Characterization of Acorn Fruit Oils Extracted from Selected Mediterranean Quercus Species

a Department of Applied Science, Al-Huson University College, Al-Balqa’ Applied University, Jordan b Faculty of Agriculture, University of Jordan, Jordan c Department of Clinical Nutrition and Dietetics, College of Health Sciences, University of Sharjah, Sharjah, UAE d Dept. of Nutrition and Food Technology, Faculty of Agriculture, Jordan University of Science and Technology, Jordan * Corresponding author: Walmm_53@yahoo.com


INTRODUCTION
Oak acorns, one of the species of Quercus genus, are of vital importance for both humans and animals.Homo sapiens have been using acorns as food for thousands of years, wherever oaks exist.Oak is also considered an edible fruit in some Mediterranean countries as it used in ice cream and other desserts and liqueurs (León-Camacho et al., 2004;M'Hrit et al., 1998;Bainbridge, 1986).In Algeria, Morocco, and in the eastern U.S.A, acorn oil has been used as cooking oil and as a salve for burns and injuries (Smith, 1950;Hedrick, 1919).In Jordan, oak acorns have been used as food either directly or as an ingredient in products such as bread, cake, and coffee (Rababah et al., 2008;Jacknis, 2004).In addition, oak acorns have been extensively under exploitation as a fodder for cattle (Bouderoua and Selselet-Attou, 2003;Nowar et al., 1994;Al-Jassim et al., 1988).
Starch is the main component of acorns, amounting to over 55% of the kernel (Rababah CHARACTERIZATION OF ACORN FRUIT OILS EXTRACTED FROM SELECTED MEDITERRANEAN QUERCUS SPECIES samples collected were healthy, ripe fruits that had fallen to the ground without mechanical damage or insect deterioration.The collected samples were shelled, the kernels (cotyledons) were crushed and dried in an oven at 50 °C for 3 days, and then the dried cotyledon were ground into ball meal and homogenized with pestle and mortar.

Oil extraction
The oils of the dried powder of cotyledon from the three species were extracted by the Soxhlet method using petroleum ether (bp 40-60 °C), and were expressed on a dry-weight basis.(1995) methods were used to determine the iodine, density and saponification values.The refractive index at 20 °C was determined with a refractometer (Reichert.Abbe Mark III.USA), K 232 and K 270 extension coefficients were calculated from the absorption at 232 nm and 270 nm, respectively with a spectrophotometer (Spectro UV-VIS Double beam PC, UVD-2950.Labomed, INC.USA) using a 1% solution of oil in cyclohexane.

Fatty acid methyl esters
The fatty acid composition was analyzed by GLC after derivatization to fatty acid methyl ester (FAMEs) with 2M KHO in methanol According to (IUPAC, 1995).The analysis of FAMEs was carried out with a GLC (model GC-2010, Shimadzu.Inc. Koyoto, Japan) equipped with a flame ionization detector and capillary column (Restek, Rtx-225, USA, crossbond 50%-cyanopropylmethyl 50%-phenylmethyl polysiloxane, 60 m, 0.25 mm/D, 0.25µm df).The column oven temperature was 165 °C for 10 min, increased to 185 °C, 1 °C min -1 and kept at 185 °C for 1 min, then increased to 220 °C, 3°C min -1 and kept at 220 °C for 20 min.The injector temperature was 240 °C and the flame ionization detector temperature was 260 °C.The flow rate was 0.8 mL min -1 with a helium and split ratio of 80.The (FAMEs) were identified using a chromatogram of fatty acid standards (12-24 carbon atoms) (Sigma)
Several studies have reported that the oil content of various white species of Quercus, did not exceed 12% (Rababah et al., 2008;Bernardo-Gil, 2007;Özcan, 2007;León-Camacho et al., 2004;M'Hrit et al., 1998), which indicates that acorn oil cannot be considered as commercial edible oil, Bernardo-Gil et al., (2007) reported that the Portuguese legislation classified the acorn oil as an alimentary oil, although no industrial oil has been produced.However, some of Q. Erthrobalanus spp.(black and red oak group) contain 30.8-31.3% oil, being similar to or higher than the best oil olives (Ofcarcik, 1971).In addition, acorn oils have good nutritional quality with a flavor comparable to that of olive oil (Özcan, 2007;Lopes et al., 2005;Bainbridge, 1986).It has been reported that the oleic acid and linoleic acid content in 16 acorns of Quercus taxa from Turkey were between 53-65% and 24.2-49.1%,respectively (Özcan, 2007).This justifies the fact that acorn oil should enjoy the respect of dietary reference for fatty acid intake, particularly linoleic acid, which is considered the most significant and valuable benefit to human health (Horrobin, 1983).
Furthermore, phenolic compounds and tocopherols, as natural antioxidants, are present in remarkable amounts in acorn fruits (Rakic´ et al., 2007;Lopes et al., 2005).Therefore, acorn oils might exhibit oxidative stability and nutritional values similar to those of olive oil.
Oaks (Quercus spp.) are widely distributed throughout the Mediterranean region, and Jordan, has recently designated the Quercus aegilops as the national tree (MoA, 2003).The nutritional profile of oak acorn species in Jordan is lacking in the literature and its exploitation so far is not sufficient, particularly in human diets.Therefore, this study attempts to document the oak acorn oil content, crude oil physical and chemical constants, fatty acid profile, and some minor components, that commonly exist in oils for three species of acorns widely distributed throughout Jordan Quercus aegilops (QA), Quercus infectoria (QI), Quercus calliprinos (QC).It is also aimed at evaluating the nutritional value of the oil from the three acorn species as well as the potential use of these oils as a natural source of antioxidative substances, phytochemicals for the pharmaceutical, and food industry.

Raw materials
Samples of acorn fruits were randomly collected from white oak trees of three species (QA, QI and QC), from their natural distribution areas in the north of Jordan during the maturation season between November and January of 2009-2010.The W.M. AL-ROUSAN, R.Y. AJO, K.M. AL-ISMAIL, A. ATTLEE, R. R. SHAKER AND T. M. OSAILI from 200 to 300 °C at a rate of 3 °C min -1 and held at this temperature for 10 min; helium was the carrier gas at a flow rate of 0.8 mL min -1 .The injector and detector temperatures were 300 °C.

Statistical Analysis
A statistical analysis of the experiment results was performed on the Microsoft office excel program.All experiments were carried out in triplicate; results are expressed as means ± standard deviations (SD).

RESULTS AND DISCUSSION
The oil contents of acorn cotyledons of the species QA 7.51%, and QI 6.57%, were about two times higher than that of QC 3.40%.This result agrees with the results reported by (Özcan, 2006 andLopes et al., 2005) who found that the oil content of some species of acorn ranged between 3.8 and 9.1%.This indicates that the oil content of these acorn species is low for commercial production as cooking or frying oils.However, there is a possibility of using this kind of oil as a supplement ingredient in products such as baked goods that may help improve their quality and considerably reduce their costs .In addition, acorn oil might be considered similar to other plant oil sources because of its health benefits or industrial and pharmaceutical applications, as in the case of amaranth and wheat germ, whose oil contents are about 6.34% and 10%, respectively (León-Camacho et al, 2001;Sonntag, 1979).
The physical and chemical constants estimated in the oil extracted from the three Q.species are shown in Table1.As revealed, acorn oil is similar to olive oil in most specifications, except UV absorption at 270 nm, which ranged from 1.495-2.037.This range is several times higher than that reported for olive oil (0.3).This can be attributed to the high positive correlation between the oil content of bitter compounds and the UV absorbance (García-Mesa et al., 1992).Some of these compounds are tannins which might be dissolved in the acorn oil during extraction, thus the UV absorption at 270 nm which we obtained is rational.
Based on iodine value, which ranged from 75-88, acorn oil might be classified as a non-drying oil; Duel (1951) proposed that an iodine value above 100 means an oil is to be classified as drying while below 100 is non-drying.The obtained oils were clear yellow in QI, while the color was yellow-brown for QA, and QC.On the other hand, the saponification value for the three species was slightly higher than that of olive oil, a result which is in good agreement with the reported findings by (Mamedova et al., 1993).
Table 2 gives an overview of the content of individual fatty acids (FAs) and their proportions in the three species of Quercus under study.As shown, the most abundant fatty acids were oleic 55.6, 56.1, 53.3%, followed by linoleic 21.7, 21.3,

Phenolic compounds
The total phenolic content in acorn oils was determined using the Folin-Ciocalteu method (Gutfinger, 1981).Briefly, 10 g acorn oil were dissolved in 50 mL hexane.Twenty mL of aqueous methanol (60%) were added and mixed vigorously for 2 minutes.The methanolic phase was removed and placed in a beaker each time after the two phases were separated.The combined extracts were laid out to dryness in a vacuum rotary evaporator at 70 °C.The residue was dissolved in 1 mL methanol.0.1 mL from the methanolic extract was placed into a 10 ml volumetric flask.5 mL distilled water and 0.25 mL Folin Ciocalteau (2N) were added and mixed well for 3 min.One mL of Na 2 CO3 (about 35%) was added and the flask was filled with distilled water up to the mark.The specific absorbance of the blue color formed was measured, after 1 hour, at 725 nm (Spectro UV-VIS Double beam PC, UVD-2950.Labomed, INC.USA).A reference curve was prepared using gallic acid as the most representative phenolic standard, and expressed as mg gallic acid g -1 of oil.

Sterols
Acorn oil samples (five grams) were saponified according to Frega et al., 1992, with 1 mg α-cholestanol as internal standard with 50 mL of 2N KOH in methanol for 1 h under reflux.The unsaponifiable fraction was extracted three times with 80, 70 and 60 mL diethyl ether.The pooled extract was then washed several times with 50 mL water until the washing solution became colorless with phenolphthalein.The solvent was removed under low pressure using a vacuum rotary evaporator.The unsaponifiable fraction was dried under nitrogen flow, dissolved in chloroform (10% solution, w/v) and loaded onto 10 cm of a silica TLC plate (about 20 µL of chloroformic solution/ cm); a spot containing sterol standards ((β-sitosterol) was loaded onto the same TLC plate, so as to correctly identify the sterol band.The mobile phase was a mixture of n-hexane-diethyl ether (65:35, v/v).The sterol TLC band was visualized at 254 nm, after being sprayed with a 0.2% ethanolic solution of 2',7'-dichlorofluorescein sodium salt.The sterol band was then scraped off, extracted twice with 5mL of diethyl ether and the solvent was evaporated under nitrogen flow at room temperature.The solution containing the sterol TLC band was then silylated with 200 µL of a pyridine-hexamethyldisilazanetrimethylchlorosilane (9:3:1, v/v) mixture; after 15-20 min at room temperature, the sample was evaporated to dryness under nitrogen flow and dissolved in 100 µL of n-hexane.The derivatized unsaponiables were analyzed with a Shimadzu GLC (model 2010, Japan) coupled to an FID detector.They were separated on a GC non-polar column (Restek, USA, cross-bond 5%-diphenyl 95%-dimethyl polysiloxane, 30 m, 0.25 mm id, 0.1 µm df).The oven temperature was programmed CHARACTERIZATION OF ACORN FRUIT OILS EXTRACTED FROM SELECTED MEDITERRANEAN QUERCUS SPECIES particularly, in terms of the three abundant FAs (oleic, linoleic and palmitic acid), which together constituted 95% of the total fatty acids.The linoleic acid content of acorn oil samples is slightly higher than that of olive oil, therefore, from a nutritional point of view, acorn oil may be considered healthier for the human diet.
The ratio of the unsaturated fatty acids to the saturated fatty acids was about 3.9:1; this high ratio may be attributed to the high content of linoleic acid.
It is well-known that natural antioxidants such as tocopherols have a positive correlation with the unsaturated fatty acids (León-Camacho et al., 2004). 23.4%, and palmitic 18.7, 17.8, 18.4% in the acorn oil of QA, QI, and QC species, respectively.The minor variations in fatty acid levels observed between our three species and other species (performed by other investigators) might be related to the difference in acorn varieties, environmental conditions, and oak acorn maturity.
The oleic and linoleic acid percentages were in accordance with earlier findings (León-Camacho et al., 2004;Bouderoua and Selselet-Attou, 2003), whereas the palmitic acid content was higher than reported.
As listed in table 2, the fatty acid composition of acorn oil is comparable to that of olive oil,  respectively.Certainly, this amount of phenolics will increase the oxidative stability of the oil.Since no prior experiment on the acorn oil content of phenolic compounds has been reported, the reliability of the results in the present study is difficult to establish.However, a related review of the literature depicts that the usual value for virgin olive oil ranges between 100 and 300 mg kg -1 (Andrewes et al., 2003).This indicates that the acorn oils contain significant amounts of phenolic compounds.Sterols are the major constituents of the unsaponifiables in vegetable oils.They have cholesterol-lowering properties, and may protect from heart disease.Recently they are incorporated into a growing spectrum of functional foods, as they are added to dairy products, bakery goods, sausages, and fruits juices (Garcia-Llatas et al., 2011).
Table 4 delineates the distribution of sterols in the oil from the three acorn species in this study and other oils.The total sterol contents were Table 3 shows a comparison of the tocopherol contents in the oil from the three spp.selected in the current study and other vegetable oils.The findings reveal that the total content of tocopherols in the present study ranged from 1440-1783 mg kg -1 oil, which is higher than its content in other vegetable oils.This implies that the oil from the three species is expected to have resistance to oxidation.The main tocopherol was γ-tocopherol, forming almost 90% of the total tocopherol content.This difference in the total tocopherol contents of the three species with olive and peanut oils may be attributed to the higher content of γ-tocopherol in acorn oils.On the other hand, α-tocopherol content was comparable with the content in olive or peanut oil.
Phenolic compounds are a natural antioxidant agent that exists in oil.The amount of phenolic compounds extracted during production is fundamental for the oxidative and nutritional quality of the oil.The total phenolic compound contents were 84, 95, 109 mg kg -1 for the oils of QA, QI, QC,

Table 1 Physical and chemical parameters of Quercus spp. oils
Each value is the mean of three replications followed by SD.