1. Introduction
The cultivation of olive trees is a widespread practice in the Mediterranean region, accounting for about 98% of the world’s olive cultivation [
1]. The olive tree is gradually expanding, with nearly 18 Mt of olives harvested yearly around the world. Olive tree culture and the olive processing industry produce large amounts of byproducts. It has been estimated that pruning alone produces 25 kg of byproducts (twigs and leaves) per tree, annually [
2]. Olive leaves are considered to be an easily available agricultural byproduct [
3,
4]. They represent around 10% of the total weight of olives upon harvesting [
5,
6]. Olive leaves are a very rich source of bioactive compounds such as secoiridoids, flavonoids, and triterpenes [
7]. They can potentially have a higher added value if their fate is reconsidered.
Valorization of the residual biomass derived from the agricultural and food sector is nowadays regarded as central to the emerging bioeconomy. This biomass is definitely underrated, despite its richness in valuable substances [
8]. Olive leaves are usually disposed as waste. Otherwise, their infusion can be used in folk medicine [
9]. The secoiridoid oleuropein is the main compound, along with other secoiridoids derived from tyrosol and flavonoids [
5,
6,
9,
10,
11]. Other olive byproducts such as olive mill waste and mill wastewater, and wet olive pomace, have also been investigated for their polyphenols content [
12].
Phenolic compounds, plant secondary metabolites, are gaining more and more interest in the agro-industrial sector. They are being extensively studied, majorly due to their biological effects. Therefore, they are the subject of numerous extraction techniques used to recover them out of their original matrices. In this regard, conventional extraction using organic solvents is the most widely used method. Nevertheless, environmental toxicity, long duration of processing, and consumption of large quantities of organic solvents are the major concerns arising from this method [
13]. These major drawbacks led the researchers to seek new technologies to be applied or to be combined with pre-existing ones.
Many extraction technologies were used for the intensification of polyphenols recovery from plant materials, such as ultrasound assisted extraction [
14,
15], microwave assisted extraction [
16,
17], pressurized liquid assisted extraction [
18], supercritical fluid extraction [
19,
20], and others. Optimization of any given extraction technique goes obviously through a maximization of polyphenols recovery while maintaining their chemical integrity and, subsequently, their functional activities.
Infrared irradiation is one of these alternatives introduced as a ‘green’ energy source [
21,
22] used to boost the extraction of natural products. The infrared assisted extraction apparatus Ired-Irrad
® is a new generation of ecofriendly machines that enhance the extraction of bioactive compounds from natural matrices using a ceramic infrared emitter [
22]. Recently, this technique has been explored on pomegranate peels [
14],
Prunus armeniaca L. pomace [
23], apricot pomace [
24,
25], and
Saussurea lappa [
26], and permitted the intensification of polyphenol recovery compared to conventional extraction methods. Infrared-assisted extraction is easy to use, economical, requires low energy consumption [
23], and has a great potential to be scaled-up to an industrial level.
The innovation of this study relies on the use of a new patented technique based on infrared apparatus (IR) irradiations for the recovery of bioactive compounds, while preserving their biological properties. To our knowledge, no previous studies have investigated this effect on olive leaves. Our ultimate aim is to optimize extraction of total phenolic content from olive leaves using IR irradiation, and to compare the results with the ones obtained using water bath conventional extraction. Moreover, quality of both extracts will be inspected by testing their antioxidative and antiradical activities, their antibacterial effect against 20 strains of Staphylococcus aureus and 7 strains of Escherichia coli, and their antifungal effect not only against Aspergillus flavus growth, but also against its production of aflatoxin B1.
2. Materials and Methods
2.1. Plant Material
Olive leaves were provided by a local olive mill in northern Lebanon El Koura in September 2018. The leaves were washed with water to remove impurities such as dust, then dehydrated in an airflow oven at 40 °C for 48 h. Dried leaves were ground (Philips, United Arab Emirates, MEA) and then sieved using a vibrating multi sieve separator (ELE International, Loveland, CO, USA). Ground leaves, from 0.85 to 2 mm in size, were packed in plastic bags and stored at ambient temperature in the dark for further use.
2.1.1. Dry Matter
Initial and final moisture contents were determined by drying the leaves for 24 h in a ventilated oven at 105 °C. The dry matter (DM) of raw material was 91 ± 0.4%.
2.1.2. Chemicals
All chemicals used in the experiments were analytical grade. Folin-Ciocalteu reagent, sodium carbonate, gallic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, sulfuric acid, sodium phosphate, ammonium molybdate, oleuropein, and hydroxytyrosol were purchased from Sigma-Aldrich, Darmstadt, Germany.
2.2. Experimental Methods
2.2.1. Water Bath Extraction
The conventional extraction was carried out in a digital water bath (JSR JSWB-22T, Gongju-city, Korea) (
Figure 1a).
2.2.2. Infrared-Assisted Extraction
The infrared-assisted extraction apparatus (Ired-Irrad
®, Beirut, Lebanon) was designed and patented in collaboration between the faculties of Sciences of Saint-Joseph University (Beirut, city, Lebanon) and the University of Balamand (Kelhat, city Lebanon) [
22]. The extraction prototype consists of a ceramic infrared emitter, linked to a proportional-integral-derivative (PID) control and temperature adjustment system. The sample consisting of olive leaves and solvent was placed in a round bottom flask connected to a condenser at a 1 cm distance from the ceramic IR emitter (Rotfil, Pianezza, Italy) (
Figure 1b).
2.2.3. Extraction Procedure
An amount of 5 g of ground leaves was added to 100 mL of solvent consisting of varying amounts of aqueous ethanol. Extractions were carried out at predetermined temperatures and time periods. The fixed particle size (0.85–2 mm) and solid to liquid ratio (1:20
w/
v) were chosen based on a preliminary set of experiments (
Figure 2a,b). Once the extraction was complete, the extracts were filtered through glass wool, then centrifuged for 10 min at 4500 rpm and stored at −20 °C until analyses. Prior to HPLC analyses, the supernatants were filtered using a 0.45 μm syringe after centrifugation [
27].
2.2.4. Total Phenolic Compounds
The total phenolic content was determined according to the Folin-Ciocalteu method [
28,
29]: 0.2 mL of each extract were mixed with 1 mL of ten-fold diluted Folin–Ciocalteu reagent (Sigma-Aldrich, Darmstadt, Germany), and 0.8 mL of sodium carbonate (Na
2CO
3) (75 g/L) (Sigma-Aldrich, Darmstadt, Germany) were added to the mixture. The absorbance was then measured by a UV-Vis spectrophotometer (Biochrom Ltd., Cambridge, England) at 750 nm. The total phenolic content was expressed as mg of Gallic Acid Equivalents per gram of dry matter mg Gallic Acid Equivalent/g DM.
2.3. Experimental Design
Response surface methodology (RSM) is an assemblage of statistical and mathematical methods used for products developing, improving, and optimizing processes [
30]. It permits to measure the linear and quadratic effects of parameters, as well as the probable interactions between the variables.
Optimization of phenolic compounds extraction from ground olive leaves was carried out using RSM. A rotatable central composite design (2
3 + star) (22 runs: 8 factorial design points, 6 star points and 8 center points with 12 degrees of freedom) was created to evaluate the main impact of three experimental factors: Solvent mixture, time, and temperature on the response parameter: Total Phenolic Compounds (TPC). The same design was applied twice: (1) For the extraction process using the conventional water bath (WB), and (2) for the infrared-assisted (IR) extraction apparatus. Ethanol percentage values varied between 40% and 80%, time between 60 and 180 min, and temperature between 38 °C and 77 °C (considered as −1 and +1 levels, respectively). Solvent mixture, time, and temperature are independent variables that were coded at five levels (−α, −1, 0, +1, +α). Considering three parameters and one response, experimental data were fitted to obtain a second-degree regression equation of the form:
where
Y is the predicted response parameter;
β1 is the mean value of responses at the central point of the experiment;
β2,
β3 and
β4 are the linear coefficients;
β5,
β6 and
β7 are the quadratic coefficients;
β8,
β9 and
β10 are the interaction coefficients; E is the solvent mixture; t is the extraction time; and T is the extraction temperature. Experimental design and statistical treatment of the results were performed using STATGRAPHICS Centurion XVII (Statgraphics 18, The Plains, Virginia).
2.4. High Performance Liquid Chromatography
Polyphenol (oleuropein and hydroxytyrosol) identification and quantification were conducted by HPLC, using an HPLC-DAD (diode array detection) (Waters Alliance, Milford, MA, USA), a quaternary Waters e2695 pump, an UV−vis photodiode array spectrophotometer (Waters Corporation, Milford, USA), a control system, and a data collection Empower 3 software. Analyses were carried out on a Discovery HS C18, 5 μm, 250 × 4.6 mm, column (Supelco, Bellefonte, PA, USA) with a HS C18, Supelguard Discovery, 20 × 4 mm, 5 μm, precolumn (Supelco, Bellefonte, PA, USA). The column temperature was maintained at 25 °C. Separation of 10 μL was performed at a flow rate of 0.8 mL min
−1. Mobile phase A consisting of 0.5% (
v/
v) acetic acid in water and mobile phase B consisting of 100% acetonitrile were used. Solvent gradient changed according to the following conditions: From 0 to 10 min, 95% (A): 5% (B) to 70% (A): 30% (B); from 10 to 12 min, 70% (A): 30% (B) to 67% (A): 33% (B); from 12 to 17 min, 67% (A): 33% (B) to 62% (A): 38% (B); from 17 to 20 min, 62% (A): 38% (B) to 50% (A): 50% (B); from 20 to 23 min, 50% (A): 50% (B) to 5% (A): 95% (B); from 23 to 25 min, 5% (A): 95% (B) to 95% (A): 5% (B); from 25 to 35 min, 95% (A): 5% (B) to 95% (A): 5% (B). Spectrophotometric detection wavelength was carried out at 280 nm. Identification of the compounds was based on retention time of standards and comparison of spectra [
31].
2.5. Antioxidant Activity
The total antioxidant activity of the extracts was determined using the phosphomolybdenum reduction essay [
32]. The principle of this method is the formation of a green complex phosphate Mo (V). A quantity of 100 µL of each extract were mixed with 1 mL of the reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). Samples were incubated for 90 min at 95 °C. Absorbance was then measured at 695 nm. Antioxidant activity was expressed as µg of Ascorbic Acid Equivalent per milliliter (µg AAE/mL).
2.6. Antiradical Activity
Free radical scavenging activity was measured by the capacity of the phenolic compounds to reduce DPPH (2,2-diphenyl-picrylhydrazyl), according to [
33]. 1.45 mL of DPPH (0.06 mM) (Sigma-Aldrich, St-Quentin Fallavier, France) radical was added to 50 μL of olive leaf extracts or Trolox (positive control) (Sigma-Aldrich, St-Quentin Fallavier, France). After 30 min of incubation at room temperature in the dark, the absorbance was measured at 515 nm using pure methanol as a blank. The inhibition percentage of the DPPH free radical is calculated as follows: Inhibition Percentage = [(absorbance of negative control − absorbance of sample)/absorbance of negative control] × 100. Antiradical activity was expressed as µg of Trolox Equivalent per milliliter (µg TE/mL) [
34].
2.7. Antifungal Activity
Aspergillus flavus NRRL (Northern Regional Research Laboratory) 62477 isolates from spices were grown in Petri dishes containing malt extract agar (MEA) at pH 5.5 ± 0.3 for 7 days at 27 °C. A spore suspension was then prepared using Tween 80 solution. The spores were counted on a Neubauer haemocytometer (Superior, Marienfeld, Lauda-Konigshofen, Germany). The final concentration of the spore suspension was adjusted to 105 spores/mL.
2.8. Fungal Growth Inhibition
Olive leaf extracts (125 and 250 µg) were added to the MEA medium. A final volume of 20 mL of MEA was transferred in petri dishes. For the control culture, 20 mL of MEA without polyphenols were poured in a petri dish. Afterwards, 10 µL of the previously prepared spore solution (10
5 spores/mL) were placed in the center of each petri dish. All the dishes were left for 7 days in the incubator at 27 °C. On the seventh day, the diameters of all the cultures were measured. The
A. flavus inhibition percentage was calculated as follows:
2.9. Aflatoxin B1 (AFB1) Inhibition
Aflatoxin B1 (AFB1) inhibition was detected using reverse phase HPLC (diode array detection) (Waters Alliance, Milford, MA, USA) coupled with a fluorescence detector and a C18 column 5 μm, 250 × 4.6 mm, column (Supleco, Bellefonte, PA, USA) fitted with a HS C18, Supelguard Discovery, 20 × 4 mm, 5 μm, precolumn (Supelco, Bellefonte, PA, USA). The column temperature was maintained at 40 °C. The mobile phase was composed of HPLC water: Methanol: Nitric acid 4M (55:45:0.35 v/v/v) and 119 mg/L KBr prepared and filtered on the same day of HPLC analysis, performed with a flow rate of 0.8 mL/min. The injection volume was 100 µL, the cycle duration was 35 min, and the wavelengths for excitation and emission were 360 and 430 nm, respectively.
2.10. Antibacterial Activity
2.10.1. Microorganisms Used
Twenty bacterial strains (American Type Culture Collections ATCC, Newman and clinical strains) of Gram-positive S. aureus and seven strains of Gram-negative E. coli (one E. coli 25921 DSM 1103 and the others are strains of different profiles of resistance) that were isolated from patients at the Centre Hospitalier Du Nord Hospital (CHN, Zghorta, Lebanon), were used in this study.
2.10.2. Determination of Minimal Inhibitory Concentration for Extracts
The macro-dilution broth method was used for the determination of the minimum inhibitory concentration (MIC) of
Olea europea extracts, as described by the Clinical and Laboratory Standards Institute [
35]. A standardized bacterial inoculum was prepared and adjusted to 0.5 McFarland, then diluted to 10
6 CFU/mL. Leaf lyophilized extracts were diluted with DMSO to produce two-fold serial dilutions ranging from 0.39 to 50 mg/mL. 1 mL of broth was added to each tube of the macro-dilution tray. 300 µL of plant extract suspension were added to the first tube in each series, after removing the same volume of broth, in order to achieve the final desired concentration. 1 mL of bacterial inoculum was added to each tube to reach 2 mL of final volume. The final extract concentration in each tube is presented in
Table 1. Broth (2 mL) was used as a negative control, whereas 1 mL of Mueller-Hinton broth and 1 mL bacterial suspension were used as a positive control. The tray was then incubated for 24 h at 35 °C. Thereafter, the test tubes were checked for turbidity and MIC was determined by observing the lowest concentration of extract where there is no visible bacterial growth compared to the negative and positive control. The antibacterial analyses were repeated twice and gave the same MIC values.
2.11. Statistical Analysis
All experiments and measurements were done in triplicates. The mean values and the standard deviations were calculated. Error bars, in all figures, correspond to the confidence level 95%. Variance analyses (ANOVA) and Least Significant Difference (LSD) tests were done by STATGRAPHICS® Centurion XV (Statgraphics 18, The Plains, Virginia).
4. Conclusions
This study revealed the efficiency of Ired-Irrad® technology for the intensification of polyphenol recovery from olive leaves. Time and temperature were shown, by response surface methodology, to be the most significantly-affecting infrared-assisted extraction parameters. Compared to the conventional methods, IR allowed the extraction of a higher yield of polyphenols and improved many of their biological activities—i.e., antioxidant, antiradical, and anti-AFB1 secretion. Compared to WB, IR technology enhanced the recovery of both oleuropein and hydroxytyrosol, the two main polyphenols present in olives leaves. IR seems to be a very promising new generation of ecofriendly machines that can enhance the extraction of polyphenols with less energetic and solvent consumptions.
5. Patents
Rajha, H.N., Debs, E., Maroun, R.G., Louka, N. (2017). System for extracting, separating or treating products through infrared radiation. Adequacy between the properties of infrared radiation and those of the processed products. Invention patent number 2017 / 11-11296L granted on 29/11/2017.