PAPER 166 Ital. J. Food Sci., vol. 27 - 2015 - Keywords: Extra Virgin Olive Oil, Diglycerides,1,2/1,3-DGs ratio, GC, Storage conditions - EXTRA VIRGIN OLIVE OIL STORED IN DIFFERENT CONDITIONS: FOCUS ON DIGLYCERIDES Z. AYYAD1, E. VALLI*2, A. BENDINI1,2, S. ADROVER-OBRADOR3, A. FEMENIA4 and T. GALLINA TOSCHI1,2 1Department of Agricultural and Food Sciences, Alma Mater Studiorum - University of Bologna, Cesena (FC), Italy 2Interdepartmental Centre for Agri-Food Industrial Research, Alma Mater Studiorum - University of Bologna, Cesena (FC), Italy 3Institute of Agricultural and Fishing Research and Training (IRFAP) of the Government of the Balearic Islands, c/ d’Eusebi Estada, 145, 07009, Palma, Spain 4Department of Chemistry, University of the Balearic Islands, Ctra. Valdemossa, km 7.5, 07122, Palma, Spain *Corresponding author: Tel. +390547338121, Fax +390547382348, email: enrico.valli4@unibo.it ABSTRACT The effects of storage conditions of extra virgin olive oil (EVOO) on the isomerization of diglycerides (DGs) have been investigated. Aliquots of EVOO were stored for 14 months under four different conditions: at 20°C in darkness and in light, at 4-6°C in light and at 20°C in light with argon in the headspace. Samples were analysed bimonthly: 12 DGs with C34 and C36 (1,2 and 1,3 isomers) were tentatively identified and quantified by GC-FID. After 14 months, a clear tendency towards a decrease of 1,2-DGs and a significant increase of 1,3-DGs during storage was observed for all samples. 1,2-DGs were always predominant compared to 1,3-DGs and, for both types, C36 DGs were prevalent compared to C34 DGs. Overall, EVOO stored at 4-6°C in light showed the highest preservation of 1,2-DGs. Ital. J. Food Sci., vol. 27 - 2015 167 INTRODUCTION Extra virgin olive oil (EVOO) is fresh olive (Olea europaea L.) juice obtained by mechani- cal and physical processes (Lozano-Sanchez et al., 2012), and it is well known as one of the major components of the diet of Mediterranean countries. EVOOs consist of triglycerides as the main components (about 98%) and other minor components including diglycerides, free fatty acids, squalenes, sterols, phospholipids, phe- nolics and different volatile compounds (BOSK- OU, 1996). Some of these minor components, in addition to a high content of mono-unsat- urated fatty acids, play a major role in keep- ing EVOO more stable against oxidation during storage compared to other vegetable oils (BEND- INI et al., 2009a). Elimination of air in the head space, either by fully filling the EVOO bottles or by its replacement with inert conditioning gas, has been found to add marked improve- ment in terms of oxidation quality, stability, shelf life and slow down the oxidation process of EVOO (URDA- ROMACHO, 2009; GIOVACCHI- NO et al., 2002). Newly produced EVOO contains a low con- centration of diglycerides (DGs) (1-3%), which are formed as intermediate products of the in- complete biosynthesis of triglycerides (SPY- ROS et al., 2004) and partial hydrolysis of tri- glycerides. During storage many changes may occur in DG composition due to isomerisation of 1,2-DGs, the predominant form in fresh EVOO, to 1,3-DGs (SACCHI et al., 1991). The effects of storage temperature and exposure to light during different periods of time on the quality of EVOO have been investigated by different au- thors (VELASCO and DOBARGANES, 2002; MEN- DEZ and FALQUE, 2007), while other studies have assessed the amount of DGs as an indic- ative parameter of the freshness of EVOO. CAT - ALANO et al. (1994) investigated DGs isomeri- sation occurring in EVOO stored in darkness, at room temperature and at 4°C. In particular, the results revealed that the 1,2-DGs remained less than 1.5 % after one year of storage for all samples analysed, while about 10% and 45% of the samples stored at room temperature and at 4°C, respectively, contained less than 0.4% 1,3-DGs. Furthermore, PÉREZ-CAMINO et al. (2001) studied the evolution of the two DG iso- mer classes in oils obtained from olives of dif- ferent qualities stored at different tempera- tures, concluding that triacylglycerol hydroly- sis and DG isomerisation depended not only on the value of free acidity, but also on the storage temperature. In addition, the 1,3/1,2-DG ratio was a useful parameter for assessing the genu- ineness of EVOOs with low free acidity during early storage stages. Another interesting study was carried out by SPYROS et al., (2004), assessing olive oil through investigation of 1,2 and 1,3-DG isomerisation during 18 months of storage at room tempera- ture, at 5°C with light and in darkness. The re- sult of the isomerisation process was mainly de- pendent on the initial quality parameters of the oil, and in particular the free acidity. Another study based on the evaluation of olive oil quali- ty in relation to storage conditions through the analysis of DG isomerisation was carried out by COSSIGNANI et al. (2007) on samples produced from different olive cultivars stored at 15°C and at 30°C in darkness for 12 months. The re- sults showed important differences in the per- centage of each individual DG and in the ratio among classes; in particular, samples analysed at time zero exhibited the highest percentage of 1,2-DGs and the lowest of 1,3-DGs, where- as samples stored at 30°C showed the highest content of 1,3-DGs suggesting that tempera- ture plays an important role in the isomeri- sation process. More recently, a study carried out by CAPONIO et al. (2013) investigated the effects of storage of EVOO in green glass bot- tles in light and darkness for 24 months, pro- viding evidence that the degree of isomerisa- tion was affected by the initial hydrolysis level of the oil and by the storage time, although oth- er storage conditions did not show any effect. Overall, these results suggest that the content of DGs and the ratio between isomers might be considered as possible markers to establish the freshness state of an EVOO alongside with oth- er quality parameters defined by official regu- lations (EU Reg. 61/2011). Therefore, the main aim of this study was to investigate the isomerisation processes related to diacylglycerols, and in particular the amounts of 1,2- and 1,3-DGs and relative C34 and C36 sub-classes as well as the 1,2/1,3-DG ratio in EVOO during storage under different conditions for 14 months. The purpose was to investigate how these compounds were influenced by dif- ferent variables such as temperature, light and headspace gases. MATERIALS AND METHODS Samples EVOO samples used in this study were pro- duced from olives of the Arbequina cultivar (Coop. Sant Bartomeu, Soller, Spain) using an industrial plant working with a three-phase de- canter. Once in the laboratory, the EVOO was poured into 250 mL transparent glass bottles. The headspace in each bottle was about 2 mL. The bottles were hermetically sealed and divid- ed into four batches. The first batch was stored in darkness inside a thermostatic chamber at 20°C (Cond. 1); the second batch was stored at 20°C under diffuse light (600 Lux for 12 h/ day 11 W; 595 lm; 6400°K) simulating the con- ditions of a supermarket shelf (Cond. 2); the 168 Ital. J. Food Sci., vol. 27 - 2015 third batch was stored in a refrigerated cham- ber at 4-6°C with diffuse light (Cond. 3); final- ly, the fourth batch was stored with argon in the headspace of bottles at 20°C with diffuse light (Cond. 4). Samples were analysed in trip- licate after 2, 4, 6, 8, 10, 12 and 14 months of storage after production. Basic chemical analysis Free acidity, peroxide value and UV absorp- tion (K 232 , K 270 ) were determined according to the official methods described in EEC Reg. 2568/91 for all samples at the initial period of storage (2 months) and after the end of storage simulation (14 months). Gas chromatographic (GC) determination of diglycerides The silylated samples were prepared accord- ing to a previous work (SWEELEY et al., 1963) and DGs were determined according to a mod- ified version of the method suggested by SERA- NI et al., (2001) using a GC Carlo Erba MFC500 with a Rtx-65TG (Restek, Bellefonte, PA) fused silica capillary column (30 m length x 0.25 mm i.d. x 0.10 μm f.t.) coated with 35 % dimethyl-65 % diphenylpolysiloxane. The oven temperature was programmed from 250 to 320°C at a rate of 2°C min-1 and then increased to 365°C at a rate of 5°C min-1. The final temperature was main- tained for 21 min. The injector and FID tem- peratures were both set at 360°C. Helium was used as carrier gas at a pressure of 130 kPa. The split ratio was 1:70. Identification of DGs was carried out by comparing peak retention times and GC traces with those of DG standards and chromatograms reported in the literature (SER- ANI et al., 2001; BENDINI et al., 2009b). The re- sults, expressed as mg of each DG per 100 mg of oil, were quantified with respect to dilaurin, added as internal standard (0.5 mL of a solu- tion 2 mg mL-1 of dilaurin dissolved in chloro- form, added to 100 mg of oil). Statistical analysis The software XLSTAT 7.5.2 version (Addinsoft, USA) was used to elaborate the data by analy- sis of variance (ANOVA, Fisher LSD, p < 0.05). RESULTS AND DISCUSSION The free acidity, peroxide values and extinc- tion coefficients (K 232 and K 270 ), shown in Table 1, indicated that at the end of the storage pe- riod all samples were within the accepted lim- its established by EU regulations for the EVOO category (EU Reg. 61/2011). Fig. 1 shows a comparison between the gas chromatography traces of DG fractions of EVOO stored for 2 and 14 months in dark at 20°C. Twelve different DGs were tentatively identified and quantified as 1,2 and 1,3 isomers with 34 or 36 carbon atoms (C34, C36). Only a co-elu- tion was present (peak 11) between 1,3 isomers of the oleic-linoleic and linoleic-linoleic couples. The peaks numbered from 1 to 6 (Fig. 1) were relative to C34 DGs whereas from 7 to 11 be- longed to C36, and palmitic-oleic (PO) and ole- ic-oleic (OO) were the most abundant DGs for the two classes, respectively. Observing the GC traces (Fig. 1), it is also possible to note that the 1,2 isomers eluted before the 1,3 ones for both groups with 34 and 36 carbon atoms. Fig. 2 illustrates the evolution of 1,2/1,3- DG ratios, and Tables 2-5 highlight the trends of 1,2-DGs (C34, C36) and 1,3-DGs (C34, C36) for EVOOs stored under the four different ex- perimental conditions. For the samples kept at 20°C in darkness (Cond. 1), a rapid and signif- icant decrease was observed in the 1,2/1,3-DG ratio for the first 8 months; this ratio continued to decrease slowly until the end of storage period (Fig. 2). A similar trend was also seen for the 1,2- DGs C34 and C36 under the same condition (Ta- ble 2), and the rapid decrease continued for up to 8 months. At the end of storage period, total 1,2-DG remained about 60 % (data not shown) of Table 1 - Results for free acidity (FA, g of oleic acid per 100 g of oil), peroxide values (PV, Meq O 2 Kg -1) and extinction coefficient at 232 and 270 nm (K 232 , K 270 ) at time zero and after 14 months of storage under the four different conditions (Cond. 1 - 4)*. * Cond. 1, stored at 20°C in dark, Cond. 2, stored at 20 °C in light, Cond. 3, stored at 4-6°C in light, Cond. 4 stored at 20°C in light with argon in the headspace. Different letters (a-e) represent significant differences among mean values for a same parameter during the storage time (from 2 to 14 months). Different letters (x-z) indicate significant differences among the four storage conditions after 2 and 14 months of storage. 2 months of storage 14 months of storage FA PV K232 K270 FA PV K232 K270 Cond. 1 0.15 ± 0.01 b,x 11.63 ± 1.29 a,xy 2.11 ± 0.03 b,x 0.10 ± 0.00 b,z 0.20 ± 0.01 a,x 12.74 ± 0.55 a,y 2.34 ± 0.02 a,x 0.15 ± 0.01 a,y Cond. 2 0.15 ± 0.01 b,x 14.00 ± 0.04 a,x 2.00 ± 0.09 b,xy 0.17 ± 0.01 b,x 0.20 ± 0.01 a,x 14.74 ± 1.02 a,xy 2.19 ± 0.07 a,y 0.18± 0.01 a,x Cond. 3 0.15 ± 0.01 b,x 10.59 ± 0.01 b,y 1.94 ± 0.12 b, y 0.13 ± 0.00 b,y 0.17 ± 0.01 a,y 15.47 ± 0.80 a,x 2.19 ± 0.06 a,y 0.14 ± 0.00 a,y Cond. 4 0.16 ± 0.01 b,x 14.00 ± 0.15 a,z 2.15 ± 0.04 b,x 0.17 ± 0.00 b,x 0.20 ± 0.01 a,x 14.70 ± 0.40 a,xy 2.24 ± 0.03 a,xy 0.18 ± 0.01 a,x Ital. J. Food Sci., vol. 27 - 2015 169 Fig. 1 - Example of full chromatogram of the EVOO sample at 20°C in dark. A) GC tracing of the diglyceride fraction of EVOO stored for 2 months at condition 1; B) GC trace of the diglyceride fraction of EVOO stored for 14 months at condition 1. 1, 1,2-PO; 2, 1,2-PoO; 3, 1,2-PL; 4, 1,3-PO; 5, 1,3-PoO; 6, 1,3-PL; 7, 1,2-OO; 8, 1,2-OL; 9, 1,3-OO; 10, 1,2-LL; 11, 13-OL + 1,3-LL. P = palmitic acid; Po = palmitoleic acid; O = oleic acid; L = linoleic acid. Fig. 2 - Trends of 1,2/1,3 DGs during the EVOO storage of 14 months at the four different conditions (Cond 1-4)*. The con- centration of DGs was calculated as mg dilaurin per 100 mg of oil. Different letters (a-e) represent significant differences among mean values for a same condition during the storage time (from 2 to 14 months). Different letters (x-z) indicate sig- nificant differences among the four storage conditions after 14 months. * Cond. 1, stored at 20°C in dark, Cond. 2, stored at 20°C in light, Cond. 3, stored at 4-6°C in light, Cond. 4 stored at 20°C in light with argon in the headspace. total DGs with higher amounts of C36 isomers, in particular diolein, which is considered the predominant DG in ol- ive oil (BOSKOU, 1996). A comparable behaviour was ob- served for samples stored at 20°C in light (Cond. 2). Accordingly, the ratio of 1,2/1,3-DGs decreased significant- ly from 5.43 to 1.69 after 10 months (Fig.2). Moreover, the 1,2-DG C36 iso- mer (Table  3) decreased significantly from 0.79 to 0.57 mg per 100 mg oil at the end of storage period, although this decrease slowed after 10 months. On the other hand, the 1,3-DG C36 isomer showed steady significant in- crease up to 12 months (Table 3) and then remained with slight changes, until the end of storage. However, 1,3- DG C34 isomers showed a significant slight change toward increases, after 6 months of storage, reaching about 0.14 mg per 100 mg sample after 14 months of storage (Table 2). The results for samples stored at low 170 Ital. J. Food Sci., vol. 27 - 2015 temperature (4-6°C) (Cond. 3) showed that, at the end of the storage period, the 1,2/1,3-DG ra- tio remained about 2 times higher than the val- ues for EVOO samples stored at 20°C (Fig. 2). Furthermore, the 1,2-DGs isomers C36 and C34 showed a significant decrease from 2 to 14 months (Table 4). Regarding the samples stored with argon in the headspace (Cond. 4), the 1,2/1,3-DGs ratio decreased significantly during the first 8 months of storage, and minor changes were detected up to the end of storage (Fig. 2). Similarly, 1,2-DGs for both C36 and C34 classes decreased after 14 months of storage compared to the initial val- ue, with a fluctuation trend ( Table 5), while 1,3- DG C36 isomers showed a significant increase throughout the entire storage period. By comparing the different conditions, after 2 months of storage the highest 1,2/1,3-DG ratio corresponded to the sample stored at low tem- perature (4-6°C), followed by the sample stored under light at 20°C with argon in the headspace Table 3 - Evolution of 1,2 and 1,3 isomers of C34 and C36 diglycerides during the EVOO storage of 14 months under con- dition 2 (at 20 °C in light). The concentration of DGs was calculated as mg dilaurin per 100 mg of oil. Different letters (a-e) represent significant differences among mean values for a same isomer during the storage time (from 2 to 14 months). Dif- ferent letters (x-z) indicate significant differences among the four storage conditions after 14 months of storage. Cond. 2 Months of oil storage 1,3 C34- DGs 1,3 C36- DGs 1,2 C34- DGs 1,2 C36- DGs 2 0.06 ± 0.00 e 0.15 ± 0.01 e 0.35 ± 0.01 ab 0.79 ± 0.15 cd 4 0.09 ± 0.02 d 0.21 ± 0.02 d 0.38 ± 0.05 a 1.06 ± 0.14 a 6 0.12 ± 0.02 cd 0.25 ± 0.02 d 0.37 ± 0.02 a 0.98 ± 0.05 ab 8 0.15 ± 0.01 ab 0.30 ± 0.01 c 0.32 ± 0.01 b 0.85 ± 0.07 bc 10 0.15 ± 0.01 a 0.36 ± 0.01ab 0.22 ± 0.01 c 0.69 ± 0.02 de 12 0.13 ± 0.00 bc 0.39 ± 0.00 a 0.23 ± 0.00 c 0.68 ± 0.00 de 14 0.14 ± 0.01 ab,y 0.32 ± 0.06 bc,y 0.21 ± 0.01 c,z 0.57 ± 0.04 e,z Table 2 - Evolution of 1,2 and 1,3 isomers of C34 and C36 diglycerides during the EVOO storage of 14 months under con- dition 1 (at 20oC in dark). The concentration of DGs was calculated as mg dilaurin per 100 mg of oil. Different letters (a-e) represent significant differences among mean values for a same isomer during the storage time (from 2 to 14 months). Dif- ferent letters (x-z) indicate significant differences among the four storage conditions after 14 months of storage. Cond. 1 Months of oil storage 1,3 C34- DGs 1,3 C36- DGs 1,2 C34- DGs 1,2 C36- DGs 2 0.09 ± 0.01 f 0.19 ± 0.03 e 0.48 ± 0.06 a 1.25 ± 0.14 a 4 0.11 ± 0.01 e 0.25 ± 0.02 de 0.47 ± 0.05 a 1.27 ± 0.16 a 6 0.13 ± 0.01 de 0.26 ± 0.01 d 0.38 ± 0.05 b 0.89 ± 0.07 b 8 0.13 ± 0.00 cd 0.33 ± 0.01 c 0.28 ± 0.01 c 0.77 ± 0.02 b 10 0.15 ± 0.00 bc 0.40 ± 0.07 b 0.28 ± 0.01 c 0.75 ± 0.05 b 12 0.16 ± 0.01 b 0.37 ± 0.03 bc 0.27 ± 0.01 c 0.74 ± 0.10 b 14 0.19 ± 0.02 a,x 0.49 ± 0.02 a,x 0.27 ± 0.02 c,yz 0.73 ± 0.05 b,y Table 4 - Evolution of 1,2 and 1,3 isomers of C34 and C36 diglycerides during the EVOO storage of 14 months under con- dition 3 (at 4-6°C in light). The concentration of DGs was calculated as mg dilaurin per 100 mg of oil. Different letters (a- e) represent significant differences among mean values for a same isomer during the storage time (from 2 to 14 months). Different letters (x-z) indicate significant differences among the four storage conditions after 14 months of storage. Cond. 3 Months of oil storage 1,3 C34- DGs 1,3 C36- DGs 1,2 C34- DGs 1,2 C36- DGs 2 0.08 ± 0.01 c 0.14 ± 0.01 d 0.58 ± 0.08 a 1.09 ± 0.18 ab 4 0.08 ± 0.00 c 0.17 ± 0.01 c 0.46 ± 0.02 b 1.25 ± 0.06 a 6 0.12 ± 0.01 ab 0.18 ± 0.01 c 0.41 ± 0.02 bcd 1.01 ± 0.11 b 8 0.12 ± 0.01 ab 0.16 ± 0.01 cd 0.34 ± 0.04 d 0.9 ± 0.12 b 10 0.10 ± 0.03 bc 0.22 ± 0.00 b 0.38 ± 0.01 cd 1.08 ± 0.03 ab 12 0.14 ± 0.02 a 0.25 ± 0.02 b 0.45 ± 0.02 bc 1.03 ± 0.17 b 14 0.13 ± 0.01 a,y 0.28 ± 0.03 a,y 0.39 ± 0.04 cd,x 1.07 ± 0.03 ab,x Ital. J. Food Sci., vol. 27 - 2015 171 Table 5 - Evolution of 1,2 and 1,3 isomers of C34 and C36 diglycerides during the EVOO storage of 14 months under con- dition 4 (at 20 °C in light with argon in the headspace). The concentration of DGs was calculated as mg dilaurin per 100 mg of oil. Different letters (a-e) represent significant differences among mean values for a same isomer during the storage time (from 2 to 14 months). Different letters (x-z) indicate significant differences among the four storage conditions after 14 months of storage. Cond. 4 Months of oil storage 1,3 C34- DGs 1,3 C36- DGs 1,2 C34- DGs 1,2 C36- DGs 2 0.07 ± 0.00 c 0.14 ± 0.02 d 0.41 ± 0.08 a 1.07 ± 0.18 ab 4 0.07 ± 0.01 c 0.18 ± 0.01 d 0.32 ± 0.02 bc 0.82 ± 0.13 cd 6 0.14 ± 0.00 b 0.31 ± 0.06 c 0.46 ± 0.05 a 1.09 ± 0.11 a 8 0.15 ± 0.01 b 0.37 ± 0.04 bc 0.29 ± 0.01 c 0.82 ± 0.02 d 10 0.21 ± 0.01 a 0.46 ± 0.04 ab 0.38 ± 0.01 ab 1.06 ± 0.21 abc 12 0.17 ± 0.01 b 0.48 ± 0.06 a 0.25 ± 0.01 c 0.70 ± 0.05 d 14 0.21 ± 0.04 a,x 0.53 ± 0.10a,x 0.31 ± 0.06 bc,y 0.84 ± 0.15 bcd,y (Fig. 2). Moreover, during the first 4 months, when EVOOs were stored at 20°C under light without headspace modification (Cond. 2), the sample exhibited a lower ratio than the respec- tive sample stored in darkness (Cond. 1). The re- sults also highlighted the positive effect of using inert gas in the head space. The total 1,2-DGs remained after 14 months (data not shown) of storage was about 1.5 times higher, in compar- ison with their presence in EVOO stored under the same conditions, but with air in the head space. The findings are in accordance with SPY- ROS et al. (2004), suggesting that the length of storage time plays an important role in isomer- isation changes of DGs, which is accelerated by temperature. The formation of oxidation products by pho- to-oxidation was confirmed by the high values of K 270 obtained for samples stored under diffuse light, especially for those stored at 20°C after 14 months of storage (Table 1). It should be noted that, at the end of storage period, all the sam- ples remained within EVOO category parame- ters. As expected, free acidity (Table 1), which is considered to be the main driving factor af- fecting DG isomerisation (PÉREZ-CAMINO et al., 2001), showed only a minor increase after 14 months of storage. The results of this study showed that the isomerisation of DGs in EVOOs depends not only on the length of storage, but also on the tempera- ture of storage. This finding is in agreement with the studies of PÉREZ-CAMINO et al. (2001) and COSSIGNANI et al. (2007). Moreover, the results showed that after 14 months of storage at 20°C (Cond. 1, 2 and 4) there were slight but not sig- nificant differences in the 1,2/1,3 ratio among samples stored under diffuse light (Cond. 2 and 4) and for those stored in darkness (Cond. 1), in spite of the fact that light exposure has an ad- verse effect on the oxidation of EVOO (signifi- cantly higher K 270 values were found for sam- ples stored under diffuse light). This result is in agreement with considerations noted by AF- ANEH et al. (2013). CONCLUSION The results of this study confirmed that the isomerisation of DGs in EVOO depends not only on the length of storage, but also on the tem- perature. 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