Polyphenolic Profile and Antioxidant Activity of Green Extracts from Grape Pomace Skins and Seeds of Italian Cultivars.

The possibility of industrial exploitation of winemaking products, as for all byproducts of vegetal origin, constantly deals with a raw material (grape pomace, GP) whose chemical composition and functional properties vary over time depending on the varietal and geographical origin of the grapes, the climatic conditions (vintage effect), and the winemaking technique. This work studied the compositional variability of polyphenolic skin and seed extracts from GP derived from white and red winemaking of different Italian grape varieties. The total polyphenolic content (GAE), the main classes of polyphenolic compounds, and the DPPH index were determined. Seed extracts were always richer in total polyphenols and condensed tannins and had higher antiradical activity (DPPH) than skin extracts: 144-298 mg GAE/g d.w. extract for skins and 327-540 mg GAE/g for seeds; the DPPH values were 1.77-3.40 mg AAE/g for skins and 3.10-10.48 mg AAE/g for seeds. Furthermore, it was verified that the evaluation of the GAE index of seed extracts, offering a good estimate of the antiradical properties (DPPH index), could represent a simple and rapid method for selecting the best lots of seeds to be used. Conversely, GP skins could be used as flour in the food industry due to their high content of dietary fiber and the presence of flavonols, which possess very interesting functional properties. Important differences in the flavonols profile were observed both between cultivars and between unfermented and fermented pomace.

Polyphenolic Composition and In Vitro Antioxidant Activity of Red Grape Seeds as Byproducts of Short and Medium-Long Fermentative Macerations.

The polyphenolic composition and antioxidant activity of grape seeds, as byproducts of red winemaking, depend on various factors, such as grape cultivar, vintage effect, grape maturity and winemaking methods. In the present work, the influence of the maceration length on the polyphenolic and antioxidant characteristics of the seeds of four Italian red grape cultivars ('Barbera', 'Grignolino', 'Nebbiolo', and 'Uvalino'), sampled from the fermentation tanks after short (two days) and medium-long (7-21 days) macerations, was studied with spectrophotometric methods, high-performance liquid chromatography (HPLC), and three different antioxidant assays (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), ferric reducing antioxidant power (FRAP) and 2,2 diphenyl-1-picrylhydrazyl (DPPH)). The total polyphenolic content (gallic acid equivalent (GAE)) of the seeds sampled after short macerations ranged between 24.5 and 60.1 mg/g dry weight (DW), and it dropped to 20.0-37.5 mg/g DW after medium-long macerations. The polyphenolic profile of the shortly macerated seeds was related to the varietal characteristics, while, after longer macerations, the influence of the maceration length prevailed on the cultivar effect. The multiple in vitro antioxidant activity tests (ABTS, FRAP and DPPH), although based on different mechanisms capable of highlighting behavioral differences between the different polyphenolic compounds, were highly correlated with each other and with the polyphenolic parameters; the qualitative differences between the matrices in the polyphenolic profile were probably less important than the quantitative differences in the polyphenolic content. The relations with the polyphenolic content were linear, except for the Efficient Concentration (EC20) parameter, whose relation was better described by a hyperbolic equation.

Use of potassium polyaspartate for stabilization of potassium bitartrate in wines: influence on colloidal stability and interactions with other additives and enological practices.

The research was aimed at verifying the efficacy of potassium polyaspartate (KPA), added at bottling, for prevention of the precipitation of potassium bitartrate (KHT). The stability of KHT was determined with the mini-contact test and the cold test. The effect of KPA on the colloidal stability of white and red wines was also studied. Turbidity, color, and total anthocyanins and flavonoids (red wines only) were monitored. In the first experiment, the stabilizing effect of KPA added at bottling, with or without Arabic gum, tannins, and filtration (0.45 µm cut-off), was studied in comparison with metatartaric acid (MTA). KPA proved to have good stabilizing efficacy for prevention of KHT precipitation and maintained its effect in all trials after 1 year of bottle aging. On the contrary, all the trials with MTA became unstable after 6 months. Moreover, KPA did not modify wine turbidity and color. The filtration (0.45 µm cut-off) did not reduce its stabilizing efficacy. In the second experiment, the effect of KPA added to a white and a red wine, respectively clarified with casein and gelatin or vegetal protein and/or bentonite, was studied. KPA did not cause any increase in turbidity or appreciable changes in the color of the white wines of the different trials. A preliminary treatment with bentonite can prevent the possible formation of haze in red wines after the addition of KPA. PRACTICAL APPLICATION: KPA prevented the precipitation of KHT in unstable white and red wines. The stabilizing effect persisted after 1 year of bottle aging. The oenologist does not need to adopt special prescriptions when using KPA in combination with Arabic gum or tannins in prebottling operations. A treatment with bentonite prevented the appearance of turbidity in red wines after the addition of KPA.

Relationship between polyphenolic content, antioxidant properties and oxygen consumption rate of different tannins in a model wine solution.

The polyphenolic content and the antioxidant capacity of seven tannins with different botanical origin were measured with spectrophotometric methods (Folin-Ciocalteu, Total Polyphenols Index, DPPH, FRAP), HPLC (phloroglucinolysis), voltammetric analysis (Linear Sweep Voltammetry, LSV). The oxygen consumption rate (OCR) was measured in an oxygen saturated model wine solution, containing transition metals and metabisulphite, with a noninvasive luminescence-based technology. The results showed a high variability in polyphenolic concentration related to the botanical origin of tannins. The OCR determined over 21 days was described by quadratic equations, with coefficients varying with tannin botanical origin, dose and SO2 concentration. The tannins ranked differently for antioxidant capacity, depending on the kind of test. The oxygen consumption parameters were positively correlated only with the LSV data measured with anodic current between 100 and 1200 mV (LSV1200mV) and with the FRAP index.

Polyphenolic Characterization of Grape Skins and Seeds of Four Italian Red Cultivars at Harvest and after Fermentative Maceration.

Agro-industry byproducts can still contain large amounts of phenolic compounds, and one of the richest sources are grape skins and seeds as grape pomace, both fermented (red winemaking) and unfermented (white winemaking). The residual polyphenolic content depends on various factors such as grape variety, vintage, and winemaking technique. In this work, four red grape varieties cultivated in northern Italy were studied: Albarossa, Barbera, Nebbiolo, and Uvalino. The work was aimed at studying the polyphenolic composition of skins and seeds from fresh grapes and from the corresponding pomace after fermentative maceration, to assess the actual importance of the varietal differences when processing winemaking byproducts for the extraction of phenolic compounds. The skin and seed extracts were prepared by solvent extraction with a 50% hydroalcoholic solution. The polyphenolic composition of all extracts was determined by spectrophotometry and high-performance liquid chromatography (HPLC); the content and the monomer composition of condensed tannins were determined by phloroglucinolysis; the antioxidant capacity was measured with the ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6- sulfonate)) method. The antioxidant capacity was higher for the seeds than for the skins, and it was positively correlated with the condensed tannins content. Significant differences in polyphenolic composition of fresh grape skins and seeds were observed between the different cultivars. In particular, Barbera and Albarossa skins were significantly distinguished from Nebbiolo and Uvalino skins for a higher content of anthocyanins and a lower content of vanillin-reactive flavans and condensed tannins; regarding seeds, Barbera and Albarossa had a lower content of vanillin-reactive flavans, proanthocyanidins, and condensed tannins than Nebbiolo and Uvalino. The winemaking process extracted the phenolic compounds to a different extent from skins and seeds, regardless of the cultivar. The differences between cultivars in the polyphenolic profile disappeared after fermentative maceration.

Comparison of the physicochemical and volatile composition of wine fractions obtained by two different dealcoholization techniques.

Three wines (a Rosé wine, a Pelaverga and a Barbera red wines) were dealcoholized at 5% v/v ethanol with two different techniques: membrane contactor (MC) and distillation under vacuum (D). The influence of the treatments on the physicochemical composition and aromatic profile of the dealcoholized fractions was investigated. The main difference between the two techniques was the concentration grade reached by the dealcoholized fractions, which was 5-6times higher with D than with MC due to the concomitant loss of water. The main fixed compounds (organic acids, cations, polyphenols, anthocyanins) were not lost during the dealcoholization, while the losses of volatile compounds were relevant. Finally, the δ18O isotopic ratio was studied. The δ18O isotope ratio increased with D and decreased with MC. At the same time the δ18O isotope ratio fell within legal limits when the final ethanol content was adjusted to 2% by blending with dealcoholized fractions.

Validation of a rapid conductimetric test for the measurement of wine tartaric stability.

This work was aimed at optimizing a rapid and reproducible conductivity test for the evaluation of wine tartaric stability, in order to improve the practices for the prevention of tartaric precipitations during bottle aging. The test consists in measuring the drop of conductivity in wines kept under stirring for a fixed time, at low temperature, after the addition of micronized potassium bitartrate crystals (KHT). An experimental design was planned to study three factors affecting the test: temperature, duration and dose of added potassium bitartrate. A standard protocol was defined to produce a micronized potassium bitartrate starting from available commercial products, since the dimensions of the crystals can affect the final conductivity values. After the choice of the best conditions the method was validated. Two different stability thresholds were defined for white wines and for red/rosé wines by comparing the results of the mini-contact test with those of the cold test.

Influence of solvents on the composition of condensed tannins in grape pomace seed extracts.

The extracting effectiveness of some solvents (water, ethanol, acetone, ethyl acetate), used as pure or in binary and ternary mixtures, was studied for the extraction of seeds from the fermented pomace of Nebbiolo cv. The aqueous mixtures of acetone provided the highest extraction yields in total polyphenols and total flavonoids. Polyphenolic extracts were also obtained with aqueous mixtures of ethanol or acetone at variable concentrations, and the condensed tannins were quantified with the phloroglucinolysis method. The aqueous mixtures of acetone were more effective than the corresponding aqueous mixtures of ethanol. The solvent influenced the extraction yield and the composition of the extracts: with the increase of the yield, the mean degree of polymerization (mDP) of the condensed tannins increased. A significant correlation was noticed between mDP and the molar percentages of (+)-catechin as terminal unit (negative correlation), and of (-)-epicatechin and (-)-epicatechin-3O-gallate as extension units (positive correlation).

Use of polyaspartate as inhibitor of tartaric precipitations in wines.

All additives used to stabilize wines against the precipitations of potassium bitartrate have some limits: metatartaric acid (MTA) is effective but very unstable, carboxymethylcellulose is stable and effective in white wines but affects color stability in red wines, mannoproteins have a variable effectiveness depending on wine composition. This work was aimed at testing the effect of new stabilizing products on tartaric precipitations, focusing on the use of Na and K polyaspartate salts (PASPs). The effectiveness of 4 different PASPs and 1 MTA added to red and white wines was compared using the mini-contact test and cold test. The dose effect and the stability of the products over time were also studied. The PASPs showed a similar stabilizing effect and a longer stability over time compared to MTA. PASPs can be considered interesting as additives for wine tartaric stabilization. Further work is in progress to better characterize their enological properties.

Influence of the matrix composition on the volatility and sensory perception of 4-ethylphenol and 4-ethylguaiacol in model wine solutions.

This study was aimed at investigating the influence of ethanol, wine polyphenols and yeast extract on the volatility of 4-ethylphenols in red wines. The use of solid-phase microextraction with short extraction times for the study of the headspace composition, revealed a strong influence of ethanol and polyphenols on reducing the volatility of 4-ethylphenols in a model wine. At the same time, the influence of ethanol and polyphenols on the perception of the "Brett character" (stable, manure, horse sweat and phenolic notes) due to 4-ethylphenols was evaluated by sensory analysis. Polyphenols and ethanol were found to have a significant influence on the olfactory perception of the tainted wines. On the contrary, a direct influence of yeast extract on the volatility of 4-ethylphenols was not observed, although some results suggested that the effectiveness of polyphenols in reducing the volatility of 4-ethylphenols could be limited by the presence of yeast proteins.

Effect of the closure type on the evolution of the physical-chemical and sensory characteristics of a Montepulciano d'Abruzzo Rosé wine.

The present work studied the effect of the kind of closure (a screw cap, a natural cork, and 2 synthetic closures) on the evolution of the oxygen content and on the physical-chemical and sensory characteristics of a Montepulciano d'Abruzzo rosé wine during the 1st 12 mo of bottle aging. The chemical analyses concerned the parameters more involved in the oxidative reactions (SO(2) , acetaldehyde, phenols, wine color), as well as the main fermentative volatile compounds. The kind of closure influenced the oxygen content in wines, free and total SO(2) concentration, and wine color (color intensity and hue). During bottle aging, free and total SO(2) concentration was significantly and negatively correlated with absorbance at 420 nm (A420), whereas the correlations with A520 were weak. Probably, the limited extent of the variations in red color (A520), when varying SO(2) concentration, were due to the low pH of this rosé wine. No effect of the kind of closure on phenols and the main fermentative volatile compounds was observed. The wines bottled with cork closures (N trials), after 12 mo of storage, had higher color intensity and hue, measured by spectrophotometry, and were visually distinguished from the other trials for the more intense pink reflections. On the whole, under the conditions of this work, all the used closures guaranteed a good preservability to the rosè wines during the 1st year of bottle aging, and the changes in composition did not significantly affect wine sensory characteristics. Therefore, these synthetic closures can represent an alternative to the cork closures for a medium to long term bottle aging of these wines.

Quantification by solid phase micro extraction and stable isotope dilution assay of norisoprenoid compounds in red wines obtained from Piedmont rare varieties.

A method to identify and quantify megastigmane norisoprenoid compounds in wines was developed using headspace solid phase micro extraction (SPME) coupled with gas-chromatography/mass-spectrometry (GC-MS). Three different compounds were quantified by stable isotope dilution assay (SIDA): ß-damascenone, ß-ionone and α-ionone. Particular attention was paid to maximising the method's sensitivity while reducing the extraction time. To optimise the extraction conditions, a statistically designed experiment was performed using extraction time, extraction temperature and ethanol content as operating variables. Five different SPME fibres suitable for the analysis of volatile compounds were compared. This study confirmed that the PDMS/DVB coating performs best for the quantification of ß-damascenone and ß-ionone, and the crucial influence of ethanol content of the sample on extraction effectiveness. Finally, the optimised method was applied to the study of various wines derived from rare and autochthonous grape varieties of north-western Italy.

Analytical and sensory characterization of the aroma of "Langhe D.O.C. Nebbiolo" wines: influence of the prefermentative cold maceration with dry ice.

The present work concerns the determination of the volatile compounds, as well as the description of the olfactory characteristics, of "Langhe DOC Nebbiolo" wines produced through the prefermentative cold maceration with dry ice, on full scale (30 quintals) in private cellars. The free volatile compounds and those obtained by enzymatic and chemical hydrolysis of the glycosilated precursors were analyzed by gas chromatography-mass spectrometry. Among free volatile compounds, a loss of alcohols with 6 carbon atoms in the cryo-macerated trials was observed. The cryo-maceration technique, when correctly applied, increased the floral and fruity notes (blackberry, cherry, and plum). This increase is related to the extraction of polyphenolic compounds (maceration effect), but no volatile compounds, neither in free form, nor produced by enzymatic hydrolysis of the glycosilated precursors, can discriminate the wines according to their content of polyphenols. Only some compounds produced by chemical hydrolysis of the precursors can discriminate the wines (statistically significant differences): 3-hydroxy-ß-damascone, methylvanillate, and blumenol C. These compounds, however, form during aging and do not have any influence on young wines aroma.