Antioxidant Species in Grapes andWines via Spectrophotometric Methods : No Quenching Effects by Copper ( II ) and Yeast Derivative Treatments

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy Centre for Colloid and Surface Science (CSGI), University of Florence, Via della Lastruccia 3, Sesto Fiorentino, 50019 Firenze, Italy National Interuniversity Consortium of Materials Science and Technology (INSTM), Via G. Giusti 9, 50121 Firenze, Italy Rocca di Castagnoli, Località Castagnoli, Gaiole in Chianti, 53013 Siena, Italy Operative Unit, University of Siena, CampoVerde, Calabria, Siena, Italy


Introduction
Consumption of alcohol is certainly a serious problem particularly in specific geographic areas and in younger generations.However, the production of high quality wines linked to centenary local traditions is an economic activity capable of improving the environmental and socioeconomic sustainability of specific territorial areas.e in-depth study of each production, storage, aging and refining stages of wine is essential to improve the knowledge of the chemical complexity of this product that is often present in our dinners.rough the study of chemical processes and their dynamics, it will be possible to learn more about the properties of a beverage whose very moderate use can also bring beneficial effects and the pleasure of a combination of harmonies between food and drink.
Wine is an alcoholic beverage produced since millennia by populations living in several areas of the world.e experimental practices carried out by very many chemical and biological studies recently performed, allowed to obtain wines, and other spirit drinks of superior quality [1,2].e costs of production, storage, marketing, care for consumers' health increased significantly, because the beverage requires an extreme care for each stage of production: from vines and grapes growths throughout commercialization.Wine market has a relevant role for financial budgets for several countries, and it is estimated to sum up to many tens of billions Euro/year [3,4].
Our attention focused mostly on Sangiovese variety, even though some samples that contained wines from other red varieties (Canaiolo, Merlot, and Colorino) were included in the study.
e wines were pure Sangiovese (100%) or 80% Sangiovese (minimum, without any white variety), and grapes were cultivated and wines were produced, only in a restricted area [5].During the last couples of decades, science and technology research focused much on wine analysis from chemical, biological, nutritional, agronomical, and plant/cellar treatments stand points, to improve the quality and to preserve the health of the consumers [6].Treatments of vines/grapes with copper(II) sulfate/oxychloride are allowed for organic cultivations, and copper chemically interacts with natural/bioactive molecules [7][8][9].Certain bioactive molecules present in grapes and wines are particularly relevant to human health because of their antioxidant potential.It is reported that polyphenols (like resveratrol, quercetin, and quercitrin) show high antioxidant [10][11][12][13], anticancer [14][15][16][17], and cardiovascular system protective activities [18][19][20][21] and play important roles in human oral microbiota preservation [22].Due to its anthocyanins and polyphenols components, wine is comparable, in terms of antioxidant properties to natural and enriched formulations [18,19,23].
e present work reports the chemical characterization and antioxidant indicators (trans-resveratrol, quercetin, and quercitrin) on selected grapes and wines at different winemaking stages.e main goal and the novelty of this research study was the determination of possible effects by excess copper(II) and yeast derivatives on antioxidant species in wines.
e soil nature (calcareous with stones, pebbles, and gravel), sun irradiation, general climate were the same for all yards, that received just organic management (organic fertilizer on March once/year; copper(II)-sulphate/calcium hydroxide aqueous suspension from April to July, 8-12 treatments/ year).
e alcoholic fermentation of must took place in steel tanks (max temperature, 29 ± 1 °C) with pumping over cycles for four times a day.ree samples of wine were collected at the end of the alcoholic and malolactic fermentations, respectively (1 L each; STY13-AF, STY13-MF).Wines from a second vineyard were studied (SGTY13) having same soil and vine characteristics (excluding the plantation, year 2008), as well as refined bottled wines from 2010-2012 and 2015 harvestings (STY10, STY11, STY12, SGTY12, STY15, and SCCY15).SCC vineyard consisted of Sangiovese 90%, Canaiolo 5%, and Colorino 5%.All the wine samples were collected in triplicate.
To evaluate possible effects on antioxidant parameters, samples of SCCY15 wines were treated by adding ultrapure Cu(II)-sulfate pentahydrate (5.0 mgCu/L) and/or yeast  [24] and other researchers [25].e absorbance values at λ 420, 520, and 620 nm were recorded via UV-Vis spectrophotometer on wine samples (decanted or filtered up to clearness; at 21 ± 2 °C) and wine shade (HUE), and the color index (CI) were calculated.e spectrophotometer was a double-beam Lambda-10 (Per-kinElmer, Monza, Italy): resolution, 2 nm; equipped with a continuum flux quartz cuvettes with optical length of 1 or 10 mm, depending on the absorbance values (without any dilution).Each reading was from the mean of ten scans.
e instrument was managed by the software UV WinLab, Ver.2.85 (PerkinElmer).e method was adapted from studies previously reported [26][27][28], as well as the official methods [24,25].Validation parameters and standard errors of the methods just reported were those published by the OIV [24] and the standard errors were <5% for all the methods.

Total Polyphenol Content.
e method was based on the colorimetric technique with external calibration curve using gallic acid (GAA) and Folin-Ciocalteu reactant (FCR) [24,29,30].Aliquots of 100, 500, 750, and 1000 μL of a standard solution of GAA (500 mg/100 mL) or 200 μL of wine samples (or 1000 μL for JU and MU samples) were preliminarily diluted in ultrapure water, by addinf FCR (5 mL) and Na 2 CO 3aq (20% w/v, 20 mL) and diluted again up to volume (100 mL).A blank of reactants was also tested.Incubation was done for 30 min (21 ± 2 °C, dark).Absorbance was recorded at λ 750 nm through 10 mm optical length PMMA cuvettes (semi-micro, Brand ® standard; see Section 2.3.1 for other instrumental details).e results were expressed as GAA equivalent, and LOD and LOQ values were 1.0 and 3.0 mg/L, respectively.In all the cases, calibration curves (in the range 5.0-50.0mg/L) were accepted with a R 2 > 0.990.
e QUE and QUEG quantifications were carried out using external calibration protocol (linearity range 20.0-300.0mg/L, using five different standard concentrations).Calibration curves were accepted with R 2 > 0.990.e intraand interday calibration analysis (on three different days) were carried out and revealed a RSD% of < 1% and 3%, respectively.As regards the selectivity, selected samples were analyzed after spiking with the standards to confirm peak identity.
e RES quantification was carried out by the standard addition method [34], and four aliquots of wine extract were added in different volumes of a standard solution (0.3 mg/mL) and MeOH/H 2 O (6 : 4, v/v) solvent mixture (to a fixed total volume).Calibration curves were accepted with R 2 > 0.990.
Both methods were tested for precision that was evaluated repeating injections of the sample extracts three times (each replicate, three replicates).e RSD% of peak area were <5% and RSD% of retention times were <2%.LOD and LOQ values were 0.1 and 0.3 mg/L for RES, and 4.0 and 10.0 mg/L for both QUE and QUEG.

Statistical Analysis.
All the samples were collected and pretreated (extracted/mineralized) in triplicates, and all the analytical procedures were also performed in triplicates.e final data were then reported as mean values and estimated standard deviations (esd).Statistically significant differences among data were verified carrying out the analysis of variance (ANOVA) and Tukey's test.
e data showing p values < 0.05 were considered statistically significant.Calculations were carried out using Microsoft Office Excel 2007, implemented with statistical analysis subroutine, and from Origin Pro8 SR2 (v.0891, B891; OriginLab Corporation, Northampton, USA).

Selected Nutrients in Grapes and Wine Fermentation
Parameters.
e content of total nitrogen (TN) for STY13-JU was 0.24 ± 0.03 gN/L (Table 1) corresponding to total protein content (TPC) of 1.53 ± 0.20 gALA/L.Data of TN in grapes from other works ranged 0.21-0.69gN/L [35,36] and underline that contents of TN smaller than 0.14 gN/L may be related to a significant lowering of the fermentation rate and the formation of undesired sulfur compounds.e TN value for STY13-SK + SE (dry samples) was 6.8 ± 0.7 gN/kg, ca.28 times higher than the value found for JU.As nitrogen starvation was not revealed in the study cases and to maintain the natural high quality of grape/wine, any nitrogen integrator was not added at fermentation stages.
It is well known that the value of reducing sugar (RSU) in grapes is an important harvesting-time indicator (ripeness range being 150-300 gGLU/kg), and the value for STY13-JU was 226 ± 10 gGLU/kg, which was in line with data previously reported for 98 different grape cultivars (range 93.5-253.9g/L; [37]).e overall sugar content expressed as Brix for both STY13-JU and STY13-MU was also not statistically different (21.2 ± 1.0 and 22.5 ± 1.0 Brix, respectively), and the alcoholic content (ALC) predictable from Brix value was 13.5 ± 0.6% v/v for MU (conversion factor 0.60) comparable with the value, obtained via official density method, for STY13-AF (13.9 ± 0.2% v/v; Table 2).is latter value was also in agreement with data suggested by enologists (13.5-14.5%)for allowing an ideal phenolic maturation and a lower production of astringent tannins.
Values of pH for STY13-MU averaged 3.18 ± 0.03, within the range pH 2.80-3.80,typical for Tuscany musts and dry red wines.e pH value of must influences the speed of fermentation kinetics, through the speed of yeast reproduction.
e pH value changed from 3.29 ± 0.02 to 3.42 ± 0.02 passing from STY13-AF to STY13-MF, while the total acidity (titratable, TAC) values changed from 6.9 ± 0.3 to 5.7 ± 0.1 gTAA/L.Regarding SGTY13-AF and SGTY13-MF samples, pH and TAC values did not change significantly 3.20 ± 0.02 and 3.22 ± 0.01, and 6.5 ± 0.1 and 6.3 ± 0.2 gTAA/L.e TAC is a key parameter for high-quality wine production, particularly relevant to flavor preservation properties upon time elapsing.It is mostly due to tartaric, malic, citric, and lactic acids, ranging usually between 3.5-15.0gTAA/L [38].e analyzed wines revealed fresh/ fragrant taste in agreement with TAC values of 5.7 ± 0.1-6.9 ± 0.3 gTAA/L, guaranteeing good lasting flavor and nice self-preservation.Moreover, TAC and VAC parameters (wine samples) did not reveal any starting of acetic fermentation process, VAC values being 304 ± 20 and 349 ± 41 mgACA/L, for STY13-AF and SGTY13-AF, respectively.It is well known that volatile acidity for wine is due to the overall fatty acids from the acetic series that mainly form at the beginning of fermentation process as metabolites of Acetobacter and Gluconobacter species and from poor wine-making procedures [25,39].Total SO 2 content (TSO 2 ) was low, ranging from 21.8 ± 1.5 to 66.3 ± 7.1 mgSO 2 /L, in agreement with present market requirements.In fact, good grape quality and wine-making practices did not require any chemical addition of sulfur dioxide to must or wine.
It is finally, well known that high alcohol contents (ALC) protect against the formation of undesired microorganisms.
e study wine samples showed relatively high ALC values, in agreement with other Chianti wines (average 14.2 ± 0.2% v/v, 2013 and 2015 wine samples).

Antioxidant Parameters in Wine Samples and Copper(II)/ Yeast Derivative Influence.
e contents of trans-resveratrol (RES), quercetin (QUE), and quercetin glucoside (QUEG), together with the total polyphenols content (TPP), wine shade (HUE), and color index (CI), were analyzed (Table 3 and Figure 1).Data for RES in wines ranged from 0.67 ± 0.02 to 1.26 ± 0.09 mg/L, showing no statistically significant discrepancies between values found for STY13-AF and SGTY13-MF samples (for same type of wine) and being in agreement with the values previously reported [29,30,40].
Interestingly, the values of RES in JU and MU samples were much lower than for the wine samples (0.024 ± 0.002 and 0.032 ± 0.008 mg/L, respectively; not statistically different on the basis of Tukey's test); instead, the content for SE and SK samples was 0.32 ± 0.01 and 10.5 ± 0.1 mg/kg, the last being comparable to data from other Sangiovese grapes (7.3 ± 0.2 mg/kg; [32]).As regards the wine samples, RES values for STY13-MF and SGTY13-MF were 1.0 ± 0.1 and 0.70 ± 0.09 mg/L, respectively (Tukey's test, p < 0.05), showing a ratio between the two wines, of 1.43, in agreement with the TPP ratio (1.22).
Attention was then devoted to investigate the possible influence of yeasts derivatives and/or copper(II) additions on refining wines.
e study was carried out on certain SCCY15 samples that underwent addition of yeast  1S.
derivatives (allowed by EU regulations [43], see Experimental), sixty days before analyses.e analytical results did not show any statistically signi cant change, in terms of RES values, that resulted to be 1.16 ± 0.05 and 1.24 ± 0.10 mg/L for SCCY15 + BB and SCCY15 + EGSH samples, with respect 1.18 ± 0.09 mg/L for original SCCY15 (Table 4 and Figure 1: e content of (a) total polyphenols (TPP, mgGAA/L) and (b) trans-resveratrol (RES, mg/L) in grapes and wines samples.Values (mean ± esd) obtained from three samples and three replicates each sample, di erent letters indicate signi cant di erences (p < 0.05, Tukey's test).Full experimental data sets are reported in Table 3S.
resveratrol for SCCY15 + Cu(II), 1.24 ± 0.09 mg/L, neither in case of addition of both copper(II) and yeasts derivatives and also tannins (Table 4).Also, the e ect of Cu(II) on TPP values was tested, not showing any in uence as well (i.e., 2786 ± 95 and 2762 ± 131 mgGAA/L for SCCY15 and SCCY15 + Cu(II), respectively; not statistically di erent on the basis of Tukey's test).en, the investigation was extended to quercetin (QUE) and quercetin-O-3′-glucoside (QUEG) components both in absence and in presence of copper(II) and yeast derivatives.ese molecules belong to the avonoids family and show a great antioxidant activity, reducing the risk of cardiovascular diseases and cancers [44] and a potential synergic e ect with trans-resveratrol [32].
e data for wine shade (HUE) and color index (CI) were in the range of 0.77 ± 0.02-1.02± 0.07 and 7.11 ± 0.15-11.70± 1.47, respectively, and agreed with values previously reported for other red wines [41,42].ese parameters are characteristics of the wines components, the absorbance at 520 nm being mainly due to the anthocyanins content; the absorbance at 420 nm, mainly due to tannins content; and the absorbance at 620 nm, typical of oxidized polyphenols [25].
It has to be noted that TPP values for STY13/15 and SCCY15 (range 3334 ± 60-2786 ± 95 mgGAA/L) resulted to be higher with respect to the values previous reported in selected papers [40,41].e values found for STY13-JU and -MU were much lower than those for corresponding wines, as polyphenols are more abundant in grape skins that stay (several days) in contact with must, during the initial steps of the alcoholic fermentation process.e di erence on TPP from STY13-AF to STY13-MF samples was not statistically signi cant (Tukey's test).e TPP contents for SGTY13-AF and SGTY13-MF samples resulted to be 16% (average) lower than corresponding values for STY13, and this can be explained through the di erence of vines aging, SGTY13 being 6 years younger, and with STY15 and SCCY15 (taking into account the SCC blend nature and age of 14 ± 3 years).
is value are also in agreement with ANT values for re ned wines (241 ± 10 and 197 ± 7 mgMVG/L for STY15 and SCCY15, respectively; Tukey's test, p < 0.05) and slightly higher than other bottled red wines (ca.130 mgMVG/L; [31]).e higher TPP and ANT values for nal products showed that the vine variety, the terroir (soil, climate, solar irradiation, etc.) and wine-making procedures guaranteed great quality products, with possible high antioxidant potentials.

Analysis of Re ned Bottled Wines.
For comparison purposes and to estimate the analytical parameters in nal marketable products, re ned bottled wines were analyzed (STY10, STY11, STY12, and SGTY12) and reported in Table 5. e data con rmed that SGT12 samples had lower values for RES (0.90 ± 0.06 mg/L) and TPP (2489 ± 12 mgGAA/L) when compared with STY12 (1.05 ± 0.06 mg/L and 2930 ± 77 mgGAA/L), in agreement with a lower age of vines (11 and 5 years).It is noteworthy that the values for RES and TPP for the year 2010 (2.6 ± 0.3 mg/L and 3200 ± 82 mgGAA/L) were signi cantly higher than those for all the other samples.is might be attributed to the climate for the year 2010 that was particularly rainy and highly humid (source: LaMMA DataBase 2018), inducing vines to overproduce polyphenols to counteract plant diseases.

Conclusion
is work brought about data relevant to the content of several analytes of grapes (juice and must) and wines after (a) e choice of wine-making protocol allowed the production of high quality wines, as confirmed by antioxidant parameters.e contents of total polyphenols resulted to be significantly higher than those reported for other red wines.(b) e optimization of an analytical protocol, based on a calibration via standard additions, allowed to reveal the contents of trans-resveratrol in Sangiovese grapes and wines (LOQ, 0.3 mg/L), overcoming the recent claims that this variety does not contain appreciable amounts of the stilbene [45].(c) e wines also revealed high quercetin contents (ca.20 mg/L), suggesting a significant contribution to the overall health benefits from human consumption [46][47][48][49].e work by Ramos-Pineda et al. [49] was related to astringency caused by the content of quercetin glucoside; the concentration of ca.40 mg/L was estimated as threshold value to perceive astringency taste [49].In case the effect generated by quercetin was equivalent to that estimated for its glucoside derivative, it is evident that values found in the wines from the present work were much below the threshold (QUE + QUEG < 25 mg/L).Also, it has to be noted that Sangiovese wines from nearby areas have usually strong bouquet that attenuates unpleasant smell/taste [50].In addition, high alcohol content (ca.14%) helps smoothness and possibly attenuates astringency.(d) It has to be also emphasized that the contents of quercetin and quercetin glucoside did not represent a risk for wine organoleptic properties, because of the formation of deposits and haziness, due to precipitation of quercetin.is possible event is excluded in the present wine samples, as the total QUE + QUEG value is below their solubility in wine matrix.(e) Points (a)-(d) constitute the main goal of the work-the study of possible effects of copper(II) and fermentation yeast derivatives on the contents of antioxidants in real wine-reliable and significant.
e work showed that the administration of copper(II) and/or yeast derivatives to vines/grapes, as allowed by regulation for organic cultivations, does not affect the content of antioxidant species.It can be noted that the matter of copper in wine, even though white ones, was widely studied [51][52][53][54], but it was always aimed to study the mechanisms of sulfides removal, relevant to model wine systems, and never to study the possible effects by the metal on the contents of polyphenols in general, and particularly of resveratrol, quercetin, and quercetin glucoside in the real wine matrix.

Data Availability
e analytical data used to support the findings of this study are included within the article, and the values of the triplicate analytical determinations for each replicate and three replicates are included within the supplementary information file.

Disclosure
Renzo Cini former full professor at Department of Biotechnology, Chemistry and Pharmacy, actually retired.

Figure 1 (
Figure 1(b); Tukey's test, p < 0.05 for all data sets).Furthermore, to test the e ect of the Cu(II) ions on RES and other antioxidant parameters, aliquots of SCCY15 wines (7 samples, in triplicate) were treated by adding excess Cu(II) sulfate (5 mgCu/L; incubation 21 ± 2 °C, 60 days).Also, in these cases, the results did not show any quenching of trans-

Table 2 :
Selected fermentation parameters in STY13 and SGTY13 wine samples at di erent wine-making stages and STY15 and SCCY15 re ned wines., total or titratable acidity content expressed as gTAA/L (tartaric acid equivalent); VAC, volatile acidity content expressed as gACA/L (acetic acid equivalent); VSO 2 , free or volatile sulfur dioxide content; TSO 2 , total sulfur dioxide content both expressed as mgSO 2 /L; ALC, alcoholic content percentage (% v/v).Values (mean ± esd) obtained from three samples and three replicates each sample.Di erent letters in the same column indicate signi cant di erences (p < 0.05, Tukey's test).Full experimental data sets are reported in Table2S. TAC

Table 3 :
Contents of selected antioxidant indicators in STY13 and SGTY13 wine samples at di erent wine-making stages and STY15 and SCCY15 re ned wines.
TPP, total polyphenols content expressed as mgGAA/L (gallic acid equivalent); RES, trans-resveratrol content expressed as mg/L; HUE, wine shade; CI, color index.Values (mean ± esd) obtained from three samples and three replicates each sample.Di erent letters in the same column indicate signi cant di erences (p < 0.05, Tukey's test).Full experimental data sets are reported in Table3S.