Analytical determinations for selected parameters in grapes/wines help planning technology treatments in the vineyards and cellars, improving the quality of final products and preserving consumers’ health. The study first reports a comparative analysis for selected parameters on juice, must, and wines at alcoholic and malolactic fermentation stages, from 2013 harvest and refined bottled wines from 2010–2012 and 2015 vintages. This was considered preliminary to the main goal of the work that consisted of testing if the contents of certain antioxidant principles were influenced or not by additions of copper(II) and/or selected fermentation yeasts. Particular attention was devoted to antioxidant molecule contents: total polyphenols, anthocyanins,
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. The 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. Through 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. The experimental practices carried out by very many chemical and biological studies recently performed, allowed to obtain wines, and other spirit drinks of superior quality [
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. The 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 [
The present work reports the chemical characterization and antioxidant indicators (
All reagents and analytical standards were of analytical grade, and solvents were of HPLC grade and purchased from Sigma-Aldrich (Milan, Italy): sodium thiosulfate pentahydrate, sodium carbonate (anhydrous), potassium chloride, potassium dichromate, iodine, potassium iodide, calcium chloride, copper(II)-sulfate pentahydrate, phenolphthalein (3% in ethanol:water, 1 : 1), starch suspension (1% in water), Devarda alloy (5 : 45 : 50%, zinc/aluminum/copper), sodium hydroxide, hydrochloric acid, sulfuric acid, nitric acid, acetic acid, acetonitrile, methanol, ethylacetate, Folin–Ciocalteu’s phenol reagent, Fehling’s reagents I and II for total reducing sugar determination, gallic acid,
The grape samples were collected in the year 2013, from 100% Sangiovese grapes (STY13, Chianti area, Tuscany, Italy). The main vineyard characteristics were as follows: altitude, 456–420 m a.s.l.; ground slope, 10% (average); dimension, 250 × 200 m2; plantation, 2002; rows distance, 2.5 m; distance in a row, 0.8 m; density, ca. 5000 plants/ha; Guyot and spurred cordon pruning methods. The 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).
The grapes sampling was performed along the two diagonals (ca. 1/4, 1/2, 3/4, the overall lengths, five spots; in triplicate; 1.5 kg of grapes each). The stalks were removed, and the berries were squeezed to prepare the grape juice samples (JU), stored in polyethylene bottles (−30 ± 1°C, dark) before analyses. The waste materials were manually handled (by using plastic knife over a polyethylene sheet) and portions of skins (SK) and seeds (SE) were separated and cleaned by pulp. SE and SK samples underwent freeze-dry procedure (lyophilizator 5 Pascal, LIO-5P 4 k; −51 ± 2°C, 1.5 ± 0.2 mbar; 3 days), grounded (granulometry, 0.08 mm sieve), and stored in polyethylene vessels (−30 ± 1°C, dark) before analyses.
Three samples (0.25 L each) of must (STY13-MU), obtained by mechanical removal of stalks and squeezing, were collected from the fermentation tanks (within 24 h from mixing), and the fermentation process was immediately stopped by freezing (−30 ± 1°C, dark, avoiding head-space). The alcoholic fermentation of must took place in steel tanks (max temperature, 29 ± 1°C) with pumping over cycles for four times a day. Three 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 derivatives Bâtonnage body (1.2 gBB/L and 1.2 gBB/L + 5.0 mgCu/L), Elevage Glutathione (1.2 gEGSH/L and 1.2 gEGSH/L + 5.0 mgCu/L), and Tannin Ellagitan Rouge (0.66 gBB/L + 0.66 gEGSH/L + 0.10 gTAN/L + 5.0 mgCu/L). Each treatment was tested in triplicate, and the treated samples were stored in polyethylene bottles, completely filled (21 ± 2°C, dark) for 60 days before analysis.
Several fermentation parameters were analyzed for a general characterization of the grapes, grape juice (JU and MU), and wines at different wine-making stages: total sugar (°Brix), reducing sugar (RSU, gGLU/kg), alcohol content (ALC, % v/v, by comparing
The absorbance values at
The method was based on the colorimetric technique with external calibration curve using gallic acid (GAA) and Folin–Ciocalteu reactant (FCR) [
The absorbance value at
On the basis of protocols already published [
The analytical determination of RES, QUE, and QUEG contents was carried out via isocratic HPLC instrument (Varian Prostar 210; Agilent Technologies, Rome, Italy) equipped with reverse phase C18 column (Varian Polaris C18-A, 250 mm × 4.6 mm, pore size 180 Å, particle size 5
The QUE and QUEG quantifications were carried out using external calibration protocol (linearity range 20.0–300.0 mg/L, using five different standard concentrations). Calibration curves were accepted with
Both methods were tested for precision that was evaluated repeating injections of the sample extracts three times (each replicate, three replicates). The 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.
All the samples were collected and pretreated (extracted/mineralized) in triplicates, and all the analytical procedures were also performed in triplicates. The 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. The data showing
The content of total nitrogen (TN) for STY13-JU was 0.24 ± 0.03 gN/L (Table
Selected parameters for STY13 in grape juice (JU), skin (SK), seeds (SE), and must (MU).
TN | TPC | RSU | BRIX | |
---|---|---|---|---|
JU/[MU] | 0.24 ± 0.03a | 1.53 ± 0.20a | 226 ± 10a | 21.2 ± 1.0a [22.5 ± 1.0]a |
SK + SE | 6.8 ± 0.7b | 43.2 ± 4.3b | — | — |
TN, total nitrogen expressed as gN/kg; TPC, total protein content expressed as gALA/kg (alanine equivalent); RSU, reducing sugars expressed as gGLU/kg (glucose equivalent). Values (mean ± esd) obtained from three samples and three replicates each sample. Different letters in the same column indicate significant differences (
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 93.5–253.9 g/L; [
Selected fermentation parameters in STY13 and SGTY13 wine samples at different wine-making stages and STY15 and SCCY15 refined wines.
pH | TAC | VAC | TSO2 | VSO2 | ALC | |
---|---|---|---|---|---|---|
STY13-JU | 3.13 ± 0.02a | 6.5 ± 0.4a | 279 ± 15a | 21.9 ± 1.5a | 10.8 ± 0.5a | — |
STY13-MU | 3.18 ± 0.03a | 6.4 ± 0.4a | 273 ± 15a | 21.8 ± 1.5a | 10.7 ± 0.5a | — |
STY13-AF | 3.29 ± 0.02b | 6.9 ± 0.3a | 304 ± 20a | 60.7 ± 3.4b | 44.0 ± 2.3b | 13.9 ± 0.2a |
STY13-MF | 3.42 ± 0.02c | 5.7 ± 0.2a | 450 ± 30b | 41.0 ± 1.6c | 31.0 ± 1.6c | 14.1 ± 0.2a |
SGTY13-AF | 3.20 ± 0.02a | 6.5 ± 0.1a | 349 ± 41c | 42.1 ± 1.0c | 31.0 ± 1.0c | 13.6 ± 0.1a |
SGTY13-MF | 3.22 ± 0.02a | 6.3 ± 0.2a | 380 ± 40c | 39.0 ± 1.2c | 27.0 ± 1.0c | 14.6 ± 0.1a |
STY15 | 3.43 ± 0.03c | 5.9 ± 0.3a | 440 ± 30b | 66.0 ± 3.3b | 12.0 ± 1.0a | 14.4 ± 0.1a |
SCCY15 | 3.39 ± 0.03c | 5.9 ± 0.2a | 470 ± 30b | 66.3 ± 7.1b | 14.0 ± 2.2a | 14.4 ± 0.1a |
Reference [ |
— | >3.5 | 1500 | 150 | — | 15 |
TAC, total or titratable acidity content expressed as gTAA/L (tartaric acid equivalent); VAC, volatile acidity content expressed as gACA/L (acetic acid equivalent); VSO2, free or volatile sulfur dioxide content; TSO2, total sulfur dioxide content both expressed as mgSO2/L; ALC, alcoholic content percentage (% v/v). Values (mean ± esd) obtained from three samples and three replicates each sample. Different letters in the same column indicate significant differences (
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. The pH value of must influences the speed of fermentation kinetics, through the speed of yeast reproduction. The 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.
The 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 3.5–15.0 gTAA/L [
It is finally, well known that high alcohol contents (ALC) protect against the formation of undesired microorganisms. The 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).
The contents of
Contents of selected antioxidant indicators in STY13 and SGTY13 wine samples at different wine-making stages and STY15 and SCCY15 refined wines.
TPP | RES | HUE | CI | |
---|---|---|---|---|
STY13-JU | 420 ± 10a | 0.024 ± 0.002a | — | — |
STY13-MU | 462 ± 13a | 0.032 ± 0.008a | 1.06 ± 0.02a | 3.97 ± 0.20a |
STY13-AF | 3033 ± 90b | 1.2 ± 0.1b | 0.77 ± 0.02b | 10.80 ± 0.32b |
STY13-MF | 2982 ± 52b | 1.0 ± 0.1b | 0.92 ± 0.04a | 11.70 ± 1.47b |
SGTY13-AF | 2597 ± 37c | 0.67 ± 0.02c | 1.02 ± 0.07a | 9.25 ± 0.05c |
SGTY13-MF | 2441 ± 70c | 0.70 ± 0.09c | 0.83 ± 0.01b | 10.55 ± 1.14b |
STY15 | 3334 ± 60d | 1.26 ± 0.09b | 0.95 ± 0.06a | 8.20 ± 0.03d |
SCCY15 | 2786 ± 95e | 1.18 ± 0.09b | 0.90 ± 0.09a | 7.11 ± 0.15e |
Reference | 1100–2700 [ |
0.4–2.6 [ |
0.63–1.1 [ |
7.2–12.04 [ |
1200–1500 [ |
— | — | — |
TPP, total polyphenols content expressed as mgGAA/L (gallic acid equivalent); RES,
The content of (a) total polyphenols (TPP, mgGAA/L) and (b)
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; [
Attention was then devoted to investigate the possible influence of yeasts derivatives and/or copper(II) additions on refining wines. The study was carried out on certain SCCY15 samples that underwent addition of yeast derivatives (allowed by EU regulations [
Values for RES (mg/L) in SCCY15 refined bottled wine and treated samples (incubation: 60 days, 21 ± 2°C, hermetically sealed, dark).
Sample (+added species) | RES |
---|---|
SCCY15 | 1.18 ± 0.09 |
SCCY15 + Cu(II) | 1.24 ± 0.09 |
SCCY15 + BB | 1.16 ± 0.05 |
SCCY15 + Cu(II) + BB | 1.15 ± 0.08 |
SCCY15 + EGSH | 1.24 ± 0.10 |
SCCY15 + Cu(II) + EGSH | 1.12 ± 0.04 |
SCCY15 + Cu(II) + BB + EGSH + TAN | 1.22 ± 0.05 |
Values (mean ± esd) obtained from three samples and three replicates each sample. The data resulted data were not statistically different (Tukey’s test). Full experimental data sets are reported in Table
The contents of QUE and QUEG in SCCY15 wine samples were 21 ± 2 and 3.0 ± 0.2 mg/L (Figure
Contents of quercetin (QUE, mg/L) and quercetin glucoside (QUEG, mg/L) for SCCY15 wine samples before and after additions of Cu(II) and selected yeast derivatives (BB, Bâtonnage body). Values (mean ± esd) obtained from three samples and three replicates each sample. Each set of data (QUE and QUEG) was not statistically different (Tukey’s test). Full experimental data sets are reported in Table
The 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 [
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 [
For comparison purposes and to estimate the analytical parameters in final marketable products, refined bottled wines were analyzed (STY10, STY11, STY12, and SGTY12) and reported in Table
Selected fermentation parameters and antioxidant indicators in STY10, STY11, STY12, and SGTY12 bottled wine samples.
pH | TAC | VAC | TSO2 | VSO2 | ALC | TPP | RES | HUE | CI | |
---|---|---|---|---|---|---|---|---|---|---|
STY10 | 3.26 ± 0.03a | 5.7 ± 0.2a | 450 ± 50a | 84 ± 4a | 67 ± 3a | 15.0 ± 0.4a | 3200 ± 82a | 2.6 ± 0.3a | 0.95 ± 0.5a | 10.3 ± 0.8a |
STY11 | 3.39 ± 0.04b | 6.1 ± 0.1a | 500 ± 50a | 63 ± 10b | 41 ± 6b | 14.8 ± 0.2a | 3036 ± 80a | 0.8 ± 0.1b | 0.93 ± 0.4a | 11.7 ± 0.9a |
STY12 | 3.44 ± 0.03b | 5.3 ± 0.1a | 380 ± 40b | 47 ± 3c | 33 ± 3c | 13.5 ± 0.2b | 2930 ± 77a | 1.05 ± 0.06c | 1.2 ± 0.7a | 10.0 ± 0.9a |
SGTY12 | 3.42 ± 0.01b | 5.3 ± 0.1a | 381 ± 30b | 52 ± 6c | 28 ± 3c | 13.9 ± 0.2b | 2489 ± 12b | 0.90 ± 0.06b | 1.1 ± 0.6a | 7.0 ± 0.7a |
Reference | — | >3.5 [ |
1500 [ |
150 [ |
— | 15 [ |
1100–2700 [ |
0.4–2.6 [ |
0.63–1.1 [ |
7.2–12.0 [ |
TAC, total or titratable acidity content expressed as gTAA/L (tartaric acid equivalent); VAC, volatile acidity content expressed as gACA/L (acetic acid equivalent); VSO2, free or volatile sulfur dioxide content; TSO2, total sulfur dioxide content expressed as gSO2/L; ALC, alcoholic content percentage (% v/v); TPP, total polyphenols content expressed as mgGAA/L (gallic acid equivalent); RES,
This work brought about data relevant to the content of several analytes of grapes (juice and must) and wines after alcoholic and malolactic fermentations, produced from the harvests during 2010–2013 and 2015. In conclusion, the following points were stated: The choice of wine-making protocol allowed the production of high quality wines, as confirmed by antioxidant parameters. The contents of total polyphenols resulted to be significantly higher than those reported for other red wines. The optimization of an analytical protocol, based on a calibration via standard additions, allowed to reveal the contents of The wines also revealed high quercetin contents (ca. 20 mg/L), suggesting a significant contribution to the overall health benefits from human consumption [ 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. This possible event is excluded in the present wine samples, as the total QUE + QUEG value is below their solubility in wine matrix. 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.
The 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 [
The 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.
Renzo Cini former full professor at Department of Biotechnology, Chemistry and Pharmacy, actually retired.
The authors declare that they have no conflicts of interest.
The authors thank Rocca di Castagnoli (Gaiole in Chianti, Siena, Italy) wine-maker farm for grapes and wines samples. The authors also gratefully acknowledge the Department of Biotechnology Chemistry and Pharmacy (Department of Excellence 2018–2022, University of Siena) and the CSGI (Centre for Colloid and Surface Science) for supporting the research and Dr Zeno Tabani for some experimental determinations.
Values of the three determinations for each replicate and three replicates (samples/extracts) for the analytical parameters in grapes and wines at different wine-making stages are reported: total nitrogen (Table 1Sa), total protein content (Table 1Sb), reducing sugar concentration (Table 1Sc), total sugar content (Table 1Sd), pH values (Table 2Sa), total acidity content (Table 2Sb), volatile acidity content (Table 2Sc), total sulfur dioxide content (Table 2Sd), volatile sulfur dioxide content (Table 2Se), alcohol content percentage (Table 2Sf), total polyphenol content (Table 3Sa), total anthocyanin content (Table 3Sb),