The effect of some pre-roasting treatments on quality characteristics of pumpkin seed oil

– In this study, the pumpkin seed oils used were obtained from roasted and unroasted seeds of the two varieties called “ Nev ş ehir Çerçevelisi ” and “ Ürgüp Sivrisi ” , belonging to the species Cucurbita pepo . Oil extraction, from seeds subjected to three different pretreatments (unsalted, dry salted and wet salted) before roasting and sun-dried unroasted seeds, was carried out by means of a screw press. Pumpkin seed oils were analyzed for quality and antioxidant characteristics. The p-anisidine, peroxide and TBARS (thiobarbutiric acid reactive substances) values of oils obtained from sun-dried unroasted seeds and roasted seeds after wet salting process were lower than the other sample groups ( p < 0.05). The obtained oils were rich in Zn, Fe, Mg, Se, Ca, K and Na minerals, and palmitic, stearic, oleic and linoleic fatty acids. In the oils, b þ g tocopherols content ranged from 131.78 to 881.77 mg/kg oil, d -tocopherol content ranged from 3.22 to 8.67 mg/kg oil. Total phenolic matter content ranged from 1.35 to 3.62 mg gallic acid equivalent (EAG)/g. ABTS þ radical scavenging activity of the oils obtained from sun-dried-unroasted seeds and roasted seeds after wet salting process was determined higher than the other oil samples ( p < 0.05).


Introduction
Pumpkin is a member of Cucurbitaceae family, and various cultivars are grown throughout the world.C. moschata, C. maxima, C. pepo, and C. mixta are the most common commercially important species (Saucedo-Hernandez et al., 2011;Prevc et al., 2013).C. moschata is the most important cultivar in Tropical America.It has spread in central America and Mexico and these areas have become the center of variety.The origin of C. maxima, which is considered to be one of the oldest cultivars, is South America.This variety is being cultivated since pre-Colombian times (Veronezi and Jorge, 2012).The most common pumpkin species grown in Turkey is C. pepo.This species is mostly grown in Nevşehir, which is at the centre of Cappadocia, one of the seven regions named in the world heritage list.12 746 tons of the 41 612 tons of pumpkin seed produced as snack in Turkey were grown in Nevşehir (TUIK, 2016).This corresponds to approximately 31% of the total production.
Pumpkin seeds as a rich source of bioactive components have been used as functional food or medicine.The seeds are used to overcome enterozoa and prostate problems in Austria, Slovenia, Serbia, Argentina, India, Mexico, Brazil, Korea and China (Pericin et al., 2009;Adams et al., 2012;Kim et al., 2012).In addition, the seeds have hypoglycaemic effect as well as antioxidant, anticancer and anti-inflammatory effects (Veronezi and Jorge, 2012;Prevc et al., 2013;Rabrenovic et al., 2014).Pumpkin seeds are consumed as snacks in some countries, in raw form or in salted roasted form (El-Adawy and Taha, 2001;Prevc et al., 2013).Pumpkin seed and oil are also used as an ingredient in baked products and salads (Juranovic et al., 2003;Nawirska-Olszanska et al., 2013).
Pumpkin seed oil is a type of oil that is commercially available in Austria, Slovenia and Hungary (Pericin et al., 2009;Prevc et al., 2013).This oil is also an important product in Japan, Czech Republic, Germany, Romania, Italy, North and South Ukraine (Oloyede et al., 2012a).
Pumpkin seed oil is used as cooking oil in some North African and Middle-Eastern countries (El-Adawy and Taha, 2001).As the oil is rich in oleic and linoleic acids, this oil may be used as cooking and salad oil, and it is also suitable for margarine production (El-Adawy and Taha, 2001;Stevenson et al., 2007;Jiao et al., 2014).
Traditionally, pumpkin seed oil is obtained by pressing the roasted pumpkin seeds while the cold pressed pumpkin seed oil is obtained by pressing the unroasted pumpkin seeds.These two oils have different sensory characteristics such as color, taste and odor.Studies on oils obtained from roasted seeds reveal that these oils are rich sources of mono and polyunsaturated fatty acids, vitamins, minerals, phytosterols, pigments, pyrazine derivatives and phenolic compounds (Xanthopoulou et al., 2009;Kim et al., 2012;Veronezi and Jorge, 2012).For this reason, pumpkin seed oil is not only an edible oil but it also attracts interest as a potential nutraceutical oil.An essential fatty acid, linoleic acid, has been determined in pumpkin seed oil, which is an indication that this oil is an important type of oil in terms of health.El-Adawy and Taha (2001) expressed that linoleic acid reduces serum cholesterol level.Based on experimental studies, risk of lipid peroxidation may increase with high (> 11%) polyunsaturated fatty acids consumption, particularly when tocopherol intake is low.Therefore, the recommended range for polyunsaturated fatty acids is 6-11% of energy.There are some recommendations for the balance between omega-6 and omega-3 polyunsaturated fatty acids.In the German-Austrian-Swiss recommendations, the effective ratio is 5:1 and it is mentioned that an unbalanced ratio may affect the further metabolism (FAO, 2010).
No studies have been conducted to determine the effect of different pre-treatments applied to the seeds, before the roasting process, on the characteristic properties of the pumpkin seed oil.Thus, the aims of this study were: to determine the characteristics of the oil extracted from pumpkin seeds that have been sun-dried-unroasted, unsalted-roasted, dry salted-roasted and wet salted roasted; to reveal the most suitable process.

Materials
Two types of pumpkin seeds, called "Nevşehir çerçevelisi" and "Ürgüp sivrisi", belonging to C. pepo species were used in the study.After being procured from the manufacturer, these seeds have been cleaned and divided into four portions of 20 kg each.One of these portions was sun-dried during the month of October before being packed in a polyethylene bag and stored at 15 °C until oil extraction took place.This group was used as the sun-dried unroasted group.For the second portion, 2% water was added into the seeds and stirred, and then 5% salt (w/ w) was added and stirred thoroughly again to ensure a homogenous distribution.The mixture was then roasted at 175 °C for 15 min in a multilayer belt forced air convection roasting oven (Sevval SRX90, Denizli, Turkey).The seeds layer thickness was 2.5 cm.This group was designed as the dry-salted-roasted group.To the third portion, 2% water was added into the seeds and stirred, and then the mixture was roasted at 175 °C for 15 min in a multilayer belt roasting oven.This group was designed as the unsalted-roasted group.For seeds of the fourth portion, 3.3% water was added into the seeds and stirred, and then 4.3% salt was added and stirred thoroughly again to ensure a homogenous distribution.The mixture was kept overnight, and then roasted at 175 °C for 15 min in a multilayer belt roasting oven.This group was designed as the wet salted-roasted group.After the roasting processes, seeds were packed in polyethylene bags and stored at 15 °C until the oil extraction process took place.The moisture contents of Nevşehir çerçevelisi (sun-driedunroasted), Nevşehir çerçevelisi (wet salted-roasted), Nevşehir çerçevelisi (dry salted-roasted), Nevşehir çerçevelisi (unsalted-roasted), Ürgüp sivrisi (sun-dried-unroasted), Ürgüp sivrisi (wet salted-roasted), Ürgüp sivrisi (dry salted-roasted), and Ürgüp sivrisi (unsalted-roasted) prior to oil expression were determined 5.74 ± 0.03, 3.11 ± 0.28, 1.87 ± 0.34, 1.87 ± 0.24, 5.36 ± 0.02, 3.64 ± 0.66, 2.77 ± 0.56, 2.30 ± 0.12, respectively.

Oil extraction
Oil extractions from different sample groups were made in low temperature by using a screw press (Koçmaksan, KMS10, Izmir, Turkey).The head section of the screw press was heated to 75 °C before pressing started, by using a specially designed heating ring.When the system reached this temperature, seeds went through the press to produce oil.Heating process of the head section does not reflect on the temperature of extracted oil since the heaters are turned off after optimal seeds flow has been achieved.First samples of pressed oil were not collected for the safety reason.The temperature of the resulting oil was measured by using a thermocouple and it was observed that the temperature was below 50 °C.After 24 h sedimentation, the resulting oil was separated from the sediment by decantation and then the oil was placed into lidded glass jars and stored in a dark room.Since the seeds called as control groups were not subjected to any roasting process, the oil obtained from this group was called as cold-pressed oil based on Nederal et al. (2014).
2.3 Basic characteristics of the pumpkin seed oils 2.3.1 Specific gravity Specific gravities were determined at 25 °C by using 10 ml pycnometer.

Iodine value
Iodine values were determined according to Nielson (2003).

Saponification value
Saponification values of samples were determined according to Nielson (2003).

Free fatty acid
Free fatty acid analyses were determined according to Nielson (2003).For this purpose, 5 g oil were weighed into a 250-ml Erlenmeyer flask and then 100 ml neutralized ethanol (to 500 ml of ethyl alcohol were added 2 ml of alcoholic phenolphthalein and titrated with 0.1 N NaOH until a faint permanent pink color is maintained) was added.The content was titrated with 0.25 N NaOH until faint pink color persists for 1 minute.Percent free fatty acids were expressed as oleic acid.

Viscosity
Viscosity values of oils were determined at 25 °C by using Fungilab rotating viscometer (Fungilab Alpha series, Fungilab, Barcelona, Spain) with the number of R1 spindle at 60 rpm.

Peroxide value
Peroxide values were determined according to Ockerman (1985).

TBARS
TBARS values of oil samples were determined according to Ke and Woyewoda (1979).

Color
L*, a* and b* values of oils were determined by using a colorimeter (Konica Minolta Chroma Meter CR-400, Japan).

Mineral contents
Cu, Fe, Mg, Mn and Zn contents were determined by a Scott Spray chamber (Norwalk, CT, USA) ICP-MS (Perki-nElmer ELAN DRC-e model) following the microwave digestion process.With this purpose, 0.1 g oil sample was mixed in a 6 ml HNO 3 þ 2 ml H 2 O 2 digestion vessel.Microwave digestion process has been realized in three stages in microwave digestion system (Berghof Speedwave Four Microwave Digestion System, Eningen, Germany).In the first stage, the temperature has been increased to reach 170 °C from ambient temperature in 5 minutes.In the second stage, the temperature has been increased to reach 190 °C within 5 minutes and kept at this temperature for 15 minutes.Finally, during the last stage, the temperature has been decreased to 50 °C within 21 minutes.After the cooling down to ambient temperature process, the digested samples were diluted with ultra-pure water to a volume of 25 ml.The oil-free blank samples were similarly heated and cooled down to complete in the same volume.All samples and reagent blanks had three parallels in the study.Before each trial, the tool has been tuned by using 10 mg/mL aqueous multi-element standard solution.The conventional sample introduction system, equipped with a cross flow nebulizer and a Scott Spray chamber, was used.The used instrumental conditions were as follows: autolens mode on; peak hopping measurement mode; dwell time of 50 sweeps by reading; one reading by 3 replicates.
Nickel sampler cone and skimmer cone were used.The radio frequency (RF) power was set to 1000 W, the plasma gas was 19 L argon (Ar) min À1 , the auxiliary gas flow was 1.2 L Ar min À1 , and the nebulizer gas was 0.81 L Ar min À1 .Analytical masses of the studied elements were 63 Cu, 57 Fe, 25 Mg, 55 Mn and 65 Zn.
B, Ca, K, Na, P and S contents were determined by ICP-OES (Perkin Elmer Optima 4300 DV) after the microwave digestion process.

Fatty acid composition
The methyl esters of the fatty acids were prepared according to IUPAC (Anon, 1987) and analyzed using a Shimadzu GC-2010 Plus gas chromatograph equipped with a DB-23 column (60 m x 0.25 mm I.D. and 0.25 mm film thickness) (J&W).Injector, column and detector temperatures were 230, 190 and 240 °C, respectively.Split ratio was 1:80.The carrier gas was helium at 1 ml/min ratio.A mix of 37 FAME from Supelco (C4-C24) (Bellefonte, PA, USA) were used.

Tocopherols
Tocopherol analyses of the oils were determined according to Bele et al. (2013).The oil samples weighing 50 mg were dissolved 10 times in hexane.50 ml of this solution was taken into a screw-capped tube and diluted with 1 ml of a mixture of methanol:hexane:tetrahydrofurane (80:10:10, v/v/v).The samples were vortexed for 5 min and centrifuged for 10 min at 5000 rpm (Hanil Science Industrial, Model: Combi 514R).Then, the samples were filtered using nylon filters (pore sizes 0.45 mm, Ampel, Shanghai, China).The filtrate was directly injected into an Agilent 1260 HPLC system (Agilent Corp., Santa Clara, CA, USA).Detection was done by the fluorescence detector (excitation: 290 nm, emission: 325 nm) at an Agilent Pursuit C18 column (150 x 4.6 mm, 5 mm).The mobile phase was a mixture of methanol and acetonitrile (50:50, v/v), and eluted at a flow rate of 1 ml/min.The tocopherols were identified by comparison of the retention times with standards of the a-, b-, gand d-tocopherols.The tocopherol contents were expressed as milligrams per kg oil.

Extraction and determination of total phenolic compounds
Total phenolic compounds of the pumpkin seed oils were extracted and determined according to Paradiso et al. (2016) with some modifications.Extraction was carried out on 2.5 g of oil by adding 5 ml hexane and 5 ml methanol/water (80:20 v/v).After 2 min vortexing, and centrifugation at 6000 rpm for 10 min at 4 °C (Hanil Science Industrial, Model: Combi 514R), the hydroalcoholic phase was recovered and the extraction was repeated twice.Collected hydroalcoholic phase was centrifuged at 9000 rpm for 5 min at 4 °C and filtered through nylon filters (pore size 0.45 mm, Ampel, Shanghai, China).Then, 0.5 ml of filtrate was added to 10 ml volumetric flask and diluted in 5 ml of water, followed by the addition of 0.5 ml of Folin-Ciocalteu reagent (FCR).The mixture was incubated for 3 min at room temperature, and 1 ml of 7.5% solution of Na 2 CO 3 was added to the reaction mixture and volume was made up to 10 ml with distilled water.The solutions were stored overnight and absorbance of the samples against blank read at 765 nm by a spectrophotometer (Thermo Fischer Scientific, Model: G10S UV-Vis).Total phenolics content was expressed as gallic acid equivalents (mg/g).

Antioxidant activity
ABTS þ Radical Scavenging Activity analyses of the pumpkin seed oils were conducted according to the method described by Re et al. (1999).ABTS þ radical was prepared by mixing ABTS stock solution (7 mM in water) with 2.45 mM potassium persulfate.This mixture was left in darkness at room temperature for 12-16 h before use.The ABTS þ radical solution was diluted with 0.1 M pH 7.4 phosphate buffer to an absorbance of 0.70 (± 0.02) at 734 nm.The reaction was started by the addition of 25, 50, 75 and 100 ml of the methanolic extract of the pumpkin seed oils.The absorbance of each reaction mixture was measured at 734 nm with a spectrophotometer (Thermo Fischer Scientific, Model: G10S UV-Vis) for 15 min after initial mixing.The inhibition percentage was plotted as a function of concentration.The antioxidant activity of the pumpkin seed oils was calculated as Trolox Equivalent Antioxidant Capacity (TEAC).The TEAC was calculated from the ratio of the linear regression coefficient of pumpkin seed oil samples to that of the Trolox.The results were expressed as mmol Trolox equivalents per g of samples.

Statistical analysis
The study was conducted according to randomized block design using three replicates.The acquired data were subjected to variance analysis by using SPSS Statistics 21 package program and comparison of mean values was made using Duncan's test.

Basic characteristics of the pumpkin seed oils
The specific gravity, refractive index, iodine number, saponification number, free fatty acid, p-anisidine, peroxide value, TBARS and viscosity values of the pumpkin seed oils are given in Table 1.Specific gravity is an important characteristic of vegetable oil and corresponds to the ratio of the weight of any substance given volume to the weight of an equal volume of water.Specific gravity values of the pumpkin seed oils ranged from 0.914 to 0.918, which are within the range reported in the literature for vegetable oils: olive oil (0.910-0.916), rapeseed oil (0.910-0.920), sunflower seed oil (0.918-0.923) and pumpkin seed oil (0.923-0.926) (Nichols and Sanderson, 2003).The refractive index of saturated fatty acids a linear increase with chain length whereas the refractive index of unsaturated fatty acids increases with the degree of unsaturation.The refractive index of saturated fatty acids shows a linear increase with chain length whereas the refractive index of unsaturated fatty acids increases with the degree of unsaturation.The refractive index values of the pumpkin seed oils ranged from 1.472 to 1.473, which are within the range reported in the literature for vegetable oils: olive oil (1.468À1.471),rapeseed oil (1.465À1.469),sunflower seed oil (1.467À1.469)and pumpkin seed oil (1.466À1.474)(Nichols and Sanderson, 2003).Results presented in Table 1 show that pumpkin seed oils had high iodine number.The high iodine number can be related to indication of high unsaturated fatty acid content.The saponification number is considered as a measure of the average length of the fatty acids and varies inversely with the molecular weight.The values were similar to the saponification number of sunflower oil (188)(189)(190)(191)(192)(193)(194) and canola oil (182-193) (Nichols and Sanderson, 2003).
Viscosity is a characteristic that is important in the design of industrial processes and it is also a factor that affects the stability of food.In general, saturated fatty acids have a higher viscosity value than unsaturated fatty acids.This is due to the fact that in saturated fatty acids carbon chains are arranged to be closer to each other, and hence they can establish intermolecular interactions such as Van der Waals forces.As can be seen in Table 1, the viscosity values measured in the present study (except for wet salted-roasted Nevşehir çerçevelisi) were lower than those reported by Ardabili et al. (2011), for C. pepo (93.66 cp), while higher than those reported by Tsaknis et al. (1997) for C. maxima and C. pepo (72 cp).The viscosity values determined in pumpkin seed oils were higher than those reported by Nichols and Sanderson (2003) for olive oil (63.28), and maize oil (51.44).Values in a column with the same superscript lowercase letters are not significantly different (the genotype was compared within itself) (p Values in a column with the same superscript uppercase letters are not significantly different (between genotypes) (p Free fatty acid values of the pumpkin seed oils are shown in Table 1.As can be seen from Table 1, free fatty acid values were found higher (p < 0.05) in oils obtained from sun-driedunroasted seeds.The increase in the free fatty acid value of cold-pressed oils might be attributed to hydrolysis of triacylglycerols.Free fatty acid value in edible oils is one of the important qualitative parameter.There is an inversely proportional relationship between the edibility value of oil and the total amount of free fatty acid.A low amount of free fatty acid is a sign that the degree of edibility of the oil is high and the storage life is also high.The values of the free fatty acid in Table 1 represent that the oil samples maintained good quality as their acid values did not exceed (taking into account that the free fatty acid value is equal to the acid value divided by 1.99) the maximum limit of 4.0 mg KOH/g of oil according to the Codex Alimentarius Commission (1999).
Peroxide value, which is based on the reduction of hydroperoxide group with iodine ion (I -), provides information about hydroperoxide concentration.Among the oils extracted from differently treated pumpkin seeds, the highest peroxide value was observed in the unsalted-roasted and dry saltedroasted pumpkin seeds (Tab.1).Peroxide values were found higher than 8 in these oil samples.However, peroxide values close to each other were obtained in the oils obtained from the wet salted seeds before roasting and the oils obtained from the sun-dried unroasted seeds (Tab.1).This may be interpreted that the wet salting process is an effective process in preventing oxidation occurring during roasting process.The reason for this situation is considered to be fact that pumpkin seeds are kept for overnight in mixture of water and salt after adding water and salt to the seeds.The added water will have had enough time to penetrate overnight and will be absorbed by the seeds.The water absorbed by the shell of the seeds will the first evaporate during the roasting process and as the water evaporates this process will cause the shell to cool down.This will cause the heat effect to be perceived at a lower level within the seeds.This will ensure that the interior of the seed is less affected by the roasting conditions and that the moisture in the interior will evaporate towards the end of the roasting process.The reason for why this does not happen in salted and roasted pumpkin seeds is the fact that the time is not enough of the water to penetrate.Codex Alimentarius Commission (1999) states that peroxide value in refined vegetable oils should be no more 10 meq O 2 /kg oil, and the maximum value in cold-pressed and virgin oils should be 15 meq O 2 /kg oil.The reason for this situation is considered to be fact that pumpkin seeds are kept for overnight in mixture of water and salt after adding water and salt to the seeds.
p-anisidine value is a method used to determine the quantity of aldehydes (particularly 2-alkenals and 2.4alkadienals).In the studied oils, p-anisidine values were low in the oils obtained from the wet salted seeds before roasting and the oils obtained from the sun-dried unroasted seeds (Tab.1).On the other hand, higher values were obtained in oils extracted from dry salted-roasted and unsalted-roasted samples (p < 0.05).
The determination of TBARS is a common method to determine secondary oxidation products.The TBARS values obtained in all samples were below 1 (Tab.1), which is the limit of acceptance of a product as rancid.Yoshida et al. (2006) reported that microwave roasting of pumpkin seeds resulted in only small increases after a long roasting period in peroxide value, carbonyl value, p-anisidine value and TBARS.

Color
The L*, a* and b* values of the oils are shown in Table 2.As seen in Table 2, roasting process resulted in increased darkness (lower L* value) (p < 0.05).Positive a* value is an indication of redness and this value was observed higher in oils extracted from the wet salted seeds before roasting (p < 0.05).On the other hand, positive b* value is an indication of yellow color intensity and in general, the yellow color intensity was found higher in roasted samples (p < 0.05).In a research conducted by Rezig et al. (2012) on oils extracted from C. maxima seeds, L*, a* and b* values were found to be 44.8,À0.18 and 28.88, respectively.As a result of a study of oil obtained from C. pepo seeds, L*, a* and b* values were found around 22, 10 and 40, respectively (Nyam et al., 2009).

Mineral contents
Mineral contents of pumpkin seed oils are shown in Table 3. Juranovic et al. (2003) used inductively coupled plasma atomic emission spectrometry (ICP-AES) to study mineral content of pumpkin seed oils.Compared to their findings, the amount of Cu was higher than in our results, while the Fe and Zn contents were at about the same level.Bandoniene et al. (2013) observed that pumpkin seed oils from different geographic origins had different elemental characteristics.The content of metals found in oils differs depending on various factors.Metals can be incorporated into the seeds and oils from the soil or be introduced to oil during the production process.The presence of trace elements such as Cu, Fe, Mn, Ni and Zn affect the oxidative stability of oils in different ways (Juranovic et al., 2003).The high levels of Fe and Cu concentrations significantly contribute to the deterioration of oil as they have redox potential and pro-oxidative effect.The phosphorus content in oils correlates positively with the severity of roasting process (Prevc et al., 2015).As a result of this research made by us, it was found that pumpkin seed oils are a rich source of Zn.Zn is an essential micronutrient element for human health.Wessells and Brown (2012) reported that approximately 17.3% of the world's population was faced with the risk of Zn deficiency.Zn is an element that is present in the majority of the metabolic processes.It is the structural element of protein and biomembranes, as well as the role of the cofactor of various enzymes, also playing a role in the fulfillment of biological functions without the risk of inducing and oxidizing during the process of gene formation and transfer (Ovca et al., 2011).Because it is involved in so many different processes, Zn deficiency can lead to various disorders in the human body.There is no particular organ storing Zn in human body, therefore sufficient amounts of Zn must be taken from foods.The main source of many metals in foods is soil.Zn is found in much lower quantities in calcareous and sandy soils when compared to clay and volcanic soil.
Another important outcome of the study is that the oils is a rich source in terms of Se content too (Tab.3).Similar to Zn, selenium is an important micronutrient element.Hurst et al. (2013) reported that, for both males and females, 55 mg/day Se intake is necessary.Kubachka et al. (2017) emphasized that Se is not only an antioxidant, but it is also of vital importance for the development and functions of organs.

Fatty acids
The fatty acid compositions of the oils are given in Table 4.As seen in the table, the samples contain high levels of unsaturated fatty acid.The results are similar to the results acquired by Stevenson et al. (2007).It is known that vegetable oils containing high levels of monounsaturated fatty acid (MUFA) (particularly oleic acid) are very effective in decreasing both cardio-vascular disease risk and infection (El-Adawy and Taha, 2001).Nutritional value of vegetable oils is determined by the ratio of MUFA/PUFA (polyunsaturated fatty acid) and theoretically MUFA offers more benefits than PUFA (Rabrenovic et al., 2014).Linoleic acid is an essential fatty acid and it is necessary for the formation of cellular membrane and different hormones.Pumpkin seed oil is a rich source both in oleic and in linoleic acid quantities, therefore it can be used as cooking and salad oil, as well as being suitable for margarine production.Having a high level of oleic acid is vital in terms of nutrition and it ensures high stability against Values in a row with the same superscript lowercase letters are not significantly different (the genotype was compared within itself) (p Values in a row with the same superscript uppercase letters are not significantly different (between genotypes) (p < 0.05).
cooking-frying.It has been reported that linoleic acid is oxidized 50 times faster than oleic acid and oil industry is trending towards sources with high oleic acid content (Rezig et al., 2012).Palmitoleic acid is described as "not detected" for the authenticity needs of pumpkin seed oil in the regulations of many European Union (EU) countries.In this study, as well as in other studies (Stevenson et al., 2007;Nederal et al., 2012;Nederal et al., 2014), presence of palmitoleic acid was observed.Therefore, as emphasized by Nederal et al. (2014) it is necessary to make a new regulation for this fatty acid in the pumpkin seed oils.

Tocopherols
The content of tocopherols of the oils are given in Table 5.As a result of this research, the content of a-tocopherol content could not be determined.As seen in Table 5, b þ g tocopherols content ranged from 131.78 to 881.77 mg/kg oil.On the other hand, d-tocopherol content ranged from 3.22 to 8.67 mg/kg oil.Tocopherols are natural antioxidants and their contents vary depending on the type of oil.Several factors, such as the climatic conditions of the material from which the vegetable oils are obtained, extraction process of the oil and the method of determining tocopherols affect the content (Rabrenovic et al., 2014).The results of this study were similar to those acquired by Rabrenovic et al. (2014).The researchers reported that the content of b þ g tocopherols ranged from 29.92 to 53.60 mg/100 g oil, while the d-tocopherol content, which is an important form for oxidative stability, ranged from 3.39 to 10.44 mg/100 g oil.Murkovic et al. (2004) and Stevenson et al. (2007) emphasized that roasting process increased tocopherol content, while Yoshida et al. (2006) did not observe any significant changes and Gemrot et al. (2006) observed that 5 minutes of roasting at 140 °C lead to significant reductions in tocopherol content.In this study, similar results were obtained by Gemrot et al. (2006) and in general, higher tocopherol contents were observed in sun-dried-unroasted pumpkin seed oils and the wet salted samples before roasting (p < 0.05).Butinar et al. (2011) stated that the content of d-tocopherol increases when the roasting time is increased from 10-12 min to 17-19 min.As a result of this research carried out by us, no increase was detected in the tocopherol content due to roasting process.The short roasting time (15 min) is considered as the reason for this situation.Considering the tocopherol contents in Table 5 and the peroxide values in Table 1, it can be seen that the peroxide values were increased by decreasing the content of tocopherols.This may be explained by the fact that heat treatment increases oxidation in polyunsaturated lipids and leads to a consumption of antioxidants.

Total phenolic compounds
The total phenolic compounds of the oil samples were given in Table 5.As seen in the table, the treatments applied to the seeds before the oil were extracted had an insignificant effect on total phenolic compounds (p > 0.05).Veronezi and Jorge (2012) stressed that the roasting process resulted in a significant decrease in total phenolic compounds.However, such a result could not be obtained in this research.The total phenolic compounds found in the studied oils displayed Values in a column with the same superscript lowercase letters are not significantly different (the genotype was compared within itself) (p < 0.05).
Values in a column with the same superscript uppercase letters are not significantly different (between genotypes) (p < 0.05).
similarities to the results acquired by Veronezi and Jorge (2012).They found that the total phenolic compounds varied between 1.35 and 3.62 mg EAG/g in the lipid fraction of four different pumpkin seeds grown in Brazil.Andjelkovic et al. (2010) emphasized that total phenolic compounds varied depending on climatic and storage conditions, and time also had an important effect on content.

ABTS þ radical scavenging activity
The radical scavenging activity values of the pumpkin seed oils are given in Table 5.As can be seen from Table 5, radical scavenging activity values in oils obtained from sun-driedunroasted seeds and roasted seeds after wet salting process were found to be higher than other oils.This is an indication that dry salted and unsalted roasting process leads to a significant decrease (p < 0.05) in radical scavenging activity.Similar results were reported by Xanthopoulou et al. (2009) and the researchers emphasized that radical scavenging activity varied depending on the total phenolic content, and extracts rich in phenolics had higher radical scavenging activity.Oloyede et al. (2012a, b) emphasized that climatic conditions had a effect on the concentration of bioactive compounds, and mature pumpkin seeds had higher antioxidant activity than non-mature ones, and increased fertilizer quantity led to lower antioxidant activity.Nawirska-Olszanska et al. (2013) reported that the seeds of the pumpkin varieties that belong to the species C. pepo exhibit better antioxidant properties than C. maxima, regardless of the extraction solvent used.The researchers also stated that 50% ethanol was more efficient than 80% methanol when used as an extracting agent, and the antioxidant activity values obtained with 50% ethanol were higher than those achieved with 80% methanol.

Conclusion
Results obtained from this study have shown that, if the pumpkin seeds are subjected to wet salting process overnight and then roasting process is carried out, both the quality criteria and antioxidant characteristics of the oil are maintained in the best way possible.Among the analyzed oil samples, four fatty acids (palmitic, stearic, oleic and linoleic acid) with high amounts were determined.From the results obtained, it is possible to indicate that pumpkin seed oil is a rich source in linoleic and oleic acid.In addition, because of the high content of the linoleic acid, this oil may be used as an essential oil acid source.

Table 1 .
Basic characteristics of the pumpkin seed oils.

Table 2 .
L*, a* and b* values of the pumpkin seed oils.

Table 3 .
The results of the mineral contents analysis of pumpkin seed oils (mg/kg).

Table 4 .
The fatty acid compositions of the oils extracted from differently treated pumpkin seeds.

Table 5 .
The tocopherol, total phenolics contents and antioxidant activity values of the oils extracted from differently treated pumpkin seeds.Standard deviation of three replicates.Values in a column with the same superscript lowercase letters are not significantly different (the genotype was compared within itself) (p < 0.05).