Evaluación química de algunas semillas de cítricos, un residuo agroindustrial como nueva fuente potencial de aceites vegetales; Chemical evaluation of citrus seeds, an agro-industrial waste, as a new potential source of vegetable oils

313 0.8 and 21.0 mg/100 g. The predominant isomers were αand γ-tocopherol, with approximate equal amounts between about 0.4 and 17.5 mg/100 g. The total sterol contents of the oils were found between 1310.54 and 3986.58 mg/kg, with β-sitosterol as the predominant sterol that accounted for more than 70% of the total amount of sterols. Other sterols, campesterol (8.03-15.26%), stigmasterol (2.557.69%), ∆5-avenasterol (1.80-5.67%), cholesterol (0.832.70%) and chlerosterol (0.93-1.78%) were detected in most of the oils. The results of the present study indicate that the seed oils of Citrus fruits are considered to be a potential oil source due to their fatty acid composition and important tocopherol and sterol, and might be used for edible applications as well as the production of potential value-added products.


INTRODUCTION
The Citrus species, belonging to the family Rutaceae, is an annual plant that is widely distributed in the Mediterranean countries of the Middle East and Southern Europe but also grows abundantly in other warm climates worldwide.The plants of most species of Citrus are large evergreen shrubs or small trees, and their fruits are among the most important tree fruit crops in the world.Citrus fruits are of great economic importance because of their varied uses (Saidani et al., 2004).

Chemical evaluation of citrus seeds, an agroindustrial waste, as a new potential source of vegetable oils.
The seed oils from seven Turkish and ten Vietnamese varieties of Citrus fruits were examined for their fatty acid composition, tocopherols and sterol contents.The oil contents of the samples varied between 32.1 g/100 g and 58.8 g/100 g.The major fatty acid of the extracted seed oils was oleic (12.8-70.1%),followed by linoleic (19.5-58.8%)and palmitic (5.1-28.3%).Stearic, vaccenic, linolenic and arachidic acids were found at low levels.The total content of vitamin E active compounds in the oils ranged between B. MATThAUS AND M.M. ÖzCAN available resources and result in the production of various products for food or feed (Abdel-Rahaman, 1980;Lazos and Servos, 1988).
Plant seeds are important sources of oils for nutritional, industrial, and pharmaceutical applications.Lipids are important for the development of cells as structural components, functional compounds and for the storage of energy.The study of oilseeds for their minor constituents is useful in order to use both oil and the minor constituents effectively (Kamel et al. 1982;Aitzetmüller, K. 1993).Some seed oils from other plants are already used for several purposes like blending with modified nutritional values, as ingredients in paint and varnish formulations, lubricants, pharmaceuticals, organic pesticides, plastics, dispersants, textiles, soaps, surface coating and oleo-chemicals, as well as oils for cosmetic purposes (Muuse et al., 1992;Watkins, 1999;hosamani and Sattigeri, 2000).Two of the main constituents of Citrus seeds are seed meal, consisting of protein and seed oil.Citrus seeds contain about 36% oil and 14% protein (Braverman, 1949;El-Adawy et al., 1999).The high oil content makes the seed material interesting for the production of oil.The fatty acid composition of some Citrus seed oils has been identified by Saidani et al., 2004;El-Adawy et al., 1999;Abdel-Rahaman et al., 1980;Lazos and Servos, 1988;habib et al., 1986.Previous studies on some Rutaceae seed oils have been reported by several authors (henderson and Kesterson, 1963;Abdel-Rahaman, 1980;habib et al., 1986;Lazos and Servos, 1988;Trandjiiska and Nguyen, 1989;helmy, 1990;Ajewole and Adeyeye, 1993;hassanein, 1999).Limited studies on the occurrence and variability of have been conducted (Abdel-Rahaman, 1980;habib et al., 1986;Lazos and Servos, 1988;Trandjiiska and Nguyen, 1989;Saidani et al., 2004;Reda et al., 2005).In many plants the seed oil composition differs considerably from lipids contained in green photosynthetic tissue.Unususal fatty acids are often present in significant percentages in the seed oil (Smith, 1970).
The aim of this study was to investigate and compare the chemical properties of the seed oils of some Citrus species provided from several locations in Turkey and Vietnam concerning the fat content and the composition of fatty acids, tocopherols, and sterols., 2006.The skin and pulp were removed from the seeds, and the seeds were washed and cleaned in an air screen cleaner to remove immature and broken seeds.Air conditioning was used for the drying of the seeds.The skins of maturated seeds were decorticated by hand and then stored in polypropylene bags at 4 °C temperature.

Oil content
The oil content was determined according to the method ISO 659:1998(ISO, 1998).About 2 g of the seeds were ground in a ball mill and extracted with petroleum ether in a Twisselmann apparatus for 6 h.The solvent was removed by a rotary evaporator at 40 °C and 25 Torr.The oil was dried by a stream of nitrogen and stored at -20 °C until use.

Fatty Acid Composition
The fatty acid composition was determined following the ISO standard ISO 5509:2000(ISO 2000).In brief, one drop of the oil was dissolved in 1 mL of n-heptane, 50 μg of sodium methylate were added, and the closed tube was agitated vigorously for 1 min at 24 °C.After the addition of 100 μL of water, the tube was centrifuged at 4500 g for 10 min and the lower aqueous phase was removed.Then 50 μL of hCl (1 mol with methyl orange) was added, the solution was mixed, and the lower aqueous phase was rejected.About 20 mg of sodium hydrogen sulphate (monohydrate, extra pure; Merck, Darmstadt, Germany) were added, and after centrifugation at 4500 g for 10 min, the top n-heptane phase was transferred to a vial and injected into a Varian 5890 gas chromotograph with a capillary column, CP-Sil 88 (100 m long, 0.25 mm ID, film thickness 0.2 μm).The temperature program was as follows: from 155 °C; heated to 220 °C (1.5 °C min -1 ), 10 min isotherm; injector 250 °C, detector 250 °C; carrier gas 36 cm s -1 hydrogen; split ratio 1:50; detector gas 30 mL min -1 ChEMICAL EVALUATION OF CiTRuS SEEDS, AN AGRO-INDUSTRIAL WASTE, AS A NEW POTENTIAL SOURCE OF VEGETABLE OILS hydrogen; 300 mL/min air and 30 mL/min nitrogen; manual injection volume less than 1 μL.The peak areas were computed by the integration software, and percentages of fatty acid methyl esters (FAME) were obtained as weight percent by direct internal normalization.

Tocopherols
For the determination of tocopherols, a solution of 250 mg of oil in 25 mL of n-heptane was used for the hPLC.The hPLC analysis was conducted using a Merck-hitachi low-pressure gradient system, fitted with an L-6000 pump, a Merckhitachi F-1000 fluorescence spectrophotometer (detector wavelengths for excitation 295 nm, for emission 330 nm), and a D-2500 integration system.Samples in the amount of 20 μL were injected by a Merck 655-A40 autosampler onto a Diol phase hPLC column 25 cm 3 4.6 mmID (Merck, Darmstadt, Germany) used with a flow rate of 1.3 mL min -1 .The mobile phase was n-heptane/ tert-butyl methyl ether (99 1 1, v/v (Balz et al., 1992).

Sterols
The sterol composition of the oils was determined following ISO/FIDS 12228:1999 (E) (ISO, 1999).In brief, about 250 mg of oil were saponified with a concentration of ethanolic potassium hydroxide by boiling under reflux.The unsaponifiable matter was isolated by solid-phase extraction in an aluminium oxide column (Merck, Darmstadt, Germany) in which fatty acid anions were retained and sterols passed through.The sterol fraction was separated from the unsaponifiable matter by stationary phase for thin-layer chromatograph (Merck, Darmstadt, Germany), reextracted from the TLC material, and then the composition of the sterol fraction was determined by GLC using betulin as internal standard.The compounds were separated on an SE 54 CB (Macherey-Nagel, Düren, Germany; 50 m long, 0.32 mm ID, 0.25 μm film thickness).Further parameters were as follows: hydrogen as carrier gas, split ratio 1:20, injection and detection temperature adjusted to 320 °C, temperature program, 245 °C to 260 °C at 5 °C min -1 .Peaks were identified either by standard compounds (β-sitosterol, campesterol, stigmasterol), by a mixture of sterols isolated from rape seed oil (brassicasterol), or by a mixture of sterols isolated from sunflower oil (∆7-avenasterol, ∆7-stigmasterol, and ∆7-campesterol).

Statistical analyses
Results of the research were analyzed for statistical significance by analysis of variance (Püskülcü and İkiz, 1989).This research was performed in three duplicates.

Fatty acid composition
The fatty acid compositions of the seed oils from both the Turkish and Vietnamese citrus species are given in Table 2.According to Table 2, there is a wide variation in fatty acid compositions of citrus seeds belonging to these countries.
The major fatty acid compositions of the Turkish citrus seed oil are oleic, linoleic and palmitic acids.Turkish citrus oil contained a considerable amount of oleic acid (18.3-70.1%)and linoleic acid (19.5-58.9%).Palmitic and stearic acids were detected between 5.1% to 28.3% and 0.3% to 5.9%, respectively.A lower amount of vaccenic acid (0.6 to 1.3%) was found in Turkish citrus oils.The high content of saturated fatty acids, consisting of palmitic acid, which amounted to between 5.1% (C.aurantium (bitter) to 28.3% (C.nobilis) is nutritionally unfavorable.
Lower amounts of vaccenic and stearic acids were found.Linoleic (23.5% to 46.4%) and oleic acids (12.8% to 36.3%) were the most predominantf fatty acids found in the Vietnamese citrus seeds.Palmitic and stearic acids were detected in the Vietnam oils at 9% to 27.6% and 2.1% to 5.5% of the total fatty acids, respectively.As another quantitatively interesting saturated fatty acid, the Vietnamese oil contained linolenic acid in a range from 0.5% (C.nobilis var.nobilis) to 8.2% (C.japonica).The major fatty acids in the Vietnamese citrus oil had similar levels to those of the Turkish citrus oil.

Citrus paradisi
The citrus seed oils investigated in the present study exhibited high degrees of unsaturation.The major fatty acids found in the oils from both countries were oleic, linoleic and linolenic acids.The data indicated that some of the cultivars of citrus grown in both countries are significantly different from each other in the levels of individual fatty acids.It is well known that the fatty acid composition of citrus seeds is influenced by the climate in which they are grown.Based on the results obtained the fatty acid composition of citrus seed oil showed that it probably falls in the linoleicoleic acid oils category.

Tocopherols
In addition to essential fatty acids, seed oils are excellent sources of Vitamin E (tocopherols).Tocopherols are natural antioxidants with biological activity.The main biochemical function of tocopherols is believed to be the protection of polyunsaturated fatty acids against peroxidation (Beringer and Dompert, 1976;Kamal-Eldin and Andersson, 1997).The group of vitamin E active compounds comprises, in addition to the tocopherols, four tocotrienols and plastochromonal-8, which also have antioxidative and biological activities, but less than the tocopherols.
The tocopherol contents of citrus seed oils belonging to Turkish and Vietnamese varieties are given in Table 3.The total amount of tocopherols of Turkish citrus oil ranged between 2.0 mg/100g (C.nobilis) and 21.0 mg/100g (C.paradisi).Turkish citrus oil was characterized by much higher amounts of α-and γ-tocopherols.The highest α-tocopherol contents were in C. paradisi (17.5 mg/100g), C. limon (Kütdiken) (13.0 mg/100g) and C. limon (ınterdonato) (10.9 mg/100 g).Very high levels of γ-tocopherol were only present in C. aurantium (3.1 mg/100g).The α-tocopherol contents of bitter and sour C. sinensis oil were found high compared with other Turkish citrus oils.
All the seeds tested showed differences in their total tocopherol and tocotrienol contents.Tocopherol composition was dominated by α-and γ-tocopherol for the oils from both Vietnam and Turkey.It is obvious that α-and γ-tocopherols are the major vitamin E active components in all citrus seed oils (Beringer and Dompert, 1976;Kamal-Eldin and Andersson, 1997).The predominant tocopherols in citrus seed oils were α-and γ-tocopherol, which were found in different amounts.The contents of plastochromano-8 in the citrus seed oils from both countries was also in very small amounts.The tribe Rutaceae included two main groups for tocopherol composition, characterized by high concentrations of α-and γ-tocopherol, respectively.

Phytosterols
The sterol compositions of the citrus seed oils are presented in Table 4.The concentrations of total sterols in the Turkish citrus oils varied from 2038.1 mg/kg (C.sinensis (bitter)) to 3574.1 mg kg -1 (C.limon (interdonato).The composition of sterols in the Turkish citrus oil is dominated by β-sitosterol, which accounted for about 75% of the total sterols in the oil.This is typical of many vegetable oils in which β-sitosterol is predominant.Other sterols had campesterol (about 11% of the total sterols), ∆5-avenasterol (about 3%), and stigmasterol (about 4%).β-Sitosterol was the major sterol, present in all products and ranging from 74.9 mg/kg (C.limon (interdonato) to 78.3 mg kg -1 (C.sinensis (bitter).The campesterol contents ranged from 8.4 mg kg -1 (C.sinensis (bitter) to 15.3 mg/kg (C.paradisi).
Citrus seed oil can be utilized as a source of oil for human consumption and the production of potential value-added products.The composition of sterols in Citrus oil is dominated by β-sitosterol, which accounted for about 70% of the total sterols in the oil.This is typical of many vegetable oils.In general, the campestenol, stigmasterol, and sitostenol contents of the Vietnamese samples were higher than those of the Turkish citrus oils.As a result of the high oil content, the seeds of citrus species seem to be an interesting source for the production of vegetable oil.The results indicate that the oil contains linoleic acid as the major fatty acid accompanied by oleic acid.The content and composition of tocopherols are comparable to those of other sources such as sunflower, corn or rapeseed oil, and therefore the use of citrus oil in nutritional or technological applications is possible.In addition, minor compounds like vitamin E active compounds and sterols are available in relatively small amounts in comparison to other commonly used vegetable oils.Thus, tocopherols present in high concentrations in citrus seed oils are expected to offer some protection during storage and processing.Further studies on these unconventional tropical plant seeds will determine whether some of the seeds described may be used as an addition to indigenous food supplies.

Chemical evaluation of citrus seeds, an agro-industrial waste, as a new potential source of vegetable oils
Institute for Lipid Research, Federal Research Center for Nutrition and Food, Piusallee 68/76, 48147 Münster, Germany b Department of Food Engineering, Faculty of Agriculture, University of Selcuk, 42031 Konya, Turkey *Corresponding author: mozcan@selcuk.edu.tr By B. Matthaus a and M.M. Özcan b, * a

Table 1 Oil contents of citrus seeds Samples Oil content [g/100 g]
). C. aurantium subsp amara had the highest oil content (58.8%), followed by C. sinensis (bitter) (57.4%) and C. sinensis Osb (sour) (56.5%).In addition to these, the oil contents of Citrus limon (kütdiken), C. limon (interdonate) and C. nobilis var.nobilis had almost the same amounts.The results reveal that the seed from C. limon has the lowest oil contents.The oil contents of the seeds from Vietnam citrus were established between 32.1% (C.aurantifolia) and 54.8% (Citrus grandis).The oil contents of C. aurantifolia, C. limonia, C. grandis and C. nobilis were found to be lower than those of the other Vietnam citrus seeds.The oil contents of the seeds from Turkey Citrus were found high compared with the samples from Vietnam.It is apparent that C. aurantium (bitter orange), C. sinensis (sour), C. sinensis (Silifke), Buoi Da Gai and C. nobilis Lour var.nobilis could be characterized by their high content of oil %.Abdel-Rahaman (1980) has established that the seeds of different Citrus species grown in Egypt contained *mean (n 5 3) 6 standard deviation.

Table 2 Fatty acid compositions of citrus seed oils [%]
(Waheed et al., 2009)F CiTRuS SEEDS, AN AGRO-INDUSTRIAL WASTE, AS A NEW POTENTIAL SOURCE OF VEGETABLE OILS namely Citrus sinensis, Citrus paradisi, Citrus aurantium were investigated by thin layer and gas chromatography.Palmitic, oleic and linoleic acids were the major components in all the lipids and lipid classes studied(Waheed et al., 2009).

Table 3 Tocopherol contents of citrus seed oils (mg/100g)
β-Sitosterol contents were found in low amounts in all citrus seed oils compared with plum, apricot and peach kernel oil(hassanein, 1999); but according to the results of habib et al., (1986) they were higher.According to Munshi

Table 4 Sterol contents of citrus seed oils
B. MATThAUS AND M.M. ÖzCAN