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. 2013 Aug 28;52(3):1754–1759. doi: 10.1007/s13197-013-1153-1

Effect of amurca on olive oil quality during storage

Sana Janakat 1,✉, Anas Al-Nabulsi 1, Fwzieh Hammad 1, Richard Holley 2
PMCID: PMC4348276  PMID: 25745252

Abstract

Total phenolic compounds (TPC), antioxidant activity (AA), lipid peroxidation inhibition (percent) (LPOIP), free fatty acid and peroxide values were measured in olive oil samples over the period of 12 months in comparison with oil samples extracted from amurca (olive oil lees) and olive oil samples taken from the bottom of the canister (near amurca) after 12 months of storage. Olive oil samples taken over the period of 12 months possessed decreasing amounts of TPC, AA and LPOIP, which led to increased peroxide and free fatty acid values. In contrast, oil extracted from amurca and olive oil samples taken from the bottom of the container after 12 months of storage possessed significantly higher TPC, AA, LPOIP and consequently lower free fatty acid and peroxide values. These results show that the presence of naturally occurring amurca (sediment) in stored olive oil stabilizes olive oil quality during storage.

Keywords: Amurca, Total phenolic compounds, Lipid peroxidation inhibition percent, Free fatty acid value, Peroxide value, Olive oil quality

Introduction

The term amurca refers to the watery bitter tasting and dark coloured sedimentation which settles at the bottom of olive oil containers over time (Niaounakis and Halvadakis 2006; Smith and Secoy 1975). Olive oil content of amurca varies tremendously (12–460 mg/kg oil) depending on the type of mills used to extract olive oil (Koidis et al. 2008). The Jordanian specification for olive oil does not state a criterion for the maximum amount of amurca that is permitted in olive oil for local use (JISM 2009), and since the Jordanian consumer prefers the bitter taste and the aroma of amurca, olive oil produced for local consumption contains a high amount of amurca.

Although amurca was reported to be a very rich source of antioxidants (Frega et al. 1999; Lozano-Sanchez et al. 2010), internationally it is considered a negative factor affecting olive oil quality, and when present the oil is often discarded (Fregapane et al. 2006). Olive oil quality is affected by many factors such as: storage temperature (Gomez-Alonso et al. 2007), contact with atmospheric oxygen and type of container (Kanavouras and Coutelieris 2006). These factors stimulate lipid peroxidation (LPO) reactions. LPO refers to the oxidative degradation of lipids and formation of lipid free radicals (Gutteridge 1986), that lead to malondialdehyde formation, which is considered an endogenous source of DNA damage and yields off-flavour and other types of quality loss (e.g. changes in colour and taste) during storage which in turn can cause large economic losses (Marnett 2002).

Alba-Mendoza (2001) suggested that elimination of amurca might increase olive oil shelf life and avoid off flavour. However, Fregapane et al. (2006) reported that filtration decreases the rate of hydrolysis of triacyglcerol and consequently prolongs the shelf life of olive oil. In contrast, Tsimidou et al.(2005) demonstrated that water and small particles (amurca) in olive oil led to loss of stability and increased peroxide value.

In an earlier study amurca was shown to possess high total phenolic compounds, antioxidant activity, and anti-lipoperoxidative activity (Janakat and Hammad 2013). The objectives of this work were to determine the impact of amurca on olive oil oxidative stability during storage and to compare the oxidative stability of amurca and oil samples collected from the bottom of containers at the end of storage.

Materials and methods

Chemicals and reagents

Folin-Ciocalteu’s phenol reagent and 2, 4, 6- tripyridyl- s- triazine (TPTZ) were purchased from Sigma (St. Louis, MO, USA), gallic acid, Tris–HCl and thiobarbituric acid (TPA) were purchased from Acros Organics (Fisher Scientific, Pittsburg, PA, USA), methanol was purchased from Medex (Naseby, UK), sodium dodecyl sulfate (SDS) was purchased from Scharlau (Barcelona, Spain), ascorbic acid was purchased from Gesellschaft Deutscher Chemiker, GDCh (Frankfurt, Germany) and ferrous chloride was purchased from AppliChem (Darmstadt, Germany).

Olive oil samples

Olive oil canisters containing 20 L were purchased from one of the olive oil mills in the province of Irbid (Northern Jordan) and which had been pressed in December 2009. Olive oil samples (n = 3) were taken from the top of the canisters (about 3 cm below the surface) at 0 time (immediately after pressing), 1, 3, 5, 7, 9, and 12 months storage. Samples from the bottom of the canisters (about 3 cm above the amurca sediment) were also taken after 12 months of storage. All samples were stored at room temperature in the dark until use.

Amurca samples

Amurca samples were obtained from the same olive oil canisters. Amurca was removed from the olive oil by centrifugation at 4000 rpm (1252 X g) for 30 min and stored at −18 °C until tested.

Oil extraction from amurca

Oil was extracted from amurca by soaking amurca samples in hexane (1:3) overnight, the mixture was centrifuged at 3,000 rpm (723 X g) for 20 min, the supernatant was collected, and hexane was evaporated using a rotary evaporator.

Preparation of olive oil and amurca extracts

Fifty grams of olive oil or amurca samples were diluted in 50 ml of hexane and the mixture was washed three times with 30 ml of methanol/water mixture (60:40). The mixture was shaken for 2 min before allowing the two phases to separate in a separatory funnel. The methanolic extracts were then washed with 50 ml of hexane and finally brought up to 100 ml in a volumetric flask and stored at −18 °C until use (Favati et al. 1995).

Total phenolic compounds

Total phenolic compounds of olive oil and amurca extracts were determined according to the Folin-Ciocalteu procedure adapted from Hajimahmoodi et al. (2008) at 725 nm. Gallic acid was used as the calibration standard and results were expressed as mg gallic acid equivalent (mg GAE/100 g fresh weight).

Antioxidant activity

Antioxidant activity of sample extracts were determined spectrophotometrically using a ferric reducing antioxidant power assay (FRAP) at 593 nm (Benzie and Strain 1999). For construction of the calibration curve, 6 concentrations of vitamin E (4, 6, 8, 10, 15 and 20 mg) were used and results were calculated as mg vitamin E equivalent (mg vitamin E equivalent/100 g fresh weight).

Free fatty acid and peroxide values

Free fatty acid and peroxide values were determined for olive oil samples and oil extracted from Amurca according to the AOAC (1984, 1997).

Determination of lipid peroxidation inhibition percent

Lipid peroxidation was determined by measuring the concentration of malondialdehyde (MDA) in rat liver homogenate using the thiobarbituric acid (TBA) reactive species assay according to methods by Ohkawa et al. (1979) and Lin et al. (1998). LPO inhibition (percent) was calculated according to the following equation (Toda et al. 2000).

LPOinhibitionpercent=control−treatmentcontrol×100

where:

control

absorbance of the MDA/TBA complex in the absence of the extract

treatment

absorbance of the MDA/TBA complex in the presence of the extract

Statistical analysis

The data were statistically analyzed using the statistical package for social sciences (SPSS, version15.0, 2007, Chicago, IL, USA). One way analysis of variance (ANOVA) tests were performed to test the difference between treatments followed by Duncan’s Analysis. Significance was declared at p < 0.05.

Results and discussion

Effect of storage time on total Phenolic compounds

Figure 1 shows the effect of storage time on total phenolic compounds of olive oil samples taken from the top of the container in comparison with total phenolic compounds of amurca and olive oil samples taken from the bottom of the container after 12 months of storage.

Fig. 1.

Fig. 1

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Effect of storage time on total phenolic compounds of olive oil samples taken from the top of the container(about 3 cm below the surface) in comparison with olive oil samples taken from bottom of the container (about 3 cm above amurca sediment) and amurca samples taken after 12 months of storage. Values are expressed as mean (±S.E.M) (n = 3). (Asterisk) indicates that olive oil was taken from bottom of the container (about 3 cm above amurca sediment) after 12 months of storage); (Number sign) indicates 1:10 dilution. P-values were calculated using ANOVA test. Means with different superscripts a,b,c,d differ significantly (p < 0.05)

Total phenolic compounds decreased from 31.6(±0.37) mg GAE/100 g oil in samples taken immediately after pressing (0 time) to 6.7(±0.19) mg GAE/100 g in samples taken after 12 months storage, which is a 78.8 % decrease of phenolic compounds. These results are in agreement with those of Gomez-Alonso et al. (2007) who reported that the reduction of total phenolic compounds ranged from 43 % to 73 % during 21 months of storage. In the present work most of the total phenolic compound loss (62 %) occurred within the first month of storage. By 3 months an additional non-significant loss of 3.2 % occurred, but by 5 months a further significant loss of 10.2 % was observed. Between 5 and 12 months storage an additional non-significant loss of 3.3 % was found.

Olive oil samples taken from the bottom of the container (near the amurca) after 12 months of storage contained 17.1(±0.30) mg GAE/100 g oil, which is a 45.8 % decrease in total phenolic compounds normally found in freshly pressed olive oil. Sedimentation of amurca at the bottom of the container with time (Koidis et al. 2008) is likely why there were higher total phenolic compounds near the bottom of the container. A cloudy appearance of freshly pressed olive oil with higher total phenolic compounds that disappears during storage was reported earlier (Tsimidou et al. 2005). The cloudy appearance may persist for several months before amurca settles at the bottom of the container (Fregapane et al. 2006). The total phenolic compounds of oil samples extracted from amurca after 12 months of storage was 289 mg GAE/100 g of amurca, which was 9.1 times higher than freshly pressed olive oil and 16.9 times higher than that of olive oil taken from the bottom of the container (at 12 months) and 43.1 times higher than the total phenolic compounds of olive oil samples taken from the top of the container after 12 months of storage. This is a clear indication that amurca is a reservoir for phenolic compounds.

Antioxidant activity

Figure 2 depicts the effect of storage time on antioxidant activity of olive oil samples taken from the top of the container in comparison with that of amurca samples and olive oil samples taken from the bottom of the container after 12 months of storage. Antioxidant activity decreased significantly with time from 1.29(±0.01) mg vitamin E equivalent/100 g oil in freshly pressed olive oil samples taken from the top of the container to 0.39(±0.003) mg vitamin E equivalent/100 g oil (p < 0.05) in samples taken from the same container location after 12 months of storage. The antioxidant activity of samples taken from the bottom of the container after 12 months of storage was 1.12(±0.09) mg vitamin E equivalent/100 g), while the antioxidant activity of oil extracted from amurca samples after 12 months of storage was 22.3(±0.16) mg vitamin E equivalent/100 g.

Fig. 2.

Fig. 2

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Effect of storage time on antioxidant activity of olive oil samples taken from the top of the container (about 3 cm below the surface) in comparison with olive oil samples taken from bottom of the container (about 3 cm above amurca sediment) and amurca samples taken after 12 months of storage. Values are expressed as mean (±S.E.M) (n = 3). (Asterisk) indicates that olive oil was taken from bottom of the container (about 3 cm above amurca sediment) after 12 months of storage; (Number sign) indicates 1:10 dilution. P-values were calculated using ANOVA test. Means with different superscripts a,b,c,d differ significantly (p < 0.05)

The correlation between total phenolic compounds and antioxidant activity of olive oil samples was found to be a linear positive relationship (R2 = 0.873) (Fig. 3), which is in agreement with the findings of Hajimahmoodi et al. (2008) and Qusti et al. (2010). Frega et al. (1999) suggested that amurca dispersed in olive oil might have some antioxidant activity.

Fig. 3.

Fig. 3

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Linear correlation between total phenolic compounds measured according to the Folin-Ciocalteu procedure and antioxidant activity measured according to the FRAP assay of olive oil samples

Lipid peroxidation (LPO)

Figure 4 shows the effect of olive oil storage time on LPO inhibition percent. LPO was expressed as malondialdehyde (MDA) formation in rat liver tissue using an ascorbate/FeCl2 model. All extracts (hexane/methanol/water) taken at 0 time to 9 months caused significant LPO inhibition percent in comparison with the control (containing all reagents except the sample extracts), which was correlated with phenolic compounds and antioxidant activity. Amurca extracts caused a 95.7(±2.5) % LPO inhibition ratio, and this could be attributed to the fact that phenolic compounds and antioxidants inhibited MDA formation (Gavino et al. 1981; Janakat and Al-Thnaibat 2008). Moreover, there was a significant decrease in LPO inhibition percent over time, from 53.13(±0.29) % in freshly pressed olive oil to 2.70(±0.33) % in olive oil samples taken from the top of the container after 12 months of storage. This is believed to be due to the fact that amurca takes several months to settle at the bottom of the container (Fregapane et al. 2006). Olive oil samples taken after 12 months of storage (from the bottom of the container) caused 53.7(±1.5) % LPO inhibition, while samples taken from the top container after 12 months of storage caused 2.7(±0.33) % LPO inhibition percent. This could be attributed to the high total phenolic compounds and antioxidant activity of amurca at the bottom of the container (Frega et al. 1999).

Fig. 4.

Fig. 4

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Effect of storage time of olive oil on LPO inhibition percent in comparison with LPO inhibition percent induced by oil extracted from amurca samples after 12 months of storage expressed as malondialdehyde (MDA) concentration at 532 nm. Values are expressed as mean (±S.E.M) (n = 3). (Asterisk) indicates that olive oil was taken from bottom of the container (about 3 cm above amurca sediment) after 12 months of storage; (Number sign) indicates 1:10 dilution. P-values were calculated using ANOVA test. Means with different superscripts a,b,c,d differ significantly (p < 0.05)

Free fatty acid value

Table 1 depicts the effect of storage time on olive oil free fatty acid value in samples taken from the top of the container in comparison with that of olive oil samples taken from the bottom of the container and oil samples extracted from amurca after 12 months of storage. A significant increase in free fatty acid value was observed with time from 1.39(±0.03)% for samples taken at 0 time from the top of the container to 3.99(±0.08)% in samples taken after 12 months storage from the container location (p < 0.05). The increase in free fatty acid value comes as a result of hydrolysis of triglyceride to free fatty acids (Gutiearrez and Fernaandez 2002; Pristouri et al. 2009; Ciafardini, and Zullo 2002). The Jordanian specification for olive oil requires that the free fatty acid value for olive oil produced for local use is ≤ 3.3 % (JISM 2009). Olive oil samples taken from the bottom of the container after 12 months of storage possessed a significantly (p < 0.05) lower free fatty acid value (2.61(±0.01)%) than samples taken from the top of the container at the same time (3.99(±0.08)%). Furthermore, the free fatty acid value in oil extracted from amurca samples after 12 months of storage was lower at 1.62 (±0.03 %) (p < 0.05), but was not significantly different from the free fatty acid value of oil samples taken at 0 time. This was due to the stabilizing role of suspended amurca against oxidative degradation (Frega et al. 1999; Lozano-Sanchez et al. 2010). On the contrary, Fregapane et al. (2006) reported that freshly pressed olive oil which had a cloudy appearance, had a high free fatty acid value in the oil and that after filtration the rate of hydrolysis of the triglycerides decreased.

Table 1.

Effect of storage time on olive oil free fatty acid and peroxide values in comparison with that oil extracted from amurca after 12 months of storage

Storage Time Free fatty acid value [Oleic Acid %] Peroxide Value [meqO2/Kg]
0 h 1.39(±0.03)a 11.69(±0.01)c
1 month 3.58(±0.16)c 12.72(±0.33)c
3 months 3.31 (±0.14)c 12.58(±0.25) c
5 months 3.33(±0.13)c 16.51(±0.61)d
7 months 3.35(±0.15)c 19.09(±0.24)e
9 months 3.66(±0.18)c,d 25.61(±0.20) f
12 months 3.99(±0.08)d 25.61(±0.20)f
12 months* 2.61(±0.01)b 9.27(±0.18)b
Amurca 1.62(±0.03)a 1.78(±0.03)a
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(*) indicates that olive oil was taken from bottom of the container (about 3 cm above amurca sediment) after 12 months of storage. P-values were calculated using ANOVA test. Means with different superscripts a,b,c,d differ significantly (p < 0.05)

Peroxide value

Table 1 also depicts the effect of storage time on peroxide values of olive oil samples taken from the top of the container in comparison with peroxide value of olive oil samples taken from the bottom of the container, and the peroxide value of oil extracted from amurca after 12 months of storage. The peroxide values were lower than 20 meq O2/kg oil (p < 0.05) in all samples except for those taken at 9 and 12 months from the top of container. Peroxide values were stable for 3 months and ranged between 11.69(±0.01) to 12.72 (±0.33) meqO2/Kg, then a significant increase was observed after 5 months of storage onwards. Peroxide values reached 16.51(±0.61) meqO2/Kg at 5 months, 19.09(±0.61) meqO2/Kg at 7 months, and 25.61(±0.20) meqO2/Kg at 9 months which is consistent with many previous studies (Tsimidou et al. 2005; Pristouri et al. 2009; Mendez and Falque 2007).

Olive oil from the bottom of the container (about 3 cm above the amurca sediment) taken after 12 months of storage had a significantly (p < 0.05) lower peroxide value 9.27(±0.18) meqO2/Kg oil than samples taken from the top of the container (about 3 cm below the surface) at the same time (25.6(±0.20) meqO2/Kg oil), which can be explained by the presence of high antioxidant activity at the bottom of the container which inhibits initiation of auto-oxidation of free fatty acids (Kahl and Hildebrandt 1986; Gutteridge 1986). This result is in accordance with the results of Fregapane et al. (2006) who reported that filtration of olive oil caused an increase in the rate of peroxide formation. Moreover, the peroxide value for oil extracted from amurca samples was 1.78(±0.03) meqO2/Kg oil which was significantly (p < 0.05) lower than the peroxide value of oil samples taken at 0 time. The high total phenolic compounds and antioxidant activity of amurca can be responsible for this low peroxide value.

Conclusions

Sedimentation of amurca during olive oil storage causes a significant decrease in total phenolic compounds as well as antioxidant activity and it causes some loss of olive oil anti-lipoperoxidative activity, which yields increased acid and peroxide values leading to loss of olive oil quality during storage. Thus, amurca, which is a natural source of phenolic compounds and antioxidant activity in olive oil should not be removed from olive oil because this might reduce olive oil shelf life.

Acknowledgment

We would like to thank the Deanship of Research at Jordan University of Science and Technology (JUST) for providing financial support for this project.

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