The Effects of Different Irrigation Treatments on Olive Oil Quality and

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The effects of different irrigation treatments on olive oil quality and composition: A comparative study between treated and olive mill wastewater Samia Ben Brahim, Boutheina Gargouri, Fatma Marrakchi, and Mohamed Bouaziz J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05030 • Publication Date (Web): 25 Jan 2016 Downloaded from http://pubs.acs.org on February 2, 2016

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Journal of Agricultural and Food Chemistry

The effects of different irrigation treatments on olive oil quality and composition: A comparative study between treated and olive mill wastewater

Samia Ben Brahim 1, Boutheina Gargouri1, Fatma Marrakchi 1, Mohamed Bouaziz 1,2 *

1

Laboratoire d’Electrochimie et Environnement, Ecole Nationale d’Ingénieurs de Sfax BP «1173» 3038, Université de Sfax, Sfax, Tunisia

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Institut Supérieur de Biotechnologie de Sfax, Université de Sfax, BP «1175» 3038, Sfax, Tunisia *Corresponding author: Dr. Mohamed BOUAZIZ, Tel: +216 98 667 581; Fax: +216 74 674 364. E-mail: [email protected]

Running title: Olive mill wastewater and treated wastewater irrigation: effect on olive oil

1 ACS Paragon Plus Environment


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Abstract

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In the present paper, two irrigation treatments were applied to olive trees cv. Chemlali:

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irrigation with treated waste water (TWW) and olive mill waste water (OMW) which was

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spreading at three levels (50, 100 and 200 m3 /ha). This work is interested in two topics: (1)

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the influence of different irrigation treatments on olive oil composition and quality and (2) the

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comparison between OMW and TWW application using different statistical analyses. The

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obtained variance analysis (ANOVA) has confirmed that there are no significant differences

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on oil quality indices and flavonoids between the control and treatments amended by OMW

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or TWW (p>0.05). However, the irrigation affected some aspects of olive oil composition

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such as the reduction in palmitic acid (16.32%) and increase in linoleic acid (19.55%).

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Furthermore, the total phenols and α-tocopherol contents increased significantly following

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OMW and TWW treatments. PCA and HCA analyses defined three irrigation groups: OMW

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50 and 100 m3/ha, OMW 200 m3/ha and control and TWW treatment. The full factorial design

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revealed that OMW amendment by 100 m3/ha is the best irrigation treatment. Thus, the

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optimal performances in terms of olive oil quality and composition were shown by olive oil

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extracted from olive grown under irrigation with 100 m3/ha of OMW.

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KEYWORDS: olive oil quality, irrigation, olive mill wastewater, treated wastewater,

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polyphenols, fatty acids.

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INTRODUCTION

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In the Mediterranean basin, the olive tree (Olea europaea L.) is the most important evergreen

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tree. Tunisia ranks the fourth world producer of olive oil after Spain, Italy and Greece. It is

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considered among the important olive oil producers with a yearly average production of

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180.000 tons in 2011/2012 crop season (IOC)1. However, there are many factors, namely the

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availability of fresh water, which could negatively affect the production. In this context, a

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shocking study has revealed that in 2025, in most parts of the Mediterranean countries, per

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capita water availability will be decreased by 60% compared to 1990, leading to the decrease

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of irrigated surfaces and the gradual deterioration in water quality 2.

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As an arid country with limited water resources, Tunisia depends heavily on irrigated

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agriculture. Thus, a non-conventional water resource has become crucial to satisfy different

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agricultural needs. In fact, the development of irrigation strategies is of vital for the

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improvement of the long-term profitability within the concept of sustainable olive growing.

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Along with the low availability of fresh water, Tunisia suffers from a major environmental

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problem resulting from the huge wastewater quantities produced yearly during a short period.

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Indeed, the use of treated wastewater (TWW) in Tunisia has been adopted since the 1965s for

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citrus fruits and is authorized for different cultures, such as the fruit trees and, in particular,

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the date palms, the citrus fruits and the vines 3. The treated wastewater still retains a

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substantial amount of organic and metallic compounds (C, N, P and K) which have a

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favorable effect on the growth of certain crops 4,5. On the other hand, olive mill wastewater is

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a by-product obtained after olive oil extraction process. This effluent is characterized by high

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concentrations of several organic compounds, such as organic acids, sugars, tannins, pectins

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and a complex consortium of phenolic substances 6,7.

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Aware of the problems engendered by water scarcity and environment damaging, Tunisia has

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developed policies to conserve water resources and encourage demand management in the



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irrigation sector. Considerable strategies adopted not only for increasing water supply in arid

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and semi-arid countries, but also for developing alternative water resources, such as the use of

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TWW and OMW in olive agriculture irrigation. Although several studies have focused on the

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effect of OMW and TWW irrigation on physical, chemical and biological properties of soil8–

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quality12–14.

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Therefore, the scope of this work is to study the effect of irrigation treatments using OMW

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and TWW on Chemlali olive oil quality and composition. The aim of chemometric analysis

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was to evaluate the feasibility and efficiency of each treatment on olive oil quality. The best

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irrigation method was chosen by applying full factorial design in order to preserve fresh water

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reserves in Tunisia and improving olive oil production without affecting negatively the

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quality.

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MATERIALS AND METHODS

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Experimental site

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The experimental site is located in the south west of Sfax city in central eastern Tunisia. The

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climate in this region of the country is Mediterranean: dry summers and relatively cold

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winters. The annual rainfall and temperature averages during the studied year 2012 were 180

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mm and 18-40 °C, respectively. The sandy soil of the experimental orchard (84.4% of sand,

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9.8% of clay and 5.8% of silt) was characterized by 1.3% as organic matter, 10.5% CaCO3;

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1.2% Nt and 7.9 as pH.

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Olive mill waste water (OMW) source and characteristics

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The original OMW used in the present study was obtained from the discontinuous process for

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olive oils extraction plant located in Sfax city. The main characteristics of the OMW used for

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irrigation were: pH: 5.1; electrical conductivity (EC): 9.1 dsm-1; salinity: 6.37 g l-1; COD: 93

, little information is available on the effects of this two irrigation treatments on olive oil


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g l-1; N: 1340 mgl-1; P: 720 mgl-1; K: 6200 mgl-1; phenols: 8400 mgl-1 and glucose 1200 mgl-1.

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The major phenolic monomers in the fresh OMW ethyl acetate extract were identified and

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quantified by HPLC. (See Fig. 1).

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Treated waste water (TWW) source and chemical characteristics

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The treated wastewater comes from domestic and industrial sources and is typically reclaimed

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at the secondary level using biological processes. These processes consist in the

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transformation of biodegradable matters into microbial residues. Aerobic biological treatment

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is performed in the presence of oxygen by aerobic microorganisms that metabolize the

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organic matter in the wastewater, thus producing more microorganisms and inorganic end-

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products (CO2, NH3 and H2O). TWW used for irrigation, whose characteristics are reported in

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Table 1, was neutral and highly saline.

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Plant material

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OMW amendments were realized in 2012 in one application. In all cases, fresh OMW (after

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24 h sedimentation at the olive mill) was applied between the rows of olive trees at a distance

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of 70 cm from the trunk using a tractor with tank trailer (spreading machine). Concerning

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TWW, Chemlali olive trees, they were irrigated with drip system with four drip nozzles (two

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per side) of 4.3 m3 day-1 per tree set in a line along the rows (at 0.5m from the trunk).

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Chemlali (Olea europaea L.) olive trees cultivated at the density of 17 trees ha-1 were selected

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to be similar in potential yield. The Olive’s production is illustrated in Table 2. The following

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treatments were applied on olive trees:

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No application of OMW (control).

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• Annual application of OMW at 50 m3 ha-1.

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• Annual application of OMW at 100 m3 ha-1.

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• Annual application of OMW at 200 m3 ha-1. 5 ACS Paragon Plus Environment


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• Application of TWW.

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Oil extraction process

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About 02–2.5 kg of healthy fruit samples from Chemlali variety were hand harvested in

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triplicate from all the olive trees for each treatment. To avoid the well-known relationship

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between quality olive oil and ripeness index, all the fruit samples were collected with a

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similar ripeness index (4.5). Samples were immediately brought to the laboratory for oil

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extraction using a laboratory olive Bench Hammer Mill (Abencor Analyzer, MC2 Ingenierias

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y Sistemas, Sevilla, Spain). The extractor is equipped with a metal crusher, a mixer whose

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internal partition is in rustproof steel, having a vertical or horizontal axis and a basket

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centrifuge. Fruits were cleaned from leaves, milled in the hammer crusher; the olive paste was

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then mixed for 30 min at 28 ◦C. The oil was separated by a vertical centrifuge (3500 rpm over

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3 min), transferred into dark glass bottles then stored at -4°C until analysis.

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Chemicals

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Methanol, hexane, acetone, acetic acid and cyclohexane HPLC-grade solvents were

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purchased from Riedel-deHaen (Switzerland). The solvents were of appropriate purity.

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Double distilled water was used in the HPLC mobile phase. Folin–Ciocalteu reagent was

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obtained from Fluka (Switzerland).

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Analytical methods

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Quality parameters

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The determination of free acidity, peroxide value and UV absorption characteristics at 232

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and 270 nm were carried out following analytical methods described by the International

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Olive Council.


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Pigment content

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Carotenoids and chlorophylls (mg/kg of oil) were determined at 470 and 670 nm,

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respectively, in cyclohexane using the specific extinction values according to the method of

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Minguez-Mosquera et al15.

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• Chlorophyll (mg/kg) = (A670 × 106) / (613 × 100 × d)

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• Carotenoid (mg/kg) = (A470 × 106) / (2.000 × 100 × d)

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where A is the absorbance and d is the spectrophotometer cell thickness (1 cm). The

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chlorophylls and carotenoids concentrations are expressed as mg of pheophytin and lutein per

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kg of oil, respectively.

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Fatty acid methyl ester analysis

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The fatty acid composition of the oils was determined by gas chromatography (GC) as fatty

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acid methyl esters (FAMEs). FAMEs were prepared by saponification/methylation with

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sodium methylate according to the EEC Reg 2568/9116. A chromatographic analysis was

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performed in a SHIMADZU set 17 A Series II gas chromatography using a capillary column

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(stabilwax, Restek, length 50 m, internal diameter 0.32 mm and film thickness 0.25 µm). The

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column temperature was isothermal at 180 °C, the injector and detector temperatures were

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230 and 250 °C, respectively. Generally, fatty acids were identified by comparing retention

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times with standard compounds, and in the present study, ten fatty acids were considered.

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These were palmitic (16:0), palmitoleic (16:1), heptadecanoic (17:0), heptadecenoic (17:1),

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stearic (18:0), oleic (18:1), linoleic (18:2), linolenic (18:3), arachidic (20:0) and gadoleic

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(C20:1) acids expressed as percentages of fatty acid methyl esters.

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Tocopherol determination



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HPLC analysis for α-tocopherol was performed with an Agilent (1100) Series HPLC system

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chromatograph (Hewlett-Packard, Waldbronn, Germany) apparatus. Detection was performed

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at 290 nm for α-tocopherol. The column was (C-18, 4.6 x 250 mm, particle size 5µm, shim-

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pack, VP-ODS, Shimadzu, Kyoto, Japan). The elution solvents used were A (2% acetic acid

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in water), B (methanol), C (acetonitrile) and D (isopropanol). The samples were eluted

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according to the following gradient: 95% A/5% B in 2 min; 60% A/ 10% B/30% C in 8 min;

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25% B/75% C in 22 min, and this percentage was maintained for 10 min; 40% C/60% D in 10

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min; and this percentage was maintained for 15 min; 25% B/75% C in 2 min, and finally, 95%

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A/5% B in 3 min. The flow rate was 1 ml/min and run time was 70 min. The run was

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performed at 32°C. The sample injection volume was 20 µl. The identification of α-

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tocopherol was achieved by comparing its retention time values with its standard

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tocopherol content was determined by diluting approximately 1100 mg of olive oil in one ml

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n-hexane mixture and analyzing the sample solution by HPLC. The concentrations of α-

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tocopherol were then calculated from the integrated peak areas of the samples and the

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calibration curve of α-tocopherol standard. Acceptable linearity was achieved in the range

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300–1100 mg kg-1 (y=1.30x, R2=0.899).

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Determination of total polar phenol content

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The determination of the total phenolic compounds included the use of the Folin–Ciocalteau

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reagent which the method of Bouaziz et al18. Briefly, a 50 mL aliquot of the extracts was

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assayed with 250 mL Folin reagent and 500 mL sodium carbonate (200 g/L). The mixture was

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vortexed and diluted with water to a final volume of 5 mL. After incubation for 30 min at

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room temperature, the absorbance was read at 765 nm; total phenols were expressed as gallic

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acid equivalents (GAE) using a calibration curve of a freshly prepared gallic acid solution

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(y= 0.0012x - 0.0345, R2 = 0.9997).

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.The α-


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Total flavonoids contents

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Total flavonoids were measured by a colorimetric assay developed by Bouaziz et al18. One ml

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aliquot of appropriately diluted sample or standard solution of catechin (20, 40, 60, 80 and

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100 mg.l-1) was added to a 10 ml volumetric flask containing 4 ml double distillate water

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(ddH2O). At zero time, 0.3 ml 5% NaNO2 was added to the flask. After 5 min, 0.3 ml 10%

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AlCl3 was added. At 6 min, 2 ml of 1 M NaOH was added to the mixture. Immediately, the

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reaction flask was diluted to volume with the addition of 2.4 ml of ddH2O and completely

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mixed. The absorbance of the mixture -pink colour- was determined at 510 nm compared to

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control water. The total flavonoid contents were expressed as mg/kg of oil catechin

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equivalents (CE). Samples were analyzed in triplicates.

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Statistical analysis

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The results were expressed as mean ± standard deviation (SD) of three measurements for the

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analytical determination. Significant differences between the values of all parameters were

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determined at p0.05) except some fatty acids. In fact, heptadecanoic, heptadecenoic, stearic, linoleic and

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linolenic fatty acids presented a significant variation only at the dose of 200 m3/ha OMW.

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Moreover, oleic acid was the most abundant one with a percentage value varying from 56.15

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to 59.57% of the total fatty acids content. For palmitic acid, a significant reduction (p

The Effects of Different Irrigation Treatments on Olive Oil Quality and

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