Fatty Acid Methyl and Ethyl Esters As Well As Wax Esters for Evaluating the Quality of Olive Oils

Eur Food Res Technol (2008) 228:65–74 DOI 10.1007/s00217-008-0907-x ORIGINAL PAPER Fatty acid methyl and ethyl esters as well as wax esters for evaluating the quality of olive oils Maurus Biedermann · Annette Bongartz · Carlo Mariani · Koni Grob Received: 27 April 2008 / Revised: 22 May 2008 / Accepted: 3 June 2008 / Published online: 19 June 2008 © Springer-Verlag 2008 Abstract A promising correlation between chemical Introduction analysis and sensorial evaluation was conWrmed: extra virgin olive oils with low contents of methyl and ethyl esters The quality of olive oils is sensorially tested by elaborate of fatty acids as well as straight chain wax esters were sen- and well established methods (EU regulation 2568/91/EEC sorially evaluated as being of high quality, whereas some and amendments). It enables the classiWcation of the with high contents were even devaluated as not being of pressed oils into the classes of extra virgin oil, virgin oil extra virgin quality. Methanol and ethanol formed during and lampant oil [1]. Nonetheless, it would be convenient to fermentation in degrading olives are esteriWed, largely by have chemical analytical methods for screening oils or sup- transesteriWcation with fatty acids from the triglycerides, porting sensorial analysis, perhaps even tracing back deW- and in this way transferred into the pressed oil. The pres- ciencies. ence of high contents of methyl and ethyl esters in degrad- A number of approaches were investigated to determine ing olives was conWrmed. Wax esters from the skin of the deWciencies of extra virgin olive oils resulting from inap- olives are extracted at low yields, whereby the yield propriate technological treatments, such as spectrophoto- increases when the olives are soft and possibly degrading. metry for the determination of conjugated fatty acids, High wax ester contents may, therefore, stand for mild oils, stigmastadiene for the detection of decoloration or high but also for deWcient oils. temperature deodoration [2] and the formation of pyrophe- ophytine from chlorophyll [3–6] or the isomerization of Keywords Olive oil · Ethyl oleate · Wax esters · 1,2-diglycerides to 1,3-diglycerides [7, 8] for the recogni- Sensorial evaluation · Degrading olives tion of weak thermal treatments. These methods primarily aimed at detecting adulteration of extra virgin oils by treated oils of minor quality, which does not necessarily aVect the sensorial quality. A diVerent approach uses the proWle of the Xavor components for the characterization of olive oils, e.g., [9]. The content of free fatty acids is used as a quality crite- M. Biedermann · K. Grob (&) rion for extra virgin olive oils. If present at elevated con- OYcial Food Control Authority of the Canton of Zurich, centrations, free fatty acids indicate lipase activity P.O. Box, 8032 Zurich, Switzerland hydrolyzing triglycerides in the olives, i.e., decay of cell e-mail: [email protected]; [email protected] compartmentation. It is a qualiWcation of oil related to the A. Bongartz quality of the olives, in contrast to, e.g., peroxide values University of Applied Sciences Zurich (ZHAW), serving as indicator of oil oxidation. P.O. Box 335, 8820 Waedenswil, Switzerland The straight chain wax esters (long, straight chain fatty alcohols esteriWed with fatty acids) were shown to be useful C. Mariani Stazione Sperimentale per le Industrie degli Oli e dei Grassi, quality indicators for olives and oils obtained from these Via Giuseppe Colombo 79, 20133 Milan, Italy [10–13]. They are located in the waxy surface layer of the 123 66 Eur Food Res Technol (2008) 228:65–74 olive and are poorly extracted by the oil pressed from the Experimental fruit. The amount extracted is higher the more soft, possibly degraded the olives are. Typically oils of inferior quality Materials (lampant oils) contain these wax esters at increased concentrations. Solvent (hexane) extracts even higher amounts On-line LC-GC-FID and LC-GC-MS were performed on from the pression residues (pomace), which is used for Thermo ScientiWc equipment (Milano, Italy), consisting of detecting the admixture of pomace oil to pression oil. A a TriPlus autosampler, a Phoenix 40 dual syringe pump legal limit was established for the wax esters C40–C46 in with three switching valves and a TRACE gas chromato- extra virgin olive oils (250 mg/kg) and reWned olive oils graph equipped with on-column injector, Xame ionization (350 mg/kg; Commission Regulation 702/2007 amending detector and a switching valve for the regulation of the Regulation 2568/91). However, Mariani [14, 15] repeatedly transfer. Mass spectrometry (MS) involved a Polaris Q ion warned that this limit may be exceeded without such adul- trap (Thermo ScientiWc). teration, if aging of the oil causes free alcohols to be esteri- Pentane and methyl tert. butyl ether (MTBE) from Wed. They may also be formed during raYnation, in Brenntag, Schweizerhall AG (Basel, Switzerland) were particular during deodoration. redistilled. Heptadecanoic and nonadecanoic acid as well as Cert et al. [16] recently proposed using fatty acid methyl octadecenol, methyl cis-11,14-eicosadienoate, docosanoyl and ethyl esters as quality markers. Methanol and ethanol are chloride, the dimethyl polysiloxane PS-255 and trihepta- formed during fermentation of olives; they cannot be ana- decanoin were from Fluka (Buchs, Switzerland), heneicosa- lyzed as such in the oil, since they are largely removed with nol and ethyl arachidate from Sigma (via Fluka). Edible oils water during pression, and even the most gentle deodoration and preserves in cans or jars containing olive oil were from would eliminate them. However, they form methyl and ethyl the local market. The wax ester 21-22:0 was prepared as esters of fatty acids, which are transferred into the oil. described in [10]. Mariani and Bellan [17] took this idea and indeed found a good correlation of the content of these esters with the Analytical method quality of olive oils determined by other means. They con- cluded that in extra virgin oil the ratio of methyl to ethyl Wax esters were isolated using HPLC in normal phase esters of fatty acids should not be below 0.9–1, i.e., the (NPLC): to 25 mg edible oil weighed into an autosampler methyl esters should predominate the ethyl esters. The con- vial, 25 l internal standard and veriWcation standard centration of ethyl oleate should not exceed 15 mg/kg: the solution was added (esters Me-17:0, Et-20:0, Me-20:2 summed concentrations of the methyl and ethyl oleate not and 21-22:0, 20 g/ml in MTBE, corresponding to exceed 30–40 mg/kg. Methyl and ethyl oleate can be 20 mg/kg in the sample) and the vial Wlled up with 1.5 ml removed by deodoration at elevated temperatures, but this hexane. Of this solution, 10 l was injected into a leaves behind other traces, such as stigmastadiene, and the 250 £ 2 mm i.d. HPLC column packed with Spherisorb ratio of the methyl to ethyl esters remains low. Si 5 m (Grom, Rottenburg-HailWngen, Germany) and The presence of the methyl and ethyl esters of fatty acids chromatographed with 4% MTBE/pentane at 300 l/min. in olive oils was detected long ago. In 1986, Mariani and The elution window of the wax ester fraction usually Fedeli [10] isolated the “nonpolar fraction” of olive oil by comprised 2–4 min in retention time, i.e., amounted to means of column liquid chromatography and analyzed it by 600 l. It was adjusted by transferring smaller fractions, GC. This method was devised for the broader investigation including those adjacent to that of interest. The fraction of the “minor components”, such as alcohols, sterols, triter- was transferred on-line into GC through the on-column pene alcohols, tocopherols and esters of these, as described interface by the concurrent eluent evaporation technique. in, e.g., [11, 14, 18, 19]. The vapor exit was closed 0.1 min after the end of the The work described in this paper started out from the on- transfer. line LC-GC-FID method for analyzing minor components The GC system consisted of a 40 cm £ 0.53 mm i.d. [20, 21] and the wax ester fraction [13] and optimized it for precolumn, in the laboratory statically coated with a 0.03- the combined analysis of methyl/ethyl oleate and selected m Wlm of OV-1701-OH, connected via a T-piece union to straight chain wax esters. Extra virgin olive oils from the a solvent vapor exit and a 20 m £ 0.25 mm i.d. separation market were analyzed and those with the highest and lowest column coated in the laboratory with a 0.12-m Wlm of PS- concentrations of these markers sensorially tested to check 255 (a dimethyl polysiloxane). Helium was used as carrier the correlation between the sensorial quality and the analyt- gas at 60 kPa inlet pressure; during transfer pressure was ical data. Additional experiments were performed on the reduced to 40 kPa. The oven temperature was initially at formation of the esters, aiming at clarifying their origin and 50 °C (3 min), then programmed at 30 °C/min to 130 °C signiWcance for oil evaluation. and at 7 °C/min to 360 °C (4 min). 123 C was used to isolate the esters interest,of and the ofthewaxesters [ the analysis acids. of fatty esters small in oil B. The chromatogram ends with the sterol ar olive.They ofthe the skin from the chain followed originating by wax esters, straight esters originating from the pulp the of [ olive W half of the chromatogram start with the diterpene esters, second the in Thewaxes squalene. dominant by the lowed fol- early, eluted are esters ethyl and methyl acid fatty The rather high content of the ofesters interest, oil B alow one.

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