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J. Nat. Prod. Plant Resour., 2014, 4 (2):16-23
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Experimental study of solid waste olive’s mill:
Extraction modes optimization and physicochemical characterization
R. Ferhat
*1, S. Laroui
1, B. Zitouni
2, A. Lekbir
2, M. Abdeddaim
2, N. Smaili
2and Y. Mohammedi
21Laboratory of the Biotechnology of Bioactives Molecules and the Cellular Physiopathology, Hadj Lakhdar University, Batna, Algeria
2
Food Technology Department, Institute of Veterinary and Agronomic Sciences, Hadj Lakhdar University, Batna, Algeria
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ABSTRACT
The main goal of this paper is to study the extraction of the oil from the solid waste after the extraction of virgin olive oil (pomace) of two various mills collected in Batna area. Also, two extractions modes are used: a physical extraction mode (Hydraulic press) and a chemical one. Olive pomace oil is a by-product from the olive oil industry that is still being used in the food industry as a low value vegetable oil. The results obtained are quantitative and qualitative. The first ones show that the best extracted oil quantity obtained is by the chemical way and especially with the mixture "Chloroform/Methanol". This result is the same (12,59-15,91%) with either samples of solid residues from traditional hydraulic pressing system or three-phase centrifugation separation process. The qualitative result obtained shows that the oil extracted by these two modes reveals the significant difference in all the physical and chemical parameters such as the relative density at 20°C, the refractive index, the acid value, the saponification value, the peroxide value and the iodine value. Except the unsaponifiable matter which haven’t a difference Crude olive pomace oil needs to be refined and is blended with virgin olive oils before being used as edible oil.
Keywords: Extracted oil, pomace, mill, valorization, physicochemical, characterization, rate
_____________________________________________________________________________________________ INTRODUCTION
The olive (Olea europea L.) is an evergreen tree, traditionally cultivated for the production of oil and table olives. As regards both wealth and tradition, the olive oil industry is a relevant one, especially in the Mediterranean countries where 97% of the world’s olive production is harvested [1].
Olive oil is produced from olives either by means of conventional systems using hydraulic presses or by resorting to modern horizontal axis centrifuges. Both systems produce olive pomace [2]. Olive pomace is the solid residue resulting from the olive oil production process [3], it consists of olive skin, pulp and pits [4,5], containing approximately 5-8% of residual oil [2] that can be economically recovered in the pomace oil industry through extraction with n-hexane [3].
Nowadays, one of the most problematic olive oil waste products is pomace (also known as cake) generates a great environmental impact due to the production of high polluting residues [6]. Several studies have stated the negative effects of these forms of waste on soil’s microbial populations [1], aquatic ecosystems [7] and even on the air [8], and constitutes one of the most polluting agricultural by-products in the Mediterranean region [3].
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Actually, it is used for animal feedenergy recovery, soil amendment or the extraction of valuable polyphe its low cost make it an important raw material for biodiesel production and is used for soap [2].
Olive pomace oil is the oil obtained by treating olive pomace with solvents or other physical treatments to obtain crude olive pomace oil, which is not suitable to be used as edible oil.
pomace oil is obtained, whose free acidity, expressed characteristics has to correspond to those defined in E
The oil extraction is obtained by acting on the pomace with solvents the method of extracting the most effective
Olive mill solid residue samples
The raw material used in this work was t pressing system and three-phase cen olive mills in N’gaous aera (Batna-Algeria)
Figure 1: Area sampling «
The olive residue was collected just after the pressing
rustic variety represents more than 40% of the national orchard. It is chara considered as being better quality oil.
reaching constant weight [13].
Reactants
The chemical reactants were used as hydrochloric acid were purchased from obtained by “Fluka-Chemika”.
Cyclohexane, Wijs reagent, thiosulfate sodium “Fisher-Scientific”, “Redel-de-Haën”
“Sigma” and “Merk-Darmstadt”.
Extraction modes
Physical process mode
The pomace oil is obtained with
hydraulic press designs has been thoroughly studied and described in the literature
Department. Institute of Veterinary and Agronomic Sciences. Hadj Lakhdar University, Batna. Algeria
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Actually, it is used for animal feed [9], a raw material for glycolipids biosynthesis [10]
energy recovery, soil amendment or the extraction of valuable polyphenols [11]. Pomace oil is a non
its low cost make it an important raw material for biodiesel production [12], also, it is considered an inferior grade
Olive pomace oil is the oil obtained by treating olive pomace with solvents or other physical treatments to obtain crude olive pomace oil, which is not suitable to be used as edible oil. After this crude oil refining, a refined olive pomace oil is obtained, whose free acidity, expressed as oleic acid, has to be not more than 0,
characteristics has to correspond to those defined in European Regulations [5].
extraction is obtained by acting on the pomace with solvents. From where the problem of this work most effective pomace olive oil in terms of quantity and quality?
MATERIALS AND METHODS
The raw material used in this work was two different samples of solid residues, each from phase centrifugation separation process for obtaining olive oil. They
Algeria) (Figure 1).
Figure 1: Area sampling « N’gaous » (Batna town, Algeria)
was collected just after the pressing operation and immediately packaged
rustic variety represents more than 40% of the national orchard. It is characterized by its small fruits (2, considered as being better quality oil. The initial moisture content was determined by drying in a oven
The chemical reactants were used as well as their purity so, petroleum ether, hexane, ethanol, methanol and hydrochloric acid were purchased from “Biochem-Chemopharma”, phenolphthalein and potassium hydroxide were
, Wijs reagent, thiosulfate sodium, chloroform, acetic acid and potassium iodide
Haën”, “Windsor Laboratories Limited-Berkshire”, “Cheminova International”
a vertical manual screw hydraulic press (Figure 2). hydraulic press designs has been thoroughly studied and described in the literature [14]
Department. Institute of Veterinary and Agronomic Sciences. Hadj Lakhdar University, Batna. Algeria
Algeria
Tunisia Morrocco
Batna
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[10] residual oil extraction, Pomace oil is a non-edible oil and , also, it is considered an inferior grade
Olive pomace oil is the oil obtained by treating olive pomace with solvents or other physical treatments to obtain After this crude oil refining, a refined olive acid, has to be not more than 0,3 g per 100 g and other
. From where the problem of this work: What is quality?
residues, each from traditional hydraulic for obtaining olive oil. They were collected from
operation and immediately packaged plastic containers. This cterized by its small fruits (2,5 g) and it is determined by drying in a oven at 70° C until
, petroleum ether, hexane, ethanol, methanol and Chemopharma”, phenolphthalein and potassium hydroxide were
and potassium iodide were purchased from “Cheminova International”,
). The construction of this [14], at our Food Technology Department. Institute of Veterinary and Agronomic Sciences. Hadj Lakhdar University, Batna. Algeria.
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Figure 2: Manual screw press for oilseeds MC 2000 AUF (Scale: 1:7) (a) Lever, (b) Spindle, (c) Framework, (d) Supply tank, (e) Oil collecting tray [14]
To press, the olive pomaces are left sodden in order to facilitate their pressing. This residue was put in a small cotton sack, introduced in the extraction component in the manual screw press (AC AUF 2000) and pressing was carried out as described by [14]. The characteristics of this press have been reported in a previous work [14]. The oil obtained was separated by decantation, and then the yield is calculated.
Chemical process mode
The oil extraction is carried out by “Soxhlet” method for the determination of fat in dried solid foods [15] with a “Gerhardt Soxtherm 2000” device. It is done by repeat extraction with several non-polar organic solvents on dry olive residue.
As already stated, initial water content of three-phase mill pomace are lower than those of traditional hydraulic press mill residue 22,26±0,39% and 44,98±0,36% respectively (Figure 4).
The olive oil pomace (20 g) was defatted with petroleum ether, hexane and mixture of chloroform/ methanol with the following proportion respectively: 100-0, 75-25, 50-50, 25-75, 0-100%.
Physicochemical characterization of extracted oils
Physical parameters measurement
Relative density was measured according to Kohl Method [16]. It is the ratio of the oil’s mass to the water’s mass of a same volume under specified conditions of pressure and temperature.
Refractive index was determinate according to NFT 60-212 [17]. Measurements are done using a suitable refractometer at a constant temperature of 20°C.
Chemical indices measurement
Determination of the acid value
For the determination of the total free fatty acid content in the oil, the titrimetric method described in Method NFT 60-204 [18] was applied. The free fatty-acids were neutralized with potassium hydroxide (0,2 mol/l).
Determination of the saponification value
The determination was estimated according the NFT 60-206 [19]. The oil pomace was poured into the bottle and heated in a boiling water-bath under reflux with an ethanolic solution of hydroxide potassium. The excess of hydroxide potassium is titrated with hydrochloric acid (0,2 mol/l).
Determination of the iodine value
It is measured according the AOCS Cd Id-92 [20], by adding to the oil residue an excess of iodine monochlorure solution (or Wijs reagent) and a mixture (acetic acid and cyclohexane). After 15 minutes of the reaction time given in dark, the iodine released in a mixture of potassium iodide and water is titrated by a thiosulfate sodium solution.
Determination of the peroxide value
The peroxide value was determinate according the NFT 60-220 [21]. Treatment of the oil in solution with acetic acid and chloroform by a potassium iodide solution. Then, titration of the released iodine with a thiosulfate sodium (0,01 N).
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Determination of unsaponifiables matter
The term "unsaponifiable matter" refers to those substances present in oils that are not saponified by alkali hydroxides and are extractable into ether. They are estimated by the NFT 60-205 [22]. After complete oil saponification, they were extracted using a solvent, and then evaporated under vacuum until obtaining a dry residue.
Data processing
The data obtained after deferent analyses are treated statistically by analysis of variances and multiple comparison of Duncan test. Software SPSS Statistics 20 is used.
RESULTS AND DISCUSSION
Oil yield
The result showed that the oil content of the two residue samples are understood 4,36±0,45 and 15,91±0,13% for all extraction methods (Figure 4). These values are conformed to those published by the European Comission [23] (8-12%), (Table 1).
These results show that methanol, a polar solvent extracts more oil than, ether, hexane and chloroform. The oil yield increases with the addition of methanol in chloroform. This yield is better with the mixture “Chloroform/Methanol 25-75%” for both samples: 15,91±0,13% for the three-phase mill pomace and 12,59±0,16% for the traditional hydraulic press one.
It was found that the most effective process (Method) is the chemical one.
Table 1: Humidity and extraction rate of the pomace olive
Residue origin Humidity Extraction methods Extraction rate
Three-phase mill 22,76±0,26 Physical 05,21 ± 0,33 Chemical •Hexane •Petroleum ether •Mixture« Chloroform/Methanol » 100/0 75/25 50/50 25/75 0/100 12,05 ± 0,11 08,31 ± 0,35 09,25 ± 0,22 12,60 ± 0,66 13,97 ± 0,18 15,91 ± 0,13 13,35 ± 0,29
Traditional hydraulic press mill 44,64±0,40
Physical 04,36 ± 0,45 Chemical •Hexane •Petroleum ether •Mixture « Chloroform/Methanol » 100/0 75/25 50/50 25/75 0/100 09,90 ± 0,20 05,94 ± 0,17 07,52 ± 0,43 09,40 ± 0,15 10,39 ± 0,19 12,59 ± 0,16 11,47 ± 0,17
Physicochemical characteristics of extracted oils
This physicochemical characterization was carried for oils extracted by hydraulic press (Physical method) (Figure 2) and the mixture “Chloroform/Methanol 25-75%” (Chemical method). This characterization is presented in Table 2.
Table 2: Several oils extracted
Oils Signification
E.O.1 Oil extracted by pressing the pomace coming from the three-phase mill
E.O.2 Oil extracted by pressing the pomace coming from the traditional hydraulic press mill
E.O.3 Oil extracted by the mixture “Chloroform/Methanol 25-75%”, the pomace coming from the three-phase
E.O.4 Oil extracted by the mixture “Chloroform/Methanol 25-75%”, the pomace coming from the traditional hydraulic press mill
Physical parameters: Relative density and refractive index
The results of relative density and refractive index of the four oils studied were presented in Table 3. In the analyzed oils, the refractive indices at 20◦C were 1,4680 to 1,4655 (p<0,05). While referring to the vegetable oils classification [24] , we note that this index fell in the range of (1,468-1,472) oils rich in oleic acid, major fatty acid in olive oil, which ranges between 63,5 and 77,5% (IOC, 2001) [25].
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These values are consistent with those found for conventional oils such as cotton (1,458–1,466) and maize (1,465– 1,468) [26].
They revealed a significant difference (p<0,05) at the interval [0,910-0,916] for the relative density and at the interval [1,4680-1,4707] for refractive index of the olive-residue oil [27].
The values of the relative density of the extracted oils by press (Mechanical method) for (E.O.1 and E.O.2) both of them presented the higher value compared to the two other oils (E.O.3) and E.O.4) extracted by chemical process (p<0,05) (Figure 5).
Chemical parameters
Table 3 and figure 6 present the evaluated chemical characteristics in oils extracted from solid waste olive.
Table 3: Physicochemical characterization of extracted olive pomace oils
Origin Extraction method Extracted oil Physicochemical characterisation
RD RI AV SV IV PV UR Three-phase mill Physical E.O.1 0,914a ± 0,00 1,470c ± 0,00 6,76d ± 0,02 185,72c ± 0,15 76,91b ± 0,12 6,5c ± 0,16 1,00a ± 0,02 Chemical E.O.2 0,910b ± 0,00 1,469b,c ± 0,00 6,86c ± 0,05 186,29c ± 0,77 77,86c ± 0,08 7,12d ± 0,13 1,07a ± 0,06 Traditional hydraulic press mill
Significance level Physical E.O.3 0,914a ± 0,001 1,467a ± 0,00 6,31a ± 0,02 180,58a ± 0,16 78,43d ± 0,12 5,99ab ± 0,12 1,06a ± 0,03 Chemical E.O.4 0,913b ± 0,00 1,469b ± 0,00 6,50b ± 0,00 184,08b ± 0,56 76,29a ± 0,14 5,25a ± 0,09 1,14b ± 0,01 0,001 0,001 0,000 0,000 0,000 0,000 0,13 RD : Relative Density at 20°C, RI : refractive Index, AV: Acid Value, SV: Saponification Value, PV: Peroxide Value, IV: Iodine Value, UR:
Unsaponifiables Matter
Acid value is an important index of physicochemical properties, being indicative of age, quality, edibility and suitability of oils [28]. The oils (E.O.1) and (E.O.2) content showed free fatty acids significantly higher than that of the oils (E.O.3) and (E.O.4) (p<0.05).
The determined acid value was less than 6,51 mg KOH/g, for (E.O.3) and (E.O.4) oils, and it is in accordance with the Codex Alimentarius Committee [26], which allows a maximum of 6,6 mg KOH/g to crude vegetable oils, although, (E.O.1) and (E.O.2) oils.
Their high acid value is indicative of oil becoming rancid due to the degradation of triacylglycerols caused mainly by enzymes, and catalyzed by physical agents such as light energy and/or heat, or injuries in the fruits from which the oil was extracted [29].
Saponification value (mg KOH/g) gives an idea of the average molecular weight or the chain length of all the acids present. Higher the molecular weight, the lower the saponification value and being inversely related [28]. The saponification value of the four oils was in accordance with the Codex Alimentarius Committee [26], which defines an interval [182-193] for refined olive-pomace oil. This high saponification value is suggestive of the presence of high molecular weight fatty acids in it.
The iodine value is an empirical test that indicates the unsaturation degree of oil [29]. The variation of this value due to the samples origin and the extraction mode, is highly significant (p<0,01). The four extracted oils present different unsaturation values are below than 79 g I2/100g oil.
The peroxide value (mEq/kg) of oil is used as a measurement of the extent of rancidity reactions. Air, or specially oxygen in the air, can react with the oil and form various peroxide components which eventually affect odor, flavor and quality. Lower the peroxide value, the fresher the oil would be [29].
The International Olive Council [25]determines for refined olive-pomace oil a maximum peroxide value of 10 meq O2/kg. The all analyzed oils presented peroxide value below 10 meq O2/kg (5,25 to 7,12 meq O2/kg). So, all oils didn’t expose to oxidative process either during the preparation of the raw material, extraction or storage.
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P.E.M.: Physical extraction method using hydraulic press C.E.M.(H): Chemical extraction method using hexane
C.E.M.(P.E.): Chemical extraction method using petroleum ether
C.E.M.(Ch.-Met.100/0): Chemical extraction method using the mixture “Chloroform C.E.M.(Ch.-Met.75/25): Chemical extraction met
C.E.M.(Ch.-Met.50/50): Chemical extraction method using the mixture “Chloroform C.E.M.(Ch.-Met.25/75): Chemical extraction method using the mixture “Chloroform C.E.M.(Ch.-Met.0/100): Chemical extraction method using the mixture “Chloroform
The unsaponifiable matter corresponds to compounds present in oils which after saponification with alkali are insoluble in aqueous solution. These compounds may include impurities, such as mineral oil or substances naturally present in oils as sterols, tocopherols and carotenoids
0 2 4 6 8 10 12 14 16 18 E x tr a ct io n r a te ( % ) 0.908 0.909 0.91 0.911 0.912 0.913 0.914 0.915 E.O.1 R el a ti v e d en si ty
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Figure 3: Humidity of olive residue
Figure 4: Oil yield (%) of olive residue
Physical extraction method using hydraulic press Chemical extraction method using hexane
Chemical extraction method using petroleum ether
Chemical extraction method using the mixture “Chloroform-Methanol” with 100/0 Chemical extraction method using the mixture “Chloroform-Methanol” with 75/25 Chemical extraction method using the mixture “Chloroform-Methanol” with 50/50 Chemical extraction method using the mixture “Chloroform-Methanol” with Chemical extraction method using the mixture “Chloroform-Methanol” with 0/100
Figure 5: Physical parameters of extracted oils (a) Relative density at 20°C, (b) Refractive index
The unsaponifiable matter corresponds to compounds present in oils which after saponification with alkali are insoluble in aqueous solution. These compounds may include impurities, such as mineral oil or
in oils as sterols, tocopherols and carotenoids [29]. 0 5 10 15 20 25 30 35 40 45 50 Three-phase Mill's Residue Taditionnal system press Mill's Residue H u m id it y ( % )
Modern Mill's Residue Taditionnal Mill's Residue
E.O.2 E.O.3 E.O.4
1.465 1.4655 1.466 1.4665 1.467 1.4675 1.468 1.4685 1.469 1.4695 1.47 1.4705 E.O.1 E.O.2 R ef ra ct iv e in d ex (a)
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. Methanol” with 100/0 Methanol” with 75/25 Methanol” with 50/50 Methanol” with 25/75 Methanol” with 0/100 .The unsaponifiable matter corresponds to compounds present in oils which after saponification with alkali are insoluble in aqueous solution. These compounds may include impurities, such as mineral oil or
Modern Mill's Residue Taditionnal Mill's Residue
E.O.3 E.O.4 (b)
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The minor compounds composition do not reveal a significant difference (p>0,05). All oils present a content less than 2,5% in according with the International Olive Council(a) Acid and Saponification value, (b) Iodine Value, (c) Peroxide
From the results of the present study, it may be
parameters of the extracted essential oils could be attributed to the Compared the two oil extraction methods from solid
2000”) and chemical one (using petroleum ether, hexane and m provided high extraction yield.
In fact, the highest quantity is obtained
0,16 for the three phase centrifugation and hydraulic system press
04,36 ± 0,45 of the three phase centrifugation and hydraulic system press residues extracted by press.
Despite the performance development amount of oil, which can go up to 15,91 ± Physicochemical parameters analysis
between these results, which reveals the heterogeneity of the two extraction 20°C, refractive index, acid value, saponification
oils.
Also, this analysis indicates that the only parameter that showed no significant unsaponifiable matter for all the extracted oils.
because it would allow positive impact of economic benefit to local populations.
[1] M.H. Ahmad-Qasem, E. Barrajon Engineering, 2013, 119, 516–524. 0 20 40 60 80 100 120 140 160 180 200 E.O.1E.O.2E.O.3 (m g K O H /g ) 0 1 2 3 4 5 6 7 8 E.O.1 E.O.2 P er o x id e v a lu e (m eq O 2 /K g )
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The minor compounds composition do not reveal a significant difference (p>0,05). All oils present a content less than 2,5% in according with the International Olive Council [25].
Figure 6: Chemical parameters of extracted oils
(a) Acid and Saponification value, (b) Iodine Value, (c) Peroxide Value, (d) Unsaponifiables Matter
CONCLUSION
From the results of the present study, it may be concluded that the variations in % oil yield and physicochemical parameters of the extracted essential oils could be attributed to the extraction methods.
Compared the two oil extraction methods from solid-waste olive residue; physical mode (Hydraulic press
) and chemical one (using petroleum ether, hexane and mixture "Chloroform / methanol"), the chemical way is obtained by the mixture "Chloroform / Methanol 25-75%" is 15,91 ± 0,
16 for the three phase centrifugation and hydraulic system press residues respectively, against 05,21 ± 0,33 and three phase centrifugation and hydraulic system press residues extracted by
performance development exhaust systems for olive oil extraction, pomace still contains t of oil, which can go up to 15,91 ± 0,13, which makes their valorization necessary and very interesting.
analysis of olive residue oils extracted reveals a significant difference (p <0, between these results, which reveals the heterogeneity of the two extraction methods. Par
, saponification value, peroxide value, iodine value) influence the quality of these
indicates that the only parameter that showed no significant difference
for all the extracted oils. The effort of valorization of this vegetable oil must be continued, because it would allow positive impact of economic benefit to local populations.
REFERENCES
. Barrajon-Catalan, V. Micol, A.J. Cárcel, J.V. Garcia-E.O.3E.O.4 Acid value (mg KOH/g) Saponificatio n value (mg KOH/g) 74.5 75 75.5 76 76.5 77 77.5 78 78.5 79
E.O.1 E.O.2 E.O.3
Io d in e V a lu e (I2 /1 0 0 g )
E.O.2 E.O.3 E.O.4
0.85 0.9 0.95 1 1.05 1.1 1.15 1.2
E.O.1 E.O.2 E.O.3
U n sa p o n if ia b le s m a tt er ( % ) (a) (c)
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The minor compounds composition do not reveal a significant difference (p>0,05). All oils present a content less
.
.
Value, (d) Unsaponifiables Matter
concluded that the variations in % oil yield and physicochemical
waste olive residue; physical mode (Hydraulic press “AC AUF ixture "Chloroform / methanol"), the chemical way 75%" is 15,91 ± 0,13 and 12,59 ± espectively, against 05,21 ± 0,33 and three phase centrifugation and hydraulic system press residues extracted by “AC AUF 2000”
pomace still contains a significant tion necessary and very interesting.
a significant difference (p <0,05) methods. Parameters (relative density at , iodine value) influence the quality of these
difference (p <0,05) is the The effort of valorization of this vegetable oil must be continued,
-Perez. Journal of Food E.O.3 E.O.4
E.O.3 E.O.4 (b)
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Antioxidant and anticholinesterase activities of Algerian pomaceolive oil
Radhia Ferhat
*1, Maria Ferhat
2, Mehmet Ӧztürk
3, Mehmet Emin Duru
3, Yunus Çetintaş
3,
Gulsen Tel Çayan
3and Salah Laroui
11
Biotechnology of Bioactive Molecules and Cellular Physiopathology Laboratory (LBMBPC),
Batna -1- University, Algeria
2
Laboratory of Therapeutic Substances (LOST), Department of Chemistry, Constantine -1-
University, Algeria
3
Department of Chemistry, faculty of Sciences and Arts, Mugla University. 48121 Mugla, Turkey
_____________________________________________________________________________________________ ABSTRACTIn this study, pomace olives coming from different mills (Press process, continuous process two-phases and three-phases) were used for extraction of oils. The highest oil yield (12.92%) was obtained with pomace olives coming from press process. Total contents of phenolics (13.47 - 21.25 mg GAE/100 g oil) and flavonoids (5.90 - 12.52mg QE/100 g oil)were determined spectrophotometrically. The pomace olive oil “POO3” (Pomace olive coming from 3-phases system) presented the highest phenolic, flavonoid contents and showed the highest DPPH, ABTS scavenging, metal chelating activity. In vitro anticholinesterase activity, the olive pomace oils showed moderate inhibition against AChE and BChE which are the key enzymes taking place in pathogenesis of Alzheimer’s disease. These results showed that the tested oils can be considered as sources of natural antioxidant, as well as moderate anticholinesterase agents.
Keywords: Antioxidant, anticholinesterase, flavonoid, phenolic content, oil,pomace olive.
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INTRODUCTION
Olive (Olea europaea L.) cultivation and olive oil production have been with humankind since ancient times. The olive tree played an integral part in the life of Mediterranean basin’s people, since the third millennium B.C., and olive oil has always been central to the economy of this region[1]. Over the centuries, the benefits of olive oil have been documented and the consumption of olive oil has increased throughout the world. In recent years, the increasing popularity of olive oil has been mainly attributed to its high content of oleic acid, which may affect the plasma lipid/lipoprotein profiles and its richness in phenolic compounds acting as natural antioxidants, which may contribute to the prevention of human disease[2].
The olive oil industry is one of the agro-industrial activities that produce a significant amount of by-products. As regards the olive pomace, today two kinds of process are mainly used to separate oil from olive pastes: the three-phase centrifugation system, which produces a relatively dry solid waste named three-three-phase pomace and a large volume of olive mill waste waters, and the two-phase system in which the extraction water injection is carried out only in the final vertical centrifugation step, reducing by one-third on average the volume of liquid effluent[3].
Pomace olive can reach up to 30% of olive oil manufacturing, depending on the milling process which, after oil extraction, is generally distributed by means of controlled spreading on agricultural soil. However, a large quantity of olive mill solid residue remains without actual application because only small amounts are used as natural
Radhia Ferhat et al
J. Nat. Prod. Plant Resour., 2016, 6 (4):8-14
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fertilizers, combustible biomass and additives in animal feeding. Many researchers have also studied the use of olive pomace in direct combustion and in the production of chemical compounds, as animal feed or soil conditioner, and activated carbon[4].This solid waste is of heterogeneous nature and can be found along with many chemical compounds, such as alkaline (potassium) and alkaline-earth (calcium and magnesium metals), sugars and polyphenols, which come from the vegetation water [5]. Since, only 2% of the phenolic compounds are transferred to the oil and as much as 98% retained in the cake, olive pomace has been considered to be an interesting source of phenolic compounds [6].
Until now, only a few papers in the literature have focused on the evaluation of the phenolic content of solid olive oil residues from different milling processes. In the past decade, several researchers have studied the replacement of Cocoa Butter with refined pomace olive oil to a certain level may reduce the costs of confectionary manufacture[7], the presence of contaminants (pesticides) in the pomace and the detoxification of this residue by the use of microorganisms or the reuse of olive pomace as metal ion adsorbent[4].
Oxidation processes are considered as major contributors to the induction and/or progress of many diseases such as cancer, Alzheimer’s, Parkinson’s, and heart diseases. Antioxidants can interfere with oxidative processes by reacting with free radicals, chelating catalytic metals, and also by acting as O2 scavengers. Among the synthetic antioxidants, the most frequently used to preserve food are butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). The use of these antioxidants is restricted due to their carcinogenicity. Thus, research efforts to identify alternative, natural, and probably safer sources of food antioxidants are an important issue. In this case, many plants can play an important role in adsorbing and neutralizing free radicals due to their high content of antioxidants such as polyphenols[8].
The objectives of this study were: (i) to determine total phenolics and flavonoids of pomace olive oils coming from different system mills, using spectrophotometric methods, (ii) to determine their antioxidant activity on DPPH free radical, ABTS cation radical decolorization and ferrous ions chelating, (iii) to determine the anti- Alzheimer(Anticholinesterase)activity.
MATERIALS AND METHODS
Plant material
The raw material used in this work was pomace olive from a different process for extraction of olive oil (Press process, continuous process two-phases and three-phases), provided by an oil factories located in Batna (Estern Algeria). The pomace was collected just after the pressing operation. The initial moisture content was determined by drying in a vacuum chamber at 70°C until reaching constant weight[9].
Spectral measurements and chemicals
The antioxidant and anticholinesterase activity measurements were carried out on a 96-well microplate reader, SpectraMax 340PC384, Molecular Devices (USA); at the Department of Chemistry, Mugla University. The measurements and calculations were evaluated by Softmax PRO v5.2 software.
Potassium persulfate, n-hexane, ferrous chloride, ferric chloride, copper (II) chloride and ethylenediaminetetraacetic acid (EDTA) were obtained from E. Merck (Darms-tadt, Germany), quercetin, Folin–Ciocalteu’s reagent (FCR), 3-(2-pyridyl)-5,6-di(2-furyl)-1,2,4-tri-azine-5’,5’’-disulfonic acid disodium salt (Ferene), neocuproine and ammonium acetate butylated hydroxytoluene (BHT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), Electric eel acetylcholinesterase (AChE, Type-VI-S, EC 3.1.1.7, 425.84 U/mg), horse serum butyrylcholinesterase (BChE, EC 3.1.1.8, 11.4 U/mg), 5,50-dithiobis (2-nitro-benzoic) acid (DTNB), acetylthiocholine iodide and butyrylthiocholine chloride, galantamine were obtained from Sigma Chemical Co. (Sigma–Aldrich GmbH, Stern-heim, Germany). 2.20-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was obtained from Fluka Chemie (Sternheim, Germany). Hexane was purchased from “Biochem-Chemopharma”. All other chemicals and solvents were in analytical grade.
Extraction oil
The oil extraction is carried out by “Soxhlet” method for the determination of fat in dried solid foods[10]with a slight modification. 20 g of dry pomace olive was put into cellulose extraction thimbles which covered with cotton and then transferred into a Soxhlet apparatus “Gerhardt Soxtherm 2000”. 150 ml of hexane was added to each flask, which was connected to the extractor. Each extraction was performed in triplicate during 3 hours. The temperature of extraction was 180°C. After extraction was completed, the excess solvent was eliminated by using drying procedure at 40ºC until reaching constant weight.
Radhia Ferhat et al
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Preparation of methanol extract
The liquid/liquid extraction was performed according to the procedure described by Ollivier et al. [11]. 1 g of pomace olive oil was weighed into a centrifuge tube, to which 1 ml of methanol/water (80/20, v/v) was added. The mixture was stirred for 10 min in a vortex apparatus, and the tube was centrifuged at 3800 rpm for 15 min. The methanol layer was then separated and the extraction repeated twice. The methanolic extracts were combined to be used for colorimetric determination of total phenols and flavonoids.
Determination of total phenolic content (TPC)
Total phenolic constituent of the methanol extracts was determined by employing the methods given in the literature [11] involving Folin–Ciocalteu reagent and gallic acid as standard. 0.5 ml of methanolic extract solution was added to a volumetric flask. 5 ml distilled water and 1 ml Folin–Ciocalteu reagent was added and flask was shaken vigorously. After 4 min, a 0.8 ml of Na2CO3 (7.5%) solution was added and the mixture was allowed to stand for 2 h by intermittent shaking. Absorbance was measured at 640 nm. The concentrations of phenolic compounds were determined using the calibration curve of gallic acid standard.
Determination of total flavonoids content (TFC)
Total flavonoid content was determined using the method as adapted by Bahorunet al.[12]. Briefly, 1ml of 2% aluminum trichloride (AlCl3) in methanol was mixed with the same volume of the methanolic extracts. Absorption readings at 430 nm were taken after 10 min against a blank sample consisting of a 1 ml extract solution with 1 ml methanol without AlCl3. The total flavonoid content was determined using the calibration curve of quercetin standard.
Determination of antioxidant activity
DPPH free radical-scavenging assay
The free radical scavenging activity was determined spectrophotometrically by the DPPHassay [13]with slight modification. In its radical form, DPPHabsorbs at 517 nm, but upon reduction by an antioxidant or a radical species, its absorption decreases. Briefly, 0.1 mM solution of DPPH in methanol was prepared and 4 ml of this solution was added to 1ml of sample solutions in methanol at different concentrations. Thirty minutes later, the absorbance was measured at 517 nm by using a 96-well microplate reader. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The capability to scavenge the DPPHradical was calculated using the following equation [14].
Where: AControl is the initial concentration of the DPPH and ASample is the absorbance of the remaining concentration of DPPH in the presence of the extract and positive controls. The extract concentration providing 50% radical scavenging activity (EC 50) was calculated from the graph of DPPH radical scavenging effect percentage against extract concentration. BHA, α-tocopherol, (+)– catechin and quercetin were used as antioxidant standards for comparison of the activity.
ABTS cation radical decolorization assay
The spectrophotometric analysis of ABTS scavenging activity was determined according to the method of Re et al. [15], with slight modifications. The ABTS was produced by the reaction between 7 mM ABTS in water and 2.45 mM potassium persulfate, stored in the dark at room temperature for 12 h. Oxidation of ABTS commenced immediately, but the absorbance was not maximal and stable until more than 6 h had elapsed. The radical cation was stable in this form for more than 2 days in storage in the dark at room temperature. Before usage, the ABTS solution was diluted to get an absorbance of 0.708 ± 0.025 at 734 nm with ethanol. Then, 160 µl of ABTS solution was added to 40 µl of sample solution in ethanol at different concentrations. After 10 min the absorbance was measured at 734 nm by using a 96-well microplate reader. The percentage inhibitions were calculated for each concentration relative to a blank absorbance (ethanol). The scavenging capability of ABTS was calculated using the following equation:
Where: AControl is the initial concentration of the ABTS and ASample is the absorbance of the remaining concentration of ABTS in the presence of sample. The extract concentration providing 50% radical scavenging activity (EC50) was
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calculated from the graph of ABTS radical scavenging effect percentage against extract concentration. BHA, α-tocopherol, (+)– catechin and quercetin were used as antioxidant standards for comparison of the activity.
Ferrous ions chelating activity
The chelating activity of the extracts on Fe2+ was measured by using Ferrin [16] with slight modifications. The extracts solution (80 µl dissolved in ethanol in different concentrations) was added to 40 µl 0.2 mM FeCl2. The reaction was initiated by the addition of 80 µl 0.5 mM ferene. The mixture was shaken vigorously and left at room temperature for 10 min. After the mixture reached equilibrium, the absorbance was measured at 562 nm. The metal chelation activity was calculated using the following equation:
Where: AControl is the absorbance of control devoid of sample and ASample is the absorbance of sample in the presence of the chelator. The extract concentration providing 50% metal chelating activity (EC50) was calculated from the graph of Fe2+ chelating effects percentage against extract concentration. EDTA was used as antioxidant standards for comparison of the activity.
Determination of anticholinesterase activity
Acetylcholinesterase and butyrylcholinesterase inhibitory activities were measured by slightly modifying the spectrophotometric method of Ellman et al.[17]. AChE from electric eel and BChE from horse serum were used, while acetylthiocholine iodide and butyrylthiocholine chloride were employed as substrates of the reaction. DTNB (5.5’-Dithio-bis (2-nitrobenzoic) acid was used for the measurement of the cholinesterase activity. Briefly, 130 µl of 100 mM sodium phosphate buffer (pH 8.0), 10 µl of sample solution dissolved in ethanol at different concentrations and 20 µl AChE (5.32×10-3 U) or BChE (6.85×10-3 U) solution were mixed and incubated for 15 min at 25°C, and then 10 µl of DTNB (0.5 mM) was added. The reaction was then initiated by the addition of 20 µl of acetylthiocholine iodide (0.71 mM) or 20 µl of butyrylthiocholine chloride (0.2 mM). The hydrolysis of these substrates was monitored spectrophotometrically by the formation of yellow 5-thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine iodide or butyrylthiocholine chloride, respectively, at a wavelength of 412 nm utilizing a 96-well micro-plate reader. Percentage of inhibition of AChE or BChE enzymes was determined by comparison of reaction rates of samples relative to blank sample (Ethanol in phosphate buffer pH 8) using the formula (E-S)/ E×100, where E is the activity of enzyme without test sample, and S is the activity of enzyme with test sample. The experiments were carried out in triplicate. Galantamine was used as a reference compound.
Statistical analysis
The experimental results were performed in triplicate. The data were recorded as mean ± standard deviation and analyzed by SPSS statistical software (Version 20.0. Armonk, NY: IBM Corp.). The data obtained are treated statistically by analysis of variances, multiple comparisons of Duncan test and p<0.05 was regarded as significant.
RESULTS AND DISCUSSION
Ash (Humidity) and oil yield
The olive pomace is produced in miller (oil mills) processing of olives for receiving the olive oil. Removal of olive oil, the vast majority of mills, made by centrifugation in a centrifugal separators two or three phases. Thus, we determined humidity and oil yield of olive pomace coming from three different process trituration of olive oil. According to the results shown in Table 1, humidity ranged from 34.92 ± 0.86 to 61.72 ± 0.98 %. Oil yield ranged from 10.22 ± 0.33 to 12.97 ± 0.70 g oil/100 g pomace olive.
Pomace produced from 2-phases system were characterised by higher humidity (61.96 ± 0.70%) comparing to that of 3-phases (48.60 ± 0.82%) and hydraulic pressing system (34.98 ± 0.14%). This was explained that two-phases system (Named ecological system) did not produce wastewater during oil extraction and generated margines with pomace coming out of decanter. However, it creates a high humidity pomace, which is difficult to handle [18].
In contrary, in the 3-phases system water with specific heat is added to decanter during pressing. Pomace humidity is two times lower than the two-phases pomace, because wastewater and pomace comes out of the decanter separately 18].
