Screening of Chemical Composition, Antimicrobial and Antioxidant Activities of Essential Oil and Volatile Fraction from Olive Mill Wastewater

: Oil mill wastewater is the main by-product of the olive oil industry resulting mainly from the treatment and pressing of olives in mills. It is a rich source of nutrients and phytochemicals with a wide spectrum of biological properties. The present study focuses on the chemical analysis and evaluation of the antimicrobial and antioxidant activity of the essential oil (EO) and the volatile fraction (VF) obtained, respectively, by hydrodistillation. Chemical analysis by gas chromatography coupled to mass spectrometry (GC/MS) and a ﬂame ionisation detector (GC/FID) revealed the predominance of phenolic compounds (25.71%, 60.36%) and fatty acids (62.37%, 38.25%) for the VF and EO, respectively. It was also shown that the main compounds were oleic acid (24.9%) for the VF and 4-ethylphenol (28.5%) for the EO. The results of the antimicrobial activity in terms of MIC values against twelve microorganisms showed that, overall, the VF was more active than the EO. The antioxidant activity of the VF and EO was evaluated using the DPPH assay and expressed as half-maximal inhibitory concentration (IC50), where the EO (218 µ g/mL) showed better antioxidant activity than the VF (244 µ g/mL). The results also revealed that the antimicrobial activity and antioxidant activity values for both oils were signiﬁcantly lower than the standards used.


Introduction
The Mediterranean region constitutes the best environment for the growth and the development of olive trees and the largest producer of olive oil. More than 98% of the world's olive oil is produced there, with Spain, Italy, Greece, Turkey and Portugal being the main producing countries [1]. The International Olive Council (IOC) reported that for the 2020-2021 harvest periods, world olive oil production was estimated at 3,034,000 tons. In addition, Algeria is the ninth-largest olive oil producer in the world, with about 35 million trees whose production of olives and olive oil is estimated at about one million and 80,000 tons per year, respectively [2,3]. Nowadays, numerous olive-oil-extraction processes are used, including the traditional extraction, press system, two-phase and three-phase extraction. The two-phase centrifugation process generates effluents in smaller quantities as compared to the former process and the three-phase centrifugation system [4]. This later produces from 10 to more than 30 million cubic meters annually of olive mill wastewater (OMW) [5], which includes 83 to 92% water coming from the olives themselves and the water used in the olive-oil-extraction process [6]. It is characterised by a dark colour (red to

Preparation of Crude Extract
First, 300 g of crude OMW (pH = 5) were centrifuged for 10 min at 4500 rpm and 5 • C, then the pH was adjusted to 2 using HCl (0.5 N). The obtained solution was treated with 100 mL of hexane twice to remove the lipid fraction. The aqueous phase was collected for further liquid-liquid extraction using 100 mL of ethyl acetate. The organic phase obtained after five repeated extractions was dried over anhydrous sodium sulphate and the solvent was evaporated to give 1.5 g of a crude extract, which corresponds to 0.5% (w/w) yield.

Extraction of Volatile Fraction
The VF of the OMW was prepared using a modified Dean-Stark apparatus. First, 300 mg of crude extract were steam-distilled with 500 mL of distilled water for 1 h.
The VF was separated from the water by liquid-liquid extraction with ethyl acetate. The organic phase was dried over anhydrous sodium sulphate and the solvent was removed using a rotary evaporator resulting in 20 mg of VF, corresponding to a yield of 6.6% (w/w).

Extraction of Essential Oil
First, 400 g of the centrifuged OMW were mixed with 1 L of distilled water. The hydrodistillation process was carried out for 3 h. The separation of the EO from the aqueous phase was carried out in a similar way to that of the VF. The extraction yield of the EO was 0.0075% (w/w). The obtained EO was weighed and stored in brown vials at 4 • C. It should be noted that the extraction time for the preparation of the VF and EO by hydrodistillation was chosen on the basis of preliminary experiments and in reference to our previous work [31].

Chromatographic Analyses of VF and EO
The coupled gas chromatography/mass spectrometry (GC/MS) and gas chromatography/flame ionisation detection (GC/FID) analyses were performed for qualitative and quantitative purposes, respectively. The analysis was performed using a GC-QP2010 system (Shimadzu, Kyoto, Japan) coupled to an FID detector. The analysis system was equipped with an auto-sampler injector and a separation capillary column HP-5 fused silica column (30 m × 0.25 mm i.d., df 0.25 µm). The oven temperature was held at 60 • C for 5 min, and then increased to 250 • C at a rate of 3 • C·min −1 . The injector temperature was set at 250 • C. Helium was used as a carrier gas at a constant rate of 28.8 mL/min and a linear velocity of 33.3 cm/s corresponding to an inlet pressure of 43.6 kPa. The interface and ion source temperatures were 250 • C and 200 • C, respectively. The acquisition was made in full scan mode in the mass range of 40-500 m/z, with a scanning rate interval of 0.2 s. Samples were dissolved in ethyl acetate (10% w/w) and then, 1 µL was injected with a split ratio 1:90. The mass range scanned was 35-550 m/z. The delay time was set at 5 min. The GC/FID analysis was also performed under the same conditions. To identify the chemical composition, the following databases were used: W11N17 (DB1) (Wiley11-Nist17, Wiley, Hoboken, NJ, USA; and FFNSC 3.0 (DB2) (Shimadzu, Kyoto, Japan). The identification was performed by applying two filters, namely spectral similarity match over 85% and linear retention index (LRI) match calculated using a C 7 and C 40 saturated n-alkane homologue series (1000 g/mL, 49452-U) supplied by Merck KGaA (Darmstadt, Germany) with a filter window of ±10 LRI units. Further identification was achieved based on mass spectra reported by specialized [32].

ATR-FTIR Spectroscopy Analyses of VF and EO
An FTIR spectrometer, model Nicolet iS10, was used for ATR dry film measurements. It was equipped with a deuterated triglycine sulphate (DTGS) detector, 0.5 µL liquid samples (EO or VF) were dried over the diamond ATR crystal of mono-reflection Dura Sample IR II accessory for liquids, from Smiths Detection Inc. (Andover, MA, USA). The OMNIC 9.8 software, from Nicolet iS10, was used for instrument control and data treatment. The absorbance was recorded over the range 4000-525 cm −1 at a resolution of 4 cm −1 with an average of 32 scans per spectrum.

Determination of the Minimum Inhibitory Concentration of VF and EO
The minimum inhibitory concentration (MIC) values were determined according to the CLSI guidelines. Inoculums were prepared by transferring one colony of each strain from the agar plate into flasks containing the nutrient broth. They were then incubated for 18 h at 37 • C. The microbial suspensions were adjusted to 1 × 10 8 CFU/mL for bacteria and 1 × 10 5 CFU/mL for C. albicans (ATCC 10231) and S. cerevisiae (ATCC 9763) using a UV-visible spectrometer at a wavelength of 625 nm. The 96-well plates were prepared by adding 100 µL of the VF and EO into the first well. Then, 50 µL of nutrient broth were added to each well, from the second to the twelfth well. A series of two-fold dilutions of the EO was performed in a concentration range from 0.0488 to 50 mg/mL by successively transferring 50 µL from the first to the eleventh well. The last well containing 50 µL of nutrient broth without the VF and EO was used as a negative control, and then 50 µL of the suspensions were added to the wells of each strip leading to a final volume of 100 µL. The plates were incubated at 37 • C for 18 to 24 h for bacteria and 36 to 48 h for the antifungal test. Kanamycin and chloramphenicol were used as references; their highest concentration was 10 mg/mL. All tests were performed in triplicate under sterile conditions.

Antioxidant Activity of VF and EO
The DPPH free-radical-scavenging test was performed to determine antioxidant activity [33]. A 0.4 mL volume of the methanolic solution of the VF and EO at different concentrations (from 7.81 to 4000 µg/mL) was mixed with 1.6 mL of DPPH methanolic solution (0.1 mM). The mixture was incubated at 37 • C and left in the dark for 30 min, and then the absorbance was measured at 517 nm, using a UV-visible reader. Butylhydroxytoluene (BHT), butylhydroxyanisole (BHA) and α-tocopherol were used as antioxidant standards. Results were expressed as IC 50 . The scavenging activity was calculated using Equation (1): where A control is the absorbance of the negative control and A sample is the absorbance of the sample oils at 517 nm.

ATR-FTIR Analyses of VF and EO
The analysis of the infrared spectrum of the EO and VF ( Figure 1) revealed the presence of strong absorption bands at 1463 cm −1 , 2923 cm −1 , 2853 cm −1 , most likely corresponding to methyne, methylene and methyl groups. The IR spectrum also showed a strong absorption band between 1600 and 1800 cm −1 centred around 1700 cm −1 . This broad band could regroup the elongation movements of the C=C at 1600 cm −1 , indicating the presence of aromatic compounds and those of the carbonyl group C=O at 1720-1740 cm −1 , in relation to the presence of compounds bearing a carbonyl function. The presence of aromatic compounds is most likely associated with the presence of phenolic compounds. This hypothesis is most plausibly confirmed by the presence of OH groups of phenolic compounds, but also of alcohols, at 3358 cm −1 [34]. The correlation between the VF and EO spectra was estimated at 71.69%.

ATR-FTIR Analyses of VF and EO
The analysis of the infrared spectrum of the EO and VF (Figure 1) revealed the presence of strong absorption bands at 1463 cm −1 , 2923 cm −1 , 2853 cm −1 , most likely corresponding to methyne, methylene and methyl groups. The IR spectrum also showed a strong absorption band between 1600 and 1800 cm −1 centred around 1700 cm −1 . This broad band could regroup the elongation movements of the C=C at 1600 cm −1 , indicating the presence of aromatic compounds and those of the carbonyl group C=O at 1720-1740 cm −1 , in relation to the presence of compounds bearing a carbonyl function. The presence of aromatic compounds is most likely associated with the presence of phenolic compounds. This hypothesis is most plausibly confirmed by the presence of OH groups of phenolic compounds, but also of alcohols, at 3358 cm −1 [34]. The correlation between the VF and EO spectra was estimated at 71.69%.

GC/MS and GC/FID Analyses of VF and EO
First, it should be noted that VF and EO displayed different chromatographic profiles. Forty-one and thirty-nine compounds accounting for 90.17 and 99.26% of the total compositions were identified in the VF and EO, respectively ( Table 1). As described above, GC/MS allowed the identification of the different compounds on the basis of the MS database. The GC/FID analysis has a double objective including the calculation of retention indices and the determination of the percentage content of different compounds.

GC/MS and GC/FID Analyses of VF and EO
First, it should be noted that VF and EO displayed different chromatographic profiles. Forty-one and thirty-nine compounds accounting for 90.17 and 99.26% of the total compositions were identified in the VF and EO, respectively ( Table 1). As described above, GC/MS allowed the identification of the different compounds on the basis of the MS database. The GC/FID analysis has a double objective including the calculation of retention indices and the determination of the percentage content of different compounds. The chemical composition of the VF and EO can be divided into four chemical classes (Table 1 and Figure 2): terpenes (VF-0.65%; EO-0.53%), phenolic compounds (VF-25.71%; EO-60.36%), fatty acids (VF-62.37%; EO-38.25%) and others (variously functionalised compounds; VF-1.44%; EO-0.12%). Previous studies have already reported the presence of fatty acids [35], phenols [36], and terpenes [37,38] as the main chemical composition of olive oil and OMW. A closer look at Figure 2 shows that the sum of the percentages of phenolic compounds and fatty acids in the VF and EO are 88.08% and 98.61%, respectively. Thus, it can be seen that almost the entire chemical composition of both oils is phenolic compounds and/or fatty acids at approximately similar percentages. The findings are most probably related to a bioconversion/degradation process of phenols into fatty acids and vice versa. As mentioned above and according to Figure 2, phenolics and fatty acids are the predominant chemical classes in the VF and EO, respectively. This result is rather surprising given that the two oils, obtained from OMW, should, in all likelihood, have a relatively similar chemical composition, especially with regard to the chemical nature of the main class. It seems, therefore, that the hypothesis of the intervention of a degradation process is more than likely. Thus, as the EO was directly prepared from the OMW while the VF was obtained following two steps including the preparation of the extract and then its hydrodistillation, it is plausible that the phenols, which are very sensitive to heat, within the VF underwent degradation reactions into fatty acids. It is possible that the degradation process followed the same chemical mechanism as the bioconversion of phenols to fatty acids. Thus, the simultaneous coexistence of the four main classes in the chemical composition of the VF and EO could most likely be related to biosynthetic considerations, as illustrated in Figure 3 [39], which describes the biosynthetic pathways of the different chemical classes and some plausible pathways suggested based on the chemical composition of the VF and EO. The results also revealed that the main chemical class in the EO was made up of phenolic compounds (60.36%), of which 4-ethylphenol (28.5%) was the predominant one. On the other hand, and unexpectedly, the volatile fraction was characterised by the presence of fatty acids as the predominant class (62.37%), with oleic acid A closer look at Figure 2 shows that the sum of the percentages of phenolic compounds and fatty acids in the VF and EO are 88.08% and 98.61%, respectively. Thus, it can be seen that almost the entire chemical composition of both oils is phenolic compounds and/or Chemosensors 2022, 10, 491 7 of 13 fatty acids at approximately similar percentages. The findings are most probably related to a bioconversion/degradation process of phenols into fatty acids and vice versa. As mentioned above and according to Figure 2, phenolics and fatty acids are the predominant chemical classes in the VF and EO, respectively. This result is rather surprising given that the two oils, obtained from OMW, should, in all likelihood, have a relatively similar chemical composition, especially with regard to the chemical nature of the main class. It seems, therefore, that the hypothesis of the intervention of a degradation process is more than likely. Thus, as the EO was directly prepared from the OMW while the VF was obtained following two steps including the preparation of the extract and then its hydrodistillation, it is plausible that the phenols, which are very sensitive to heat, within the VF underwent degradation reactions into fatty acids. It is possible that the degradation process followed the same chemical mechanism as the bioconversion of phenols to fatty acids.
Thus, the simultaneous coexistence of the four main classes in the chemical composition of the VF and EO could most likely be related to biosynthetic considerations, as illustrated in Figure 3 [39], which describes the biosynthetic pathways of the different chemical classes and some plausible pathways suggested based on the chemical composition of the VF and EO. The results also revealed that the main chemical class in the EO was made up of phenolic compounds (60.36%), of which 4-ethylphenol (28.5%) was the predominant one. On the other hand, and unexpectedly, the volatile fraction was characterised by the presence of fatty acids as the predominant class (62.37%), with oleic acid being the main compound at 24.9%. The low content of terpenes in the VF and EO is probably related either to their loss during the preparation process of the OMW extract by liquid extraction, or to their degradation under the effect of heat, although this last hypothesis remains less probable. In the present study, we noticed the presence of cis-limonene oxide, cis-pyran linalool oxide, isomenthol and camphor as monoterpenes and farnesene as a sesquiterpene. which is also the precursor of terpenes via mevalonic acid and of phenolic compounds via malonyl Coenzyme A. It is quite permissible that the contents of the four chemical classes are most likely inter-related. Thus, it is worth noting the presence of linalool and limonene in the volatile fraction of olive oil [40,41], while, in the present study, they were described in the EO as limonene oxide and linalool oxide (furan and pyran). This finding is most likely related to their chemical transformation under the effect of steam or their bioconversion by specific microorganisms present in the medium. In this context, the biotransformation of linalool to  As illustrated in Figure 3, shikimic acid is the biosynthetic precursor of the majority of phenolic compounds, whereas acetyl Coenzyme A is the precursor of fatty acids, which is also the precursor of terpenes via mevalonic acid and of phenolic compounds via malonyl Coenzyme A. It is quite permissible that the contents of the four chemical classes are most likely inter-related.
Thus, it is worth noting the presence of linalool and limonene in the volatile fraction of olive oil [40,41], while, in the present study, they were described in the EO as limonene oxide and linalool oxide (furan and pyran). This finding is most likely related to their chemical transformation under the effect of steam or their bioconversion by specific microorganisms present in the medium. In this context, the biotransformation of linalool to limonene oxide via limonene, as well as to furanoid and pyranoid linalool oxide was reported [42] using different strains of A. niger, a fungus found and isolated in OMW [43]. In the same order, plausible pathways (PPs) have been proposed for obtaining camphor and isomenthol from limonene. Additionally, the biosynthesis of neomenthol (isomer of isomenthol) has been described from limonene in Mentha arvensis L. essential oils [44]. The previous several reactions are illustrated by Figure 4, which clearly explains the presence of limonene oxide pyran and furan linalool oxide in the EO.
Chemosensors 2022, 10, x FOR PEER REVIEW 9 of 13 by hydrodistillation and solid-phase microextraction (SPME) [38]. It should be pointed out that monoterpenes, sesquiterpenes and benzenoids have been described as being more abundant in the volatile fraction of virgin olive oil obtained by hydrodistillation than by SPME [45], and vice versa for the abundance of their fatty acids. A further examination of the chemical composition (Table 1) revealed the presence of oleic acid and its isomer elaidic acid (identified as an ethyl ester), tyrosol and its derivative tyrosol acetate (considered a secoiridoid); both compounds have previously been reported in virgin olive oil [46]. Furthermore, the presence of farnesene, even in trace amounts, in the EO, and not in the VF, is linked to the relatively long hydrodistillation time (3 h) which favours its entrainment since it is a compound with a high molecular weight and low vapor pressure. The presence of farnesene and limonene has previously been proven in olive oil treated by hydrodistillation and solid-phase microextraction (SPME) [38]. It should be pointed out that monoterpenes, sesquiterpenes and benzenoids have been described as being more abundant in the volatile fraction of virgin olive oil obtained by hydrodistillation than by SPME [45], and vice versa for the abundance of their fatty acids.
A further examination of the chemical composition (Table 1) revealed the presence of oleic acid and its isomer elaidic acid (identified as an ethyl ester), tyrosol and its derivative tyrosol acetate (considered a secoiridoid); both compounds have previously been reported in virgin olive oil [46].

Antimicrobial Activity
According to Table 2, the VF exhibited strong antimicrobial activity compared to the EO. Thus, both the VF and EO inhibited the growth of all the tested microorganisms at different concentrations. The MIC values were in the range of 195.30-1562.5 µg/mL and 390.62-3125 µg/mL for the VF and EO, respectively. Based on the above data, it is evident that the VF exhibited a stronger inhibitory effect with respect to B. subtilis, M. luteus, E. coli, A. tumefaciens, E. feacium, C. albicans and S. cerevisiae. Furthermore, both oils showed the same activity against B. cereus, S. aureus, L. monocytogenes and P. aeruginosa. However, the EO was ahead of the VF only once against E. cloacae. Kanamycin and chloramphenicol showed the lowest MIC values, i.e., a very strong inhibitory effect against all the microorganisms tested in comparison with the VF and EO. It must be noted that B. subtilis, B. cereus, L. monocytogenes and P. aeruginosa were the most resistant microorganisms. Conversely, E. coli and S. cerevisiae were the most sensitive ones. The high antibacterial activity of the VF could be related to its chemical composition, which is particularly rich in fatty acids, especially unsaturated ones. The latter are also famous for their high biological activity and are involved in various biosynthetic processes. In this regard, long-chain unsaturated fatty acids are endowed with antibacterial activity [47]. In addition, it has been reported that fatty acids inhibit bacterial growth by disrupting bacterial membranes or by inhibiting fatty-acid synthesis [48]. According to the GC/MS analysis of the VF, the fatty acid fraction consisted of oleic acid and its isomer elaidic acid, linoleic acid, their ethyl ester and stearic acid ethyl ester, which are known for their antibacterial activity against several microorganisms such as E. coli, P. aeruginosa, B. subtilis and S. aureus, among others [49].
Furthermore, synergism between these fatty acids could take place and boost the antimicrobial activity of the VF. In this regard, it was found that oleic and linoleic acid exhibited a synergistic antimicrobial effect when they were mixed [50]. In addition, the presence of tyrosol in the VF (2%) composition at a higher amount as compared to the EO (0.3%), and tyrosol acetate (0.2%), which was only found in the VF could also be reasons for its high activity. In this scenario, it has been reported that these two components are endowed with good antimicrobial activities [51].

Antioxidant Capacity
The capacity of scavenging the DPPH free radicals by the VF, EO and standards was obtained graphically using a linear curve by plotting radical-scavenging percentages with respect to the sample concentrations. The antioxidant activity of both the oils and standards was expressed in terms of IC 50 (µg/mL). The results in Table 3 show that the antioxidant activity of the VF and EO was significantly lower than all the controls used. The recorded IC 50 for the EO was relatively better than for VF. It has previously been reported that OMW alkyl aromatic alcohols show significant antioxidant properties and that the best result was associated with the presence of polyphenols [51,52]. This result is mainly due to the high phenolic content in the EO as they represent the main chemical class. The difference in antioxidant activity was most probably related to the presence of gaiacol, phenol and 5-tertbutylpyrogallol, which were only present in the EO. In addition, benzyl alcohol (4.9%), benzene ethanol (7%) and 4-ethylphenol (28.5%) were comparatively much more present in the EO than in the VF.

Conclusions
In the present work, the chemical analysis of the VF and the EO of OMW revealed the presence of four chemical classes including phenolics, fatty acids, terpenes and a group of variously functionalised compounds. The antimicrobial test revealed that the VF showed a strong inhibitory effect compared to the EO against most of the microorganisms tested, including B. subtilis, M. luteus, E. coli, A. tumefaciens, E. feacium, and especially the yeasts, C. albicans and S. cerevisiae. This high antimicrobial activity is most likely related to its richness in fatty acids, especially unsaturated fatty acids, which are known to have strong antimicrobial properties. The antioxidant capacity of the two oils by the DPPH method, through the free-radical-scavenging capacity, showed that the EO had a relatively higher antioxidant activity than the VF in terms of IC 50 .
In general, the in vitro assays carried out showed that the essential oil and the volatile fraction of OMW provide an important source of natural antimicrobial and antioxidant agents that could be used in the food, cosmetic or pharmaceutical industries, while reducing their hazardous impact on the environment.