Olive trees are known as trees of the subtropical region, which can survive and live for a few decades. The original homeland of olive trees is the Mediterranean region, including Iraq [1
]. The estimated area of planted olive trees is approximately eight million hectares. In addition, many studies pointed out that the cultivation of olive trees began around 3500 years ago [2
]. The olive leaf is one of the tree byproducts, which are obtained through either the pruning and harvesting process or fall due to different climatic factors. There is an ancient and prodigious history of olive trees because of their nutritional, medicinal, and traditional uses [3
]. An essential part of the Mediterranean diet is olive products because of their ability to modulate and control the oxidative balance in vivo; indeed, they can monitor and inhibit pathogenic bacteria [4
Lipid oxidation has been one of the major concerns of the scientific community for centuries. Scientists are persistently looking for antioxidants that will adequately work against oxidation in both fats and oils [6
]. Synthetic antioxidants are used in most countries, and their usage level is regulated and the safety of the compounds has been tested based on long-term toxicity studies. Moreover, most of the synthetic antioxidants are stable either under processing conditions or under the storage status of oils and fats [7
]. However, these synthetic antioxidants can cause carcinogenic and toxicological effects [8
]. There are no decisive results on the safety of these materials, thus utilizing antioxidants from natural sources has arisen throughout the world [9
There is an intensive investigation of olive leaves because of their many positive and beneficial effects for human health. For instance, olive leaves can work as anti-inflammatory, anti-microbial, anti-diabetic, anti-atherosclerotic, and anti-carcinogenic agents [10
]. Nowadays, people are consuming and taking olive leaf extracts that can be in either liquid or capsule form. Oleuropein is considered one of the most abundant phenolic compounds in olive leaf extracts [12
]. In fact, Oleuropein possess a spectacular effect against many foodborne pathogens such as Staphylococcus aureus
, Helicobacter pylori
, and Campylobacter jejuni
]. Haddadin [13
] found that olive tree leaves possessed large amounts of phenolic compounds such as vannilin, vannilic acid, verbascoside, luteolin-7-glucoside, and catechin; and their qualities were similar to those that are found in olives and other products derived from them, thus possessing the ability to prevent oxidation in food.
The main objectives of this study are; (1) to investigate the antioxidant and antimicrobial properties of olive leaf extract (OLE), (2) to determine the olive leaf extract chemical composition, and (3) to monitor the effects of using olive leaf extract on the quality of raw sheep meat slides.
2. Materials and Methods
2.1. Preparation of the Olive Leaf Extracts
Olive leaves were collected from an olive orchard located in Najaf province, Iraq. They were placed in polyethylene bags and kept in the refrigerator until use, and then the olive leaves were characterized by specialists at the Department of Horticulture, College of Agriculture, University of Basra. The olive leaves were cleaned and properly washed from extraneous matter. Then, they were dried in a hot air-oven for 48 h at 37 °C. A powder form of the dried olive leaves was obtained after crushing them in a blender. Thereafter, the olive leaf extracts were prepared according to Pereira et al. [10
] by using the mixed solvents as follow:
Ethanol/H2O (80:20 v/v %)
Methanol/H2O (80:20 v/v %)
Diethyl ether/H2O (80:20 v/v %)
Hexanol/H2O (80:20 v/v %)
Five grams of powdered olive leaves was weighed and placed in a 500 mL beaker with the addition of 200 mL of each solvent separately. The solvent extraction process was performed at a temperature of 38 °C using a stirrer speed of 180 rpm for 20 min followed by filtration using Whatman no.1. Centrifugation was performed at 5000 r/min for 15 min, and then the filtrates were evaporated at 38 °C using a rotary evaporator for 4 h. The crude extracts were kept in dark containers in the refrigerator before use.
2.2. Determination of Total Phenolic Compounds
The total phenolic compounds were measured according to Lako et al. [14
] with slight modifications. One mL of crude olive extract was mixed with 20 mL of (DMSO) dimeyhyl sulfuoxide. Afterwards, 2.5 mL of Folin-Ciocalteu was added with the addition of 10 mL of distilled water. The mixture was left for 2.5 min at room temperature. Then 2 mL of sodium carbonate (7.5%) was added, and the mixture was kept for one hour at room temperature in a dark place. The absorbance was read at a wavelength of 725 nm, and it was compared to a calibration curve prepared with known amounts of Gallic acid (Roth, Karlsruhe, Germany). The results were expressed as mg Gallic acid/g extracts.
2.3. Ferric Thiocyanate
The antioxidant activity of the olive leaves was measured according to Elmastas et al. [15
], which included measuring the effectiveness of the anti-oxidation of linoleic acid by mixing 1 mL of the crude extract with 4 mL of ethanol (95%), 4.1 mL of linoleic acid (2.5%) in ethanol, and 8 mL of phosphate buffer (0.05 M, pH 7). The mixture was incubated at a temperature of 45 °C for 24 h, and then 0.1 mL of this mixture was added to 9.7 mL of ethanol (75%), 0.1 mL of ammonium thiocyanate (30%), and 0.1 mL of ferrous chloride (0.02 M). All conditions were the same for preparing the control sample except for mixing 1 mL of distilled water instead of the sample extracts. The synthetic antioxidant butylated hydroxytoluene (BHT) was used as a model for comparison. The absorbance was measured at a wavelength of 500 nm using a UV-Spectrophotometer. The percentage of antioxidant activity was calculated as follows:
% Antioxidant activity = 100 − (A/B) × 100
A: Absorbance of thesample
B: Absorbance of thecontrol
2.4. Ferric Reducing Antioxidant Power
The antioxidant ability of the olive leaves was determined according to [16
]. One mL of olive leaf extract was dissolved in 1 mL of distilled water, and 2.5 mL of K3
(1% w/v) with 2.5 mL of 0.2 M phosphate buffer (pH 6.6). The mixture was kept at 50 °C for 20 min, and then about 22.5 mL of trichloro acetic acid (10% w/v) was added. Afterwards, centrifugation at 3000 rpm for 10 min was performed in order to obtain an upper layer (2.5 mL). Then 2.5 mL of distilled water and 0.5 mL of FeCl3
(0.1%, w/v) were mixed with the obtained upper layer. All conditions were the same for preparing the control sample except for mixing 1 mL of distilled water instead of the sample extracts. The synthetic antioxidant (BHT) was used as a model for comparison. The absorbance was measured at 700 nm using a spectrophotometer. The ferric reducing antioxidant power was calculated as follows:
% Ferric reducing antioxidant power = 100 − (As/Ac) × 100
As = absorbance of the sample
Ac = absorbance of the control
2.5. Free Radical Scavenging Activity
Firstly, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution was prepared by dissolving 2 mg of this indicator in 100 ml of methanol. Secondly, the DPPH solution (3 mL) was added to 1 mL of olive leaf extract and was then mixed gently according to [17
]. The mixture was incubated in a dark place for 30 min. The control sample was prepared by mixing 1 mL of methanol with 3 mL of the prepared DPPH solution. The synthetic antioxidant (BHT) was used as a model for comparison. The absorbance was measured at 517 nm for each sample using a spectrophotometer. The inhibition of DPPH activity was calculated using the following formula:
% Inhibition of DPPH activity= [Ac − As/Ac ] × 100
Ac: absorbance of the control
As: absorbance of the sample
2.6. Studying the Stability of the Olive Leaf Extract
The preliminary results indicated that methanol extraction showed the highest values for the tested performance among the used solvents. For this reason, methanol extraction was used in the remainder of the experiments. The storage times and temperatures for the olive leaf were tested to ensure that there was minimal change for the total phenolic compounds, in order to avoid degradation of the phenolic compounds. Concentrated olive leaf extracts were stored at −18 °C, 5 °C, 25 °C, and 35 °C for 75 days. The total phenolic compounds were monitored every 15 days.
2.7. Determination of the Olive Leaf Extract Profile
The reversed phase HPLC with silica-based C18 was used as the stationary phase for determination of oleuropein, tyrosol, and verbascoside from the olive leaf extract. The mobile phase was a mixture of water and acetonitrile (80/20 volume ratio) containing 1% acetic acid at a flow rate of 1.0 mL/min. Oleuropein, tyrosol, and verbascoside in the crude olive leaf were identified by comparing their retention times with the corresponding standards [18
2.8. Application of the Olive Leaf Extract on the Sheep Meat Slides
2.8.1. Preparation of Sheep Meat Samples
The sheep meat was obtained from local markets in Basra province. The sheep meat was cut into different sizes of slides (approximately 15 × 15 cm3). The weight of the sheep meat slides was about 10 g for each slide. The sheep meat slides were divided into five batches; three batches of sheep meat slides were immersed in solutions containing 0.5%, 1.5%, and 2.5% OLE (w/v) in a 1:1 ratio (sheep meat:distilled water w/v) for 15 h at 5 °C. After treatment, the samples were drained. The fourth batch was used as a control sample by immersion in distilled water. Ziploc bags were used to keep the sheep meat slides in five portions and were stored at 5 °C for 12 days.
2.8.2. pH Determination of the Sheep Meat Slides
The pH of the sheep meat slides was estimated according to Aytul et al. [19
]. Approximately 2–2.5 g of sheep meat slides were weighed and mixed with distilled water (1:10 w/v). Centrifugation was performed at 12,000 rpm for 4 min in order to homogenize the mixture of sheep meat slides and distilled water. The pH of treated and non-treated samples was measured in triplicate by a pH meter.
2.9. Determination of Oxidative Stability of the Sheep Meat Slides
2.9.1. Thiobarbituric Acid (TBA) Assay
The TBA assay was carried out according to Bekhit et al. [20
] with slight modifications. Firstly, the solution of TBA (0.45 %) and trichloroacetic acid (TCA) (prepared in 0.25 N HCl) was prepared. Then 2.5 grams of treated sheep meat slides (0.5%, 1.5%, and 2.5% w/v) were mixed and homogenized at 10,000 rpm for 5 min in a beaker containing 25 mL of the prepared solution of TBA and TCA. Then, in order to develop a pink color, approximately 5 mL of the homogenized sample was placed in a water bath at 100 °C for 12 min. The boiled samples were cooled under tap water. Thereafter, centrifugation at 5000 rpm for 15 min was performed. The whole procedure was repeated by using distilled water instead of the olive leaf extract (control sample), while BHT was used for comparison. Finally, the absorbance of the treated samples was measured at 532 nm using a spectrophotometer. The TBA value was calculated as follows: TBA [mg Malonaldehyde (MDA) equivalent/kg sheep meat] = A × 7.8, where A: the absorbance.
2.9.2. Antimicrobial Activity of the Olive Leaf Extract on Sheep Meat Slides
The antimicrobial activity of the olive leaf extract was determined according to Andrews [21
] with slight modifications. Approximately 10 g of treated sheep meat slides (0.5%, 1.5%, and 2.5% w/v) was mixed and homogenized with 90 mL of 0.1% sterile peptone water under sterilized conditions for 2 min at 25 °C. The serial dilution of the mixture was made using 0.1% peptone water. The diluted samples were transferred to a petri dish containing nutrient agar and MacConkey agar in order to determine both the total count bacteria and total coliform bacteria, respectively. Microbial counts were expressed as log10
CFU/g of the sample.
2.10. Statistical Data Analysis
The SPSS statistical software program (SPSS for Windows version 17, Spss Inc., Chicago, IL, USA) was used to analyze the data. Data were expressed as mean ± standard deviation (SD). The obtained results were considered significant at p < 0.05.