Valorization of Lyophilized Olive Mill Wastewater: Chemical and Biochemical Approaches

Abstract

Lipid composition and antioxidant activity have been carried out in order to valorize the composition of olive mill wastewater extracts with different solvents (supercritical carbon dioxide, n-hexane, dichloromethane and ethanol). The antioxidant activity was evaluated using ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) and DPPH (2,2-diphenyl-1-picrylhydrazyl) tests. The total phenolics and flavonoid contents were, also, determined. The chemical composition of the extracted oil was established, respectively, by gas chromatography–flame ionization detector and high-performance liquid chromatography methods. The results showed that the oleic acid and equivalent carbon number of forty-eight were the major compounds of the analyzed oils. Residual olive mill wastewater from the Sousse region displayed the highest DPPH radical-scavenging activity (31.10 ± 0.10 μg/mL). The chemical analysis of extracts of OMWs showed that the n-hexane fraction contained an abundance of oleic acid (61.62%) and an equivalent carbon number of forty-eight (53.14%). The best antioxidant activity was determined for the ethanol fraction (14.5 μg/mL). The final results showed a significant difference and variations in polar and apolar components. Moreover, n-hexane extracts showed high percentages of Monounsaturated fatty acids (MUFA) with 64% of OMWs oil composition and the dichloromethane extracts contained the largest amount of flavonoids (160.30 ± 1.70 mg EQ/g DM).

1. Introduction

In Tunisia, more than 30% of the cultivable lands are planted by olive trees [1]. However, large amounts of liquid by-product denoted olive mill wastewater (OMW) was engendered during the extraction processes [2]. In fact, these effluents are spread into the soil or discharged into onshore evaporation ponds. Nevertheless, this liquid is extremely harmful for the environment due to the presence of high phenol content, organic acids and lipidic compounds with their acidic character. OMW is a mixture of various inorganic and organic compounds dissolved in water with a variable amount of oil and greases. Moreover, a portion of leaves, twigs and olive fruits constitute the solid particles present in OMW. Olive mill wastewater is characterized by a high concentration of polyphenols [3,4]. The presence of organic acids, amino acids and sugars leads to the growth of microorganisms contributing to the unpleasant smell produced by evaporation ponds used for the treatment of this effluent [5]. OMW is characterized by high organic load, high salinity, suspended solids toxic phenols and several other components that may have possible negative effects on chemical and physical soil properties, as well as soil biological activities [5,6]. Moreover, high organic load, polyphenols and other pollutants can cause environmental problems for the ecosystem such as soil contamination and water pollution [6]. When OMW is not properly treated, it can have negative effects on soil and water quality, including the contamination of rivers, streams, and groundwater. OMW can also harm aquatic life and contribute to the eutrophication of water bodies. Additionally, the high organic content of OMW can lead to the formation of odors and the creation of breeding grounds for insects and other pests [3,4,5,6].

It is well known that OMW derivatives may prevent the growth of microorganisms [3]. However, these active compounds could be exploited and recycled using suitable management strategies in order to minimize the ecological impact of olive mills. Consequently, global attention has been shown to resolve this environmental problem. In order to avoid, or at least to reduce, the high toxicity of this byproduct (OMW), numerous treatment methods have been proposed [3,5,7]. The proposed methods of OMW treatment include physicochemical methods (e.g., membrane processes coagulation, evaporation, flocculation), biological treatment (anaerobic and aerobic digestion), and oxidation processes (e.g., Fenton and wet air oxidation, ozonation). However, traditional methods of treatment (storage in lagoons and summer season evaporation) remain to be the most common [7]. In his review, Sehar (2019) [8] suggested that constructed wetlands is reliable, environment-friendly and profitable approach technology for the treatment of OMW effluents [8].

In order to improve the quality of the OMW and decrease its toxicity levels, numerous treatment options have been investigated [3,9,10]. Among them, biological, physical and chemical technologies as well as combined approaches were described [3,11]. Caporaso et al. (2018) reported an integrative approach to valorize OMW phenolics in food processing. Moreover, the authors described several examples on the use of these extracts as a food additive (vegetable oils), in food emulsions and for milk products or other model systems. Their possible use as antimicrobial agents applied to meat product conservation is also another promising approach [11].

Polyphenols are antioxidant compounds with high added value for human health. Polyphenols have been shown to play a vital role in health and a variety of beneficial effects were demonstrated including: the regulation of metabolism, weight, chronic disease and cell proliferation [12,13]. According to the same authors [13], various polyphenols have antioxidant and anti-inflammatory properties that could have preventive and/or therapeutic effects for cancer, neurodegenerative disorders, obesity and cardiovascular disease. The antioxidant activity of various natural extracts is attributed to phenolic compounds which constitute a major source of natural antioxidants. These products were in some cases used in many industrial products as a naturally occurring antioxidants [14]. They are valuable in the food industry [15] not only as preservative agents but also due to their advantageous effects on human health [16]. Consumer requests for unrefined or natural products are increasing and the use of synthetic antioxidants is restricted [17].

Fatty acids and triglycerides are the most important constituents in the lipophilic OMW fraction. Long-chained unsaturated fatty acids have been used in a large range of applications in the pharmaceutical industry and foodstuffs. It has been revealed that they could have a beneficial impact against various diseases [18]. In order to find the best method to isolate and recycle the active compound, a large amount of research has taken place. Therefore, the aim of this research was to extract oil with a high percentage of fatty acids and triglycerides from OMW using the supercritical fluid extraction (SFE) method.

Supercritical carbon dioxide (SC-CO₂) extraction is one of the most used green techniques for extracting active compounds. It helps preserve the quality of the active compounds. In fact, carbon dioxide presents many advantages such as low toxicity, low cost and non-explosive character [19].

The extraction of these compounds from OMW depends on the used solvent polarities, which represents a key factor. In supercritical or liquid–liquid approaches, the solvents could be used to recover compounds with different polarities. SC-CO₂ and n-hexane are the most usually used solvents to remove the lipid fraction [20,21]. While ethanol and dichloromethane are referred to as interesting for phenols recovery [19].

SC-CO₂ extraction was achieved to remove the maximum amount of lipid compounds. The chemical composition of the obtained extracts was analyzed by high-performance liquid chromatography (HPLC) and by the gas chromatography–flame ionization detector (GC) methods. Furthermore, the residue obtained after SC-CO₂ was evaluated by two methods: 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). The polyphenols flavonoids content was determined by spectrophotometry. Otherwise, to make the investigation more in depth and to valorize the OMW benefits, conventional solid–liquid extractions were performed on lyophilized OMW using solvents with a wide range of polarities. For comparative reasons and for the purpose of assessing the potential of the OMW extracts as a functional food, the phenolic compounds content and the antioxidant activity of the obtained fractions were determined.

2. Materials and Methods

2.1. Materials

OMW was collected from olive oil extraction factories from 12 different localities in Tunisia: 6 in the Sousse region and 6 in the Sfax region. These samples were collected in glass containers (2 L), directly after the oil-separation process and stored at 4 °C.

Analytical quality solvents, ACS grade (≥95%), were supplied by Sigma Aldrich (absolute n-hexane, ethanol and dichloromethane) and carbon dioxide by Messer Tehnogas (Serbia) with a purity of 99.95%.

The reagents DPPH^•, ABTS⁺, methyl ester samples, ascorbic acid, gallic acid and sodium carbonate anhydrous (Na₂CO₃) were supplied by Sigma Aldrich with high purity (≥95%).

2.2. Methods

2.2.1. Solvent Extraction

To compare the SFE method and conventional solvent-extraction processes, the lyophilized OMW was subjected to solvent maceration with an increasing solvent polarity (n-hexane/dichloromethane/ethanol). Five grams of the OMW samples were mixed with 50 mL hexane for 30 min on an orbital rotary plate (150 rpm). The extraction procedure was repeated twice with the posterior n-hexane solvent removed from the combined fractions by a rotary evaporator at 50 °C and drying in the nitrogen atmosphere. The extracted oil was weighed for yield determination, and the tubes with extracts were kept frozen (−20 °C) until analysis. The same procedure was repeated with the same sample, respectively, for the dichloromethane and ethanol solvents.

2.2.2. Supercritical CO₂ Extraction

Supercritical CO₂ extraction experiments were performed using a laboratory system (Eurotechnica GmbH, unit HPEA 500, Belgrade, Serbia). These extraction conditions were selected according to the extracted lipids from lyophilized OMW raw materials (

Table 1. SC-CO₂ extraction conditions.

Parameters	Temperature	Pressure	SC-CO2 Flow Rate
Supercritical CO2 extraction	[40–60 °C]	[25–35 MPa]	3.39–5.99 g/min

) [21,22].

2.2.3. Fatty Acid Composition Analyses

Fatty acid composition analyses permit the evaluation of saturated fatty acids (SFA) and unsaturated fatty acids (UFA). Following the method described in the literature [23], the esterification reaction of the extracted oil was established to be converted to methyl esters. The extracted oils were mixed with heptane (0.5 g in 4 mL) with 0.5 mL of 2 N methanolic KOH. After centrifugation, the obtained sample was analyzed using the GC-FID technique.

The samples were separated on a Carbowax capillary column 30 m × 0.32 mm × 0.32 μm. The used carrier gas was helium, with a flow of 1 mL/min. The temperature of the injector was 230 °C and the injected volume sample was fixed (5 μL). The temperature of the oven was programmed at 165 °C for 8 min then at 210 °C at 2 °C/min. The temperature of the detector was 250 °C.

The peaks were determined by the co-elution of standard methyl ester samples under the same GC conditions [24]. The composition of the fatty acids was expressed as a relative area percentage. All the analyses were performed in triplicate.

2.2.4. Analyses of Triglycerides (TAGs)

The objective of the experiment was fixed to identify the triglycerides l possessing a carbon number between 42 and 50. Therefore, equivalent carbon numbers (ECN) were determined (ECN42, ECN44, ECN46, ECN48 and ECN50). The analyses of the triglycerides were performed by a direct analytical technique using HPLC [25].

The samples (10 μL) were separated directly into octadecylsilane column 250 mm × 4.5 mm × 5 µm. The elution solvent was constituted by petroleum ether/ethyl ether 87/13 (v/v). The triglycerides were detected using a differential refractometer as a detector. The identification was performed using standards and by comparison with the data from the literature (NIST/NBS, Wiley libraries).

2.2.5. Total Phenolic Contents

The polyphenols contents in studied samples were performed using the Folin and Ciocalteu method [26]; Gallic acid (GA) was used as a standard. Briefly, the phenolic extract (100 μL) was mixed with distilled water (5.9 mL). The Folin–Ciocalteu reagent (500 μL) was added. After 3 min, 1.5 mL of anhydrous sodium carbonate solution (20% in water) was added. The volume was completed to 10 mL with distilled water and mixed. After 2 h of incubation, the absorbance measurement was performed using a UV–vis spectrophotometer at 760 nm.

2.2.6. Total Flavonoids Contents

The measurement of the total flavonoid contents in OMW extracts was achieved by the method of Loots et al. (2007) [27]. The flavonoid assay method depends on the ability of the flavonoid compounds to form chromogenic complexes with aluminum chloride.

A total of 0.5 mL of extract was mixed with 2 mL of distilled water and 0.15 mL of 15% sodium nitrite solution (NaNO₂). After 6 min, 0.15 mL of 10% Aluminum chloride (AlCl₃) is added and incubated for 6 min, and then, 2 mL of 4% NaOH (Sodium hydroxide) is added. The volume was adjusted to 5 mL with distilled water and the absorbance is measured after 15 min at 510 nm.

2.2.7. Antioxidant Activity

DPPH Radical-Scavenging Method

Antioxidant scavenging activity was analyzed using the DPPH method [28]. A total of 100 μL of the solution extracts were stirred with 900 μL of 0.1 mM 1,1-diphenyl-2 picrylhydrazyl methanolic solution. The mixture was incubated for 30 min at 25 °C, the absorbance was recorded at 520 nm using a spectrophotometer (Aquarius, Cecil, CE 7400).

The results were expressed as µg extract/mL. The antioxidant activity values were presented as IC₅₀: The concentration necessary to reduce or inhibit 50% of the initial concentration of DPPH. Ascorbic acid was used as a standard for DPPH radical-scavenging activity.

ABTS Radical-Scavenging Assay

The total antioxidant activity of the tested extracts was estimated by using ABTS radicals [28]. A total of 100 μL of the tested samples were added to 900 mL of diluted ABTS solution. After 6 min of incubation, the absorbance was measured using a spectrophotometer at 734 nm.

For ABTS antioxidant activity, Ascorbic acid was used as a standard. The antioxidant capacity of the tested samples was expressed as IC₅₀ (µg/mL).

3. Results and Discussion

3.1. Composition of SC-CO₂ Extracts: Oil Composition Analyses

The objective of the experiment is to estimate, for the first time, the composition of fatty acids (FA) and triglycerides (TAG) in the extracted oils. As mentioned before, the extraction procedure was supercritical CO₂ of freeze-dried OMW and the used raw materials were sampled from two different regions of Tunisia: Sousse and Sfax.

3.1.1. Fatty Acids

The results of the fatty acid analyses for OMW oil samples from two different localities collected in Sousse and Sfax and for the different operating conditions are summarized in

Table 2. FA content (expressed as a percentage of total fatty acids) in oils obtained by the supercritical carbon dioxide extraction method, by origin and SC-CO₂ conditions adopted.

	Sousse						Sfax
	S1	S2	S3	S4	S5	S6	F1	F2	F3	F4	F5	F6	t-Test	Significance (0.05)
	25/40	30/40	35/40	25/60	30/60	35/60	25/40	30/40	35/40	25/60	30/60	35/60
C16:0	18.60	18.75	19.11	19.56	19.73	19.79	17.10	17.21	17.35	18.32	18.43	18.53	4.168	1.92 × 10−3 *
C16:1	2.35	2.37	2.39	2.45	2.48	2.51	2.60	2.63	2.65	2.71	2.73	2.77	−6.857	4.4 × 10−5 *
C17:0	0.07	0.07	0.07	0.07	0.07	0.07	0.08	0.08	0.08	0.08	0.08	0.08	---	Ns
C17:1	0.07	0.07	0.07	0.08	0.08	0.08	0.07	0.07	0.07	0.07	0.07	0.07	2.236	0.049 *
C18:0	2.67	2.67	2.68	2.69	2.70	2.72	2.23	2.24	2.25	2.27	2.26	2.26	43.911	9.01 × 10−13 *
C18:1	61.08	61.01	60.97	60.54	60.50	60.45	59.10	59.02	58.95	58.84	58.8	58.77	14.186	5.96 × 10−8 *
C18:2	13.93	13.87	13.62	13.44	13.30	13.26	17.43	17.36	17.30	16.36	16.26	16.19	−11.924	3.10 × 10−7 *
C18:3	0.54	0.52	0.51	0.48	0.46	0.45	0.82	0.80	0.78	0.75	0.77	0.76	−15.915	1.97 × 10−8 *
C20:0	0.45	0.43	0.42	0.43	0.43	0.42	0.33	0.35	0.33	0.35	0.35	0.32	13.036	1.33 × 10−7 *
C20:1	0.24	0.24	0.25	0.26	0.25	0.25	0.24	0.24	0.24	0.25	0.25	0.25	0.877	0.40 ns
FA	21.34	21.49	21.86	22.32	22.50	22.58	19.41	19.53	19.68	20.67	20.77	20.87	5.276	3.6 × 10−4 *
MUFA	64.19	64.12	64.10	63.76	63.74	63.71	62.34	62.31	62.24	62.22	62.20	62.18	17.639	6.20 × 10−9 *
PUFA	14.47	14.39	14.13	13.92	13.76	13.71	18.25	18.16	18.08	17.11	17.03	16.95	−12.319	2.2813 × 10−7 *
UFA	78.66	78.51	78.23	77.68	77.50	77.42	80.59	80.47	80.32	79.33	79.23	79.13	−5.209	3.6 × 10−4 *

ns; Not significant: confidence interval percentage 95% *; Significant: confidence interval percentage 95%. S1, S2…S6: Site factories of OMW collection from Sousse; F1, F2…F6: Site factories of OMW collection from Sfax.

. Each of these series gives a complete representation of the composition of the oil.

Ten fatty acids were identified (C16 to C20) for all the studied samples, and OMW oils can be considered dominated by oleic acid. The major component (oleic acid) accounts for nearly 60% of fatty acids. The percentage of this fatty acid varied between 58.77% to 61.08%. Moreover, OMW collected from Sousse presented higher percentages of oleic acid (60.45–61.08%). Nonetheless, samples collected from Sfax showed percentages of oleic acid lower than 60% and varied between (58.77–59.1%) of the total percentage of FA. Linoleic acid (C18: 2) and palmitic acid (C16: 0) showed percentages lower than 20%. Furthermore, the highest percentages of linoleic acid were observed for Sfax (17.43–16.19%) and the lowest for Sousse (13.26–13.93%). The third most representative fatty acid in the chemical profile of OMW was found to be for palmitic acid which showed percentages ranging between 19.79–18.60% for Sousse and 17.10–18.53% for Sfax.

This study presented a significant variation in the composition of fatty acids. This may be due to the variation of olive cultivars and geographical areas, and different climatic conditions including pluviometry rate, seasonal variation of temperature and soil types humidity.

The chemical composition of OMWs could be compared to the chemical composition of virgin olive oil profiles. Olive oil has a unique composition of fatty acids: its oleic acid content varies from 55% to 83%, while linoleic acid (C18: 2) constitutes 2.5–21% and palmitic acid constitutes 7.5−20% [29]. Fatty acids are a valuable parameter in determining the quality and authenticity of olive oil [30].

The rest of the fatty acids (C16: 1, C17, C18: 0, C18: 3 and C20: 0) are poorly represented in the chemical composition of OMW with percentages varying between 0.07% and 2.77%; in addition, their percentages varied accordingly to the origin of OMW collection.

The results in Table 2 showed an evaluation of saturated fatty acids (SFA) and unsaturated fatty acids (UFA).

The proportion of saturated fatty acid (SFA) in the analyzed samples of OMW varied according to the origin of the collection. The samples of OMW collected from Sousse SFA varied between 21.34–22.58% and for those collected from Sfax, the percentages of SFA ranged between 19.41–20.87%.

It varies slightly depending on the localities of OMW sample collection sites: between 78.66% and 77.42% for the Sousse and between 80.59% and 79.13% for Sfax. The highest level of unsaturated fatty acids (UFA) is recorded in Sousse. This composition allows for the reduction in cholesterol levels by allowing preventive action, especially against cardiovascular diseases [31].

Moreover, the total fatty acid composition is affected by other principal known factors such as: latitude, climatic conditions and the stage of ripening of the fruit at harvest, particularly the oleic acid content [32].

Pressure and temperature play an important role on the economy of the process because they strongly affect productivity. The effects of temperature and pressure on the percentage of total fatty acids in the extract were studied for T = 40 and 60 °C and P = 25, 30 and 35 MPa. The results are shown in Table 2 and Figure 1 and Figure 2.

In the process of the extraction, all variations of (T, P) have no effect on C16: 1, C17: 0, C17: 1, C18: 0, C18: 3, C20: 1 and C20: 1. While, only C18: 1, C18: 2 and C16: 0 have been influenced. Furthermore, the percentage of fatty acid changed slightly as the temperature and pressure varied. The increasing operating conditions induces augmentation of the selectivity between saturated fatty acids. Whereas, for the unsaturated fatty acid, the opposite has been observed. UFA decreased with increasing temperature and pressure (Figure 1 and Figure 2).

The dependence on pressure is expected and SFA increases as the temperature and pressure are higher. This can be elucidated by the increase in the density of CO₂, which lead to an increase in the solvation power. However, UFA decreases with increasing temperature and pressure. It is concluded that the selectivity of fatty acids varies with temperature and pressure [33].

3.1.2. Triglycerides (TAGs)

Table 3. Composition of triglycerides by number of carbons (%).

	Sousse						Sfax
	S1	S2	S3	S4	S5	S6	F1	F2	F3	F4	F5	F6	t-Test	Significance (0.05)
	25/40	30/40	35/40	25/60	30/60	35/60	25/40	30/40	35/40	25/60	30/60	35/60
ECN42	3.62	3.50	3.41	3.13	3.07	2.95	1.40	1.33	1.25	1.13	1.08	1	10	1.36 × 10−8 *
ECN44	8.92	8.90	8.92	8.92	8.91	8.92	10.77	10.75	10.77	10.77	10.76	10.77	10	3.62 × 10−22 *
ECN46	31.23	31.33	31.39	31.69	31.8	31.91	34.71	34.83	34.87	35.09	35.22	35.28	10	4.72 × 10−10 *
ECN48	51.10	51.24	51.31	51.55	51.62	51.71	48.48	48.55	48.61	48.74	48.82	48.90	10	5.20 × 10−10 *
ECN50	5.13	5.03	4.97	4.71	4.60	4.51	4.64	4.54	4.50	4.27	4.12	4.05	10	7.94 × 10−3 *

* Significant: confidence interval percentage 95% S1, S2…S6: Site factories of OMW collection from Sousse; F1, F2…F6: Site factories of OMW collection from Sfax.

summarizes the results of SC-CO₂ extraction process with respect to triglyceride composition (%). The analysis in Table 3 distinguishes, essentially, five molecular species of TAGs in OMW oils: ECN42, ECN44, ECN46, ECN48 and ECN50.

The highest percentages of TAGs for the sample collected from Sousse were ECN48 with a proportion exceeding 51%. The percentage of OMW collected from Sfax varied between 48.48 and 48.90%. The second valuable triglyceride in the composition of the analyzed OMW samples corresponds to ECN46. The percentage values ranged between 34.71–35.28% for Sfax and 31.23–31.91% for Sousse. The significate difference between the samples of OMW collected from Sousse and Sfax could be attributed to many factors, such as climatic conditions, cultivar types and fruit-ripening stage, which may affect the TAGs and FAs composition [32].

As shown in Table 3, the effects of temperature and pressure on the percentage of TAGs in the OMW extracts were studied. The obtained results (

Table 4. Total phenolics contents, flavonoids and antioxidant activity of lyophilized OMW samples.

Origin of OMWs	Operating Condition [P (MPa)/T (°C)]	Total Phenolic Content mg GAE/g	Content Flavonoïdes mg EQ/g	DPPH IC50 μg/mL.	ABTS IC50 μg/mL.
Sousse	S1 (25/40)	181.70 ± 1.60	104.00 ± 0.90	55.90 ± 0.10	29.60 ± 1.00
	S2 (30/40)	188.20 ± 2.70	108.40 ± 1.40	53.40 ± 0.10	25.60 ± 0.00
	S3 (35/40)	201.50 ± 0.90	112.90 ± 0.80	52.10 ± 0.10	22.10 ± 0.30
	S4 (25/60)	211.70 ± 1.00	112.50 ± 1.10	50.50 ± 0.30	18.20 ± 0.10
	S5 (30/60)	216.10 ± 1.90	121.50 ± 1.60	50.10 ± 0.00	16.90 ± 0.10
	S6 (35/60)	196.00 ± 0.90	103.20 ± 1.00	52.70 ± 0.20	23.30 ± 0.40
Sfax	F1 (25/40)	79.10 ± 1.00	25.60 ± 0.30	287.50 ± 2.70	116.20 ± 0.10
	F2 (30/40)	87.30 ± 2.70	27.00 ± 0.10	243.30 ± 0.20	82.20 ± 0.40
	F3 (35/40)	85.80 ± 1.70	33.70 ± 0.90	229.40 ± 0.00	75.60 ± 0.50
	F4 (25/60)	94.80 ± 1.50	38.10 ± 1.50	214.20 ± 0.10	60.40 ± 0.20
	F5 (30/60)	104.20 ± 0.50	43.90 ± 0.50	207.10 ± 3.10	51.40 ± 0.30
	F6 (35/60)	85.90 ± 2.00	21.00 ± 1.00	266.90 ± 0.20	100.90 ± 0.00
t-test		16.868	17.663	−14.843	−5.777
Significance (0.05)		1.13 × 10−8 *	7.20 × 10−9 *	3.86 × 10−8 *	1.79 × 10−4 *

* Significant: confidence interval percentage 95%. S1, S2…S6: Site factories of OMWs collection from Sousse; F1, F2…F6: Site factories of OMW collection from Sfax.

) revealed non-significant effects of the variables (T, P) on ECN44.

The TAGs percentages changed slightly as the temperature and pressure increases. The selectivity between ECN46 and ECN48 changes significantly with the increase in temperature and pressure, respectively, during the operating conditions. Whereas, for ECN42 and ECN50, the opposite is observed. ECN42 and ECN50 decrease with increasing temperature and pressure.

Extracted oils have a unique character profile of TAGs that can be used to determine the origin and detect adulteration. At this stage, it is then possible to classify the vegetable oil based on the fatty acid and triglyceride compositions of the oils. The two determinations (TAGs and FAs) are complementary and they give better information on the lipid compositions of the oils than those resulting from the analysis alone of the fatty acids or triglycerides separately.

3.2. Polar Fraction: Analysis of Lyophilized OMW Residue after the SFE

3.2.1. Total Phenolic Content

The results of the total phenolic content of the different solvent extracts from freeze-dried OMW samples, obtained from the residue using extraction with SC-CO₂ are determined by the Folin–Ciocalteu method. The results were presented in Table 4.

Our results revealed that the total content of phenolic compounds in OMW samples collected from Sousse and Sfax varied significantly. The phenolic contents of the extracts obtained from OMW samples collected from Sousse are higher than those collected from Sfax. The ranges of total phenolic compounds in different regions varied between 181.70 (±1.60) to 216.10 (±1.90) mg GAE/g DM for Sousse and from 79.10 (±1.00) to 104.20 (±0.50) mg GAE/g DM) for Sfax. These values are lower than those recorded in the study of Gueboudji et al. (950 ± 14.2 µg GAE/mg of extract) [34]. Niaounakis and Halvadakis (2004) [35] reported that the concentration of highly variable total phenols in a complex raw OMW liquid matrix could range from 0.5 to 24 g/L.

The levels of phenolic compounds in OMW raw material are influenced by cultivars, cultivation sites, and the influence of climatic conditions [36].

The two tested variables (T, P) have a positive effect on polyphenol contents, except for (T = 60 °C, P = 35 MPa) a decrease in the content of polyphenols is observed compared to previous experiments. As shown by Figure 3, an optimum extraction process (T = 60 °C and P = 30 MP) is achieved for all samples of OMW regarding the origin of collection sites (both for Sousse and Sfax).

The polyphenol contents for optimal operating conditions were 216.10 ± 1.90 mg GAE/g DM for OMW samples collected from Sousse and 104.20 ± 0.50 mg GAE/g DM for OMW samples collected from Sfax.

3.2.2. Total Flavonoids

The concentrations of flavonoids are determined according to the aluminum trichloride (AlCl3) method using spectrophotometer. The summary of flavonoid content analyzed in the tested extracts is shown by (Figure 4).

The total flavonoids contents of various freeze-dried OMW samples varied from 103.20 ± 1.00 to 121.50 ± 1.60 mg EQ/g DM, for Sousse and from 21.00 ± 1.00 to 43.90 ± 0.50 mg EQ/g DM, for Sfax (Table 4). The flavonoids contents, for the samples collected from Sousse and for the different extraction conditions, is higher than those collected from Sfax. These values found in Sfax are lower than those recorded in the study of Gueboudji et al. (80.6 ± 17.27 µg QE/mg of extract) while those found in Sousse are higher [34].

The effect of pressure and temperature on the content of flavonoids were studied following SC-CO₂ extraction. (Figure 4)

The two tested variables (T, P) have a positive effect on flavonoids, except for (T = 60 °C, P = 35 MPa), where a decrease in flavonoid contents is observed compared to previous experiments. An optimum is reached both for the samples of Sousse series and for that of Sfax for T = 60 °C and P = 30 MP as indicated in (Figure 4). The flavonoids contents for this optimal pair of experimental conditions were 121.50 ± 1.60 mg EQ/g of OMW powder and 43.90 ± 0.50 mg EQ/g of OMW powder, respectively, for the samples collected from Sousse and Sfax.

3.2.3. Free Radical-Scavenging Ability by the Use of a Stable DPPH Radical

The results obtained in the study of the antioxidant power of the freeze-dried OMW extracts by the DPPH method are illustrated in Table 4. In view of these results, it is noted that the capacity of the trapping of the free radical DPPH^• is remarkable in the extracts obtained from the samples of OMW collected from Sousse (IC₅₀ = 50.10 ± 0 to 55.90 ± 0.10 μg/mL). This capacity remains lower than that of ascorbic acid (IC₅₀ = 3.10 ± 0.10 μg/mL). Low activity was recorded for the extracts obtained from the samples collected from Sfax (IC₅₀ = 207.10 ± 3.10–287.50 ± 2.70 μg/mL). The order of the strongest antioxidant activity to the lowest was vitamin C, Sousse extracts and those obtained from Sfax. These values are lower than those recorded in the study of Gueboudji et al. (9.62 ± 0.28 µg/mL) [37].

The difference between the antioxidant extracts could be explained in terms of the different quality of the OMW, geographical origin, date of harvest, climatic conditions and different operating conditions [38]. The effects of temperature and pressure on the antioxidant potency in the extract are shown in (Figure 5).

The variables (T, P) showed a negative effect on the antioxidant activities, except for (T = 60 °C, P = 35 MPa). As shown in (Figure 5), the experimental conditions T = 60 °C and P = 30 MP for all tested samples (Sousse and Sfax) led to the most potent extracts regarding DPPH antioxidant activity. The highest values of DPPH antioxidant activity (Sousse: IC ₅₀ = 50.10 ± 0.50 μg/mL; Sfax: IC₅₀ = 207.10 ± 0.50 μg/mL) correspond to the previously described experimental conditions (T = 60 °C and P = 30 MP).

3.2.4. ABTS Free Radical-Scavenging Activity

The results of the antioxidant activity of freeze-dried OMW extracts by the ABTS method are shown in Table 4. The results corroborate those obtained for DPPH antioxidant activity and the extracts obtained for the samples collected from Sousse exhibited the highest antioxidant activity (Sousse: lowest value= 16.9 μg/mL; Sfax: lowest value = 51.4 μg/mL).

For the samples collected from Sousse, antioxidant activity ranged from 16.90 to 29.60 μg/mL and from 51.40 to 116.20 μg/mL for the extracts obtained from the samples collected from Sfax. The ABTS antioxidant values of the extracts obtained from OMW samples are significatively higher than ascorbic acid value (IC₅₀ = 1.60 ± 0.00 μg/mL). The results are higher than those recorded in the study by Gueboudji et al. (7.10 ± 0.11 µg/mL) [37]. This result is consistent with many previous studies [39].

In addition, OMW extracts have been demonstrated to be a more efficient scavenger of the ABTS radical. Thus, the nature of the phenolic compounds present in olive fruits could be a determining key which explains the reason why can react with ABTS^•+ better than DPPH^• in these results.

The effect of pressure- and temperature-operating conditions on the antioxidant activity of the extracts are shown in (Figure 6).

The two tested variables (T, P) have a negative effect on the antioxidant activities of the extracts, except for (T = 60 °C, P = 35 MPa), a decrease in antioxidant activity is observed compared to previous experiments. As shown by Figure 6, an optimum extraction process (T = 60 °C and P = 30 MP) is achieved for all samples of OMW regarding the origin of collection sites (both for Sousse and Sfax). The antioxidant activity of the extracts for optimal operating conditions were 16.9 20 μg/mL for OMW samples collected from Sousse and 60.4. mg μg/mL for OMW samples collected from Sfax.

3.3. Composition of the Oil Extracted by the Conventional Solvent Extraction

3.3.1. Evaluation of the Extracted Oil Yield

The lyophilized OMW samples were subjected to successive macerations with organic solvents with increasing polarity (n-hexane, dichloromethane and ethanol). The obtained results were expressed as a percentage of the plant material used, are presented in (Figure 7).

The yields of the lyophilized OMW samples obtained from Sousse were 25.54%, 17.09% and 5.55%, respectively, for hexane, dichloromethane and ethanol. However, for the region of Sfax, the best-obtained yield was for hexane extract with 33.87%, followed by ethanol (11.12%) and dichloromethane (2.17%). The lowest yield is observed for the dichloromethane extract with a percentage of 2.17%.

These variations in extraction yields could be due to the difference in polarity and solubility for compounds present in OMWs.

It is essential to realize that this extraction yield can be obtained after a long extraction period (more than 270 min) and considerable consumption of toxic solvents that make it unfit for the environment.

The extraction of bioactive compounds such as polyphenols, and lipids from lyophilized OMWs are done using different solvents. Lipids are isolated by using more apolar solvents, such as n-hexane; while polyphenols are commonly obtained by ethanol (polar solvent) and dichloromethane (medium polar solvent). The extraction of polyphenols, using ethanol, is more efficient [19].

3.3.2. Analysis of Hexane Extracts

Fatty Acids

The results of the analyses of the fatty acid fraction for the samples of OMW collected from Sousse and Sfax are summarized in

Table 5. Fatty acid composition of hexane extracts.

FA (%)	Sousse	Sfax
C16:0	18.64	14.37
C16:1	2.32	2.42
C17:0	0.03	0.22
C17:1	0.13	0.09
C18:0	2.76	2.44
C18:1	61.62	61.20
C18:2	13.36	17.75
C18:3	0.40	0.46
C20:0	0.53	0.83
C20:1	0.21	0.22
SFA	21.43	17.03
MUFA	64.81	64.76
PUFA	13.76	18.21
UFA	78.57	82.97

.

Ten fatty acids have been identified (C16 to C20) from the hexane extracts of OMWs collected from Sousse and Sfax. OMW oil can be considered as an oleic oil because of the abundance of oleic acid (C18:1). The oleic acid content showed a percentage of 61.20% for Sfax while it is the highest for Sousse (61.62%). The highest percentage of linoleic acid is observed for Sfax (17.75%) and is the lowest for Sousse (13.36%). Palmitic acid has respective rates of 18.64% for samples from Sousse and 14.37% for Sfax [40,41].

Triglycerides

Table 6. Composition of triglycerides by number of carbons (%) in n-hexane extracts.

	Sousse	Sfax
ECN42	3.73	1.61
ECN44	8.85	10.64
ECN46	30.04	34.13
ECN48	53.14	49.72
ECN50	4.24	3.90

summarizes the results of n-hexane extraction with respect to the yield of triglycerides (% by weight). The TAGs composition was different for the two OMW oil extracts.

The analyses in Table 6 essentially distinguish five molecular species of TAGs in OMW oil: ECN48, ECN46, ECN44, ECN50 and ECN42.

The highest percentage of TAGs in Sousse and Sfax oils was ECN48; these represented about 50% of total TAGs (Sfax: 49.72%; Sousse 53.14%). The second compound in order of quantitative importance was ECN 46, (34.13% for Sfax), while Sousse oil contained a lower percentage with 30.04% of total TAGs.

3.3.3. Polar Fraction: Analyses of Ethanol and Dichloromethane Extracts

Total Phenolic Content

In this work, we studied the antioxidant potential of the extracts of OMW. The contents are shown in

Table 7. Total phenolics, flavonoids contents and antioxidant activity in OMW extracts according to the nature of the extraction solvent (ethanol/ dichloromethane).

Extract	Origin of OMW	Total Phenolic Content mg GAE/g	Flavonoids Content mg EQ/g	DPPH CI50 μg/mL.	ABTS CI50 μg/mL.
Ethanol	Sousse	170.60 ± 0.90	160.30 ± 1.70	31.10 ± 0.10	14.50 ± 0.10
	Sfax	67.60 ± 0.70	52.20 ± 0.90	60.80 ± 0.30	55.90 ± 0.80
dichloromethane	Sousse	124.20 ± 0.50	113.50 ± 2.40	98.70 ± 0.30	21.60 ± 0.30
	Sfax	61.60 ± 0.20	48.50 ± 1.10	213.00 ± 0.00	59.20 ± 0.70
t-test		3.539	3.687	0.748	101.81
Significance (0.05)		0.071 (ns)	0.066 (ns)	0.478 (ns)	0.01 *

ns; Not significant: confidence interval percentage 95% *; Significant: confidence interval percentage 95%

. As measured by the Folin–Ciocalteu test, the OMW samples of Sousse showed the highest amounts of total polyphenols.

In view of the results, it is clear that the amount of total polyphenols varies signigicantly depending on the nature of the used extraction solvent. The total phenolic content of OMW extracts obtained from the samples collected from Sousse was more than two times higher than the phenols content obtained from the samples collected from Sfax. Table 7 showed that the ethanol extract has the best extracting power. The highest phenols contents 170.60 and 67.60 mg GAE/g DM were obtained, respectively, for Sousse and Sfax followed by the dichloromethane extracts with respective values of 124.20 and 61.60 mg GAE/g DM for Sousse and Sfax. These values are lower than those recorded in the study of Gueboudji et al. (0.950 mg GAE/mg of extract) [34]. The variation in the total phenolic contents could be the result of the changing solubility of these compounds according to the solvent polarity [42].

The solvents used can be classified according to a decreasing extracting capacity as follows: ethanol, dichloromethane. Indeed, ethanol has a great polarity t to extract polar molecules, such as polyphenols. Thus, it can be concluded that ethanol solvent allows the most suitable extraction of phenolic compounds from OMWs. The results found are consistent with the levels of polyphenols found in the literature. A study by Mulinacci et al. indicates that the polyphenols content of OMWs ranged from 12.40 to 401.70 mg GAE/g DM [43,44].

Total Flavonoids

Table 7 showed the results of analyses of total flavonoid compounds. The amounts of flavonoid compounds fluctuate enormously in the various extracts analyzed and depend on the polarity of the extraction solvent and the source. The effect of the solvent on the solubility of total flavonoids showed approximately the same classification for total polyphenols. Table 7 reveals that the ethanol extract of OMWs collected from Sousse contained the largest amount of flavonoids (160.30 ± 1.70 mg EQ/g DM), while the lowest was found in the dichloromethane extract of OMWs collected from Sfax (48.50 ± 1.10 mg EQ/g DM) and their amount is lower than that of total polyphenols.

The ethanol extracts of Sousse and Sfax appear to be the most flavonoid-rich solvents (160.30 ± 1.70 and 52.20 ± 0.90 mg EQ/g DM). The dichloromethane extracts have a lower level (113.50 ± 2.40 and 48.50 ± 1.10 mg EQ/g DM, respectively). These values found for OMWs collected from Sfax are lower than those recorded in the study of Gueboudji et al. (80.6 ± 17.27 µg QE/mg of extract) while those found for the extracts obtained for OMWs collected from Sousse are higher [34].

Free Radical-Scavenging Ability by the Use of a Stable DPPH Radical

The results of the free radical-scavenging activity of the various extracts are presented in Table 7. These results showed that the best free radical-scavenging activities (DPPH) were recorded by ethanol extracts with IC₅₀ values = 31.10 and 60.80 μg/mL, for extracts of OMWs collected from Sousse and Sfax, respectively. The dichloromethane extracts showed the lowest anti-radical activity (IC ₅₀ = 98.70 and 213.00 μg/mL). This capacity remains lower than that of ascorbic acid (IC₅₀ = 2.60 ± 0.10 μg/mL). These values are lower than those recorded in the study of Gueboudji et al. (9.62 ± 0.28 µg/mL) [28]. The nature of the solvent has a significant effect on the antioxidant activity of OMW samples.

ABTS Free Radical-Scavenging Activity

The IC₅₀ values shown in Table 7 exhibited that OMWs have antioxidant activities that differ significantly depending on the nature and the extraction solvent. The results reported for the study of the antioxidant power of OMWs by the ABTS method are also shown in (Figure 8).

The antioxidant activity of the extracts obtained for OMWs collected from Sousse varies between 14.50 μg/mL and 59.20 µg/mL. Extracts of ethanol are always the most active among the extracts tested with IC₅₀ values of 14.50 μg/mL for OMWs collected from Sousse and 55.90 μg/mL for OMWs collected from Sfax. Moderate activity is also recorded for the dichloromethane extracts (IC₅₀ = 21.60 for OMWs collected from Sousse and 59.20 μg/mL for those collected from Sfax). The IC₅₀ values for ascorbic acid were 1.60 ± 0.10 μg/mL. The results of the antioxidant activity of OMW extracts are lower than those recorded in the study of Gueboudji et al. (7.10 ± 0.11 µg/mL) [28].

In view of the results, it is mentioned that the antioxidant activity of the various extracts found for the radical ABTS follows about the same trend, but we note that it is higher than that found for the radical DPPH. Our results corroborate with many previous studies [28,30].

The antioxidant activity of the OMW extracts depends on the conditions as well as the extraction method and polarity of the organic solvent. For that reason, different classes of simple and complex phenolic compounds can be generated and, therefore, exhibited different behavior regarding antioxidant potency [45].

4. Conclusions

The chemical profiles of the supercritical CO₂, n-hexane, dichloromethane and ethanol extracts of lyophilized OMW were identified. SC-CO₂ extraction using different pressure and temperature provide oil, which contained, mainly, oleic acid followed by linolenic and palmitic acid. The percentage of fatty acids and triglyceride is affected by the temperature and the pressure during the optimization of the extraction process. Nevertheless, for a pressure of 30 MPa and temperature of 60 °C, the amount of fatty acid, triglycerides and antioxidant activity are the most effective. However, successive organic solvents maceration with increasing polarity (n-hexane, dichloromethane and ethanol) was carried out for OMW from two distinct regions (Sousse and Sfax). Hexane extracts were characterized by the dominance of oleic acid, followed by linolenic and palmitic acid. Thus, the choice of solvent used for the extraction has also an important impact in determining the chemical composition. Extraction using SC-CO₂ presents a real alternative that replaces conventional extraction methods which allows us to preserve a good quality of OMW extracts.