT, ascorbic acid and trolox).

Statistical analysis
The results were presented as mean ± standard deviation of three dependent determinations. Significant differences between means of phenolic contents, LC-MS analysis, and antioxidant potential results were determined by analysis of variance (ANOVA) and Duncan's multiple ranges. Differences considered significant at p < 0.05. The correlation was analyzed using Pearson's coefficient. Statistical analyses were performed using XLSTAT software (www. xlstat. com).

Physicochemical criteria
The physicochemical properties of the studied OMW are presented in Table 1. The OMW obtained from the Zlitni variety were acidic effluents (pH = 5.05) loaded with mineral and organic matter showing a high value of electrical conductivity (EC = 13.51 mS/cm). Indeed, the OMW possessed high dry matter, chemical oxygen demand (COD) and biological oxygen demand (BOD 5 ) and were found to be, respectively, 110.67, 208 g/L and 75 g/L. However, the fatty matter was detected at a low level (0.99%). Results of physicochemical criteria of OMW were consistent with several earlier studies reporting that OMW was an acidic liquid, with pH values varying from 3 to 5 and an electrical conductivity value of 16.79 mS/cm. Generally, it is composed of water (83-94%), organic matter (4-16%), lipids (1 to 14%), COD (40-220 g/L), and BOD 5 (35-110 g/L) (Alique et al. 2020;Değirmenbaşı and Takaç 2018). It has been reported that the quantity, as well as the physicochemical properties of OMW, depend on several factors, including production process type, olives verities, use of pesticides and fertilizers, ripening stage, climatic conditions, and geographic area (El-Abbassi et al. 2017).

Total phenolic, flavonoid, and tannin contents
The total polyphenol, flavonoid, and tannin contents extracted by maceration and liquid-liquid extraction methods are presented in Fig. 1. Statistical analysis showed significant differences between means of total polyphenol, total flavonoid, and tannin contents extracted by the two methods. The OMW extract from maceration showed significantly higher polyphenol (22.97 versus 6.47 g GAE/100 g DM), flavonoid (2.34 versus 1.10 g QE /100 g DM) and tannin contents (2.47 versus 0.847 g CE /100 g DM) than liquid-liquid extraction method. When compared to the liquid-liquid extraction technique, these contents increased by 255.02, 112.73, and 191.62%, respectively. Similar results were reported by Romeo et al. (2020) and Değirmenbaşı and Takaç (2018) who obtained, respectively, TPC values of 788.96 ± 1.41 mg/100 mL and 0.5-24 g/L.

Identification and quantification of phenolic compounds by LC-MS analysis
The contents of individual phenolic compounds obtained by LC-MS are shown in Table 2. Different compounds in OMW extracts were detected from each extraction method. A total of sixteen components were identified in the extracts 110.67 ± 6.03 COD: chemical oxygen demand (g/L) 208.00 ± 10.00 BOD 5 : biological oxygen demand (g/L) 75.00 ± 4.36  Fig. 1 Total polyphenol, flavonoid, and tannin contents of Zlitni OMW extracts by two methods of extraction (TPC, total polyphenol content; TFC, total flavonoid content; TTC, total tannin content; GAE, gallic acid equivalent; QE, quercetin equivalent; CE, catechin equivalent); for each content with different letters differ significantly (p < 0.05), a, b: homogeneous groups obtained through maceration, and only 12 compounds were found in the liquid-liquid extracts. Four phenolic acids were identified in the macerated extract and were caffeic acid, luteolin-7-O-glucoside, 4,5-di-O-caffeoylquinic acid, and apigenin-7-O-glucoside. The results indicated that the maceration method gives higher values than the liquid-liquid extraction method in quinic acid, rutin, hyperoside, luteolin-7glucoside, 4,5-di-O-caffeoylquinic acid, and salviolinic acid. It was found that extracts from the maceration method showed significantly high quinic acid, rutin, hyperoside, luteolin-7glucoside, 4,5-di-O-caffeoylquinic acid, and salviolinic acid amounts more than the liquid-liquid extraction method. The macerated extracts showed the highest phenolic level with an average value of 45.11 ppm, compared to the liquid-liquid method (9.84 ppm). The quinic acid was the major phenolic compound with an average value of 4.82 and 35.10 ppm in extracts obtained by liquid-liquid and maceration techniques, respectively. The richness of OMW in phenolic components was widely discussed. The HPLC analysis and after liquid-liquid revealed several compounds such as gallic acid, hydroxytyrosol-4-β-glucoside, hydroxytyrosol, tyrosol, caffeic acid, p-coumaric acid, and oleuropein aglycone (El-Abbassi et al. 2012). Moreover, Romeo et al.
(2020) characterized ten compounds including chlorogenic acid, vanillic acid, caffeic acid, p-coumaric acid, verbascoside, luteolin, and apigenin. Some phenolic compounds frequently prevalent in OMW, such as gallic acid and p-coumaric acid, were not detected in our extracts which could be attributed to the fast oxidation of these compounds (Belaid et al. 2002). The presence of caffeic acid, luteolin-7-O-glucoside, 4, 5-di-O-caffeoylquinic acid, and apigenin-7-O-glucoside only in the extracts obtained by the maceration method might be due to their oxidization and therefore their rapid transformation. It was noted that the difference in the quantity and quality of the phenolics was due to a certain loss of these compounds that remain trapped in hexane and ethyl acetate phases during the extraction process. It was confirmed that not all phenolic compounds were extracted by ethyl acetate, especially the phenolic compounds of high molecular weight as tannins (El-Abbassi et al. 2017). In addition, the freeze-drying method was recommended to preserve the stability of the phenolic fraction of the olive oil mill wastewaters.
According to Turkmen et al. (2007), phenolic compounds were generally extracted using suitable solvents such as methanol, ethanol and N,N-dimethylformamide dineopentyl acetal. Therefore, methanol was a polar solvent that allow the extraction of polyphenols. The maceration method was a simple, high-efficiency, and economical technique for polyphenol extraction. The efficiency of the method was mostly influenced by the solvent, the pH of the extraction medium that determined the compound solubility, the temperature,

Antioxidant potentials
The free radical scavenging activity determined by DPPH . , ABTS, . and FRAP was widely used to estimate the antiradical/antioxidant capacity of phenolic compounds of OMW extracted with two methods and compared the data to many reference standards to obtain more useful and arguably essential results. One-way ANOVA analysis revealed a significant difference in antioxidant potential depending on the extraction methods (Table 3). The results of DPPH radical scavenging activity showed that the macerated extracts exhibited the highest antioxidant activity as indicated by the lower IC 50 value (7.55 μg/mL) higher than that of BHT (IC 50 = 11.11 μg/mL), ascorbic acid (IC 50 = 12.28 μg/mL), and trolox (IC 50 = 16.12 μg/mL). Similarly, the analysis data of the ABTS assay showed that the extract obtained from the maceration extraction method give the best activity with (IC 50 = 6.08 μg/mL) lower than that of ascorbic acid and BHT (IC 50 = 1.52 and 2.20 μg/mL), respectively, and higher than that of trolox (IC 50 = 9.06 μg/mL) and the liquid-liquid extract (IC 50 = 13.51 μg/mL). From the results of FRAP, extracts of the maceration extraction method were exhibited the highest antioxidant activity (IC 50 = 3.12 µg/ mL) than ascorbic acid (IC 50 = 9.94 µg/mL), and extracts from liquid-liquid extraction (IC 50 = 11.56 µg/mL), and much higher activity than trolox (IC 50 = 17.06 µg/mL) and BHT (IC 50 = 20.05 µg/mL) (Table 4). This is supported by the findings of Romeo et al. (2020) who reported that the olive mill wastewater showed strong antiradical DPPH (IC 50 = 114.37 mmol TE/100 mL) and ABTS scavenging activities (IC 50 = 2569.19 mmol TE/100 mL). The obtained results showed that the total polyphenol content was highly and positively correlated with the antioxidant capacity evaluated by the DPPH, ABTS, and FRAP assays with a p-value of 0.001 and a correlation coefficient (r = 0.999). According to De Marco et al. (2007), the phenolic compounds of OMW were characterized by a strong antioxidant potential. The in vitro antioxidant activity of natural extracts has received much more attention. These methods involved the presence of oxidizing species such as free radicals and metal complexes (Alam et al. 2013). Several studies have shown that the antioxidant activity depends on the concentration of total polyphenols, the antioxidant structures, as well as the reaction time (Abramovič et al. 2018;Gueboudji et al. 2021b;Leouifoudi et al. 2015). The antioxidant potential of the studied extracts might be attributed to the total and individual phenolic compounds especially quinic acid, kaempferol, and cirsiliol that are identified at high amounts. Therefore, the present results of the antioxidant activity of phenolic extracts were in accordance with their phenolic composition. Indeed, the ability to reduce free radicals is largely influenced by the phenolic composition of the sample.

Conclusion
The phytochemical contents and the antioxidant potentials of olive mill wastewater were deeply investigated. The phenolic compounds, as well as the antioxidant activities, were highly and significantly dependent on the extraction method. The OMW extract from the maceration method showed significantly higher polyphenol, flavonoid, and tannin contents than the liquid-liquid extraction method. The LC-MS analysis revealed a total of 16 and 12 phenolic compounds were identified respectively in the macerated and liquid-liquid extracts mostly predominated by the quinic acid. Furthermore, DPPH·, ABTS· + , and FRAP antioxidant activities showed a significant variation between the two different methods and the maceration extraction gives the highest antioxidant potential. Finally, it should be noted that OMW presented a natural source of phenolic compounds with more potent antioxidant potential that can be used in various industries such as food and pharmaceutical. The recovery of polyphenols offers the double opportunity to obtain biomolecules with high additive value on the one hand and to reduce the pollutant effects of OMW on the other hand, particularly in the countries bordering the Mediterranean Sea.