294 DOI 10.1002/ejlt.200300922 Eur. J. Lipid Sci. Technol. 106 (2004) 294 300 Juan Carlos Bada a Manuel León-Camacho b Manuela Prieto c Leocadio Alonso a a Instituto de Productos Lácteos de Asturias (CSIC), Villaviciosa, Asturias, Spain b Instituto de la Grasa (CSIC), Sevilla, Spain c Escuela Técnica Superior de Ingenieros Industriales, Universidad de Oviedo, Gijón, Asturias, Spain Characterization of oils of hazelnuts from Asturias, Spain Nine varieties of virgin hazelnut oil from different autochthonous cultivars from distinct locations in Asturias (Spain) were obtained by extraction under a pressure of 280 kg/ cm 2 at a temperature of 45 7C. The extracted oils were separated and filtered. The overall composition (percentages of husk, oil and moisture) of unhusked seeds was determined as well as moisture, ash, total sulfur and heat power of the husks for their possible use as biomass. Recovery of the oil extraction procedure for its application to the production of extra virgin edible hazelnut oil was studied. Fatty acid, tocopherol and sterol compositions as determined by capillary column gas chromatography and triglyceride compositions as determined by high performance liquid chromatography are reported in this study. All values were compared with a Turkish and a French hazelnut oil, with solvent-extracted hazelnut oil and with a 75/25 blend. A cluster analysis was performed as a criterion to differentiate the different hazelnut oils as groups. Keywords: Oil, hazelnut, fatty acid, tocopherols, sterols, triglycerides. Research Paper 1 Introduction Spain is one of the main hazelnut-producing countries in the world after Turkey and Italy. World production is in the order of 775 000 t, with Spain producing approximately 18 000 t, which represents 2.32% of worldwide production. With a production of 16 400 t, Catalonia is the leading region in Spain [1]. The negret variety is the most representative, followed by Gironella and Cupla [2]. Asturias represents a production of 250 t originating from different autochthonous varieties such as Amandi, Casina, Espinaredo and Quirós as well as from other international varieties principally centered around the localities of Piloña, Amieva, Caso and Llanes [3]. Several authors have studied the physico-chemical characteristics and nutritional values of different hazelnut oils [2, 4 7], the influence of environmental conditions and geographical origin [2, 6, 7], and the oxidative stability of virgin oil extracted by cold pressure [8]. Enzymatic activity [9] has also been reported. The present work presents a detailed and comprehensive study of hazelnut oil from Asturias (Spain). The overall composition (oil, moisture and percentage of the seed) was determined as well as moisture, ash, total sulfur and heat power of the husks for their possible use as biomass. Fatty acid, tocopherol and sterol compositions as determined by capillary gas chromatography (GC) and triglyceride compositions as determined by high performance liquid chromatography (HPLC) are reported in this study. Correspondence: Juan Carlos Bada, Instituto de Productos Lácteos de Asturias-CSIC, Carretera de Infiesto s/n, 33300 Villaviciosa, Asturias, Spain. Phone: 134-985892131, Fax: 134-985892233; e-mail: jcbg@ipla.csic.es 2 Materials and methods 2.1 Samples Different autochthonous varieties harvested in Asturias (Spain) were collected from the locations of Piloña and Llanes, Asturias (Spain). 2.2 Pressing oil extraction Hazelnut samples without the husks, which had been previously removed by hand, were ground in an electrical grinder until a fine powder was obtained. All hazelnuts were of optimal ripeness and physical condition. Hazelnut powders were heated at 45 7C and pressed between twelve vertically stacked cylindrical steel plates, each of 12 cm i.d. and 1 cm thickness. Hazelnut powders were placed between the plates to a film thickness of 8 mm. All plates were pressed together at a maximum pressure of 280 kg/cm 2. The extracted oil was separated and filtered at 45 7C. 2.3 Solvent oil extraction Hazelnut samples were blanched and ground in an electrical grinder. Oil was extracted in a Soxhlet glass apparatus using hexane as solvent [10]. 2.4 Water content Moisture was determined by weight loss after heating in an oven at 105 7C in accordance with IUPAC methods [11].
Eur. J. Lipid Sci. Technol. 106 (2004) 294 300 Hazelnut oil from Asturias, Spain 295 2.5 Fatty acid analysis Fatty acid methyl esters (FAME) were analyzed by GC. FAME were extracted with n-heptane after cold methylation with 2 N KOH in methanol [12]. GC was performed with a HP-5890-II apparatus (Hewlett-Packard, Palo Alto, CA, USA) using a fused silica capillary SP-2380 column (60 m 6 0.25 mm, 0.2 mm film thickness). The oven temperature was kept at 160 7C for 13 min and was then raised to 190 7C at a rate of 1.5 7C/min and held isothermally for 20 min. The injector temperature was kept at 225 7C, while the detector temperature was 250 7C. Hydrogen (131 kpa inlet pressure) was used as carrier gas, while the make-up gas was nitrogen. 2.6 Triglyceride analysis Triglycerides were quantified by HPLC [13, 14]. The HPLC setup was composed of a low press quaternary pump HP-1050, a Rheodyne valve with a 20-mL loop, a thermostatic system of columns, and a refraction index detector HP-1074. The Lichrospher 100 RP-18 column (250 mm 6 4 mm, 4 mm) (Merck, Darmstadt, Germany) was used with an acetonitrile : acetone (1:1) mobile phase at 1.15 ml/min isocratic elution flow. The temperature of oven and detector was 40 7C. 2.7 Saponinification Oil samples were saponified following the procedure described by the Official Journal of the Commission of the European Communities [15] using potassium hydroxide and 5-a-colestane as internal standards. After saponification, the unsaponifiable matter was isolated by an extraction method using diethyl ether for the isolation of sterols. 2.8 Derivatization of the unsaponifiable matter Sterols were analyzed after trimethylsilyl derivatization (TMS) using pyrydine-hexamethyldisilazane-trimethylclorosilane as derivatization agents as proposed by the Official Journal of the Commission of the European Communities [15]. apparatus equipped with a split-splitless injector and a flame ionization detector. A fused silica capillary column HP-5 (30m 6 0.32 mm i.d., 0.25 mm film thickness) was used, with hydrogen (48.2 kpa inlet pressure) as carrier gas and nitrogen as the make-up gas. The oven temperature was isothermally maintained at 265 7C for 30 min. The injector temperature was 280 7C, while the detector was kept at 300 7C. The internal standard was a-cholestanol (Fluka, Buchs, Switzerland). 2.10 Tocopherol analysis Tocopherols were quantified by an HPLC instrument composed of a low press quaternary pump HP-1050, a Rheodyne valve with a 20 ml loop, a thermostatic system of columns, and a fluorescence detector RF-235 (Shimadzu, Kyoto, Japan). The column Lichrospher Si-60 (250 mm 6 4 mm, 5 mm thickness) (Merck, Darmstadt, Germany) was used with n-hexanol : iso-propanol (99:1) as mobile phase at 1 ml/min isocratic elution flow. Quantification was carried out by a calibration system based on standards [17]. 2.11 Squalene analysis Squalene was isolated with the waxes by a modified process of the standard analysis of waxes [18]. A low polarity solvent (n-hexane : diethyl ether, 98.5:1.5) was used in a silica gel column to separate squalene from esters of aliphatic alcohols and sterol esters [19]. Squalene (Sigma, St. Louis, MO, USA), with a response factor of 0.963, was the internal standard. This was quantified by GC [15] after oil fractionation by means of silica gel column chromatography (silica gel 60, particle size 0.063 0.2 mm) (Merck). An HP-5890-II apparatus equipped with a cold on-column injector with oven track system and a flame ionization detector was employed. The column used was a TRB-5 (15 m 6 0.32 mm i.d., 0.1 mm film thickness) (Tracer, Barcelona, Spain). The initial temperature of 70 7C was raised to 180 7C at a rate of 45 7C/min and then raised at a rate of 5 7C/min to 310 7C and held isothermally for 7 min. Hydrogen was the carrier gas (145 kpa inlet pressure), while nitrogen was the make-up gas. The detector temperature was 350 7C. 2.9 Sterol analysis Sterols were purified by thin-layer chromatography [16], with all series of compounds being quantified by GC. Quantification by GC was carried out with an HP-5890-II 2.12 Statistical analysis Statistica release 5.5 (Statsoft, Tulsa, OK, USA) was used in the statistical analysis of hierarchical clusters and factor analysis. Euclidean distance and Ward algorithms
296 J. C. Bada et al. Eur. J. Lipid Sci. Technol. 106 (2004) 294 300 were used to compute the cluster analysis. Factor analysis was applied under the general conditions of principal components analysis. 3 Results and discussion 3.1 Lipid composition Tab. 1 shows the overall composition (percentages of husk, oil and moisture) of the hazelnuts derived from the different production cultivars from Asturias. There is some variation in the oil contents of the different cultivars (50.84 67.14%). This variation is predominantly due to differences in location (including soil condition, elevation, rainfall and temperature) but may also be caused by the extraction procedure. Reports in the literature showed the oil content in niger seed to be in the range of 30 50% and 43.6 66.19% [5 9]. Results reported in this study are slightly higher than those reported by these authors. The husk composition of a representative sample of all the hazelnuts studied, including moisture, ash, total sulfur and heat power, is given by 12.05%, 0.84%, 0.02% and 4.93% of the dry sample, respectively. The major fatty acids determined by capillary GC are palmitic (C16:0), stearic (C18:0), oleic (C18:1) and linoleic (C18:2) acid (Tab. 2). The oleic acid content ranged between 78.54% and 83.47%. This range is within the data Tab. 1. Overall composition [%] of hazelnut samples. Values reported are the means of duplicate analyses. Sample Husk Oil Moisture 1 56.65 53.30 6.72 2 55.59 56.91 6.15 3 55.30 53.28 8.02 4 52.96 49.25 6.27 5 50.45 51.47 6.82 6 51.18 56.98 6.42 7 48.89 62.10 6.39 8 51.16 56.15 8.84 9 49.14 65.40 5.20 given in a study on the influence of variety and geographical origin on the lipid fraction of hazelnuts from Tarragona [7], which is the largest (80%) hazelnut-producing region in Spain. Linoleic (7.5 13.19%) and linolenic (0.1 0.25%) acids would be the most susceptible to lipid oxidation and would contribute to poor shelf life. These results are similar to those reported in a study on the analysis of factors influencing lipid oxidation in hazelnuts [20]. These authors reported values for linoleic acid (9.36 11.18%) and for linolenic acid (0.10 0.22%). These differences in fatty acid contents in oilseeds are specific and often depend on cultivar species [21]. Fatty acids were identified by comparing their retention times with those of standard fatty acids. Tab. 2. Total fatty acids composition of lipid fractions extracted from hazelnut samples {. Values reported are the means of duplicate analysis. Sample C16:0 C18:0 C16:1 C18:1 C18:2 C18:3 C20:0 C20:1 C22:0 [%] 1 5.38 2.84 0.19 79.05 12.14 0.15 0.15 0.14 0.02 2 5.52 2.50 0.22 80.41 10.94 0.13 0.14 0.14 0.02 3 5.53 2.81 0.21 80.90 10.16 0.13 0.14 0.13 0.03 4 5.48 2.81 0.23 80.89 10.22 0.13 0.14 0.13 0.02 5 5.14 2.74 0.17 80.92 10.62 0.14 0.13 0.14 0.03 6 5.01 3.05 0.17 80.71 10.64 0.12 0.16 0.15 0.03 7 4.80 2.21 0.17 82.43 10.02 0.12 0.11 0.14 0.02 8 4.85 2.26 0.17 82.35 10.00 0.12 0.11 0.14 0.03 9 5.27 2.15 0.20 81.68 10.32 0.12 0.12 0.14 0.02 10 5.28 2.15 0.22 82.98 9.04 0.11 0.11 0.14 0.02 11 5.27 2.19 0.20 81.35 10.61 0.12 0.12 0.14 0.03 12 5.38 2.21 0.21 83.03 8.83 0.13 0.11 0.14 0.02 13 5.28 2.16 0.18 81.98 10.03 0.11 0.11 0.13 0.02 14 5.25 2.26 0.21 78.54 13.19 0.25 0.13 0.17 0.03 15 5.43 2.76 0.20 83.47 7.55 0.08 0.13 0.16 0.20 { For samples 1 10 oils were extracted by pressure. For samples 11 and 12 oils were extracted by solvents. Sample 13 is a mixture oil 75/25 of samples 10 and 11. Samples 14 and 15 are commercially available French and Turkish oils.
Eur. J. Lipid Sci. Technol. 106 (2004) 294 300 Hazelnut oil from Asturias, Spain 297 Tocopherols (vitamin E isomers) are well-known natural antioxidants, and their presence in seeds is often correlated with a relatively high abundance of unsaturated fatty acids, as occurs in hazelnut oils. a-tocopherol was the major component of total tocopherols in the sample investigated. Its level varied from 207.98 to 618.76 mg/kg oil, constituting between 80 90% of total tocopherol content (Tab. 3). These results are in line with those reported in some hazelnut varieties harvested in Oregon (USA) [22] compared with other hazelnut oils from other countries (211.4 654.9 mg/kg oil). Although a-tocopherol was the predominant tocopherol isomer, b-tocopherol and g-tocopherol were also detected. Tocopherol levels are of interest as determining antioxidant activity [2], and the high content in unsaturated fatty acids in the hazelnut oil in this study would make the oil prone to oxidation. As regards the sterol content (Tab. 4), the predominant component was b-sitosterol, constituting 76.45 86.51% of total sterol. The next major components were campesterol and d-5-avenasterol, each constituting between 3 5% of total sterol. Other components such as stigmasterol, chlerosterol and d-7-avenasterol were each present at a level of 0.4 1.5%. A small amount of campestanol (0.2 0.4%) was present in all samples analyzed. There were no significant differences between the samples in terms of sterol composition. b-sitosterol was the predominant sterol in all hazelnut varieties studied [20], followed Tab. 3. Squalene [mg/kg oil] and tocopherol [mg/kg oil] composition of lipid fractions extracted from hazelnut samples {. Values reported are the means of duplicate analyses. Sample Squalene a-tocopherol b-tocopherol g-tocopherol 1 137.73 397.19 21.86 33.26 2 173.31 423.37 19.87 33.96 3 186.80 400.25 18.97 35.52 4 122.30 404.99 18.46 35.06 5 103.33 407.66 18.91 31.59 6 132.91 405.86 20.61 40.92 7 139.63 380.12 15.63 23.66 8 93.64 375.19 17.13 24.47 9 126.10 361.22 15.58 28.09 10 176.84 367.75 17.45 30.62 11 148.35 394.79 17.15 32.27 12 175.39 399.66 18.35 34.09 13 104.96 522.95 33.56 65.27 14 126.66 207.98 10.95 49.63 15 392.49 618.76 30.82 131.16 { For samples 1 10 oils were extracted by pressure. For samples 11 and 12 oils were extracted by solvents. Sample 13 is a mixture oil 75/25 of samples 10 and 11. Samples 14 and 15 are commercially available French and Turkish oils. Tab. 4. Total sterols and sterol compositions of lipid fractions extracted from hazelnut samples {. Values reported are the means of duplicate analyses. Sample Campesterol Campestanol Stigmasterol Chlerosterol b-sitosterol Sitostanol d-5- Avenasterol d-5-stigmastadienol d-7-stigmastenol d-7- Avenasterol Total sterols [%] [mg/kg oil] 1 4.67 0.23 1.07 1.91 83.29 2.16 4.11 0.91 0.89 0.76 2261 2 4.04 0.19 0.77 0.82 77.67 1.95 3.51 0.72 0.56 0.55 2490 3 4.68 0.21 1.08 0.89 85.27 1.79 3.47 1.04 0.66 0.69 2548 4 4.19 0.17 0.76 0.80 76.44 1.68 3.79 0.73 0.73 0.65 2273 5 4.37 0.18 0.82 0.97 78.95 1.77 2.99 0.63 0.55 0.57 3469 6 4.23 0.21 0.74 0.79 76.98 1.82 2.96 0.69 0.70 0.68 2102 7 4.48 0.24 0.90 0.87 85.22 3.08 3.12 0.71 0.81 0.56 1956 8 4.46 0.27 0.77 0.89 85.99 2.81 3.04 0.71 0.58 0.49 1575 9 4.32 0.23 0.78 0.81 85.80 2.34 3.25 0.79 0.68 0.42 1458 10 4.47 0.26 0.78 0.91 86.51 2.11 3.39 0.72 0.39 0.43 1772 11 4.46 0.26 0.78 0.91 86.44 2.10 3.38 0.71 0.39 0.43 1921 12 4.32 0.23 0.78 0.81 85.80 2.34 3.25 0.80 0.68 0.42 1642 13 4.35 0.25 0.74 0.89 87.16 1.70 3.31 0.82 0.42 0.37 2062 14 5.46 0.35 1.04 1.18 81.53 3.28 4.06 0.92 1.46 0.77 1534 15 4.88 0.40 0.85 1.07 82.46 2.92 4.01 0.97 1.56 0.87 1411 { For samples 1 10 oils were extracted by pressure. For samples 11 and 12 oils were extracted by solvents. Sample 13 is a mixture oil 75/25 of samples 10 and 11. Samples 14 and 15 are commercially available French and Turkish oils.
298 J. C. Bada et al. Eur. J. Lipid Sci. Technol. 106 (2004) 294 300 by campesterol, d-5-avenasterol and stigmasterol. Peaks were identified by comparing their retention times with those of standard sterols and standard oils such as olive, sunflower and soy [15]. Triglyceride analysis with critical pairs is defined as those structures that have the same equivalent carbon number (ECN), which is formulated as ECN = CN = 2n, where CN is the carbon number and n is the number of ethylenic double bonds of the triglyceride. The separation of triglyceride critical pairs is applicable to all hazelnut oils containing long-chain fatty acid triglycerides. We identified and quantified twelve triglycerides: LLL, OLLn, PLL 1 LnOO, POLn, OLO 1 POO, PLO, OOO, SOL 1 POO, PLS 1 POP, GOO, AOL 1 SOO and POS (P = C16:0; S = C18:0; O = C18:1; L = C18:2; Ln = C18:3; A = C20:0; B = C22:0). Peaks were identified by comparison of logarithms of selectives (log a) relative to the triolein in relation to the corresponding values of standard homogeneous triglycerides and standard oils such as olive, sunflower and soy [23]. The values for every variety are shown as percentages in Tab. 5. The main triglycerides are triolein (50.97 60.16%), followed by OLO 1 POO (12.57 19.05%). Triglycerides that have two or three saturated fatty acids show the lowest proportions, e.g. PLS (0.28 0.69%) and PLS 1 POP (0.46 0.81%). These results are comparable with those obtained in a study on the influence of geographical origin on the lipid fraction of hazelnuts from Catalonia [6]. 3.2 Statistics A multivariate exploratory technique was used to obtain a meaningful description of the similarity between virgin hazelnut oils and other oils (solvent-extracted, 75/25 blend of oil, and Turkish and French hazelnut oils). Cluster analysis, which is specifically designed to identify patterns in a multivariate data set, was applied to the major fatty acids, tocopherol, sterol and triglyceride compounds. As can be seen in Fig. 1, there are two main groups among all the variables with a high classification level. The first group (samples 1 6), extracted under pressure, has a similarity aggregation between samples and this group with the French and Turkish oils. In the second group, the hazelnut oils extracted using a solvent and the 75/25 blend (samples 11 13) had a higher level of aggregation in accordance with the mathematical representation. Basically, the profile of the virgin hazelnut oils differs from that of the solvent-extracted oils and the 75/25 blend. The results presented in this initial survey suggest that the fatty acid, sterol, tocopherol and triglyceride compounds could be used collectively as a criterion to differentiate hazelnut oils. 4 Conclusion From the results of the present work, it is clear that hazelnut oil from Asturias is a good source of polyunsaturated fatty acids. In addition, it is also a good source of vita- Tab. 5. Triglyceride composition of lipid fractions extracted from hazelnut samples {. Values reported are the means of duplicate analyses. Samples LLL OLLn PLL1 LnOO POLn OLO1 POO PLO OOO SOL1 POO PLS1 POP GOO AOL1 SOO POS [%] 1 0.96 0.10 4.86 0.75 18.59 3.59 50.97 13.29 0.67 0.19 5.45 0.53 2 0.84 0.11 4.02 0.68 17.81 3.46 53.60 13.17 0.51 0.22 5.07 0.48 3 0.71 0.10 3.79 0.68 16.34 3.15 54.77 13.26 0.77 0.35 5.50 0.53 4 0.65 0.10 3.81 0.69 16.22 3.18 54.19 13.19 0.66 0.40 6.01 0.65 5 1.03 0.12 4.03 0.74 15.66 3.23 54.68 13.09 0.86 0.12 5.94 0.44 6 0.89 0.13 4.19 0.70 18.04 3.34 52.68 12.43 0.52 0.28 6.11 0.69 7 0.80 0.12 3.77 0.55 15.86 1.82 58.66 12.56 0.51 0.39 4.42 0.46 8 0.76 0.16 3.75 0.56 15.95 2.68 57.09 13.46 0.46 0.23 4.42 0.38 9 0.72 0.10 3.25 0.63 14.52 2.66 58.79 13.30 0.81 0.20 4.61 0.36 10 0.77 0.12 4.13 0.86 16.64 3.01 56.39 12.05 0.47 0.43 4.60 0.49 11 0.73 0.11 3.88 0.58 16.55 3.02 56.01 13.36 0.49 0.34 4.48 0.39 12 0.69 0.10 3.34 0.56 15.00 3.28 58.27 12.90 0.51 0.20 4.64 0.45 13 0.80 0.11 3.81 0.70 16.33 2.98 56.63 12.85 0.54 0.23 4.58 0.51 14 1.67 0.23 5.58 0.82 19.05 3.97 51.70 11.18 0.65 0.30 4.51 0.27 15 0.60 0.07 2.73 0.40 12.56 2.40 60.16 13.56 0.60 0.25 6.03 0.56 { For samples 1 10 oils were extracted by pressure. For samples 11 and 12 oils were extracted by solvents. Sample 13 is a mixture oil 75/25 of samples 10 and 11. Samples 14 and 15 are commercially available French and Turkish oils.
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