Enrichment of pomace olive oil in triterpenic acids during storage of “Alpeorujo” olive paste

Research Paper

Enrichment of pomace olive oil in triterpenic acids duringstorage of “Alpeorujo” olive paste

Aranzazu García, Manuel Brenes, Ma Carmen Dobarganes, Concepción Romero andMa Victoria Ruiz-Méndez

Instituto de la Grasa (CSIC), Seville, Spain

Triterpenic acids are natural compounds present in plants and foods with beneficial properties for humanhealth and thus they are desirable in the food, cosmetics and pharmaceutical industries. Pomace olive isconsidered a good source of these substances. In this study, oleanolic and maslinic acids were found to bethe main triterpenic acids identified in pomace olive oil obtained from stored “Alpeorujo”. Determinationof the two acids was achieved by a new procedure that consists of extracting the acids from the oil with amixture of methanol/ethanol, and then separating and quantifying them by HPLC. Results showed thattheir concentration increased up to 16 g/kg of oil during storage of the pomace in large ponds for7 months. The concentration of both triterpenic acids was similar in the pomace olive oil obtained by usingthe centrifugation system. By contrast, a much lower concentration of maslinic than oleanolic acid wasdetected in pomace olive oils obtained by solvent extraction from the previously centrifugated “Alpeorujo”paste. These triterpenic acids also contributed to the acidity of the crude oil. Likewise, the oil of thepomace paste was enriched in other substances such as 4-ethylphenol and aliphatic alcohols during thestorage of the paste in large ponds. Consequently, crude pomace olive oil can be considered a good sourceof triterpenic acids when obtained from a stored olive paste.

Keywords: 4-Ethylphenol / Aliphatic alcohols / Maslinic acid / Oleanolic acid / Pomace olive oil / Triterpenic acids

Received: March 12, 2008; accepted: June 9, 2008

DOI 10.1002/ejlt.200800070

1136 Eur. J. Lipid Sci. Technol. 2008, 110, 1136–1141

1 Introduction

The method for obtaining virgin olive oil consists in crushingthe fruits, a malaxation step of the olive paste for 30–60 minand, finally, separation of the oil by centrifugation. At present,most of the olive oil-producing countries use a two-phasecentrifugation system to separate the oily phase from the wetolive paste, which is called “Alpeorujo” in Spain.

This olive pomace can be composted and spread on fields[1], but it still retains a significant amount of residual oil [2]and can also be a source of valuable compounds [3, 4]. Hence,several extraction plants receive the fresh olive pomace, elim-inate most of the moisture and extract the oil from the cake bysolvent extraction using hexane. Alternatively, other factoriesstore the “Alpeorujo” in large ponds for months and the resi-dual oil is extracted periodically. As a first step, the stored

“Alpeorujo” is malaxed and the oil is separated in a three-phase centrifugation decanter. The new olive pomace is thensubmitted to a drying process, followed by a solvent extractionprocess with hexane in order to exhaust the cake. In summary,two crude pomace olive oils are obtained by centrifugationand solvent extraction means from stored “Alpeorujo” [5].

Very few studies are available on the chemical character-istics of this crude olive pomace, but it is obvious that twoprocesses take place during the storage period: (i) a fermen-tation of the olive paste [6] and (ii) an accumulation of lipo-philic compounds in the oily phase of the paste [7, 8]. Inaddition, the pomace olive oil obtained from fresh pomace hasa higher concentration of lipophilic compounds (waxes, ali-phatic alcohols, triterpenic acids and others) than virgin oliveoil [2]. In the present study, the focus was on the triterpenicacids because of the interest in their health properties on thepart of researchers. Besides, they are highly demanded by thefood, cosmetics and pharmaceutical industries. Oleanolic andmaslinic acids are the main triterpenic acids in olive oil andpomace olive oil, although they are found in a higher con-centration in the latter type [9]. Researchers have discovered

Correspondence: Ma Victoria Ruiz-Méndez, Instituto de la Grasa (CSIC),Avda. Padre García Tejero 4, 41012 Seville, Spain.E-mail: [email protected]: 134 954 691262

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

Eur. J. Lipid Sci. Technol. 2008, 110, 1136–1141 Triterpenic acids in POO 1137

many beneficial properties in triterpenic acids, such as theirantimicrobial and anti-hyperglycemic activities [10, 11], anti-inflammatory and anti-tumor effects [12–14], a growth-stim-ulating factor used in trout diets [15], and a hepatoprotectiveeffect [16]. Therefore, there is currently a growing interest inobtaining triterpenic acids from natural sources.

Taking into account the importance of these substances,we have studied the content of triterpenic acids in the pomaceolive oil present in stored “Alpeorujo” as a potential futuresource of these valuable compounds. Additionally, we havedeveloped a new method for analyzing triterpenic acids inolive oil based on the HPLC technique [17, 18] as an alter-native to the current GC method [9].

2 Materials and methods

2.1 Samples and treatments

Real samples of pomace olive oil were taken from “Oleícola elTejar, SCA” extraction facilities located in the southern regionof Spain. This company stored 50,000 tons of “Alpeorujo” ina big pond at ambient temperature for 6 months, and oil fromthe stored “Alpeorujo” was extracted sequentially by cen-trifugation and solvent extraction methods at industrial scaleevery month. First, a three-phase centrifugation system wasused to extract the pomace oil from the stored “Alpeorujo”and, subsequently, the extracted paste was submitted to a sol-vent extraction process with hexane [7]. Oil samples (1 L)were sent to the Instituto de la Grasa and stored at –18 7Cuntil analysis.

Pomace olive oil was also obtained in the laboratory fromone sample of fresh pomace olive by centrifugation or solventmethod [7].

2.2 Analysis of triterpenic acids

The extraction of these substances from the pomace olive oilwas made by a mixture of methanol/ethanol (1 : 1). The oil(0.8 g) and the alcoholic solvent (1.6 mL) were vortexed for1 min, centrifuged at 7500 rpm for 5 min at 20 7C, and thealcoholic phase was separated from the lipid phase. This stepwas repeated six times. Subsequently, the pooled alcoholicextract was vacuum evaporated and the residue dissolved in2.4 mL methanol, which was centrifuged at 7500 rpm for5 min at 20 7C. Finally, the solution was filtered through a0.2-mm pore size filter and an aliquot (20 mL) was injectedinto the liquid chromatograph.

The chromatography system consisted of a Waters 717plus auto sampler, a Waters 600E pump, and a Waters 996diode array detector (Waters Inc., Milford, MA, USA). ASpherisorb ODS-2 (5 mm, 25646 mm i.d.; Waters Inc.) col-umn was used. The temperature of the column was kept con-stant at 35 7C by using a Waters column heater. The mobilephase (methanol/acidified water with phosphoric acid at

pH 3.0, 92 : 8, vol/vol) was delivered to the column at a flowrate of 0.8 mL/min and the eluate was monitored at 210 nm. Ablank of methanol was injected into the chromatograph afterfive sample injections to clean the column. Triterpenic acids(oleanolic and maslinic acids) were quantified using oleanolicacid (Sigma, St. Louis, MO, USA) as external standard. Asimilar response factor was assumed for both triterpenic acidson the basis of their similar UV spectra. The standard curvewas made with a range of oleanolic acid concentrations from50 to 3000 mg/kg.

Mass spectra were acquired using a quadrupole massanalyzer (ZMD4; Waters Inc.), equipped with an electrosprayionization (ESI) probe and working in the negative mode.Cone voltage fragmentation was at 10 V, capillary voltage was3 kV, desolvation temperature was 200 7C, source tempera-ture was 120 7C, and extractor voltage was 12 V.

2.3 Analysis of phenolic compounds

Phenolic extracts of pomace olive oils were obtained followingthe procedure described elsewhere [19]. Briefly, 0.6 mL oilwas extracted using 360.6 mL N,N-dimethylformamide(DMF); the extracts were combined and then washed withhexane, and N2 was bubbled into the DMF extract to elim-inate residual hexane. Finally, the extract was filtered througha 0.45-mm pore size filter and injected into the liquid chroma-tograph. The chromatographic system was the same as usedfor triterpenic acid analysis. Mobile phase, conditions andquantification have been described elsewhere [20].

2.4 Total polar compound content

Total polar compounds were determined in oil samples byadsorption silica column chromatography, following theIUPAC standard method [21].

2.5 Other oil analysis

Wax content, fatty alcohols and acidity were determinedaccording to the EEC recommended methods [22]. Analyseswere performed in a Hewlett-Packard Model 5890-II gaschromatograph, and data storage and treatment was achievedwith the help of an IBM computer with software HPChem-Station v. A.05.01. The gas chromatographic conditions ofthe EEC regulations were slightly modified as follows:

Waxes: The gas chromatograph was equipped with a coldon-column injector with oven-track system and a flame ioni-zation detector. A TRB-5 column (5% diphenyl/95% dimethylpolysiloxane, length 15 m, 0.32 mm i.d. and 0.1 mm filmthickness; TRACER 120113) was used. Hydrogen (140 kPainlet pressure) was used as carrier gas and nitrogen as make-up gas. The oven temperature was held at 70 7C for 5 min,then increased at 45 7C/min to 180 7C and at 5 7C/min to340 7C where it was held for 7 min. The detector temperaturewas 350 7C.


1138 A. García et al. Eur. J. Lipid Sci. Technol. 2008, 110, 1136–1141

Fatty alcohols: The gas chromatograph was equipped witha split injector, and a HP-5 column (5% diphenyl/95% dime-thyl polysiloxane, length 30 m, 0.32 mm i.d. and 0.25 mmfilm thickness; HP 19091J-413) was used. Carrier gas washydrogen with nitrogen as make-up gas. The oven tempera-ture was held at 220 7C, then increased at 3 7C/min to 280 7Cwhere it was held for 23.7 min. The injector temperature was280 7C and the detector temperature was 300 7C.

Unsaponifiable matter: The oil samples were treated with a2 M solution of potassium hydroxide in ethanol/water(80 : 20, vol/vol), and this mixture was refluxed for 60 min[23]. The unsaponifiable fraction was extracted three timeswith portions of ethyl ether and, subsequently, the extract waswashed with water and then the solvent was removed. Theunsaponifiable matter was expressed as percentage of oil.

A portion of the unsaponifiable matter was then dissolvedinto chloroform, and aliphatic alcohols were separated by thin-layer chromatography.

2.6 Statistical analysis

Statistical analysis was performed using the Statistica packagesoftware (Statistica for Windows, Tulsa, OK, USA 1996).

3 Results and discussion

A new method was developed for analyzing triterpenic acidsin oil. It is well known that several solvents can be used toextract triterpenic acids from oil. We tried methanol, ethanoland a mixture of methanol/ethanol (1 : 1), the last one beingthe best solvent based on the amount of triterpenic acidsrecovered. Also, solutions of 250, 500 and 1000 mg/kg ofoleanolic acid in refined olive oil were prepared and extractedwith methanol/ethanol (1 : 1) five times each. The meanrecovery of oleanolic acid was 94.0% (SD, 5.77). Thus, themixture methanol/ethanol was accepted as a good solvent toextract triterpenic acids from pomace olive oil.

Because triterpenic acids lack a chromophore, their non-specific detection is currently carried out at 200–210 nm. In

this work, they were monitored at 210 nm, and a chromato-gram is shown in Fig. 1. There are two main triterpenic acidsin different parts of the olive fruit and olive oil, oleanolic andmaslinic acids, although traces of ursolic acid have also beendescribed in some samples [9, 24, 25]. Oleanolic acid wastentatively assigned to peak 4 because of a coincidence in theretention time with an authentic standard. However, the tri-terpenic acid extract was also injected into a chromatographconnected to a mass spectrometer detector. Thus, peak 4 hada molecular mass of 456 uma, which corresponds to that ofoleanolic acid, and the mass fragmentation was also similar forpeak 4 and oleanolic acid. Furthermore, it was found thatpeak 1 had a molecular mass of 472 uma, which is similar tothat of maslinic acid, and its fragmentation pattern was thesame as that of oleanolic acid. Consequently, peak 1 wasidentified as maslinic acid. Ursolic acid was also sought out bythe mass spectrometer technique in several extracts, but itssignal was not observed. In addition, two compounds wereeluted between maslinic and oleanolic acids, peaks 2 and 3.Compounds corresponding to these two peaks had molecularmasses of 278 and 280 uma, respectively. Because of their UVspectra and fragmentation pattern, it was assumed that thesetwo compounds were fatty acids, and it was confirmed withauthentic standards that peak 2 corresponded to linolenic acidand peak 3 to linoleic acid. None of the other peaks of thechromatogram showed a similar UV spectrum to that of thetriterpenic acids, and oleic acid was not detected in the chro-matogram. Fortunately, a good separation between linoleicand linolenic acids, and the triterpenic acids was achieved byusing the mobile phase and the chromatographic conditionsstudied.

Therefore, the extracting technique developed togetherwith the analysis of triterpenic acids by HPLC can be con-sidered a very good alternative to the current method usingsolid-phase extraction cartridges and gas chromatographyseparation [9].

With regard to the influence of the oil extraction methodon the content of triterpenic acids, the oleanolic and maslinicacids from crude pomace olive oils obtained first by cen-trifugation means and then by solvent extraction means are

Figure 1. HPLC chromatogram at 210 nm ofthe triterpenic acid extract of a pomace olive oil.Peaks: (1) maslinic acid; (2) linolenic acid;(3) linoleic acid; (4) oleanolic acid.



Figure 2. Concentrations of oleanolic and maslinic acids in thepomace olive oils obtained sequentially by centrifugation and sol-vent extraction from stored “Alpeorujo”. Bars indicate standarddeviation.

presented in Fig. 2. The concentration of both acids increasedin the oils with increasing storage time of the “Alpeorujo”paste. It must be noted that the content of maslinic acid wasalways higher than that of oleanolic acid in the oils extractedby centrifugation. A concentration of almost 8000 mg of eachtriterpenic acid per kilogram of oil was reached in oilsobtained from “Alpeorujo” stored up to May, and thereforethese oils represent a very good source of these valuable com-pounds. In fact, the concentration of triterpenic acids in virginolive oils is lower than 100 mg/kg oil [9], which means thatthese substances are concentrated in the lipophilic phase of the“Alpeorujo” paste during storage.

The pomace olive oil generated during the centrifugationextraction (three-phase centrifugation system) was subse-quently treated with hexane to extract the residual oil. It can beobserved in Fig. 2 that in this case the concentration of masli-nic acid was always much lower than that of oleanolic acid.Although the previous elimination of maslinic acid duringcentrifugation influences the amount obtained in the sub-

sequent step of solvent extraction, its low concentration wasmainly attributed to the lower lipophilicity of maslinic ascompared to that of oleanolic acid. To confirm this hypothesis,an alcohol extract of oleanolic and maslinic acids was washedwith hexane, and the solvent extracted more than 30% ofoleanolic acid and only 6% of maslinic acid. Additionally, oilwas obtained from fresh “Alpeorujo” by either centrifugationor solvent extraction (Table 1). It was again confirmed thatmaslinic acid was poorly recovered in the oil obtained by sol-vent extraction. Hence, a centrifugation extraction is prefer-able over a solvent extraction with hexane if both triterpenicacids are required in high concentration in the pomace oliveoil.

A chemical parameter of “Alpeorujo” paste that alsoincreased with storage time was the acidity in both oilsobtained by centrifugation and subsequently by solventextraction means (Fig. 3). It has been reported that oil dete-rioration occurs during olive pomace storage in large ponds[7, 8], and it was confirmed in our experiments by the greatincrease in polar compounds evaluating all the compoundswith polarity higher than that corresponding to the triglycer-ides. These compounds are degradation compounds andmainly diglycerides and fatty acids originating from the actionof lipolytic enzymes (Table 2). Thus, a release of fatty acidsfrom the triglyceride matrix can be expected and, therefore,an increase in oil acidity. The high amount of triterpenic acidsfound in the oils must be noted because they must be elimi-nated during the neutralization step of the refining process.They form the sodium salts [9] and are separated from theaqueous medium because of their solubility [24]. Conse-quently, they should be quantified as losses. However, thecontribution of triterpenic acids to this acidity increase isunknown and must not be ruled out, taking into considerationthe high amount of these substances found in the oils. Refinedolive oil was spiked with oleic and oleanolic acid, and aciditywas titrated with NaOH solution. The amount of sodium hy-droxide necessary to neutralize oleic acid was almost doublethat needed for oleanolic acid. Thus, the contribution of tri-terpenic acids to the oil acidity, expressed as percentage ofoleic acid, could be estimated as 10–20%.

As well as in triterpenic acids, pomace olive oil was alsoenriched in other minor compounds during storage of“Alpeorujo” paste, as confirmed by the increase of the unsa-

Table 1. Distribution of triterpenic acids in pomace olive oilsobtained by centrifugation or solvent extraction from a fresh olivepomace.

Maslinic acid[mg/kg]

Oleanolic acid[mg/kg]

Centrifugation 685 (6)§ 1158 (94)Solvent extraction 329 (46) 1415 (2)

§ Standard deviation of three replications.


1140 A. García et al. Eur. J. Lipid Sci. Technol. 2008, 110, 1136–1141

Table 2. Chemical parameters of the oil obtained sequentially by centrifugation and solvent extraction from the olive paste “Alpeorujo”stored for several months.

January February April May

Centrifugation Solvent Centrifugation Solvent Centrifugation Solvent Centrifugation Solvent

Total waxes [mg/kg] 330§ 129 not analyzed 155 310 569 327 602Aliphatic alcohols [mg/kg] 2798 3456 2675 3842 3345 5847 3883 6223Unsaponifiable matter [%] 1.25 1.08 1.28 1.83 2.36 2.60 2.53 3.44Polar compounds [%] 9.9 15.2 11.3 15.3 15.1 18.6 18.2 20.1Non-polar compounds [%] 89.3 83.5 81.3 83.3 84.9 81.1 81.7 79.5

§ Mean value of duplicates.

Figure 3. Acidity of the pomace olive oils obtained sequentially bycentrifugation and solvent extraction from stored “Alpeorujo”. Barsindicate standard deviation.

ponifiable matter (Table 2). Phenolic compounds were foundin both the oils either obtained by centrifugation and solventextraction means (Fig. 4); in particular, the concentration of4-ethylphenol was significant and increased with storage time.This compound was probably formed by microbial action in asimilar manner as occurs during wine fermentation [26],resulting in impregnated pomace olive oil with a strong off-odor. Although 4-ethylphenol is eliminated during the physi-cal and chemical refining of the oil [20], it is already present inthe crude olive pomace oil and could accompany the tri-terpenic acids in their extracts.

Aliphatic alcohols are other compounds detected in highconcentrations in the pomace olive oils (Table 2) that arehigher in the oils obtained by solvent extraction than by cen-trifugation means, but that in both cases increased their con-centration with storage time of “Alpeorujo”. In fact, a con-centration of up to 6 g/kg of oil was reached in pomace oliveoils extracted in May, which is a much higher concentrationthan reported in virgin olive oil [27].

In conclusion, the oily phase of the “Alpeorujo” paste wasenriched in lipophilic compounds during storage of the pastein large ponds. Among these substances, the triterpenic acids

Figure 4. Concentration of phenolic compounds (4-ethylphenoland other polyphenols) in the pomace olive oils obtained sequen-tially by centrifugation and solvent extraction from stored “Alpeo-rujo”. Bars indicate standard deviation.

must be highlighted because of their biological activitiesrecently reported and the high concentration detected in thepomace olive oils. Thus, these oils can be considered a goodsource of triterpenic acids in the future.



Acknowledgments

This research was developed under the projects AGL2006-01552and AGL2007-63-647. Thanks to Oleícola el Tejar for supplyingsamples and collaboration.

Conflict of interest statement

The authors have declared no conflict of interest.

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Enrichment of pomace olive oil in triterpenic acids during storage of “Alpeorujo” olive paste

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