J. Agric. Food Chem. 2007, 55, 7869–7873
7869
Cultivar Differences on Nonesterified Polyunsaturated Fatty Acid as a Limiting Factor for the Biogenesis of Virgin Olive Oil Aroma ARACELI SÁNCHEZ-ORTIZ, ANA G. PÉREZ,
AND
CARLOS SANZ*
Department of Physiology and Technology of Plant Products Instituto de la Grasa, C.S.I.C. Padre García Tejero 4, 41012 Seville, Spain
The relationship between the content of nonesterified polyunsaturated fatty acids and the contents of oil aroma compounds that arise during the process to obtain virgin olive oil (VOO) was studied in two olive cultivars, Picual and Arbequina, producing oils with distinct aroma profiles and fatty acid compositions. Results suggest that the biosynthesis of VOO aroma compounds depends mainly on the availability of nonesterified polyunsaturated fatty acids, especially linolenic acid, during the process and then on the enzymatic activity load of the lipoxygenase/hydroperoxide lyase system. Both availability of substrates and enzymatic activity load seem to be cultivar-dependent. KEYWORDS: Lipoxygenase pathway; polyunsaturated fatty acid; olive oil; aroma
INTRODUCTION
The benefits of virgin olive oil (VOO) consumption are related to protection against cancer and cardiovascular diseases due to its fatty acid profile and the presence of minor constituents such as phenolic compounds (1, 2). However, the increase in the demand for high-quality VOO can be attributed not only to its potential health benefits but also to its excellent organoleptic properties. The aim of increasing the quality standards for VOO is continuously stimulating the study of biochemical pathways related to organoleptic properties and the development of technological procedures to improve those organoleptic properties. In this sense, our group established a decade ago the participation of the lipoxygenase (LOX) pathway in the biosynthesis of compounds of six straight-chain carbons (C6 compounds) in olive oil aroma (3). C6 aldehydes and alcohols and the corresponding esters are the most important compounds in the VOO aroma, from either a quantitative or a qualitative point of view (4, 5). These compounds are synthesized from polyunsaturated fatty acids containing a (Z,Z)-1,4-pentadiene structure such as linoleic (LA) and linolenic (LnA) acids. In a first step of this pathway, LOX produces the corresponding 13hydroperoxide derivatives that are subsequently cleaved heterolytically by hydroperoxide lyase (HPL) to C6 aldehydes (3, 6, 7). C6 aldehydes can then undergo reduction by alcohol dehydrogenases (ADH) to form C6 alcohols (3, 8) and can finally be transformed into the corresponding esters by means of an alcohol acyltransferase (3, 9). Moreover, Angerosa et al. (5) also demonstrated the relevance of compounds of five straight-chain carbons (C5 compounds) in the aroma of olive oil. C5 compounds would be generated through an additional branch of the LOX pathway that would involve the production of a * To whom correspondence should be addressed. Tel: +34 95 611550. Fax: +34 95 616790. E-mail:
[email protected].
13-alcoxyl radical by LOX as demonstrated in soybean seeds (10). This radical would undergo subsequent nonenzymatic β-scission in a homolytic way to form a 1,3-pentene allylic radical that could be chemically dimerized to form pentene dimers or react with an hydroxyl radical to form C5 alcohols. The latter would be the origin of C5 carbonyl compounds present in the aroma of olive oil through an enzymatic oxidation by ADH as suggested to occur in soybean leaves (11). The lack of HPL activity gives rise to an accumulation of hydroperoxides and a subsequent increase of the homolytic LOX branch activity, producing higher contents of C5 compounds as demonstrated in antisense-mediated HPL-depleted tomato plants (12). There are quite a number of studies describing the way technological procedures affect VOO aroma compound profile or the biosynthetic pathway determining this profile (13–18). However, as far as we know, there is no study devoted to identifying limiting factors for the biosynthesis of VOO aroma compounds. Taking into account that nonesterified polyunsaturated fatty acids seem to be the main substrates for olive LOX activity (6), the aim of the present work was to establish the limiting factors affecting the biosynthesis of VOO aroma compounds through the LOX pathway by studying the relationship between the content of polyunsaturated fatty acids during the process to obtain VOO and the oil aroma compound profile. MATERIALS AND METHODS Plant Material. Olive fruits (Olea europaea L.) cultivars Picual and Arbequina were harvested in CIFA Cabra-Priego orchards (Cabra, Córdoba, Spain) at ripe stage, maturity index (MI) 5, according to Uceda and Frias (19). Olive Oil Extraction. Olive oil extraction was performed using an Abencor analyzer (Comercial Abengoa, S.A., Seville, Spain) that simulates at laboratory scale the industrial process of VOO production. Milling of olive fruits (1 kg) was performed using a stainless steel
10.1021/jf071202i CCC: $37.00 2007 American Chemical Society Published on Web 08/21/2007
7870
J. Agric. Food Chem., Vol. 55, No. 19, 2007
Sánchez et al.
Table 1. Composition (%) of the TG and FFA Fractions of the Oils Obtained by Addition of Different Amounts of LA (mg/kg fruits) during the Oil Extraction Process Picual
Arbequina FFA
FFA
fatty acid
TG
0 mg
50 mg
100 mg
200 mg
TG
0 mg
50 mg
100 mg
200 mg
16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:1 22:0
11.21 1.39 2.98 76.82 6.13 0.68 0.43 0.25 0.11
11.95 1.35 5.68 69.30 9.01 1.99 0.37 0.19 0.17
11.33 1.29 5.19 61.01 19.03 1.64 0.19 0.11 0.20
9.52 1.17 4.46 54.18 28.55 1.46 0.39 0.15 0.13
8.02 1.23 3.49 45.93 39.25 1.42 0.36 0.17 0.11
14.47 2.38 1.46 69.98 10.34 0.67 0.32 0.28 0.10
26.96 3.67 8.16 54.24 2.91 2.12 0.64 0.85 0.43
17.64 2.32 5.69 40.31 31.88 1.43 0.36 0.13 0.25
16.47 2.75 3.32 31.77 44.34 0.88 0.23 0.11 0.13
10.79 1.30 2.84 21.71 62.61 0.15 0.28 0.10 0.21
hammer mill operating at 3000 rpm provided with a 5 mm sieve. The resulting olive pastes were immediately kneaded in a mixer at 50 rpm for 30 min at 30 °C. Centrifugation of the kneaded olive pastes was performed in a basket centrifuge at 3500 rpm for 1 min. After centrifugation, oils were decanted and paper-filtered. Samples for volatile and fatty acid analyses (0.5 g each) were stored under nitrogen at -18 °C until analysis. To increase the proportion of polyunsaturated fatty acids during the process to obtain the oil, different amounts of either LA or LnA in the range of 0–200 mg/kg fruit were added as sodium salts (20) to the olive fruits during the milling step. Duplicate experiments were carried out for each cultivar. Fatty Acid Composition and Free Acidity. Triaglycerols (TG) and polar compounds containing free fatty acids (FFA) from the oils were fractionated by silica column chromatography according to Waltking and Wessels (21). The fatty acid composition of the different fractions was analyzed in triplicate by gas chromatography (GC) after derivatization to fatty acid methyl esters with 2 N KOH in methanol for the TG fraction according to the IUPAC standard method (22) and with diazomethane in diethylether-saturated N2 for the FFA-containing fraction (23). A HP-6890 chromatograph equipped with a HP Innowax capillary column (polyethylene glycol, 30 m × 0.25 mm i.d., film thickness 0.25 µm; Hewlett Packard, United States) was used for the analysis of the methyl esters under the following temperature programme: 180 °C (4 min), 4 °C min-1 to 230 °C (15 min). Hydrogen was used as the carrier gas at a flow rate of 1 mL min-1. The temperature of both the split injector and the flame ionization detector was 250 °C. Free acidity in control oils was determined according to Annex II in EC Regulation EEC/2568/91 (24). Analysis of Volatile Compounds. Olive oil samples were conditioned to room temperature and then placed in a vial heater at 40 °C. After 10 min of equilibrium time, volatile compounds from headspace were adsorbed on a SPME fibre DVB/Carboxen/PDMS 50/30 µm (Supelco Co., Bellefonte, PA). The sampling time was 50 min at 40 °C. Desorption of volatile compounds trapped in the SPME fibre was done directly into the GC injector. Volatiles were analyzed in duplicate using a HP-6890 gas chromatograph equipped with a DB-Wax capillary column (60 m × 0.25 mm i.d., film thickness 0.25 µm; J&W, Scientific, Folsom, CA). Operating conditions were as follows: N2 as the carrier gas; injector and detector temperatures at 250 °C; and the column was held for 6 min at 40 °C and then programmed at 2 °C min-1 to 128 °C. Quantification was performed using individual calibration curves for each identified compound by adding known amounts of different compounds to redeodorize high oleic sunflower oil. Compound identification was carried out on a HRGC-MS Fisons series 8000 equipped with a similar stationary phase column and two different lengths, 30 and 60 m, matching against the Wiley/NBS Library and by GC retention time against standards. Volatile compounds were clustered into different classes according to the polyunsaturated fatty acid and the LOX pathway branch origin (see Table 3), and data were statistically evaluated using the Microsoft Excel 2002 software program. Chemicals and Reagents. LA, LnA, and reference compounds used for volatile identification were supplied by Sigma-Aldrich (St. Louis,
MO) except for (Z)-hex-3-enyl acetate, which was purchased from Givaudan Co. (Clifton, NJ), and (Z)-hex-3-enal, which was generously supplied by S.A. Perlarom (Louvaine-La-Neuve, Belgium). RESULTS AND DISCUSSION
Two olive cultivars, Picual and Arbequina, whose oils are quite different in terms of aroma profile and fatty acid composition (25, 26), were selected to study the limitation of substrate for the biosynthesis of VOO aroma compounds through the LOX pathway. For this purpose, the relationship between the content of nonesterified LA and LnA during the oil extraction process and the VOO aroma compound profile was studied. It is generally accepted that nonesterified fatty acids are the physiological substrates of plant LOX, and particularly olive LOX was reported to oxidize esterified fatty acid at a much lower velocity (
