Relations Between the Phenolic Composition and Free Radical Scavenging, and Antibacterial Activities of Red Wines from Different Cultivars of Vitis vinifera L.

Abstract

Polyphenols are widely distributed in the plant kingdom, including the grape (Vitis vinifera L.), and are extracted during winemaking. Because of their potential beneficial health, free radical scavenging and antibacterial activities of some red wines from the Zupa viticulture and winemaking region in Serbia are produced from old autochthonous grape cultivars: Vranac, Kratosija, and Prokupac, and from Merlot, Pinot Noir, and Gamay cultivars originated from France. The results showed that the antioxidant activity was highly correlated with the content of total phenolics (r² = 0.9737), tartaric esters (r² = 0.9980), flavonols (r² = 0.8938), and monomeric anthocyanins (r² = 0.9239) in selected wines. The antibacterial activity of investigated wines against the pathogenic strain of Staphylococcus aureus significantly correlated with their phenolic constituents.

Keywords:

• Red wines
• Phenolics
• Tartaric esters
• Flavonols
• Anthocyanins
• Free radical scavenging activity
• Antibacterial activity

INTRODUCTION

Polyphenols are present in fruit, bark, leaves, and seeds of many plants, where their major function is to provide against microbiological pathogens, insect pests, and larger herbivores. Through the formation of complexes with salivary proteins, these compounds are responsible for the astringency of some fruits and beverages.[1] Phenolic compounds represent one of the most important quality parameters of grapes and wines since they contribute to organoleptic characteristics, such as color, astringency, and bitterness. The phenolic composition in wines is determined by several factors, such as the variety of used cultivars of Vitis vinifera L. and conditions under which they were grown: soil, geo-graphical location, and weather. Usually, the sunlight exposure and temperature are the main factors influencing grape phenolic composition. Winemaking techniques also play a very important role in the chemical composition of the wines: the time of maceration and fermentation in contact with the grape skins and seeds, pressing, maturation, fining and aging conditions.[2] The wine consists of different phenolic compounds, so the antioxidant and the antibacterial activity of wine are connected with a synergy of these compounds. Still, it is very important to determine which group of phenolic compounds is most influential in antioxidant and antibacterial properties of wine. Recent studies indicate that consumption of the small amounts of red wine on a regular basis reduce the risk of coronary heart disease and atherosclerosis, and this benefit is ascribed to the antioxidant properties of the polyphenolic compounds.[3–5] In both, in vitro and in vivo research trials, anthocyanins have demonstrated a noticeable ability to reduce cancer cell proliferation and to inhibit tumor formation.[6–8] Finally, the higher the concentration of polyphenols is in wines, the higher will be their antioxidant capacity and antimicrobial activity.[9–15]

Serbia is one of the middle-weight wine producers in Europe. Many of the Serbia-growing districts boast remarkably good ecological conditions for the production of high-quality wines with geographically controlled designations of origin in a large assortment. The Zupa region vineyards are considered to be the best wine-producing regions in Serbia, with a tradition of wine growing that goes back to the pre-Roman times. The Zupa wine region has a favorable geographical position and climate conditions, like other renowned vineyards in Europe, such as the French Bordeux. The aim of this study was to determine the free radical scavenging and antibacterial activities of red wines produced from autochthonous and French grape cultivars, produced in Zupa's vineyards and winemaking, and their correlation with content phenolic composition.

MATERIALS AND METHODS

Chemicals

2,2-Diphenyl-1-picrylhydrazyl free radical, gallic acid, caffeic acid, and quercetin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The used reagents were of analytical quality.

Bacterial Culture

Staphylococcus aureus (strain ATCC 28053 gram-positive) micro organism was used for the antibacterial test and was grown on Mueller–Hinton dextrose agar.

Wine Samples

The selected wines with controlled geographical origin from different grape cultivars of vintage grown in Zupa's wine region (Central Serbia) were analyzed. All of them are red wines with a definite geographical origin: Medjas (Zupa–Aleksandrovac, 2008); Crno (Zupa–Aleksandrovac, 2008); Kratosija (Zupa–Aleksandrovac, 2008); Vranac (Zupa–Aleksandrovac, 2008); and As (Zupa–Aleksandrovac, 2008).

Determination of Total Phenolics, Tartaric Esters, and Flavonols

Total phenolics, tartaric esters, and flavonols in selected wine samples were determined by spectrophotometrical methods.[16] The dilution wine sample was mixed with ethanol/HCl solvent. Approximately 15 min after the absorbance at 280 nm was used to estimate total phenolics (gallic acid was used as a standard); the absorbance at 320 nm was used to estimate tartaric esters (caffeic acid was used as a standard), and the absorbance at 360 nm was used to estimate flavonols (quercetin was used as a standard).

Determination of Monomeric Anthocyanins

The total monomeric anthocyanins in wine samples were determined using the pH-differential method described by Guisti and Wrolstad.[17] Anthocyanins have a maximum absorbance at a wavelength of 520 nm at a pH of 1.0. Their colored oxonium form predominates at pH 1.0 and the colorless hemiketal form predominates at pH 4.5. The pH-differential method is based on this reaction, and permits accurate and rapid measurement of the total monomeric anthocyanins:

The result, considered as the monomeric anthocyanin pigment, was calculated as mg of cyanidin-3-glucoside, by using a molar absorptivity (ϵ) of 26,900 and a molecular weight of 449.20:

Determination of Indices for Anthocyanin Pigment Degradation, Polymeric Color, Browning, and Hue

Indices for anthocyanin degradation of the wine can be derived using the pH-differential method described by Guisti and Wrolstad.[17] The absorbance at 420 nm of the bisulfite-treated sample serves as an index for browning. The color density of the control sample and the polymer color of the bisulfite-bleached sample wine were calculated as follows:

The value of hue was calculated as follows:

and the ratio between polymerization color and color density was used to determine the percentage of the color that was contributed by polymerized material:

Free Radical Scavenging Activity

The free radical scavenging activity of the wine samples was analyzed by using 2,2-diphenyl-1-picrylhydrazyl (DPPH^•) assay.[18–20] Antioxidant assays are based on measurement of the loss of DPPH^• color by change of absorbance at 517 nm caused by the reaction of DPPH^• with the tested sample. The reaction was monitored by a UV/VIS spectrophotometer. The diluted wine sample (wine was diluted with water, 1:10 v/v) and fresh 1 × 10⁻⁴ M DPPH^• methanolic solution were mixed at the indoor temperature. After a 20-min incubation period at room temperature, the absorbance was read against a blank at 517 nm. Scavenging capacity of DPPH^• in percentage (%) of each wine sample was calculated from the decrease of absorbance according to the equation:

where A _control is the absorbance of control reaction (methanol with DPPH^•), A _blank is the absorbance of dilution wine sample, and A _sample is the absorbance of the dilution wine sample with DPPH radical.

Antibacterial Activity

An agar well-diffusion method was used for the determination of the antibacterial activity of the wine samples.[12,15,21,22] The microorganisms mentioned above were incubated at 37°C for 24 h by inoculation into dextrose agar. The number of cells in 1 mL of suspension for inoculation measured by the McFarland nephelometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was 1 × 107 cfu/mL. A volume of 0.5 mL of this suspension was homogenized with 15 mL of melted dextrose agar and poured into Petri dishes. For screening, sterile 6-mm discs (Hi Media, Mumbai, India) were impregnated with 25 μL of wine sample. After incubation for 24 h in the thermostat at 37°C, inhibition zone diameters, ZI (including disc) were measured and expressed in mm.[22] The presence of the inhibition zone indicates the activity of tested wines against bacteria.

Statistical Analysis

All of the measurements were carried out in triplicate, and presented as mean ± standard deviations (SD). The direction and magnitude of correlation between variables was quantified by the correlation factor r ² . The P-value less than 0.01 were considered statistically significant.

RESULTS AND DISCUSSION

Phenolic Composition in Wine Samples

Vineyards of the Zupa region focus on the old autochthonous wine varieties: the red Kratosija, Vranac, and Prokupac. These indigenous varieties are often compared to Pinot Noir, Merlot, and Gamay sorts originated from France. Vranac and Kratosija grape varieties are ripe in mid-September, when the skin is dark blue in color. The oldest native Serbian grape variety, Prokupac, has been grown in Zupa for over 500 years. The juice is light red, with a sugar level of 22–24% and acidity of 6–8 g/L. This wine is very suitable for coupling with other red wines and the investigated wines Crno and As were produced coupling Prokupac, Vranac, and Gamay varieties. Then, Medjas wine was produced coupling Merlot, Gamay, and Pinot Noir varieties grown in Zupa's vineyards. The concentrations of total phenolics, tartaric esters, and flavonols of these red wines were presented in Table 1.

Table 1 Content of total phenolics, tartaric esters, flavonols, monomeric anthocyanins, polymeric color, and some relevant parameters in selected red wines ^a

Wine and vintage	Grape varieties	Alcohol content (vol%)	pH value	Total phenolics (mg/L)	Tartaric esters (mg/L)	Flavonols (mg/L)	Monomeric anthocyanins (mg/L)	Polymeric color (%)	Hue (absorb. units)
Medjas, 2008	Merlot, Gamay, P. Noir	11.5	2.85	1533.05 ± 1.14	256.65 ± 1.04	138.17 ± 1.21	42.76 ± 0.78	67.05 ± 0.51	0.9631
Kratosija, 2008	Kratošija	11.0	2.80	1497.70 ± 2.05	194.66 ± 1.55	126.39 ± 1.42	41.57 ± 0.53	67.39 ± 0.43	0.8514
Vranac, 2008	Vranac	11.0	2.78	1492.65 ± 2.03	173.07 ± 1.53	109.34 ± 0.46	41.24 ± 0.85	66.43 ± 1.03	0.9352
Crno, 2008	Prokupac, Vranac, Gamay	11.2	2.87	1482.87 ± 2.20	172.46 ± 2.04	109.05 ± 0.71	40.70 ± 0.52	67.60 ± 0.81	0.9552
As, 2008	Prokupac, Vranac, Gamay	11.0	2.86	1480.10 ± 2.06	171.30 ± 1.81	108.62 ± 1.04	40.50 ± 0.44	67.88 ± 0.42	0.9524
^aData are the means ± standard deviation values (n = 3).

It was reported that the wines Kratosija and Vranac have an average amount of phenolics as gallic acid equivalent 1,495.17 mg/L, the coupling wines Crno and As have an average amount 1,481.49 mg/L versus 1,533.05 mg/L of coupling wine Medjas from French sorts; also, the wines Kratosija and Vranac have an average amount of tartaric esters 181.86 mg/L as caffeic acid equivalent, the coupling wines Crno and As have an average amount 171.88 mg/L versus 256.65 mg/L of the coupling wine Medjas; and the wines Kratosija and Vranac have an average amount of flavonols 117.87 mg/Las quercetin equivalent, the coupling wines Crno and As have an average amount 108.84 mg/L versus 138.17 mg/L of the coupling wine Medjas produced from Merlot, Gamay, and Pinot Noir.

The differences between obtained values were influenced by the actual contents of phenolic compounds of the different variety grape. So, analyzed red wines from autochthonous varieties grapes contain lower values of total phenolics, tartaric esters, and flavonols compared to red wines from French varieties, but there are no significant differences among these red wines. In addition, the anthocyanin content as an important quality parameter of red wines was investigated. Anthocyanin compounds influence final color of the resulting wines.[7] Anthocyanins have several different ionic forms in wine that change based on wine pH. At typical wine pH, somewhere between 10 to 15% of the monomeric anthocyanins present are pigments. Anthocyanin pigment undergoes a reversible structural transformation with a change in pH manifested by strikingly different absorbance spectra. At a pH 1.0, anthocyanins are in red colored oxonium form (flavilium cation); however, at pH 4.5, a proton is lost from the anthocyanin resulting in a carbinol pseudo base structure (hemiketal), which is colorless. The concentration of monomeric anthocyanins determined by the pH-differential method is presented in Table 1. The results show that the range of monomeric anthocyanins in investigated wines was 40.50–42.76 mg/L (mean 41.35 mg/L, SD = 0.79, n = 5) as cyanidin-3-glucoside equivalent. The wines Kratosija and Vranac have an average amount 41.41 mg/L, the coupling wines Crno and As have an average amount 40.60 mg/L versus 42.76 mg/L of the coupling wine Medjas.

The concentration of monomeric anthocyanins is essential for good color of the wine produced by different grape varieties. It was observed that the hue of investigated wine samples was from 0.8512 to 0.9626 absorbance units. However, monomeric anthocyanins are subject to hydrolysis, oxidation, and polymerization in wine. Wine characteristics, such as pH, the amount of SO₂, and acetaldehyde, influence these processes and anthocyanin interactions with other phenolic compounds. Anthocyanins can become associated with polymeric molecules in wine through a complex set of reactions. Polymeric color, determined by the pH-differential method, ranged between 67.05 and 67.88% (average amount 67.07%, SD = 0.62, n = 5), in correlation with grape varieties of these young wines.

The Correlation between the Phenolic Constituents and Free Radical Scavenging and Antibacterial Activities of the Wine Samples

It is clear that the wine is a complex mixture of polyphenols and free radical scavenging capacity can be expressed as the amount of wine needed to quench a certain amount of DPPH^• free radicals.[23–26] The decrease in the absorbance at 517 nm is taken as a measure of the extent of radical scavenging. All wine samples show a higher DPPH radical scavenging activity after incubation with a free radical solution. The total content of phenolics, tartaric esters, flavonols, and monomeric anthocyanins in investigated wines contribute to its increased antioxidant activity. All investigated wines show very similar antioxidant behavior in a range from 59.55 to 63.98% scavenging ability of DPPH radical (average amount 60.75%, SD = 3.50, n = 5).

On the basis of the studies done by other researchers in which the anthocyanins are absorbed by the body and interfered with metabolism of normal and cancerous cells by interacting with the production of metabolites and with cellular receptors, it is suggested that anthocyanins could be bioactive compounds in vivo.[6,9,19,27] In this study, the antibacterial activity of the selected wine samples against Staphylococcus aureus strain is analyzed. The diameter of inhibition zone (mm) obtained from the wine samples, also reported in Table 2, show that antibacterial activity was in a range from 17 to 18.5 mm (average amount 17.8 mm, SD = 0.33, n = 5). The different activity of investigated wines against the pathogenic bacteria tested, gave a hypothetical relationship between phenolic compounds of the wines and their antibacterial effect.

Table 2 Correlation coefficients (r ²) between phenolic constituents and free radical scavenging and antibacterial activities in selected red wines

	Total phenols	Tartaric esters	Flavonols	Monomeric anthocyanins
Radical scavenging activity	0.9737	0.9980	0.8938	0.9239
Antibacterial activity against S. aureus strain	0.7617	0.5576	0.7929	0.8550

Since structural features of phenolic compounds are reportedly responsible for antioxidant and antibacterial activity, measurements of total phenolics on wines may be related to their physiological activities. The phenolic content and DPPH radical scavenging activity are very closely correlated (r ² = 0.9737, SD = 0.35, P < 0.001, n = 5) for the all analyzed wines and the significant linear correlation (r ² = 0.7617, SD = 0.32, P < 0.005, n = 5) also, was confirmed between total phenolics and antibacterial activity against S. aureus (Fig. 1). The correlation coefficient between the concentration of tartaric esters and the DPPH^• scavenging ability was very good (r ² = 0.9980, SD = 0.10, P < 0.001, n = 5), but the correlation coefficient between tartaric esters and antibacterial activity against S. aureus strain was 0.5576 (SD = 0.41, P = 0.10, n = 5) (Fig. 2).

Figure 1 Relationship between radical scavenging (▪) and antibacterial (•) activity against S. aureus strain and total phenolic content of selected wine samples.

Figure 2 Relationship between radical scavenging (▪) and antibacterial (•) activity against S. aureus strain and tartaric esters of selected wine samples.

The correlation between the concentration of flavonols and the antioxidant and antibacterial activities against S. aureus strain, also was confirmed (Fig. 3). The correlation coefficient between the concentration of flavonols and the DPPH^• scavenging ability was good (r ² = 0.8938, SD = 0.70, P < 0.01, n = 5), and the correlation coefficient between flavonols and antibacterial activity against S. aureus strain was 0.6286 (SD = 0.79, P = 0.10, n = 5). The correlation between the concentration of monomeric anthocyanins and the antioxidant and antibacterial activities against S. aureus strain was confirmed (Fig. 4). The correlation coefficient between the concentration of monomeric anthocyanins and the DPPH^• scavenging ability was very good (r ² = 0.9239, SD = 0.59, P < 0.01, n = 5), and the antibacterial activity against S. aureus strain also was good (r ² = 0.8550, SD = 0.25, P < 0.02, n = 5).

Figure 3 Relationship between radical scavenging (▪) and antibacterial (•) activity against S. aureus strain and flavonols of selected wine samples.

Figure 4 Relationship between radical scavenging (▪) and antibacterial (•) activity against S. aureus strain and monomeric anthocyanins of selected wine samples.

The results of this study showed that selected red wines have potent antioxidant and antibacterial properties in vitro as estimated by DPPH radical scavenging assay and an antibacterial test against the S. aureus strain. The correlations were very good between antioxidant activity and total phenolics, tartaric esters, flavonols, and monomeric anthocyanins of tested wines. The significant linear correlation between wine phenolic compounds and antibacterial activity against the S. aureus strain of wines was confirmed also (Table 2).

The structure–activity relationships of the antimicrobial activity of phenolic compounds are often contradictory.[28] As wine is complex of phenolic compounds, the physiological activity of selected wines is not a property of a single phenolic compound. It is important to determine which group of phenolic compounds is the most significant in determining antioxidant and antimicrobial potency of wine. Our results suggest that the amounts of monomeric anthocyanins are very important for understanding the antioxidant and antibacterial potency of tested wines.

CONCLUSIONS

According to the results obtained in this study, the free radical scavenging ability of investigated wines was strongly correlated with phenolic composition. A relatively good amount of phenolic constituents, and significant antioxidant and antibacterial activities in red wines produced from different vine varieties from the Zupa–Serbian viticulture and winemaking region, were observed. However, differences in the free radical scavenging and antibacterial activities between wine from Merlot, Gamay, and Pinot Noir grape cultivars and wines from Kratosija and Vranac indigenous varieties were not confirmed. On the basis of the studies done by other researchers, in which the anthocyanins were absorbed by the body and interfered with metabolism of normal and cancerous cells by interacting with the production of metabolites and with cellular receptors, it is suggested that anthocyanins could be bioactive compounds in vivo. Our results showed a significant correlation between the amounts of anthocyanins in investigated wines and its free radical scavenging and antibacterial activities against S. aureus strain.

ACKNOWLEDGMENT

The research was supported by the Europen Union, FP7–REGPOT–2007-3-01, KBBE: Food, Agriculture, and Biotechnology, Project “Chromlab-Antioxidant,” No. 204756.