ORIGINAL RESEARCH ARTICLES

 

Determination of bioactive compounds and mineral contents of seedless parts and seeds of grapes

 

 

M.M. Özcan^{I, *} ; F. Al Juhaimi^II; M. Gülcü^III; N. Uslu^I; Ü. Geggel^IV

^IDepartment of Food Engineering, Faculty of Agriculture, Selcuk University, 42031 Konya, Turkey
^IIDepartment of Food Science & Nutrition, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia
^IIIMinistry of Food, Agriculture and Livestock Viticultural Research Station 59100, Süleymanpas.a, Tekirdag, Turkey
^IVDepartment of Food Engineering, Faculty of Agriculture, Namik Kemal University, Tekirdag, Turkey

 

 

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ABSTRACT

In this study, phenolic compounds, minerals, total flavonoids, total phenolic contents and antioxidant activities of the seedless parts (pulp+skin) and seeds of table and wine grapes were determined. Also, the total oil, tocopherol contents and fatty acid composition of seed oils of table and wine grapes were investigated. The highest total phenolic content of the grape pulp was found in Trakya ilkeren (199.063 mg/100 g), while total flavonoid and antioxidant activity of the pulp was determined at a high level in Red Globe (6.810 mg/g, 90.948%). Antioxidant activity, and the total phenolic and flavonoid contents of grape seeds varied between 86.688 and 90.974%, 421.563 and 490.625 mg GAE/100 g, and 90.595 and 145.595 mg/g respectively (p < 0.05). Generally, the main phenolic compounds of all grape pulps and seeds were gallic acid, 3,4-dihydroxybenzoic acid, (+)-catechin and 1,2-dihydroxybenzene. In addition, the oil contents of grape seeds ranged from 5.275 (Cavus) to 13.881% (Cinarli karasi) (p < 0.05). The major fatty acids of grape seed oils were linoleic, oleic and palmitic acid. The seed oil of the Trakya ilkeren variety was rich in tocopherols in comparison with the other varieties. The major minerals of both the seedless parts and the seeds were determined as K, Ca, P, S, Mg.

Keywords: Grape, bioactive compounds, antioxidant, fatty acid composition, mineral content

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INTRODUCTION

Grapes (Vitis spp.) belong to the Vitaceae family and are used mainly for their juice, especially in the wine industry (Lutterodt et al., 2011). The bioactive compounds, including simple phenolics, flavonoids, anthocyanins, stilbenes, proanthocyanidins and vitamin E, are important phytochemicals in grapes. Simple phenolic compounds of grapes are hydroxycinnamic acid (p-coumaric, caffeic, sinapic and ferulic acids) and hydroxybenzoic acid (gallic, gentisic, protocatechuic and p-hydroxybenzoic acids) (Georgiev et al., 2014; Shi et al., 2016). Several phenolic compounds are found in the skins, pulp and seeds of grapes (Nile et al., 2013). Most of these compounds have antioxidant properties, which play a significant role in human health because they reduce the oxidation of low-density protein, thereby decreasing the risk of heart disease and preventing cancer (Yilmaz et al., 2015; Yalcin et al., 2016). The amount of phenolics is affected by the grapevine variety, climatic and geographical factors, cultural practices, and the stage of ripeness (Obreque-Slier et al., 2010; Farhadi et al., 2016).

A large amount of grape seeds are obtained from the waste of fruit products. The wastes of this industry represent about 50% to 60% of the raw fruit. On the other hand, grape seeds are used as a source of oil, with nutritional and bioactive constituents (Kamel et al., 1985; Hassanein & Abedel-Razek, 2009). Grape seed oils obtained from grape by-products can be used for pharmaceutical and nutritional purposes due to their high levels of vitamin E and unsaturated fatty acids (Hassanein & Abedel-Razek, 2009). These by-products contain some valuable substances, such as phenolics, fatty acids, tocopherols and minerals with potential applications in the food industry (Crews et al., 2006; Wie et al., 2009). Generally, grape seed oil is used in salad dressings, marinades, deep frying, flavoured oils, baking, massage oil and cosmetic products (Akin & Altmdis.li, 2011).

The aim of this study was to investigate some physicochemical properties (soluble solid content, titratable acidity and maturation index of grapes; total dry matter of grape seeds), antioxidant activities, total phenolic and flavonoid contents, phenolic compounds and mineral contents of grapes (pulp+skin) and seeds, and the oil yields, fatty acid and tocopherol contents of grape seeds due to the importance of grapes and seeds as sources of bioactive compounds, oil, tocopherol and minerals.

 

MATERIAL AND METHODS

Grapes (Cavuş, Cinarli Karasi, Kalecik Karasi, Red Globe, Trakya Ilkeren, Yalova Incisi and Yapincak) were obtained from the Viticulture Research Institute of Tekirdag in Turkey. All varieties belong to Vitis vinifera. Their colours and properties are given in Table 1.

Sample extraction

For the phenolic compounds and antioxidants, the seedless parts (pulp+skin) and seeds of the grapes were extracted according to Gomez-Alonso et al. (2007) with some modifications. About 2 g of each sample were added to 15 ml of methanol:water:formic acid (5:4.85:1.5, v/v).The mixture was sonicated for 2 min, followed by centrifugation at 4 500 rpm for 15 min, after which the supernatants were collected. Then 10 ml of n-hexane were added and mixed using a vortex apparatus. After the extract was concentrated at 50°C in a rotary evaporator, and after the dried extracts had been dissolved in 1.5 ml of methanol, it was injected into HPLC.

Physicochemical analysis

The content of soluble solids, titratable acidity, maturation index and total dry matter were analysed according to Cemeroglu (1992).

Total phenolic content

The total phenolic content of the grape pulp and seeds was determined by Folin-Ciacueltau (FC) reagent according to Yoo et al. (2004). About 10 mL of Na₂CO₃ solution and 1 ml of Folin-Ciacueltau were mixed, and then completed with 25 ml deionised water. After 1 h, the total phenolic content was measured at 750 nm in a spectrophotometer. The results were given as mg gallic acid equivalent/100 g.

Antioxidant activity

The free radical-scavenging activity of the samples was determined using DPPH (1,1-diphenyl-2-picrylhydrazyl) according to Lee et al. (1998). The extract was mixed with 2 mL methanolic solution of DPPH. The mixture was shaken vigorously and allowed to stand at room temperature for 30 min. The absorbance was recorded at 517 nm by using a spectrophotometer.

Determination of phenolic compounds

Phenolic compounds were determined by a Shimadzu-HPLC equipped with a PDA detector and an Inertsil ODS-3 (5 μm; 4.6 x 250 mm) column. As mobile phases, 0.05% acetic acid in water (A) and acetonitrile (B) mixture were used. The flow rate of the mobile phase and the injection volume were 1 ml/min at 30°C and 20 μl respectively. The gradient program was as noted: 0 to 0.10 min 8% B; 0.10 to 2 min 10% B; 2 to 27 min 30% B; 27 to 37 min 56% B; 37 to 37.10 min 8% B; and 37.10 to 45 min 8% B. The peak records were carried out at 280 and 330 nm. The total running time for each sample was 60 min. Phenolic compounds were determined according to the retention time and absorption spectra of peaks of standard compounds. The total area under the peak was used to quantify the phenolics.

Total flavonoid content

The total flavonoid content of the samples was determined using the colorimetric method according to Hogan et al. (2009). One mL of extract was mixed with 0.3 ml of NaNO₂, 0.3 ml of AlCl₃ and 2 ml of NaOH respectively. The absorbance of the mixtures was recorded at 510 nm using a spectrophotometer.

Mineral content

After 5 g of grape samples had been dried in a drying cabinet at 70°C, about 0.5 g of the sample was digested in a closed microwave system by using 5 ml of 65% HNO₃ and 2 ml of 35% H₂O₂. After 20 ml ultra-deionised water was added to the digested sample, the sample was analysed by ICP AES (Varian-Vista, Australia) for minerals (Skujins, 1998).

Oil extraction

Oil was extracted from the ground seeds with petroleum ether in a Soxhlet apparatus for 6 h. The solvent (petroleum ether) was then evaporated (AOAC, 1990). The obtained oil was kept in coloured bottles at -18°C until analysis.

Fatty acid composition

The oil sample (50 to 100 mg) was converted to its fatty acid methyl esters (FAME). The fatty acid methyl esters were identified by comparing the retention time of the samples and appropriate standards of fatty acids methyl esters (AOAC, 1990). The fatty acid methyl esters were injected into a gas chromatograph (Shimadzu GC-2010) with a capillary column, CP-Sil 88 (100 m long, 0.25 mm ID, film thickness 0.2 μm) and flame-ionisation detector (FID).

Tocopherol content

Tocopherol content was measured according to Spika et al. (2015). About 0.1 g of oil was dissolved in 10 ml of w-hexane and filtered through a 0.45 μm nylon filter. HPLC analyses of tocopherols were done using a Shimadzu-HPLC equipped with a PDA detector and LiChroCART Silica 60 (4.6 x 250 mm, 5μ; Merck, Darmstadt, Germany) column. Tocopherols were separated by isocratic chromatography using a mobile phase of 0.7% propan-2-ol in w-hexane. The flow rate of the mobile phase was 0.9 ml/min, and the injection volume was 20 μΙ The peaks were recorded at 295 and 330 nm with a PDA detector. The total running time per sample was 30 min. Standard solutions of tocopherols (α-, β-, γ- and δ-tocopherol) were constructed at concentrations of 0 to 100 mg/L. All analyses were done in triplicate.

Statistical analyses

Analysis of variance (ANOVA) using JUMP version 9.0 (SAS Inst. Inc., Cary NC, USA)and the mean ± standard deviation (MSTAT C) of the results were calculated according to Püskülcü and ikiz (1989).

 

RESULTS AND DISCUSSION

The soluble solid contents (of the pulp), total dry matter (of the seeds), titratable acidity, maturation index, harvest date and some properties of grape cultivars are shown in Table 2. The highest and the lowest soluble solid contents were found in Kalecik karasi (22.00°Brix) and Red Globe (15.20°Brix) respectively (p < 0.05). The results for titratable acidity ranged from 4.10 g/L to 7.80 g/L. Maximum titratable acidity was determined in Kalecik karasi, while the minimum value was found in Yapincak. The maturation index of the grape varieties at harvesttime varied from 23.40 (Yapincak) to 47.50 (Cinarli karasi). Water-soluble dry matter, titratable acidity, maturation index and grape colour had an effect on the maturation of the grapes. The grapes can be harvested when the maturation index is a minimum of 20 (OIV, 2008). As can be seen in Table 2, the grapes used in the current experiment were harvested at the maturation stage. According to the total dry matter assay, the highest value was found in Yapincak seeds (68.27%), while the lowest amount of dry matter was in seeds of Yalova incisi (56.38%). According to Samoticha et al. (2017), the soluble solid contents range from 10.6°Brix to 22.3°Brix in white grapes, and from 15.2°Brix to 20.6°Brix for red grapes. The average total acidity of the white and red cultivars was 0.95 and 0.93 g tartaric acid/100 g, respectively.

The antioxidant activities, total phenolic and total flavonoid contents of the grape samples are presented in Table 3. Antioxidant activities of the samples varied between 30.298% and 90.948%. The total phenolic contents of the grape samples were found to be between 44.063 and 199.063 mg GAE/100 g. Trakya ilkeren had the highest total phenolic content (199.06 mg GAE/100 g), followed by Red Globe (170.313 mg GAE/100 g) and Cavuş (146.641 mg GAE/100 g). Significant differences were observed between the total phenolic contents of the grape extracts (p < 0.05). The total flavonoid contents of the grape varieties ranged from 0.491 to 6.810 mg/g. The total flavonoid content of Red Globe (6.810 mg/g) was found to be close to that of Trakya ilkeren (6.744 mg/g). The minimum flavonoid content was determined in Yalova incisi, with a value of 0.491 mg/g. Hogan et al. (2009) observed that the total phenolic and flavonoid contents of wine grapes varied between 0.68 and 1.82 mg GAE/g and 0.48 and 1.19 mg RE/g respectively. In another study, the total phenolic contents of both grape pulp and grape seed varied from 9.26 to 62.29 mg GAE/100 g and from 162.29 to 326.18 mg GAE/100 g respectively (Yilmaz et al., 2015). In the experiments reported by Samoticha et al. (2017), the total phenolic content of white grapes varied from 1974.9 to 3 884.4 mg/100 g, while it ranged from 1 037.0 to 5 759.1 mg/100 g in red grapes. The degree of maturity and environmental conditions influence the amount of phenolic compounds. Moreover, the fruit parts, such as skin, pulp or seeds, exhibit different polyphenol concentrations. The antioxidant activities of grape varieties range from 93.80% to 95.62% in grape skin, from 88.16% to 94.68% in grape pulp, and from 88.10% to 94.46% in grape seeds (Farhadi et al., 2016).

Antioxidant activities, along with the total phenolic and total flavonoid contents of the grape seeds, are shown in Table 3. The antioxidant activities of the seeds varied between 86.688% and 90.974%, and was higher than that found in the pulp+skin of grape. The total phenolic contents of the grape seeds ranged from 421.563 to 490.625 mg GAE/100 g, and the richest variety in terms of total phenol content was Cavuş (490.625 mg GAE/100 g), followed by Cinarli karasi (487.813 mg GAE/100 g). The total flavonoid content of the grape seeds was considerably higher compared with the rest of the grapes and was found to be between 90.595 and 145.595 mg/g (p < 0.05). Concerning the results, the grape seeds contained rich bioactive substances. According to Ky et al. (2014), the total phenolic content of grape seed extracts varied from 36.6 to 49.7 mg GAE/g, and the antioxidant activity of the seed extract of grape varieties using DPPH ranged from 117.4 to 536.2 μmol Trolox/g (dry weight). The total phenolic contents of the grape seeds (825.80 to 3 313.5 mg GAE/100 g) were higher than that of the pulp (50.79 to 141.72 mg GAE/100 g) (Anastasiadi et al., 2010).

The highest oil content was determined in Cinarli karasi (13.881%), while the seeds of Cavus. variety (5.275%) contained the lowest oil content. Sabir et al. (2012) reported that the oil content of grape seeds ranged from 7.3% to 22.4%. In addition, the oil content of Red Globe and Kalecik Karasi was found to be 142.1 and 123.3 g/kg. Fernandes et al. (2013) determined that the highest and lowest oil content of grape seeds was 12.40% and 3.95% respectively. In another study, the oil content in grape seeds of wine varieties averaged at 11.60 g/100 g (Lachman et al., 2015).

The phenolic compounds of the grape pulp and seeds are presented in Table 4. Generally, the main phenolic compounds of all varieties were gallic acid, 3,4-dihydroxybenzoic acid, (+)-catechin and 1,2-dihydroxybenzene. The highest gallic acid was determined in Yalova incisi (77.832 mg/100 g), Cinarli karasi (57.325 mg/100 g) and Kalecik karasi (49.704 mg/100 g) (p < 0.05). The highest content of 3,4-dihydroxybenzoic acid was found in the Cavus. (101.462 mg/100 g) and Yalova incisi (54.093 mg/100 g) varieties. Yalova incisi (53.776 mg/100 g) had also the greatest content of (+)-catechin. The highest amount of 1,2-dihydroxybenzene was also observed in Yalova incisi (95.589 mg/100 g) and Cinarli karasi (56.357 mg/100 g). Significant differences were observed among the gallic acid, caffeic acid and rutin trihidrate contents of grape pulp extracts and between the 3.4-dihydroxybenzoic, resveratrol and isorhamnetin contents of grape seed extracts (p < 0.05). Moreover, apigenin 7 glucoside in Cavus. (22.995 mg/100 g), and the caffeic acid and quercetin contents in Yapincak (20.244 mg/100 g and 22.968 mg/100 g respectively), were found to be higher than in other varieties. The phenolic compounds of grape seeds were determined as significantly high in comparison to in the rest of the grapes. The major phenolic compounds of Red Globe were 1,2-dihydroxybenzene (445.287 mg/100 g), rutin (202.915 mg/100 g), apigenin 7 glucoside (194.638 mg/100 g) and caffeic acid (142.473 mg/100 g). (+)-Catechin (284.424 mg/100 g), gallic acid (221.492 mg/100 g) and quercetin (201.424 mg/100 g) were highest in Cinarli karasi; 1,2-dihydroxybenzene (556.198 mg/100 g and 549.264 mg/100 g respectively) in Cavu. and Kalecik karasi; (+)-catechin (480.509 mg/100 g and 309.335 mg/100 g, respectively) in Yapincak and Yalova incisi; and rutin (231.542 mg/100 g) was found in a considerable amount in Trakya ilkeren varieties. The highest resveratrol content was observed in Cinarli karasi, with a value of 197.508 mg/100 g. Rockenbach et al., (2011) studied phenolic compounds of different grape varieties (Cabernet Sauvignon, Merlot, Bordeaux and Isabel), and the rutin, kaempferol, catechin, epicatechin, resveratrol and gallic acid contents ranged from 12.00 to 41.43 mg/100 g, from 0.0 to 15.09 mg/100 g, from 94.28 to 150.16 mg/100 g, from 0.0 to 44.36 mg/100 g, from 0.0 to 6.40 mg/100 g and from 4.59 to 18.68 mg/100 g, respectively. The phenolic acids of wine grapes were found to be gallic acid (16.7 to 72.6 μg/g), chlorogenic acid (2.1 to 3.3 μg/g), vanillic acid (2.2 to 49.4 μg/g), caffeic acid (0.1 to 9.2 μg/g), coumaric acid (2.8 to 4.2 μg/g) and ferulic acid (0.1 to 0.6 μg/g) (Hogan et al., 2009). Anastasiadi et al. (2010) reported that the phenolic compounds of grape seeds were gallic acid (7.22 to 161.74 mg/100 g), catechin (143.63 to 1067.0 mg/100 g) and epicatechin (89.21 to 371.65 mg/100 g). The main phenolic compounds in the pulp of grapes were catechin (354 to 514 μg/g), epicatechin (135 to 234 μg/g), gallic acid (87 to 192 μg/g) and quercetin (87 to 198 μg/g). The gallic acid, catechin, epicatechin and quercetin contents of grape seeds were between 67 and 91 μg/g, 122 and 156 μg/g, 103 and 167 μg/g, 25 and 38 μg/g respectively (Farhadi et al., 2016).

The fatty acid composition of the grape seed oils is shown in Table 5. Grape seeds are a good source of essential fatty acids, and their linoleic acid contents varied between 66.94% and 77.359%. Significant differences were observed among the linoleic acid contents of grape seed oils (p < 0.05). The amount of oleic acids of seed oils ranged from 10.746% to 19.660%. Palmitic and stearic acids varied between 7.176% and 10.358% and 2.983% and 5.435% respectively (p < 0.05). The highest linoleic and the lowest oleic acid contents were found in the Cavus. variety. Furthermore, the linolenic acid content of seed oils was below 1%. According to Lachman et al. (2015), linoleic acid was the most prominent fatty acid and was observed at between 68.10 and 78.18 g/100 g. In another study, the major fatty acid of seed oils was linoleic acid (53.6% to 69.6%), followed by oleic (16.2% to 31.2%), palmitic (6.9% to 12.9%) and stearic (1.44% to 4.69%) acids. The oils of Red Globe and Kalecik Karasi seeds contained high levels of linoleic acid, with values of 68.8% and 63.7% respectively (Sabir et al., 2012). The oil contents of the grape seeds ranged from 12.09% (Kalecik karasi) to 17.08% (Gamay), and the main fatty acids were linoleic (66.69% to 72.50%), oleic (13.13% to 18.50%) and palmitic acids (6.87% to 9.56%) (Yalcin et al., 2016).

The tocopherol contents of seed oils are given in Table 4. The α-tocopherol content of seed oils ranged from 0.137 mg/g (Red Globe) to 0.231 mg/g (Cavuş) (p < 0.05). The amounts of β- and γ-tocopherols were 0.00 to 0.220 mg/g and 0.110 mg/g to 0.185 mg/g respectively. Cavuş seed oil had the highest α-tocopherol content, while the highest β- and γ-tocopherol contents were determined in the Trakya ilkeren variety. According to the study of Sabir et al. (2012), the α-tocopherol content of seed oils was determined at between 260.5 and 153.1 mg/kg. The α-, β-, γ- and δ-tocopherol contents were 142.2, 1.29, 25.5 and 0.87 mg/kg for Red Globe seed oil, and 172.4, 0.70, 27.5 and 0.63 mg/kg for Kalecik karasi respectively. It was reported that grape seed oils are a good source of α-tocopherol, with the value ranging between 85.5 and 244.0 mg/kg (Fernandes et al., 2013).

The mineral contents of the seedless parts and seeds of grapes are given in Table 5. The major minerals of the seedless parts of grapes were potassium (K, 1772.615 to 4569.411 mg/kg), calcium (Ca, 187.351 to 498.814 mg/kg), phosphorus (P, 145.038 to 286.042 mg/kg), sulphur (S, 154.673 to 211.982 mg/kg), magnesium (Mg, 77.638 to 133.086 mg/kg) and sodium (Na, 62.485 to 133.401 mg/kg) (p < 0.05). Other minerals, e.g. Fe, Al, B, Cu, Cd, Mo, Zn, Mn and Pb were found at lower levels. The highest potassium content was observed in the Trakya ilkeren variety (4 569.411 mg/kg), followed by Kalecik karasi (2 944.953 mg/kg). Cinarli karasi had the highest Ca content (498.814 mg/kg), while Yalova incisi had the maximum Mg content (133.086 mg/kg). The greatest amounts of P, S and Na were determined in Trakya ilkeren (286.042 mg/kg) and Yalova incisi (211.982 mg/kg). Moreover, Trakya ilkeren was the variety with the highest Fe (24.205 mg/kg) and Al (13.550 mg/kg) contents. Cd shows a toxic effect and was found below 1%, while Ni and Cr were not detected in any of the varieties.

 

Table 6

 

Macro-elements such as K, Ca, P, Mg, S and Na in the grape seeds varied between 3 537.591 and 8 042.746 mg/kg, 3 567.776 and 6 500.015 mg/kg, 1 907.320 and 2 395.649 mg/kg, 1 121.404 and 1 516.708 mg/kg, 818.120 and 1 039.520 mg/kg, and 56.268 and 247.756 mg/kg respectively (p < 0.05). The mineral contents of the grape seeds were higher than in the rest of the grape. The highest Ca, P and S were observed in Red Globe seeds, with values of 6 500.015 mg/kg, 2 395.649 mg/kg and 1 039.520 mg/kg respectively (p < 0.05). The seeds of Yalova incisi had the highest K (8 042.746 mg/kg) and Mg (1 516.708 mg/kg) contents. In addition, grape seeds did not contain Ni and Cr, although Cd was found at levels below 1%, similar to in the grape pulp+skin. In the experiments of Aykut (2002), the mineral composition of the different grape varieties (Sultani, Hamburg Misketi and Alicante Bouschet) was as follows: K (1 255 to 1 750 mg/kg), Ca (24 to 34 mg/kg), Mg (53.75 to 102.5 mg/kg), Na (31.32 to 53.30 mg/kg), P (97.20 to 168.2 mg/kg), Fe (0.71 to 1.88 mg/kg), Cu (0.8 to 2.5 mg/kg), Zn (0.35 to 0.79 mg/kg) and Mn (0.49 to 1.45 mg/kg).

Some differences were observed in the results when compared with the literature. These differences could probably be due to variety, cultural factors and analytical conditions. Grape pulp and seeds are very important for human health because of phenolics, fatty acids, tocopherols, minerals and bioactive properties.

 

CONCLUSIONS

Grape and grape products are important foods due to their richness in bioactive components. Many studies emphasise the importance of grapes and their products, which are sources of natural antioxidants, for health. Therefore, it is necessary to investigate the physicochemical properties and bioactive components of local varieties as well as international varieties. For this purpose, this study was performed to determine the bioactive compounds of both the seeds and the seedless parts of different grape varieties. In addition, the oil content, fatty acid composition and tocopherol content of seeds were investigated. Of all the grape varieties, Red Globe and Trakya Ilkeren have the highest antioxidant activities (90.948%, 90.735%), and total phenolic (170.313 mg/100 g, 199.063 mg/100 g) and total flavonoid (6.810 mg/g, 6.744 mg/g) contents. The seedless parts of the grapes (pulp+skin) exhibited lower phenolic, flavonoid and mineral contents than the seeds of the grapes.

Furthermore, the main fatty acid was linoleic acid (66.942 to 77.359%), and α-tocopherol was the most abundant isomer (0.137 to 0.231 mg/g). The mineral contents of the grape varieties also were compared. Grapes are a significant source of K, Ca and P.

 

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Submitted for publication: March 2017
Accepted for publication: July 2017

 

 

Acknowledgements: The authors extend their appreciation to the International Scientific Partnership Program ISPP at King Saud University for funding this research work through ISPP# 0015
* Corresponding author: E-mail address: mozcan@selcuk.edu.tr