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Industrial Crops and Products 31 (2010) 499–508

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Industrial Crops and Products

journa l homepage: www.e lsev ier .com/ locate / indcrop

haracterization of colored and colorless phenolic compounds in Öküzgözüines from Denizli and Elazig regions using HPLC-DAD–MS

asim Kelebeka,b,∗, Ahmet Canbasa, Michael Jourdesc, Pierre-Louis Teissedrec

University of Cukurova, Faculty of Agriculture, Department of Food Engineering, 01330 Adana, TurkeyDepartment of Food Technology, Adiyaman Vocational School, University of Adiyaman, 02040 Adiyaman, TurkeyUniversité Victor Segalen Bordeaux 2, Faculté d’Oenologie-ISVV, UMR oenologie 1219, 210 Chemin de Leysotte, CS 50008, 33882 Villenave d’Ornon Cedex, France

r t i c l e i n f o

rticle history:eceived 29 November 2009eceived in revised form 26 January 2010ccepted 30 January 2010

a b s t r a c t

Characterization of colored and colorless phenolic compounds of wines from Öküzgözü, a native grapevariety grown in Turkey and influence of different vineyard locations on these compounds wereinvestigated. High performance liquid chromatography-diode array detector (HPLC-DAD) and mass spec-trometry (MS) were used for the phenolic compounds analysis. Fourteen anthocyanins, six flavanols,

eywords:küzgözüerroirhenolic compoundsed wine

twelve phenolic acids and six flavonols were identified and quantified. It was observed that total phe-nolic content of Denizli region wines was clearly higher than Elazig wines. Malvidin-3-O-glucoside andits acylated esters was the most dominant anthocyanin, (+)-catechin was the most dominant flavanol,trans-caftaric acid was the most dominant phenol acid and myricetin-3-O-glucoside the most domi-nant flavonol in both regions’ wines. Sensory analysis has also been used in investigating wines derivingfrom the two different vineyard locations. Based upon sensory analysis, wines from Denizli region was

lor, h

nthocyanins preferred due to better co

. Introduction

Phenolic compounds are secondary metabolites naturallyresent in wine grapes and/or produced during the winemak-

ng process. They contribute to sensory characteristics such asolor, flavor, astringency and hardness of wine directly or bynteraction with proteins, polysaccharides, or other phenolic com-ounds (Singleton and Essau, 1969; Lee and Jaworski, 1987). Theain phenolic compounds deriving from grape can be divided in

wo groups, non-flavonoid and flavonoid (anthocyanins, flavan--ols and flavonols) compounds. The non-flavonoid phenoliconstituents present in wine are mainly hydroxybenzoic andydroxycinnamic acids as well as volatile phenols, stilbenes. More-ver, even if those compounds are colorless or slightly yellow, theyave an important role in red wine color since they are knowno enhance and stabilize the color of red wines by intra- andntermolecular reactions (Boulton, 2001). They furthermore con-

ribute to wine flavor (volatile phenolic acids) and some of theme.g. resveratrol) also exhibit potent biological activities (Ribéreau-ayon et al., 2000; Gómez-Alonso et al., 2007).

∗ Corresponding author at: University of Adiyaman, Department of Food Tech-ology, Adiyaman Vocational School, 02040 Adiyaman, Turkey.el.: +90 544 267 07 68; fax: +90 416 223 21 29.

E-mail address: hkelebek@adiyaman.edu.tr (H. Kelebek).

926-6690/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.indcrop.2010.01.012

armony, flavor and body attributes.© 2010 Elsevier B.V. All rights reserved.

Anthocyanins are the main pigments responsible for the color ofthe grapes and young wines. In the Vitis vinifera varieties, there arefifteen different anthocyanins divided into three groups dependingof the glucoside function in position 3 (mono-glucoside, acetyl-glucoside and p-coumaroyl-glucoside). In each groups, there isfive different anthocyanins due to their aromatic B-ring substi-tutions (malvidin, delphinidin, peonidin, petunidin and cyanidin)(Hebrero et al., 1988; Ribéreau-Gayon et al., 2000). Flavan-3-ols(monomeric catechin and proanthocyanidins) are another largefamily of polyphenolic compounds in red wine, which are mainlyresponsible for the structure of red wines such as astringency andbitterness (Singleton and Essau, 1969). The last groups of flavonoidspresent in red wine are the flavonols such as quercetin, myricetin,kaempferol, isorhamnetin and their glycosides. Those compoundsare known for their influences on red wine coloration by copigmen-tation (Boulton, 2001), they also seem to contribute to bitternessof wine (Jackson, 2002) and also display some antioxidant activity(Majo et al., 2008).

Environmental factors (topographical, agro-pedological, cli-matic), usually described by the French term “terroir”, have beenacknowledged to influence grape and wine quality. A complex rela-tionship exists between the factors that influence wine quality. This

is the results of a complex relationship between temperature, sun-light, soil, water availability and physiological process of the vinevariety (Jackson and Lombard, 1993). Soil and climate are two mainfactors taken into consideration with regards to the influence onwine quality (Prado et al., 2007). Many studies have focused on the

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ffects of climate (Jackson and Lombard, 1993; Due, 1995), becauset is considered to be the main constraining effect. The effects ofltitude (Mateus et al., 2001), water availability (Kennedy et al.,002), slope (Mazza et al., 1999) and exposure (Tsanova-Savova andibarova, 2002; Spayd et al., 2002) on vine have also been evaluated.

n the literature, the phenolic composition of wines produced ineveral regions has been extensively studied by many researcherssanova-Savova and Ribarova (2002) and Spayd et al. (2002). How-ver, the influences of environmental factors on the characteristicsf grapes grown in Turkey have not been studied.

Turkey has the world’s fourth largest grape-growing area andfth in terms of grape production after Italy, France, Spain and USA,ith 3,850,000 tonnes produced annually (Fao, 2008). Öküzgözü isative grape variety of V. vinifera, widely planted in the Denizli andlazig region and is used for the production of one of the highestuality red wines in Turkey. It is an important red grape variety forurkey, which produces well-balanced and characteristics winesith an fruity notes such as strawberry, cherry, and blackberry-

ike odors (Kelebek et al., 2007). According to our acknowledge, thenfluence of vineyard location on the colored and colorless phenolicontent of Öküzgözü wines and how these factor affects the qual-ty of Öküzgözü wines have been never investigated and reported.herefore, the aims of this research were to detect possible dif-erences in phenolics composition and wine quality manufacturedith grapes of the Öküzgözü variety, the most valued of the ones

uthorised in Turkey to manufacture red wines and to examine theffect of the vineyard location on the phenolic content of winesrom Öküzgözü cultivated in Denizli and Elazig terroirs.

. Materials and methods

.1. Chemicals

Milli-Q water (Millipore, Bedford, MA) was used in all thisork. HPLC-grade methanol, acetonitrile and formic acid (Merck,armstadt, Germany) were used after filtration through a 0.45-m pore size membrane. Delphinidin-3-O-glucoside, cyanidin--O-glucoside, petunidin-3-O-glucoside, peonidin-3-O-glucoside,alvidin-3-O-glucoside, procyanidins B1 and B2 were purchased

rom Extrasynthese (Lyon, Genay-France). Gallic acid, ferulic acid,affeic acid, p-coumaric acid, sinapic acid, myricetin, quercetin,yricetin-3-O-glucoside, quercetin-3-O-glucoside, kaempferol-3--glucoside and isorhamnetin-3-O-glucoside, (+)-catechin, and−)-epicatechin were purchased from Sigma Chemical Co. (St. Louis,

O, USA).

.2. Grape cultivation

The study was carried out in two nonirrigated vineyard plots

ocated in the Denizli (Cal, 38◦5′N, 29◦17′E) and Elazig (Sun,8◦41′N, 39◦8′E) vinegrowing region during two consecutive years2005 and 2006). The vineyards were primarily selected in ordero represent the major soil and climate types of the region and tonclude what appeared to be distinctive differences in fruit ripen-

able 1limatic parameters of the studied areas for the growth period (April–September).

Parameter Denizli

2005 2006 Average from

Total rainfall (mm) 235 174 146Degree days (base 10 ◦C) 2139 2251 2132Mean maximum temperatures (◦C) 26 27 29Mean daily temperatures (◦C) 20 20 23Mean minimum temperatures (◦C) 14 15 16Cumulative sunshine (h) 1863 1809 1791

d Products 31 (2010) 499–508

ing capacity, and wine quality potential. Denizli region (Denz) wassituated on uniform; clay mixed with loam, with 34% of silt and 37%of sand in the surface layers, situated on a flood plain at the alti-tude zone of 850 m. Elazig region (Elaz) was set on a clay soil andsituated on west-facing hill slopes of the main plain, at an altitudeof 1000 m.

Meteorological data (rainfall, temperature and hours of sun-shine) were provided by the Meteorological weather stationplaced in Denizli and in Elazig (http://www.meteor.gov.tr/veridegerlendirme/il-ve-ilceler-istatistik.aspxm). Both vineyardshave the same climatic classification, a steppe climate, with hotdry summers and cold winters and wide differentials of temper-ature between day and night (Table 1). In average between 1975and 2006, these two vineyards do not have significant climatic vari-ation. However, during the two studied vintage (2005 and 2006),some major differences were recorded between Elazig and Denizliregion in terms of rain fall and mean temperatures (Table 1).

Both vineyards were planted with V. vinifera L. cv. Öküzgözüvines and to minimize the effect of nonenvironmental factors, spe-cial care was taken to achieve maximum uniformity in viticulturalconditions. Vine was 5 years old with a planting between 4000 and5000 vines per hectare and with vine spacing of 3 m between rowsand 2 m within a row.

2.3. Grapes analyses

Grapes reach enological maturity when various factors are inbalance, giving the potential to produce the highest quality wine.Technological (sugar/acid ratio), aromatic (greatest aroma poten-tial), and phenolic maturity are independent variables that must allbe taken into account in assessing enological maturity and decid-ing when the grapes should be harvested (Ribéreau-Gayon et al.,2000; González-Neves et al., 2004a). In order to determine the totalpotential in anthocyanins (ApH1, mg L−1 malvidin-3-O-glucoside),extractable anthocyanins (ApH3.2, mg L−1 malvidin-3-O-glucoside)and phenolic richness (A280, absorbance determined at 280 nm),half of the berries of each sample were analyzed according toGlories (2001). Tannic components of the skins (dpell) and seeds(dTpep), their relative proportions (dpell% and Mp%) and the cellu-lar maturity index (EA%) were calculated as follows:

EA% =[

ApH1 − ApH3.2

ApH1

]× 100

Mp% =[

A280 − (ApH3.2 × 40)/1000A280

]× 100

depll = ApH3.2 × 401000

depll% =[

dpellA280

]× 100

dTpep = A280 − dpell

Elazig

1975 to 2006 2005 2006 Average from 1975 to 2006

84 110 1442370 2556 2660

29 30 2822 23 2114 15 14

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All the measurements were carried out with an Uvikon 922 spec-rophotometer (Kontron Instruments, Saint Quentin en Yvelines,rance), using quartz and glass cells with 1 cm pathlengths.

.4. Winemaking

Healthy grapes of cv. Öküzgözü (500 kg for each region) wereanually harvested at optimum commercial maturity in the 2005

nd 2006 vintages in Denizli and in Elazig vineyards and trans-orted to the Experimental Winery at the Department of Foodngineering, University of Cukurova.

Grapes from each terroir were destemmed and crushed on withcommercial grape destemmer-crusher (Sarksan Ltd. Sti, Ankara,urkey). They were then transferred with 30 mg kg−1 sulfur dioxidento two equal parts in tanks with a volume of 250 L stainless-steelanks for maceration. The maceration took place at 20–22 ◦C for auration of 8 days, after maceration, musts were pressed gentlyith a horizontal press. Alcoholic fermentations were conductedith Zymaflore F10 yeast culture (20 g h L−1, Lafford Oenologie,ordeaux, France) at controlled temperature (20 ± 2 ◦C). Duringlcoholic fermentation, temperature and must density were mon-tored twice a day. After the alcohol fermentation, the wines

ere racked and kept at 20 ◦C to stimulate malolactic fermenta-ion spontaneously. Malolactic fermentation was judged by theegradation of malic acid using paper chromatograph (Cartesiond Campos, 1988). When the malolactic fermentation completedhe wines were racked and 80 mg L−1 sulfur dioxide was added.inally, wines were bottled and stored at 15 ◦C for 6 months prior tonalysis.

.5. Standard chemical analysis

Titratable acidity, pH and reducing sugar analyses were per-ormed on musts and wines (OIV, 1990). Additionally, the density,thanol content, volatile acidity, free and bound SO2, ash, extract,Cl index, gelatine index, and total tannin content were deter-ined for each wine (Ough and Amerine, 1988; OIV, 1990;

ibéreau-Gayon et al., 2000).

.6. Spectrophotometric color analysis

The color intensity (CI) was calculated as the sum of thebsorbances at 420 nm, 520 nm and 620 nm and the tint was deter-ined by dividing the absorbance at 420 nm by the absorbance

t 520 nm. The contribution of each coloration (yellow, red andlue) to the overall color of wine was calculated by dividing thebsorbance at 420 nm (Ye%), 520 nm (Rd%) and 620 nm (Bl%) by theolor intensity (CI). The proportion of red coloration produced byree and bound anthocyanins under their flavylium cations formdA%) was calculated using the following formula

A% =[

1 − Abs420 + Abs620

2 × Abs520

]× 100

s describe by Glories (1984). All theses absorbance measurementsere carried out with an Uvikon 922 spectrophotometer (Kontron

nstruments, Saint Quentin en Yvelines, France).

.7. HPLC analysis of phenolic compounds

Each sample were first filtered through a 0.45 �m Nylon fil-

er and then injected on an Agilent 1100 HPLC system (Agilentechnologies, Palo Alto CA-USA), equipped with a quaternaryump, a diode-array detector, an automatic injector, and thehemStation software. The phenolic compounds were separatedn a reversed-phase Beckman Ultrasphere ODS column (Roissy

d Products 31 (2010) 499–508 501

CDG, France): 4.6 mm × 250 mm, 5 �m equipped with a precolumn4.6 mm × 10 mm (same granulometry).

The HPLC determination and quantification of the anthocyaninswas carried out as described in Kelebek et al. (2007). Identifi-cation and assignation of each anthocyanins was performed bycomparing their retention times and ultra-violet–visible spectra toauthentic standards and to previously reported data (Hebrero et al.,1988; Mazza et al., 1999) and also confirmed by HPLC–MS analy-sis (Kelebek et al., 2007). Quantification of each anthocyanins wascarried out as described in Kelebek et al. (2007).

The HPLC determination and quantification of the main col-orless phenolics was carried out as described in Kelebek et al.(2009). Identification of the main colorless phenolics compoundswere performed by comparing their retention times and UV spectrato authentic standards and also confirmed by HPLC–MS analysis.Their quantification was performed in triplicate using an exter-nal standard calibration curve for each compound. Quantificationof non-commercial compounds was made using the calibrationcurves and response factor of a structurally closer compoundsuch as procyanidins B1 for the procyanidins B3 and B4; caf-feic acid for cis and trans-caftaric acids; p-coumaric acid for cisand trans-coutaric acids. Calibration curves were obtained usingthe commercial standards in the concentrations range normallypresent in enological products (approximately 1–200 mg L−1), theobtained regression coefficients (r2) were above 0.994 in all cases.

2.8. MS apparatus and LC–MS analysis

LC–MS analyses were performed on a Micromass PlatformII simple quadrapole mass spectrometer (Micromass-Beckman,Roissy Charles-de-Gaulle, France) equipped with an electrosprayion source. The LC–MS analyses were realized using identical sol-vents, gradient and column as for the quantification analysis. Themass spectra were recorded between 100 and 2000 amu and themass spectrometer was operated in the positive-ion mode with asource temperatures set at 120 ◦C, a capillary voltage set at 3.5 kVand cone voltages set at 60 and 90 eV.

2.9. Sensory analysis

Descriptive and preference analysis were realized to esti-mate the organoleptic profile of Öküzgözü wines using attributesreported in the literature (Girard et al., 2001; Jackson, 2002). Thepanel was composed of 10 previously trained assessors from ourdepartment. Each judge was asked to separately evaluate each winein terms of 9 general descriptors such as harmony, astringencyaftertaste, astringency, bitterness, sourness, sweetness, body, fla-vor, color. For the aroma and flavor evaluations, each wine wereserved at room temperature in 250 ml tulip shaped black glasses(30 ml), whereas the visual evaluation took place in 250-ml INAOapproved crystal wine glasses (25 ml). All glasses were labeledwith three digit random numbers and presented in random order.Evaluation sessions took place in a sensory room, at ambient tem-perature under daylight in our department. For each wine obtainedfrom different region, panellists evaluated the each descriptor ona horizontal unstructured scale of 10 cm. The marks given for eachdescriptor by all the panellists were summed. Results were ana-lyzed by analysis of variance.

2.10. Statistical analysis

The statistical analysis used for the data analysis were two-wayanalysis of variance (ANOVA) to test the effect of the two factorsstudied (region and year). The statistical analyses were performedusing SPSS statistics software version 11.0 (SPSS Inc., Chicago, IL,USA).

502 H. Kelebek et al. / Industrial Crops and Products 31 (2010) 499–508

Table 2Mean values of the phenolic contents of the grapes, skins extracts and musts of Öküzgözü obtained from different regions.

Öküzgözü Denizli Elazig Factor effects

2005 2006 2005 2006 Region (R) Year (Y) R × Y

Titratable aciditya (g L−1) 7.28 ± 0.40 7.37 ± 0.30 6.84 ± 0.12 7.60 ± 0.20 ns * *

pH 3.26 ± 0.00 3.12 ± 0.00 3.57 ± 0.00 3.34 ± 0.01 ** ** **

Reducing sugar (g L−1) 216.79 ± 0.24 229.10 ± 0.36 207.55 ± 0.24 225.10 ± 0.18 * * nsA280 56.2 ± 0.0 48.8 ± 0.52 37.4 ± 0.28 56.2 ± 1.08 ** ** **

ApH1 (mg L−1) 1705.4 ± 6.22 1897.2 ± 4.16 876.9 ± 1.24 1064.0 ± 9.90 ** * **

ApH3.2 (mg L−1) 649.3 ± 7.50 789.2 ± 8.42 460.8 ± 2.27 527.6 ± 3.71 ** ** **

EA% 61.9 ± 0.30 58.4 ± 0.35 47.4 ± 0.29 50.4 ± 0.81 ** * **

dpell 26.0 ± 0.30 31.6 ± 0.34 18.4 ± 0.10 21.1 ± 0.15 ** ** **

dpell% 46.2 ± 0.53 65.17 ± 1.14 49.2 ± 0.05 37.37 ± 0.42 ** ** **

dTpep 30.0 ± 0.30 17.0 ± 0.08 18.9 ± 0.18 35.0 ± 0.21 ** ** **

Mp% 50.7 ± 0.05 34.83 ± 0.21 53.8 ± 0.53 62.6 ± 0.14 ** * **

R. Signi

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esults are the means of three repetitions.a As tartaric acid equivalent. R: region, Y: year, R × Y: region and year interaction* Significance at p < 0.05.

** Significance at p < 0.01.

. Results and discussion

.1. Grapes composition

The general compositions of grapes and must obtained fromoth regions are summarized in Table 2. Titratable acidity, pH andugar composition of must was in accordance with previous studiesarried out on Öküzgözü grapes (Cabaroglu et al., 2002; Kelebek etl., 2007). However, there were significant differences in the meanalues of general composition of the wines in relation to regions andears. As can be seen in Table 2, the titratable acidity from 2006 vin-age was found higher than that in 2005 vintage from both region.

oreover, the main phenolics content (anthocyanins and tannins)n grape from both vineyards and vintage were estimated by ana-yzing berries according to Glories (2001). The berries from 2005 to006 vintage at harvesting time presented similar skin maturity;owever the skin maturity of the berries from Elazig vineyard wereigher than Denizli vineyard as reveal by a lower cellular maturity

ndex (EA%). As reported by Glories (2001), a lower cellular matu-ity index (EA%) reveal a higher skin maturity since the differenceetween the total anthocyanins and the extractable anthocyaninsecreases when the degradation of the walls and membranes of theell of the skins increases. On the other hands, the seed maturityf Elazig berries from 2005 was higher than in 2006 at harvest-ng time as show by lower MP% index which was the opposite inenizli vineyard. As previously discuss (Glories, 2001; González-eves et al., 2004b) Mp% index represent the tannin proportion in

he seeds, which decreases during grape maturation thus a lowerp% index values imply an higher seed maturity. This differences

egarding skin and seed maturity mainly results of the complexnteraction of climatic conditions and location of the vineyards.urthermore, as reported in Table 2 for both 2005 and 2006 vin-age, berries from Denizli region present a higher anthocyaninsontent as well as higher skin tannins content than berries fromlazig region, as reveal a by higher total potential in anthocyaninsApH1), total extractable anthocyanins (ApH3.2) and total skin tan-ins (dpell) values.

.2. General wine composition

The general compositions of wines obtained from the grape har-ested in the vineyard from Denizli and Elazig is reported in Table 3.

he composition of wines were in accordance with previous stud-es carried out on Öküzgözü wines (Cabaroglu et al., 2002; Kelebekt al., 2007). The ethanol, titratable acidity, tannin level, HCl index,elatin index, color intensity and Rd% in 2006 wines were higherhan in 2005 wines which is in accordance with the analysis per-

ficance at which means differ as shown by analysis of variance.

formed on grape and might results of climatic variations observedon both vineyard.

It can be observed that the wines from the Denizli region pre-sented a higher ethanol, titratable acidity, tannin level, HCl index,gelatin index and color intensity than Elazig region which is againin agreement with the analysis performed on grape. The HCl indexrepresents the tannin polymerization level and the gelatine indexmeasures tannin reactivity towards proteins and therefore assesseswine astringency. In red wine HCl index usually ranges between 10and 30 and the acceptable values of the gelatin index is between 40and 60 in order to have a red wine with a satisfactory astringencyfor the future consumer (Ribéreau-Gayon et al., 2000).

As summarized in Table 3, red wine produce with grape fromDenizli vineyard had a higher color intensity and Rd% than winefrom Elazig. In overall, the comparison of our color index for bothwine with previously reported data on high quality red wine fromFrance (Glories, 1984; Ribéreau-Gayon et al., 2000) reveal that thered wine produced with berries from Denizli and Elazig vineyard in2005 and 2006 can be considered as a high quality wine regardingtheir color specificities.

3.3. Anthocyanin composition of the wines

Fourteen different anthocyanins, including five glucosides, fiveacetyl glucosides, and four coumaroyl glucosides were identifiedand quantified (Fig. 1a). Molecular ion and fragmentation infor-mation, together with �max values for the identified compoundsare present in Table 4a. Table 4b shows the anthocyanin com-position of the Öküzgözü wines obtained from the two differentregions. The results obtained by applying two-way ANOVA to testthe significant main effects of the two factors revealed significantdifferences for most of the quantified anthocyanins present in eachwine. It was observed that the anthocyanin profiles and compo-sition of the wines were similar, thus the vineyard location didnot produce any selective effect on individual anthocyanins. How-ever, the anthocyanins concentration in wines from Denizli region(447.64–703.88 mg L−1) was higher than in wines from Elazigregion (382.50–598.38 mg L−1), as expected due to the analysis per-formed on grapes, the anthocyanin contents in the 2006 vintagefor both regions was also higher than in 2005 (Table 4b). Thesedifferences could be attributed to influence of climatic conditions.The anthocyanin level observed in our wines is in accordance with

previously reported data such as Mazza et al. (1999) which reportthat the content of anthocyanin in wines were 316–337 mg L−1 forCabernet franc, 338–371 mg L−1 for Merlot, and 171–273 mg L−1 forPinot noir as well as the results obtained on Greek red wines fromdifferent vineyard locations (Kallithraka et al., 2001).

H. Kelebek et al. / Industrial Crops and Products 31 (2010) 499–508 503

Table 3General composition of the Öküzgözü wines obtained from different regions.

Analysis Denizli Elazig

2005 2006 2005 2006

Density (20 ◦C/20 ◦C) 0.9918 ± 0.00 0.9907 ± 0.00 0.9915 ± 0.00 0.9916 ± 0.00Ethanol (v/v; %) 12.1 ± 0.02 12.7 ± 0.04 11.5 ± 0.01 12.5 ± 0.06Titratable aciditya (g L−1) 6.12 ± 0.02 6.63 ± 0.01 5.93 ± 0.02 6.56 ± 0.04pH 3.46 ± 0.00 3.34 ± 0.01 3.52 ± 0.02 3.41 ± 0.01Tannin (g L−1) 2.78 ± 0.04 2.89 ± 0.08 2.09 ± 0.03 2.81 ± 0.01Volatile acidityb (g L−1) 0.24 ± 0.02 0.22 ± 0.02 0.26 ± 0.02 0.26 ± 0.02HCl index 37.9 ± 0.10 38.4 ± 0.12 35.6 ± 0.08 35.7 ± 0.11Gelatin index 54.62 ± 0.02 56.11 ± 0.02 50.33 ± 0.02 54.22 ± 0.02Reducing sugar (g L−1) 1.7 ± 0.06 2.1 ± 0.04 1.6 ± 0.05 2.2 ± 0.04Free SO2 (mg L−1) 13.2 ± 0.03 16.5 ± 0.05 15.1 ± 0.02 17.3 ± 0.04Bound SO2 (mg L−1) 92.4 ± 0.12 90.1 ± 0.18 88.7 ± 0.14 89.6 ± 0.15Extract (g L−1) 20.6 ± 0.10 22.6 ± 0.13 20.2 ± 0.16 22.9 ± 0.07Ash (g L−1) 2.0 ± 0.01 2.2 ± 0.03 1.9 ± 0.01 2.1 ± 0.02Color intensity 1.40 ± 0.06 2.24 ± 0.01 1.04 ± 0.03 1.87 ± 0.04Tint 0.40 ± 0.01 0.65 ± 0.02 0.34 ± 0.00 0.36 ± 0.04Ye% 26.40 ± 0.31 23.47 ± 0.02 32.83 ± 0.12 24.55 ± 0.08Rd% 65.41 ± 0.11 69.92 ± 0.03 50.52 ± 0.06 68.92 ± 0.10Bl% 8.19 ± 0.20 6.61 ± 0.00 15.65 ± 0.02 6.53 ± 0.20dA% 60.04 ± 0.79 37.98 ± 0.22 75.50 ± 0.86 55.80 ± 0.69

Results are the means of three repetitions.a As tartaric acid equivalent.b As acetic acid equivalent.

Table 4aRetention times, mass spectral details and UV data of anthocyanins identified in wines, together with cited references.

Compounds Peak no Rt (min) �max (nm) [M]+ Fragments Ref.a

Delphinidin-3-O-gluc 1 8.41 526 465 303 a, bCyanidin-3-O-gluc 2 9.60 516 449 287 a, bPetunidin-3-O-gluc 3 10.9 527 479 317 a, bPeonidin-3-O-gluc 4 12.0 516 463 301 a, bMalvidin-3-O-gluc 5 13.0 527 493 331 a, bDelphinidin-3-O-acet-gluc 6 14.9 529 507 303 a, bCyanidin-3-O-acet-gluc 7 16.2 517 491 287 b, cPetunidin-3-O-acet-gluc 8 18.4 529 521 317 a, bPeonidin-3-O-acet-gluc 9 19.8 517 505 301 a, bMalvidin-3-O-acet-gluc 10 20.1 529 535 331 a, bDelphinidin-3-O-p-coum-gluc 11 22.6 527 611 303 b, cPetunidin-3-O-p-coum-gluc 12 24.0 527 625 317 b, cPeonidin-3-O-p-coum-gluc 13 26.0 525 609 301 a, bMalvidin-3-O-p-coum-gluc 14 27.3 530 639 331 a, b

a (a) Wang et al. (2003); (b) Villiers et al. (2004); (c) Garcia-Beneytez et al. (2003).

Table 4bAnthocyanin content (mg L−1 ± standard deviation) of Öküzgözü wines obtained from different regions.

Anthocyanins Denizli Elazig Factor effects

2005 2006 2005 2006 R Y R × Y

Delphinidin-3-O-gluc 38.24 ± 0.64 (8.5%) 60.42 ± 0.33 (8.6%) 30.58 ± 0.49 (8.0%) 39.08 ± 0.30 (6.5%) ** ** **

Cyanidin-3-O-gluc 9.21 ± 1.07 (2.1%) 6.06 ± 0.05 (0.9%) 3.68 ± 0.13 (1.0%) 4.69 ± 0.07 (0.8%) ** ** **

Petunidin-3-O-gluc 75.26 ± 4.24 (16.8%) 69.77 ± 0.60 (9.9%) 43.67 ± 1.12 (11.4%) 66.30 ± 1.00 (11.1%) ** ** **

Peonidin-3-O-gluc 40.74 ± 0.99 (9.1%) 39.55 ± 0.34 (5.6%) 32.31 ± 0.15 (8.5%) 26.81 ± 0.40 (4.5%) ** * **

Malvidin-3-O-gluc 203.43 ± 1.38 (45.5%) 407.69 ± 3.49 (57.9%) 201.34 ± 3.22 (52.6%) 348.86 ± 5.25 (58.3%) ** ** **

Delphinidin-3-O-acet-gluc 7.71 ± 0.17 (1.7%) 4.12 ± 0.04 (0.6%) 3.65 ± 0.12 (1.0%) 4.79 ± 0.07 (0.8%) ** ** **

Cyanidin-3-O-acet-gluc 0.87 ± 0.04 (0.2%) 1.49 ± 0.01 (0.2%) 0.30 ± 0.35 (0.1%) 1.88 ± 0.03 (0.3%) ns ** **

Petunidin-3-O-acet-gluc 7.83 ± 0.19 (1.8%) 7.01 ± 0.06 (1.0%) 4.45 ± 0.07 (1.2%) 4.03 ± 0.06 (0.7%) ** * *

Peonidin-3-O-acet-gluc 4.61 ± 0.26 (1.0%) 5.45 ± 0.05 (0.8%) 4.10 ± 0.05 (1.1%) 4.58 ± 0.07 (0.8%) ** * nsMalvidin-3-O-acet-gluc 21.49 ± 0.06 (4.8%) 40.29 ± 0.35 (5.7%) 22.56 ± 0.05 (5.9%) 35.39 ± 0.53 (5.9%) ** ** **

Delphinidin-3-O-p-coum-gluc 2.60 ± 0.02 (0.6%) 1.65 ± 0.01 (0.2%) 1.58 ± 0.01 (0.4%) 1.87 ± 0.03 (0.3%) ** ** **

Petunidin-3-O-p-coum-gluc 5.93 ± 0.11 (1.3%) 5.06 ± 0.04 (0.7%) 4.15 ± 0.11 (1.1%) 6.92 ± 0.10 (1.2%) ns * *

Peonidin-3-O-p-coum-gluc 4.90 ± 0.09 (1.1%) 6.11 ± 0.05 (0.9%) 4.61 ± 0.10 (1.2%) 4.54 ± 0.07 (0.8%) ** * **

Malvidin-3-O-gluc-p-coum 24.82 ± 0.79 (5.5%) 49.21 ± 0.42 (7.0%) 25.52 ± 0.15 (6.7%) 48.64 ± 0.73 (8.1%) ns ** *

Total 447.64 ± 7.21 703.88 ± 5.84 382.50 ± 5.82 598.38 ± 8.71

R: region, Y: year, R × Y: region and year interaction.Significance at which means differ as shown by analysis of variance.ns: not significant.

* Significance at p < 0.05.** Significance at p < 0.01.

504 H. Kelebek et al. / Industrial Crops and Products 31 (2010) 499–508

Fig. 1. (a) HPLC chromatograms of wine recorded at 520 nm from Denizli region in 2005. (b) HPLC chromatogram of the Öküzgözü wine flavanols (�: 280 nm), phenolic acids(�: 328 nm) and flavonols (�: 360 nm).

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H. Kelebek et al. / Industrial Cro

As can be seen in Table 4a, five mono-glucoside deriva-ives of delphinidin, cyanidin, petunidin, peonidin, and malvidin,espectively, have been detected. Among all these anthocyanins,alvidin-3-O-glucoside was the most dominant in both regionsines, as it accounted for the largest proportion of the total

nthocyanin compounds (∼60%). Wines obtained from Denizliegion have 203.43–407.69 mg L−1 of malvidin-3-O-glucoside andlazig wines contained 201.34–348.86 mg L−1 of this compound.t has been stated that the proportion of malvidin derivativess usually higher in wines than in the grapes used for theirlaboration, because it is the structurally more stable antho-yanic form, and therefore the one that decreases the less duringermentation, post-fermentation standing and ageing of winesGlories, 1984). In addition to this compound, Öküzgözü winesontained significant level petunidin-3-O-glucoside (9.9–16.8%),elphinidin-3-O-glucoside (6.5–8.6%) and peonidin-3-O-glucoside4.5–9.1) and in a lesser extend cyanidin-3-O-glucoside (0.8–2.1%).s observed on berries analysis (extractable anthocyanins) and onine (color intensity, tint and Rd%), the concentration of antho-

yanin were higher in 2006 vintage in both region.Among the acylated anthocyanins, malvidin-3-O-(6-O-acetyl)-

lucoside was the most abundant anthocyanins in both regionsines. Concentration of malvidin-3-O-(6-O-acetyl)-glucoside inines from Denizli region (21.49–40.29 mg L−1) was slightlyigher than in wines from Elazig region (22.56–35.39 mg L−1) andalvidin-3-O-(6-O-acetyl)-glucoside contents of wines were sig-

ificantly higher in 2006, for both studied region. Many authorsave reported the importance of malvidin and the acylated antho-yanins in the differentiation of the various cultivars, taking intoccount the grapes’ anthocyanins profiles (Roggero et al., 1986;aló et al., 1994; Mazza et al., 1999). Caló et al. (1994) consid-red that peonidin and malvidin are highly dependent on variety,

ollowed by cyanidin and acetylated anthocyanins. Mazza etl. (1999) reports that malvidin-3-O-(6-O-acetyl)-glucoside alongith malvidin-3-O-(6-O-p-coumaryl)-glucoside is considered to be

ne of the most important derivatives for the characterizationf varieties. As expected, among the p-coumaroylated antho-

able 5aetention times, mass spectral details and uv data of phenolics identified in wines, togeth

Compounds Peak no Rt (min) �ma

Flavanols(+)-Catechin 15 21.1 278(−)-Epicatechin 16 38.2 278Procyanidin B3 17 16.2 280Procyanidin B1 18 18.9 280Procyanidin B4 19 23.7 280Procyanidin B2 20 30.6 280

Phenolic acidsGallic acid 21 4.6 272Protocatechuic acid 22 10.0 260Vanilic acid 23 23.9 262Syringic acid 24 33.2 275cis-Caftaric acid 25 11.7 328trans-Caftaric acid 26 13.8 328cis-Coutaric acid 27 19.6 310trans-Coutaric acid 28 21.1 310Caffeic acid 29 25.9 320p-Coumaric acid 30 39.1 308Ferulic acid 31 46.9 323Sinapic acid 32 53.1 310

FlavonolMyricetin-3-O-gluc 33 54.2 353Quercetin-3-O-gluc 34 58.1 355Myricetin 35 64.2 371Kaempferol-3-O-gluc 36 65.2 345Isorhamnetin-3-O-gluc 37 72.9 356Quercetin 38 79.8 372

a (a) Atanasova (2003); (b) Monagas et al. (2005).

d Products 31 (2010) 499–508 505

cyanins, malvidin-3-O-(6-O-p-coumaryl)-glucoside was the mostabundant anthocyanins. The total amount of malvidin-3-O-(6-O-p-coumaryl)-glucoside was between 24.82 and 49.21 mg L−1 inDenizli wines and between 25.52 and 48.64 mg L−1 in Elazig wines(Table 4b).

3.4. Colorless phenolic composition of the wines

A total of twenty-four colorless phenolic compounds were iden-tified and quantified in wines obtained with the grape harvestedfrom both Denizli and Elazig vineyard including flavanols (6), phe-nolic acids (12), and flavonols (6). Molecular ion and fragmentationinformation, together with �max values for the identified com-pounds are present in Table 5a. The results obtained by applyingtwo-way ANOVA to test the significant main effects of the twofactors (the interaction and the within-error terms were pooled)revealed that significant differences existed in most of the quan-tified compounds determined (Table 5b). There were significantdifferences in the mean values of all of the phenolic compoundsof the wines in relation to regions and years. Denizli region wineshave 138.36–181.43 mg L−1 of flavanols, 76.81–140.0 mg L−1 phe-nol acids, 8.78–13.6 mg L−1 of flavonols and Elazig wines contained114.48–145.80, 86.91–163.10 and 5.28–8.0 mg L−1 of these com-pounds, respectively. As can be seen, phenolic content of wineswas higher in 2005, for the both regions studied.

Six flavanols; (+)-catechin, (−)-epicatechin, procyanidin B1,procyanidin B2, procyanidin B3 and procyanidin B4 were detectedin wines (Table 5a). As can be seen in Table 5b, wines obtainedfrom Denizli region contained more flavanols than wines obtainedfrom Elazig region in both vintage. Moreover, by contrast withthe anthocyannin concentration, the level of flavanols was higherfor the 2005 vintage in both region than in 2006 vintage.

Among the flavanols, (+)-catechin was the most abundant inÖküzgözü wines. Concentration of (+)-catechin in wines from Deni-zli region (64.0–72.2 mg L−1) was higher than in wines from Elazigregion (51.8–64.4 mg L−1) and (+)-catechin contents in wines weresignificantly higher in 2005 vintage in both studied region. (−)-

er with cited references.

x (nm) [M]+ (m/z) Fragments (m/z) Ref.a

289 – a289 – a577 425, 289 b577 425, 289 b577 425, 289 b577 425, 289 b

169 125 a, 294 153 109 b

167 – b197 – b

, 294 311 179 a, 294 311 179 a, b

295 163 a296 163 a, b

, 300 179 135 a163 119 a

, 293 193 134 a223 – a

479 317 a463 301 a317 – a447 285 b478 316 a301 – a

506 H. Kelebek et al. / Industrial Crops and Products 31 (2010) 499–508

Table 5bColorless phenolic content (mg L−1 ± standard deviation) of Öküzgözü wines obtained different regions.

Compounds Denizli Elazig Factor effects

2005 2006 2005 2006 R Y R × Y

Flavanols(+)-Catechin 72.22 ± 10.12 64.04 ± 3.30 64.40 ± 5.47 51.79 ± 2.71 ** ** **

(−)-Epicatechin 34.51 ± 3.46 21.18 ± 1.70 29.30 ± 1.66 18.50 ± 1.17 ** ** **

Procyanidin B3 7.10 ± 0.12 6.02 ± 0.92 6.61 ± 0.44 5.08 ± 0.74 ** ** **

Procyanidin B1 39.90 ± 4.93 26.19 ± 2.29 26.10 ± 2.18 23.40 ± 1.12 ** ** **

Procyanidin B4 3.50 ± 0.68 2.19 ± 0.12 2.80 ± 0.50 1.73 ± 0.42 ** ** **

Procyanidin B2 24.20 ± 2.12 18.74 ± 1.22 16.61 ± 0.88 13.98 ± 1.06 ** ** **

Total 181.43 138.36 145.8 114.48

Phenolic acidsGallic acid 19.41 ± 1.32 9.03 ± 0.11 18.90 ± 0.22 8.66 ± 0.22 ** ** **

Protocatechuic acid 1.32 ± 0.07 0.27 ± 0.04 1.41 ± 0.02 0.34 ± 0.02 ** ** **

Vanillic acid 0.80 ± 0.03 0.20 ± 0.01 0.92 ± 0.10 0.22 ± 0.02 ** * *

Syringic acid 3.30 ± 0.12 0.77 ± 0.02 2.90 ± 0.18 0.76 ± 0.04 ** ** **

cis-Caftaric acid 3.90 ± 0.18 0.94 ± 0.03 4.31 ± 1.12 1.16 ± 0.03 ** ** **

trans-Caftaric acid 68.91 ± 2.51 42.50 ± 2.11 78.40 ± 2.54 51.56 ± 1.88 ** ** **

cis-Coutaric acid 6.10 ± 0.13 1.82 ± 0.08 6.60 ± 1.02 2.19 ± 0.07 ** ** **

trans-Coutaric acid 33.11 ± 3.44 20.15 ± 1.49 45.41 ± 2.42 20.77 ± 0.89 ** ** **

Caffeic acid 1.90 ± 0.13 0.50 ± 0.06 2.40 ± 0.12 0.66 ± 0.14 ** ** **

p-Coumaric acid 0.80 ± 2.23 0.23 ± 0.01 0.63 ± 0.08 0.17 ± 0.02 ** ** **

Ferulic acid 0.62 ± 0.02 0.18 ± 0.01 0.61 ± 0.04 0.19 ± 0.01 ** ** **

Sinapic acid 0.83 ± 0.02 0.22 ± 0.02 0.70 ± 0.02 0.23 ± 0.01 ** ** **

Total 140.9 76.81 163.1 86.91

FlavonolsMyricetin-3-O-gluc 6.10 ± 0.88 3.92 ± 0.56 3.60 ± 0.60 2.37 ± 0.17 ** ** **

Quercetin-3-O-gluc 3.42 ± 1.64 2.62 ± 0.02 1.81 ± 0.21 1.16 ± 0.07 ** ** **

Myricetin 0.91 ± 0.16 0.33 ± 0.01 0.60 ± 0.54 0.40 ± 0.01 ** ** **

Kaempferol-3-O-gluc 0.31 ± 0.06 0.17 ± 0.00 0.20 ± 0.16 0.13 ± 0.02 ** ** **

Isorhamnetin-3-O-gluc 0.90 ± 0.02 0.50 ± 0.02 0.70 ± 0.22 0.46 ± 0.06 ** ** **

Quercetin 2.01 ± 0.16 1.24 ± 0.08 1.11 ± 0.05 0.76 ± 0.03 ** ** **

Total 13.60 8.78 8.00 5.28

R iffer an

EpCrrbte2i(w“2wltiboiasiwbcw

: region, Y: year, R × Y: region and year interaction. Significance at which means ds: not significant.

* Significance at p < 0.05.** Significance at p < 0.01.

picatechin was the second most abundant flavanol followed byrocyanidin B1 and procyanidin B2 in wine from both regions.oncentration of (+)-catechin and (–)-epicatechin in French winesanges from 32.8 to 209.8 mg L−1 and from 22.1 to 130.7 mg L−1,espectively, and the level of procyanidins in these wines wasetween 7.8 and 39.1 mg L−1 for B1, 18.3 to 93 mg L−1 for B2, 21.4o 215.6 mg L−1 for B3, and 20.2 to 107.2 mg L−1 for B4 (Carandot al., 1999). Kilmartin et al. (2002) found values between 3.2 to23 mg L−1 of (+)-catechin, and 2.4 to 143 mg L−1 of (−)-epicatechin

n wines from New Zealand and Australia. Rodríguez-Delgado et al.2001) and Orbán et al. (2006) found similar concentrations in redines from the Canary Islands and from the Hungarian wine region

Eger”, respectively (Rodríguez-Delgado et al., 2001; Orbán et al.,006). Our data is also in accordance with the results of Greek redines from the Aegean, north and south Greece regions as pub-

ished by Kallithraka et al. (2001). Moreover, it is well know thathe concentration of flavanols is a key parameter for red wine qual-ty, since those compounds are responsible of the astringency anditterness. They also play an important role in the overall evolutionf red wine during its ageing due to their intra and intermolecularnteraction with the other phenolic constituent of the wine such asnthocyanins (Ribéreau-Gayon et al., 2000; Boulton, 2001). Thus,ince both the red wine from Denizli and Elazig region has sim-

lar range of flavanol concentration as reported in the main red

ine across the world (as detail above), the expected astringency,itterness and ageing abilities of these two wines should also beomparable with the red wine produce in the main vineyard in theorld.

s shown by analysis of variance.

The twelve phenolic acids identified and quantified during ouranalysis were gallic acid, protocatechuic acid, vanilic acid, syringicacid, cis-caftaric acid, trans-caftaric acid, cis-coutaric acid, trans-coutaric acid, caffeic acid, p-coumaric acid, ferulic acid and sinapicacid. By contrast with the flavanols concentration, wine from Elazigregion contained higher level of phenolic acid than wines obtainedfrom Denizli region in both vintage. Moreover, as observed forthe anthocyanin concentration, the level of phenolic acid washigher for the 2005 vintage in both region than in 2006 vintage.trans-Caftaric acid and trans-coutaric acid were the most domi-nant phenolic acids in Denizli and Elazig regions’ wines, as theyaccounted for the largest proportion of the total phenolic acidscontents (i.e. ∼75–80%). Concentration of trans-caftaric and trans-coutaric acid in Denizli wines ranges from 42.50 to 68.9 mg L−1

and from 20.15 to 33.10 mg L−1, whereas Elazig wines contained51.56 to 78.40, and 20.77 to 45.40 mg L−1 of these compounds,respectively. Similarly, Zafrilla et al. (2003) found trans-caftaricand trans-coutaric acids to be the major p-hydroxycinnamic acidsin Monastrell red wines, their levels in freshly fermented winesbeing in the region of 31 and 15 mg L−1, respectively. Gomez-Plazaet al. (2000) also studied freshly bottled Monastrell wines andfound trans-caftaric and trans-coutaric acid levels over the ranges171–187 and 117–124 mg L−1, respectively. Some authors consider

that the trans-coutaric acid/trans-caftaric acid ratio may charac-terize wines according to their varietals origin. Wine may alsocontain their corresponding p-hydroxycinnamic acids (p-coumaricand caffeic acid) due to the hydrolysis of the tartaric moiety in wine(Gómez-Alonso et al., 2007). Gallic and protocatechuic acids are

H. Kelebek et al. / Industrial Crops and Products 31 (2010) 499–508 507

gözü w

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mOfoftOflpQfqtwpageCtNm(cae

3

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Fig. 2. Sensory profiles of Öküz

undamentally found in wine because they originate mainly fromrape pulp, although they have also been found in small concen-rations in grape seeds containing (+)-catechin and (−)-epicatechinallates (Gómez-Alonso et al., 2007).

The six flavonols identified and quantified in both wine wereyricetin-3-O-gluc, quercetin-3-O-gluc, myricetin, kaemferol-3--gluc, isorhamnetin-3-O-gluc, and quercetin. It appears that wine

rom Elazig region contained lower level of flavonol than winesbtained from Denizli region in both vintage. Moreover, as observedor the flavanol level, the concentration of flavonol was higher forhe 2005 vintage in both region than in 2006 vintage. Myricetin-3--gluc and its aglycone form (Myricetin) were the most dominantavonol in Denizli and Elazig wines, as it accounted for the largestroportion (i.e. ∼52%). of the total flavonol contents (Table 5b).uercetin-3-O-gluc was the second most abundant flavonol and

ollowed by myricetin in wine from both regions. Concentration ofuercetin-3-O-gluc and myricetin in Denizli wines ranges from 2.62o 3.40 mg L−1 and from 0.33 to 0.90 mg L−1, respectively. Elazigines contained 1.16 to 1.80, and 0.40 to 0.60 mg L−1 of these com-ounds, respectively. The content of flavonols in red wines haslways been much higher when both aglycones and glycoconju-ates were taken into consideration in the determinations (Makrist al., 2006). In nine samples from various red cultivars, includingabernet Sauvignon, Syrah, Merlot and Tempranillo, quercetin con-ent was from 0 to 43.1 mg L−1 (Vinas et al., 2000). Another study onew York wines also showed that aglycones such as quercetin andyricetin occur at rather low levels, as they averaged 5.23 mg L−1

Goldberg et al., 2000). As summary, our results regarding flavonoloncentration in red wine from Denizli and Elazig region are inccordance with previously reported level in red wine from differ-nt wine regions.

.5. Sensory evaluation

To profile the sensory characteristics of wine, the wines werevaluated by a descriptive and preference test. Fig. 2 shows the

verage scores of the 10 descriptors. From the results of analysisariance (ANOVA) conducted on the descriptive data, bitterness,ourness and sweetness characters of wines were not found toe significantly different (p < 0.05). These characters were similaror both wines. However, color, harmony, astringency aftertaste,

ines (*significance at p < 0.05).

astringency, flavor and body for Denizli wines were found signif-icantly (p < 0.05) different from the Elazig wines. It appears thatthe average scores of the descriptors were higher in 2006 for bothregions. In the preference test, Denizli wines were preferred dueto better color, body, harmony and astringency properties by thejudges.

4. Conclusions

The results obtained from the vintage 2005 and 2006 suggestthat red wine produce with grape harvested in Denizli vineyardpresent high anthocyanin, flavanol and flavonol content than wineproduce in Elazig region. These differences in phenolics contentresults of the complex interaction between Öküzgözü vine and thevineyard specificities (climatic variation, soil and location). More-over, higher anthocyanin content and lower colorless phenoliccompounds had been observed in 2006 vintage for both studiedvineyards studied. Malvidin-3-O-glucoside and its acylated esterswere the most dominant anthocyanin, (+)-catechin was the mostdominant flavanol, trans-caftaric acid was the most dominant phe-nolic acid and myricetin-3-O-glucoside the most dominant flavonolin wine from both regions. Based upon sensory analysis, wines fromDenizli region have been prefer due to its better color, harmony,flavor and body attributes.

Acknowledgements

This research was supported by The Scientific and TechnicalResearch Council of Turkey (Project no. TUBITAK-TOVAG-105O364)and Cukurova University Research Council (Project no. ZF-2005D12).

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