Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 9
Issue 1
Food Science and Technology
Available Online: http://www.ejpau.media.pl/volume9/issue1/art-18.html


H. Hülya Orak
Thrace University, Tekirdağ Agricultural Faculty, Food Technology Department of Vocational College Tekirdağ, Turkey



Eight important red wine grape varieties (Alicante, Kuntra Karasakiz, İrikara, Gabarnet Franch, Cinsaut, Gamay, Merlot and Syrah) which are use for production for red wine by Doluca winery were analyzed. The highest AA (percentage of inhibition on peroxidation in linoleic acid system) was obtained in Gabarnet Franch and Merlot extracts as 90.25% and 90.15% respectively. The lowest AA was determined in İrikara (83.20%) which had the lowest phenol content. Total phenol content (TP) was varied between 1376 (İrikara) and 2329 µg × ml-1 GAE equivalent in methanol extracts (Merlot). Total anthocyanin content (TA) was ranged from 253.5 mg × kg-1 (İrikara) to 2488.4 mg × kg-1 (Alicante). The lowest PPO activity was found in Merlot (0.070 U × ml-1 × min) and the highest activity in Gamay (1.155 U × ml-1 × min). Total sugar content in the analyzed varieties was changed from 15.30% (Syrah) to 22.64 (Merlot). The examinations showed that AA and TP significant correlation (r2 = 0.854**) but less significantly related with TA (r2 = 0.263).

Key words: total antioxidant activity, total phenolic, total anthocyanin, polyphenoloxidase activity, red wine grape varieties.


The antioxidants are our first line of defence against free radical damage, and are critical for maintaining optimum health and well-being [26]. The phenolic compounds in fresh grapes and commercial grape juices may also be beneficial in the prevention of coronary heart disease as they also have strong antioxidant activity toward human LDL oxidation in vitro [22]. The antioxidant activity of wine, fresh grapes, or grape juice is thus attributable to different types of phenolic constituents, but the antioxidant effectiveness on LDL lipid peroxidation is correlated to distinct types of phenolics and their relative concentrations in various samples phenolic compounds in grapes and wine therefore, may be linked to the cardio protective effect of moderate wine consumption through their antioxidant activity [6,7,21,22].

According to Macheix et al. [18] grapes are among the fruits containing the highest content of phenolic substances. Grapes contains a large amount of different phenolic compounds in skins, pulp and seeds, that are partially extracted during winemaking [27]. They showed that more than 15 different phenolic molecules with antioxidant properties (flavan-3-ols, anthocyanins, cinnamic acid derivatives, flavonol derivatives and trans-resveratrol) may be separated in a single run by direct injection of red wine. Bravo [2] explained that wines in particular constitute an excellent source of dietary polyphenols, as they may contain from 1000-4000 (red wines) mg × l-1 total phenols. The phenolic compounds, anthocyanins and their antioxidant properties of grapes have been extensively investigated some researchers, and they were reported that a strong correlation between antioxidant capacity and total phenols of grapes [17,18,28,31]. According to many authors antioxidant activity of fruits, results mainly from phenolics, particularly flavonoids [6,7,15,21,40]. Flavonoids have been demonstrated to have anti-inflammatory, antiallergenic, anti-viral, anti-aging, and anti-carcinogenic activity [11,15,16].

Anthocyanins are natural pigments, responsible for a wide range of colours in grape and red wines. Wang et al. [39] reported that anthocyanins contribute greatly to the antioxidant properties of certain colourful foods, such as grapes and cranberries and cyanidin is the most common anthocyanidin, and the 3-glucoside is the most active antioxidant anthocyanin. The anthocyanins, a subclass of flavonoids and they are attract pollinators and seed dispersers and protect plant tissues from photoinhibition and oxidation resulting from photosynthesis [9]. Some researchers found a strong correlation among antioxidant capacity, total phenols and anthocyanins [15,40]. On the other hand, some investigations also indicated that anthocyanins may be less significantly correlated with the antioxidant properties [1,3,6,14]. Therefore in this work we selected some important winery red grape varieties for the determination total phenolic content, anthocyanin, antioxidant activities and their relationship between each other.

In grape juice and musts, the PPO activity causes colour and turbidity modificidations due to Macheix et al. [19] and it can damaged stability and the organoleptic characteristics, so the polyphenols oxiditation are generally controlled using SO2 [5]. We aimed to investigation polyphenoloxidase activities of red grapes because browning is probably the most severe problem in winemaking and it is known that phenolics and polyphenoloxidase (PPO) are the compounds of musts and wines responsible for browning [20]. In addition to their some chemical characteristics were analyzed which are important for wine industry as invert sugar, refractive index, acidity and pH.


Plant material
Alicante, Kuntra Karasakız, İrikara, Gabarnet Franch, Cinsaut, Gamay, Merlot and Syrah are important red wine grape varieties which are use for production for red wine by Doluca winery. These grapes were obtained from Doluca and analyzed at optimum technological maturity. Analyses were carried out on samples which were collected in September in 2004.

Sample preparation and extraction
Firstly grape berries were removed from each bunch and homogenized in an ice cooled blender (Waring 32BL 79) without leave seeds. For sample extraction, 75 g of blended grape was macerated in 100 ml of methanol containing 0.1% HCl. Then the extract was filtered over Whatman No. 1 paper under vacuum, and the residue was repeatedly extracted with the same solvent until it was colourless. Extraction continued until the extraction solvents became colourless (total solvent volume was between 500-1000 ml). The extracts of grape samples in 100 ml were separately for determination their anthocyanin content. On the other hand the rest of all extracts in methanol was removed and concentrated by a rotary evaporator at 50°C for determination total phenolic content and antioxidant activities.

Determination of total anthocyanin content (TA)
Total anthocyanin contents of samples were determined using the pH-differential method described by Giusti and Wrolstad [8]. Obtained grape extracts in methanol diluted with buffer to give an absorbance reading between 0.4 and 0.6 units. The main anthocyanins reported as being present in red grape cultivars are malvidin-3-O-glucoside (30). From this reason, the wavelength of maximum absorption was used as 520 nm with a Hitachi U-2000 spectrophotometer and the difference in absorbance values at pH 1.0 and 4.5 were recorded. The result, considered as the total anthocyanins content, was calculated as mg of malvidin-3-O-glu per 1000 ml fruit by using a molar absorptivity (ε) of 28,000 and a molecular weight of 493.5 [8].

Determination of total antioxidant activity (AA)
Total antioxidant activity of grape extract was determined according to the thiocyanate method [23]. Ten milligrams of grape extracts was dissolved in 10 ml water. One milligram of extracts in 1ml of water was added to linoleic acid in potassium phosphate buffer (2.5 ml, 0.04 mol × L-1, and pH 7.0). Fifty millilitre linoleic acid emulsion consisting of 175 mg Tween-20, 155 ml linoleic acid, and 0.04 mol × L-1 potassium phosphate buffer (pH 7.0). On the other hand, 5.0 ml control consisting of 2.5 ml linoleic acid emulsion and 2.5 ml potassium phosphate buffer (0.04 mol × L-1, pH 7.0). The mixed solution was incubated at 37°C in a glass flask and the peroxide value was determined by reading the absorbance at 500 nm, after reaction with FeCl2 and thiocyanate at several intervals during incubation. Aliquots (0.1 ml) were drawn room the incubation mixture and mixed with 4.7 ml of 75% ethanol, 0.1 ml of ammonium thiocyanate and 0.1 ml of 20 mM in ferrous chloride in 3.5% HCl and allowed to stand at room temperature for 5 min. The solutions without added extracts used as blank samples. All data are the average of triplicate analyses. The inhibition of lipid peroxidation in percent was calculated by following equation:

% Inhibition = 100-(increase in absorbance of sample/increase in the absorbance of the control reaction) × 100 [13].

Determination of total phenolic content (TP)
Total phenolic content in the grape extracts were determined with Folin-Ciocalteu reagent according to the method of Slinkard and Singleton [32] using gallic acid as a standard phenolic compound. Briefly, 1 ml of extract solution contains was mixed with 45 ml distilled water. 1 ml of Folin-Ciocalteu reagent was added and the content of the flask mixed thoroughly. After 3 min 3 ml of Na2CO3 was added then the mixture was allowed to stand for 2 h. The absorbance was measured at 760 nm. The concentration of total phenolic compounds in grape extracts was determined as microgram of gallic acid equivalent by using an equation that was obtained from standard gallic acid graph.

Determination of polyphenoloxidase (PPO) activity
Grape samples (50 g) was homogenized in an ice cooled blender (Waring 32BL 79) for 3 min of mixing with cold 100 ml, 0.2 M potassium phosphate buffer ( pH 7.0 ) for enzyme extraction. Homogenates were filtered through cheese-cloth and centrifuged for 10 min at 18 000 RPM (Hettich Centrifuge) under cold condition. PPO activity was measured by Halpin and Lee [10]. Mcllvaine’s buffer (0.2 M Na2HPO4/ 0.1 M citrate monohydrate in a 2.3:1 proportion) was adjusted to pH 6.5. For the substrate solution prepare, 1.3764 g catechol (Sigma, Chem. Co.) was dissolved in 25 ml Mcllvaine’s buffer was added to an additional 250 ml Mcllvaine buffer (1 + 10 ) and stirred to 30 min to equilibrate. Prepared enzyme extract (200 µL) was added to 2.8 ml substrate solution in a tube, mixed and the change in absorbance at 420 nm measured over time. One unit of PPO activity was expressed as a change of absorbance of 0.1 per min per ml enzyme extract (Unit × ml-1 × min ).

Other quality attributes analysis
Reduced sugar (RS) and total sugar (TS) were assayed according to the method of using 2, 4 dinitrofenol reactive and glucose as a standard. This determination was done with spectrophotometer at 600 nm [30]. For determination of soluble dry matter in water (Brix) a refractometer was used (Atago, model TYP IT) and evaluated at 20°C [1]. Determination of titrable acidity of samples was done according to TS 1125 [35] and calculated according to tartaric acids. pH value was determined according to TS 1728 [37] by using Fisher accument model 10.

Statistic analysis methods
Statistic analysis were done using by Tarist statistical packaged program and performed two replications and two parallel for randomised complete block factorial test design [24].


Anthocyanins are natural pigments, responsible for a wide range of colours in grapes and red wines. The anthocyanins in the red grapes vary greatly with the species, maturity, production area, seasonal conditions, and yield of fruit. TA contents of the varieties are presented in Table 1. The highest TA was determined in Alicante as 2488.4 mg × kg-1 fresh weight (Table 1). After then high TA content was also found as 1334.3 mg. kg-1 and 1048.8 mg × kg-1 in Syrah and Merlot respectively. TA of the Merlot was determined lower than us (550.6 mg × l-1) by Kallithraka et al. [14]. Neves [25] determined that the total potential in anthocyanins (ApH1) as 707.7 mg × l-1 malvidin glucoside in Merlot and lower than our findings. Different production area of grape varieties may cause these differences. İrikara grapes showed the lowest TA value (253.5 mg × kg-1) among the varieties. The TA content of this grape variety was found similar to Emperor, (252 mg × kg-1); and Perla Nera (201mg × kg-1) grapes varieties by finding Carreno et al. [4].

TP concentration was varied between 1379 (Cinsoult) and 2329 µg × ml-1 GAE equivalent of phenolic compounds in the 1ml of the methanol extracts (Merlot). Differences among the varieties were found statistically important (Table 1). The concentrations of TA as determined by the Folin Ciocalteu method varied from 1847 to 2600 mg × l-1 GAE, for the red wines by Teissedre and Landrault [34]. According to these researchers, the most interesting varieties for high levels of total phenol are Syrah (2266 mg × l-1 GAE) and Merlot (2000 mg × l-1 GAE). In our study Merlot was demonstrated higher phenolic content than Syrah (1906 µg /GAE). This difference may be originated from different growing region.

Table 1. Total anthocyanin (TA) content, total phenol, PPO activity and inhibition of lipid peroxidation (antioxidant activity) of grape varieties

(mg × kg-1 fresh berry weight)

(µg × ml-1 GAE extract)

Inhibition of lipid peroxidation (%)

PPO activity
(U × ml-1 × min)




88.65 b


Kuntra Karasakız



89.66 a





83.20 b


Gabarnet Franch



90.25 a





87.55 b





89.88 a












LSD 0.05





Table 1 shows inhibition of lipid peroxidation in percent of methanol extracts (AA) of grape samples. As seen Table 1, the AA of Gabarnet Franch and Merlot extracts were 90.25% and 90.15% respectively. By contrast the lowest AA was determined in İrikara (83.20%) which had the lowest TF content. Frankel et al. [6] determined the relative inhibition of LDL oxidation varied from 46 to 100% for red wines. The antioxidant activity of wines made with long maceration times exclusively from Rhone Valley Syrah and Grenache varieties was 60% relative inhibition of LDL oxidation [34]. Teissedre and Landrault [34] compeered that of these results with the different Californian red wines showed that the Rhodanian wines made from Syrah and Grenache gave values of relative inhibition of human LDL oxidation identical to those of Cabernet-sauvignon but less than those of Petite Syrah (80%) or Merlot (74%). In our research these grapes showed higher AA activity. In this study the grapes analyzed without separation of seed and these may caused the higher AA. To be known that grape seeds separate from wine during wine making.

Bowning of fruits and vegetables is usually due to the catalytic action of the enzyme polyphenoloxidase (PPO) is a well-known phenomenon. This browning o-Diphenols, present in the tissue of fruits and vegetables, are oxidized by polyphenoloxidase in the presence of oxygen. Generally, inactivation of PPO is using by SO2 in grape products such as juice, wine and raisin [20]. In this research the PPO activity was varied from 0.070 (Merlot) to 1.155 U × ml-1 × min (Tekirdağ Gamay). Weemaes [41] was found PPO activity of white grapes as 0.25ΔO.D./min.

The level of reducing sugars is also used as an estimate for total sugar in grapes, juice and wines [38]. Because the level of alcohol produced during fermentation depends on the ripeness or sugar content of the grapes. Significant differences were found among the reducing sugar content in the analyzed varieties (Table 2). The highest sugar content value (22.64%) was obtained in Merlot and the lowest value (15.30%) was in Syrah grapes. The glucose plus fructose contents were found between 9.63-12.4% in samples of grapes from five varieties (Loureiro, Trajadura, Boal, Verdelho, Touriga) by Varandas et al. [38]. When is compared the sugar content of all varieties in our research was higher than those varieties. Sanz et al. [30] found the total sugar content in grape as 21.15% and it was similar to Merlot (22.64%). The refractive index was also changed between 16.15 and 23.43% and these values obtained from Kuntra Karasakız and Merlot.

Table 2. Some chemical characteristics of crape varieties

Sugar (%)


g × 100 g-1




17.20 d



Kuntra Karasakız


16.15 e





22.05 b



Gabarnet Franch


22.20 b




17.98 c

19.04 c



Tekirdağ Gamay


22.15 b




22.64 a

23.43 a


3.41 bc


15.30 e

16.61 de



LSD 0.05





Total organic acid contents of Syrah, Alicante, Tekirdağ Gamay and Gabarnet Franch were not showed differences statistically. Acidity of these grape varieties were found as 6.42, 6.06, 6.04 and 6.00 g × l-1 tartaric acid equivalents. The pH value in the analyzed varieties was changed between 3.30 (Syrah) and 3.60 (Cinsoult). The titratable acidity was determined as 0.553 g × 100 ml-1 by Hernandez et al [12] in ‘Auttumn seedless’ table grapes at harvest.

In this research grape varieties were showed high TP content and high AA. The AA was correlated to a higher degree with TP content (r2 = 0.854**) and it was related with TA content (r2 = 0.263) less significantly. On the other hand the negative correlation was found between AA and pH (r2 = -0.680*). As it was seen Table 3, PPO activity was correlated with the phenolic content as insignificantly negative (r2 = -0.183) and insignificantly positive (r2 = 0.261) with TA. Recently, many authors [15, 17, 18, 40] found a linear correlation between phenolic content, antioxidant activity and level of anthocyanins. As regards anthocyanins, they have been proven very efficient antioxidants in a number of systems [28, 39] and in vitro LDL oxidation inhibitors [34] while other studies demonstrated a relationship between anthocyanin content and antioxidant activities of red grape extracts and grape juices [9, 21] in different model systems. On the other hand the other researchers [1, 3, 6, 14] determined that, correlation of total phenol with antiradical or antioxidant activity was even higher but poor with total anthocyanin content and our findings was showed in agreement these researchers.

Table 3. Linear correlations between the different characteristics of grape varieties
















0.263 ns













-0.326 ns







-0.417 ns








0.458 ns














This study showed important data with the AA, TA and TP contents of technologically important red wine grape varieties. All red grape varieties have high antioxidant activity but the Gabarnet Franch and Merlot have higher AA among the varieties. Furthermore, the correlation of the AA and TP was found strongly however AA and TA were determined insignificantly. Phenolic components that have strong activity alone or in combination with the anthocyanin pigments are may be responsible the AA of grape varieties. According the result of this research, similar investigation may be carried out on white grape varieties for general proof.


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H. Hülya Orak
Thrace University, Tekirdağ Agricultural Faculty,
Food Technology Department of Vocational College Tekirdağ, Turkey
Phone: +0900282 283 14 38
Fax.,+090 0282 293 14 60
email: horak@trakya.edu.tr

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