DOI: 10.14720/aas.2016.107.2.18 Agrovoc descriptors: wines, white wines, antioxidants, phenolic compounds, phenols, winemaking, lees, wine yeast Agris category code: Q01, Q02, Q04 Comparison of total polyphenols content and antioxidant potential of wines from 'Welschriesling' and 'Sauvignon Blanc' varieties during ageing on fine lees Jasna LUŽ AR1, Tjaša JUG2, Polona JAMNIK3, Tatjana KOŠMERL3 Received July 29, 2016; accepted September 30, 2016. Delo je prispelo 29. julija 2016, sprejeto 30. septembra 2016. ABSTRACT Phenolic compounds are key components of wine, since they contribute to wine characteristics such as colour, astringency and bitterness. They also act like antioxidants, with mechanisms involving free-radical scavenging that could prevent cardiovascular diseases and cancer. The aim of the present work was to compare the obtained results of total polyphenols content and antioxidant potential (AOP) of several white wines (welschriesling and sauvignon blanc) during ageing on fine lees. The total polyphenols content decreased in average for 16.1 % in welschriesling wines and for 18.7 % in sauvignon blanc wines in the period of three months of wine ageing on lees. In the same period AOP of wines decreased in average for 16.0 % in welschriesling wines and for 8.0 % in sauvignon blanc wines. Expectedly, the samples with added oak chips in grape must had higher antioxidant potential than others. Key words: white wines, antioxidant potential, phenolic compounds, DPPH, yeast lees IZVLEČEK PRIMERJAVA VSEBNOSTI CELOKUPNIH FENOLOV IN ANTIOKSIDATIVNEGA POTENCIALA VIN IZ SORT 'Welschriesling' IN 'Sauvignon Blanc' MED STARANJEM NA FINIH DROŽEH Fenolne spojine so ključne sestavine vina, ki prispevajo k značilnostim vina, kot so barva, trpkost in grenkoba. Delujejo kot antioksidanti z mehanizmi, ki vključujejo lovljenje prostih radikalov, kar lahko prepreči kardiovaskularne bolezni in raka. Namen dela je bil primerjati dobljene rezultate vsebnosti skupnih fenolov in antioksidativni potencial (AOP) belih sortnih vin laški rizling in sauvignon med zorenjem vina na finih drožeh. Trije meseci spremljanja so pokazali, da se je vsebnost skupnih fenolov zmanjšala v povprečju za 16,1 % v vinih sorte laški rizling in za 18,7 % v vinih sorte sauvignon. Vrednost AOP se je v tem času prav tako zmanjšala v povprečju za 16,0 % pri vinih sorte laški rizling in za 8,0 % pri vinih sorte sauvignon. Pričakovano so imeli vzorci z dodatkom trsk iz hrastovega lesa v mošt večji antioksidativni potencial kot ostali. Ključne besede: bela vina, antioksidativni potencial, fenolne spojine, DPPH, droži 1 INTRODUCTION Phenolic compounds are key components of wine that not only contribute to the organoleptic characteristics of wine but they also are the main cause of colour changes in wine. The light yellow colour as well as the brown colour in white wines is undesirable due to the higher content of phenolics and its oxidation. Nevertheless, phenolic compounds also act as antioxidants, with mechanisms involving free-radical scavenging that 1 University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 11, SI-1000 Ljubljana, M.Sc., email: jluzar2@gmail.com 2 Chamber of Agriculture and Forestry of Slovenia, Institute for Agriculture and Forestry, Pri hrastu 18,5000 Nova Gorica, Ph.D. 3 University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 11, SI-1000 Ljubljana, Ph.D., email: tatjana.kosmerl@bf.uni-lj.si, polona.jamnik@bf.uni-lj.si Acta agriculturae Slovenica, 107 - 2, september 2016 str. 473 - 482 Jasna LUŽAR et al. we are examining (Perez-Serradilla and Luque de Castro, 2008). White wines are characterised by lower concentrations of total polyphenols (typically 200500 mg l-1) than red wines because of a lack of red-coloured anthocyanins; white wines are additionally characterized by the predominance of hydroxycinnamic acids (Kilmartin, 2010). Phenolic compounds have received much attention in the prevention of human cardiovascular disorders and cancer due to their antioxidant properties (Paixao et al., 2007). Cells of aerobic organisms are constantly exposed to the effects of reactive oxygen species (ROS) - free radicals. Phenolic compounds have a functional role as they behave as antioxidants against the free radicals. We could say that they increase the antioxidant capacity in the human body after (especially red) wine consumption (Serafini et al., 1998). We were monitoring the total polyphenols content and antioxidant potential of wine during ageing on fine lees. Yeast cells have been shown to exert a protective effect toward polyphenol oxidation during ageing on lees (Salmon et al., 2002). They have a role as a competing substrate for oxygen in wine. During alcoholic fermentation, yeast cells require oxygen for their metabolic activity and the reactive oxygen species that are produced can potentially oxidise wine polyphenols. At the end of fermentation, phenolic compounds take part in oxygen consumption. After completion of alcoholic fermentation, yeast lees can consume oxygen for up to 3 years in contact with ageing wine. The consumption of oxygen by yeast lees has been ascribed to a mild oxidation of the membrane lipids of the yeast lees that leads to lipid peroxides and further products that may add to the wine flavour (Kilmartin, 2010). The aim of the present work was to compare the obtained results of total polyphenols content and AOP of several white wines (from 'Welschriesling' and 'Sauvignon Blanc' varieties) during wine ageing on fine lees. 2 MATERIALS AND METHODS 2.1 Samples We collected 35 samples of Slovenian wines (18 samples of welschriesling and 17 samples of sauvignon blanc wines) and then examined the content of total polyphenols and AOP of these young wines. Investigated samples were taken one week after alcoholic fermentation was completed (at time t=0). Wine ageing was performed in the cooling room of Biotechnical Faculty at temperature 4 °C. No sulphite was added to welschriesling and sauvignon blanc wines. We were monitoring content of total polyphenols and AOP of wine at different times - at time t1=0, t2=14 days, t3=28 days, t4=48 days and t5=76 days, during wine ageing on fine lees. 2.2 Folin-Ciocalteu assay Total polyphenols were determined by Folin-Ciocalteu (FC) spectrofotometric method (Singleton and Rossi, 1965). Undiluted samples of white wine and gallic acid (used as a standard) were incubated in sodium carbonate solution (20 %, w v-1) and FC reagent for 2 hours at room temperature. The absorbance was measured at wavelength 765 nm. Measured absorbance is proportional to mass concentration of phenolic compounds and it is expressed as gallic acid equivalents (mg of gallic acid per litre of wine). The determination was performed in duplicate and the results are expressed as the mean value. 2.3 Determination of antioxidant potential (AOP) of wine with the 1-diphenyl-2-picrylhydrazyl radical (DPPH') scavenging method Antioxidant potential of wine was determined by DPPH radical scavenging method (BrandWilliams et al., 1996). DPPH'solution in 99 % methanol was added to 50 ^l diluted sample (R=2). After 30 min, the absorbance was measured at 517 nm. For each sample there was a blank (methanol) and reference (DPPH'solution). When antioxidant reacts with the DPPH'radical and radical becomes a stable molecule, a decrease in 474 Acta agriculturae Slovenica, 107 - 2, september 2016 Comparison of total polyphenols ... from 'Welschriesling' and 'Sauvignon Blanc' varieties during ageing on fine lees absorbance occurs. DPPH scavenging ability is The determination was performed in triplicates and expressed as concentration of DPPH in mmol l"1. the results are expressed as the mean value. 3 RESULTS AND DISCUSSION 3.1 Antioxidant potential (AOP) of wines 3,5 ^ 3 | 2,5 ^ 2 o e 1 5 • | 1,5 IS 1 § 0,5 « 0 ^^^V^vvwvvvvvvvv Sample t1 t2 t3 t4 t5 Figure 1: AOP of welschriesling wines at different sampling times (t1 = 0, t2 = 14 days, t3 = 28 days, t4 = 48 days and t5 = 76 days) after completion of alcoholic fermentation Figure 2: AOP of sauvignon blanc wines at different sampling times (t1 = 0, t2 = 14 days, t3 = 28 days, t4 = 48 days and t5 = 76 days) after completion of alcoholic fermentation The results of the AOP of welschriesling and sauvignon blanc wines are presented in Figure 1 and Figure 2, respectively. Both Figures show the gradually decrease of antioxidant potential of welschriesling and sauvignon blanc wines as a function of wine ageing on fine lees. We noticed the increase of AOP in some of the wine samples that decreased at the next sampling. The most obvious was this phenomenon of the AOP increasing at the second time of sampling in welschriesling wines WR2, WR8, WR11, and in sauvignon blanc wines SB7, SB 10 and SB 12-SB16. The increase of AOP of wine could be caused by the antioxidant defence system of yeast -the increase in antioxidant content and increase in the levels of antioxidant enzymes including superoxide dismutase and glutathione reductase on yeast entering into the stationary phase, may Acta agriculturae Slovenica, 107 - 2, september 2016 3 Jasna LUŽAR et al. constitute adaptive response to the enhanced oxidative stress (Jakubowski et al., 1999). Table 1: Content of reducing sugars (RS), content of total polyphenols (PFT), FC index and antioxidant potential (AOP) after 76 days of wine ageing on fine lees. Sample RS (g l-1) PFT (mg GAE l-1 ) FC index (/) AOP (mmol l-1) welschriesling wines WR1 (control) 117.2 444.6 36 2.70 WR2 3.1 327.6 6 2.17 WR3 1.1 322.1 6 2.08 WR4 1.5 331.1 10 2.28 WR5 3.6 299.5 9 1.96 WR6 (AM-HT1) 1.9 353.1 9 2.28 WR7 (AM-HT1) 1.4 331.1 11 2.01 WR8 (AM-HT1) 1.4 325.8 10 2.22 WR9 1.7 324.1 7 2.07 WR10 1.2 305.4 8 1.95 WR11 7.7 334.1 7 2.27 WR12 3.4 297.6 7 1.76 WR13 2.4 320.1 10 1.93 WR14 31.4 328.6 3 1.70 WR15 1.9 328.6 10 1.95 WR16 1.5 297.1 10 1.70 WR17 1.4 309.0 11 1.77 WR18 1.7 322.1 10 1.87 sauvignon blanc wines SB1 (control) 121.7 451.1 38 2.99 SB2 2.3 320.6 7 2.22 SB 3 1.3 310.1 6 2.16 SB4 2.1 315.6 9 2.27 SB 5 3.4 298.6 9 2.11 SB6 5.0 322.2 9 2.23 SB7 (AM-MT2) 5.2 328.6 9 2.26 SB 8 1.0 284.1 4 2.06 SB 9 1.2 252.6 6 2.14 SB 10 1.3 340.6 8 2.39 SB 11 1.3 311.7 7 2.12 SB12 (AM-MT2) 1.1 294.9 4 2.33 SB13 (FR-MT3) 1.2 356.6 10 2.65 SB14 (FR-MT3) 1.7 371.6 9 2.79 SB15 (AM-MT2) 1.5 342.6 9 2.51 SB16 (AM-MT2) 1.7 342.6 9 2.54 SB 17 9.1 233.1 7 1.81 1 Addition of American oak chips (highly toasted), 2 Addition of American oak chips (medium toast), 3 Addition of French oak chips (medium toast) The highest antioxidant potential was measured in a control sample SB1, where spontaneous fermentation was performed, and sample SB 14 with addition of French oak chips (medium toast). As observed from Table 1, the control samples have the highest amount of reducing sugars (Yrs(sbi) = 121.7 g l-1 and Yrs(wri) = 117.2 g l-1; while the average amount of reducing sugars in 476 Acta agriculturae Slovenica, 107 - 2, september 2016 Comparison of total polyphenols ... from 'Welschriesling' and 'Sauvignon Blanc' varieties during ageing on fine lees other samples is yRS = 3.3 g l- ) and they interfere with free radical DPPH\ Because of that it shows greater antioxidant potential than it should. The lowest antioxidant potential had sample WR16 with addition of commercial yeast strain EC-1118 (40 g hl-1) and nutrient Naturferm (40 g hl-1). On Figure 1 and Figure 2 we can see that wines with added oak chips (samples WR6, WR7, WR8, SB7, SB10, SB12, SB13, SB14, SB15 and SB16) express stronger ability to scavenge DPPH radical than other samples. AOP of samples SB12-SB16 was even higher at sampling time t5 than at sampling to. This could be due to extracted polyphenols from oak chips to wine and therefore increasing AOP of investigated samples. 3.2 Content of total polyphenols As observed from Figure 3 and Figure 4, content of total polyphenols is constantly decreasing during wine ageing on fine lees. Figure 3: Content of total polyphenols in welschriesling wines at different sampling times (t1 = 0, t2 = 14 days, t3 : 28 days, t4 = 48 days and t5 = 76 days) after completion of alcoholic fermentation Figure 4: Content of total polyphenols in sauvignon blanc wines at different sampling times (t1 = 0, t2 = 14 days, t3 = 28 days, t4 = 48 days and t5 = 76 days) after completion of alcoholic fermentation The highest content of total polyphenols was determined in samples WR1 and SB1, produced by spontaneous fermentation. Table 1 lists concentrations of total polyphenols and reducing sugars in both welschriesling and sauvignon blanc wines at sampling time t5 = 76 days. It shows that samples with higher amount of Acta agriculturae Slovenica, 107 - 2, september 2016 477 Jasna LUŽAR et al. reducing sugars have considerably higher content (Abramovic, 2011). We could clean up samples by of total polyphenols. The disadvantage of using Folin-Ciocalteu method for determination of total polyphenols is that the mentioned reagent reacts nonspecifically with all phenolic hydroxyl groups (-OH), including aromatic amino acids, ascorbic acid, reducing sugars and organic acids a solid phase extraction (SPE) to remove other reducing compounds and to get more accurate results. Therefore our results indicate higher content of total polyphenols and the correction is needed. Table 2: Correction of total polyphenol content (mg GAE l-) with FC reagent regarding the amount of reducing sugars (g l-1) (Košmerl and Kač, 2010) Reducing sugars (g l-1) Division factor of total polyphenols concentration 0-10 / 10-25 1.03 25-100 1.06 100-200 1.10 Figure 5: Relation between total polyphenols and reducing sugars with (*) and without correction (♦) The correlation between reducing sugars and total polyphenols (PFT) was even worse after PFT-correction (R2 = 0.2182) in comparison to non-correction (R2 = 0.4597) for our investigated samples. Expectedly, addition of oak chips in grape must of 'Welschriesling' and 'Sauvignon Blanc' varieties (samples WR6, WR7, WR8, SB7, SB10, SB12-16) resulted in much higher content of total polyphenols. We can assume that polyphenols were extracted from oak chips into wine during wine ageing on fine lees and therefore have increased content of total polyphenols. 3.3 Correlation between total polyphenols and AOP of wines Figure 6 and Figure 7 presents the correlation between total polyphenols content and AOP of welschriesling and sauvignon blanc wines. It shows a strong positive dependence of AOP on the content of total polyphenols in all wines of both varieties. The linear correlation between AOP of wine and total polyphenols concentration was better for sauvignon blanc wines than for welschriesling wines. From these obtained results we can 478 Acta agriculturae Slovenica, 107 - 2, september 2016 Comparison of total polyphenols ... from 'Welschriesling' and 'Sauvignon Blanc' varieties during ageing on fine lees conclude that the correlation between AOP and total polyphenols is particularly varietal characteristic as also demonstrated Kosmerl and Cigic (2008). Phenolic composition of individual variety has different influence on correlations of total polyphenols with AOP; polyphenols with higher numbers of hydroxyl groups and those having hydroxyl groups in ortho positions in the aromatic rings usually have higher antioxidant potential (Kosmerl and Cigic, 2008). It can be also summarized that the correlation between AOP of wine and total polyphenols concentration has increased during wine ageing on lees. Table 3: Coefficient of determination (R2) for welschriesling wines at different sampling times Sampling Coefficient of determination (R2) t1 0.6538 t2 0.5850 t3 0.7224 t4 0.7398 t5 0.6530 3,4 3,2 1 3,0 I 2,8 J= ^ 2,6 £ 2,4 o ' X O 2,2 < 2,0 1,8 1,6 230 ■ ♦ ■ ♦ ^^ m ^ A m ■ ■ iSi J X > 280 330 380 430 Total polyphenols (mg GAE l-1) 480 530 t1 t2 t3 t4 t5 Figure 6: Correlation between total polyphenols and AOP of welschriesling wines at different sampling times (ti=0, t2=14 days, t3=28 days, U=48 days and t5=76 days) after completion of alcoholic fermentation Acta agriculturae Slovenica, 107 - 2, september 2016 7 Jasna LUŽAR et al. Table 4: Coefficient of determination (R2) for sauvignon blanc wines at different sampling times Figure 7: Correlation between total polyphenols and AOP of sauvignon blanc wines at different sampling times (ti=Q, t2=14 days, t3=28 days, t4=48 days and t5=76 days) after completion of alcoholic fermentation Sampling Coefficient of determination (R2) ti 0.7434 t2 0.7141 t3 0.7619 t4 0.7868 t5 0.8467 3,2 3.0 ir-2,8 i g I 2,4 = 2,2 P4 ' o <1 2,0 :,6 à /TS, X 230 280 330 380 430 Total polyphenols (mg GAE H) 480 ♦ tl t2 à :3 t4 t5 530 3.3 Correlation between FC index and total polyphenols In Table 1 are given informative values for FC index. In the first part we examined correlation between FC index and total polyphenols content and in the second part correlation between reducing sugars and FC index. As shown in Figure 8, correlation between FC index and total polyphenols content was very weak in the case of sauvignon blanc wines and higher for welschriesling wines. 480 Acta agriculturae Slovenica, 107 - 2, september 2016 Comparison of total polyphenols ... from 'Welschriesling' and 'Sauvignon Blanc' varieties during ageing on fine lees R2 = 0,6566 'Welschriesling' 'Sauvignon Blanc' 20 30 40 FC index (/) 60 Figure 8: Correlation between FC index and total polyphenols content at sampling time ti = 0 On the other hand Figure 9 presents that index was even stronger at sampling time t5 = 76 correlation between reducing sugars and FC index days (R2 = 0.8449). It means that the amount of at sampling time t1 = 0 was strong (R2 = 0.6879) of reducing sugars influence on values of FC index. both welschriesling and sauvignon blanc wines. The correlation between reducing sugars and FC 60 50 ^40 w X ij ■3 30 .=