December 2016 Issue of Wines & Vines

Oxygen's Impact on Red Wine Aging

How red wine phenolics evolve in the bottle

by Angelita Gambuti, Maurizio Ugliano, Alessandra Rinaldi and Luigi Moio

High-quality red wines generally require a period of aging in the bottle before they are ready for consumption. During this time, modifications of sensory properties occur such as the decrease of astringency1 and stabilization of color from purple-red to orange-red.

Numerous appellations outside the United States impose a minimum period of bottle aging for high-quality red wines to achieve specific sensory attributes. However, wine aging implies a significant financial cost to each winery, so it is important tounderstand the timing, factors and mechanisms of astringency and color changes during bottle aging.


practical winery vineyard

Astringency is a tactile sensation mainly elicited by the precipitation of salivary proteins that cause the mouth to feel dry.2 Wine components responsible for astringency include phenolics such as proanthocyanidins (PAs) or wood tannins (ellagitannins and gallotannins) that react with salivary proteins, causing their complexation and subsequent precipitation on the mouth epithelium.3


The color of red wine is due to anthocyanins and derived copigments. While the main reactions involved in color stabilization are known,4,5,6,7 the impact of reactions occurring during aging and involving PAs and anthocyanins on the decrease of wine astringency are not well established.1,8,9 In the past, this phenomenon was attributed to the formation of greater polymeric structures.10 Unlike anthocyanins, precise chemical analysis of changes in the tannin structures was not well known.

Many of the complex reactions involving phenolics are affected by oxygen exposure to wine, and a moderate uptake of oxygen during aging can accelerate and/or trigger specific reactions influencing sensory properties.8,11,12,13,14 For this reason, aging of red wine in oak barrels is a widely used practice. In fact, the ingress of small amounts of oxygen through the wood and between wood staves results in many chemical reactions involving wine and wood phenolics, enhancing the decrease of wine astringency and the stabilization of color.15,16

This practice and bottle aging is expensive and implies a significant financial cost in the wine’s bottle price. Micro-oxygenation (MOx) has been proposed to add a continuous flow of oxygen to a tank, simulating oxygen uptake occurring during wood aging.

Besides the winemaking process, wine can be further exposed to oxygen during bottle aging, depending on oxygen permeability of the closure. Because of the extremely low rate of oxygen ingress through a closure, this form of oxygen exposure is referred to as nano-oxygenation.21

Oxygen transmission rates (OTR) of wine closures vary widely depending on closure type and strongly influence the evolution of white and red wines during bottle aging.21,23,24,25,26,27 Additionally, the oxygen present at bottling, often referred to as total package oxygen (TPO), together with oxygen released from the closure upon insertion into the bottleneck also contribute to oxygen exposure in the bottle.28,29

Astringency evaluation
In spite of the fact that oxygen exposure has been linked to many reactions by phenolics involved in the decrease of astringency, a clear correlation between chemical transformation of phenolics and their sensory properties has not been reported. This can be due to the fact that sensorial activity of wine tannins is not easy to predict because it is influenced by their chemical nature, quantity and the inner balance with other compounds present in wine.2,30,31,32,33,34

Several analytical methods have been developed to predict wine astringency by evaluating the ability of wine to form insoluble complexes with human saliva.35,36,37 Since 2010 the saliva precipitation index (SPI), based on the electrophoretic analysis of selected salivary proteins precipitated after reaction with wine polyphenols, has been utilized to objectively evaluate changes in astringency.

Despite extensive research to evaluate the effect of micro- and nano-oxygenation on the evolution of phenolics in red wine, very few studies have evaluated the fate of micro-oxygenated wines after a long period of bottle aging.21,22

No study has dealt with the impact of closure oxygen permeability on the saliva precipitation index (SPI) of red wine.38 In this work, the SPI was utilized to objectively evaluate changes in astringency as a function of oxygen uptake before and after bottling. Sensory rating of astringency, polyphenolic composition and chromatic characteristics also were evaluated.

Micro-oxygenation trials

Cantina del Taburno Winery produced two 2006 Aglianico red wines in agreement with standard procedures used for Aglianico del Taburno DOC wine. These wines were chosen because of their different pH levels. For both wines, two micro-oxygenation treatments were applied. Each wine was transferred from the initial tank into six 50 hL tanks (3 meters tall).

Micro-oxygenation was performed on four tanks with a Microdue system. Oxygen was provided through a diffuser composed of a porous ceramic membrane. Two tanks were denoted as MO1, two others as MO2, and two tanks were control wines.

After three months of MOx treatment, each wine was bottled in 750ml glass bottles flushed with 98% N2 gas and sealed with a 44 mm natural cork. Analyses were performed at bottling and after 42 months of aging in bottle.

Nano-oxygenation trials

Two red wines were used to study the influence of closure OTR on wine phenolic composition and astringency. Wine 1 was a blend of 40% Cabernet Franc, 40% Merlot and 20% Blaufränkisch. Wine 2 was a 100% Montepulciano.

All bottles were sealed with Nomacorc co-extruded synthetic closures. Three distinct levels of oxygen exposure in the bottles were obtained by using closures with different oxygen ingress profiles: Select 300 (W1low and W2low), Select 500 (W1medium and W2medium) and Select 700 (W1high and W2high). The values of total oxygen exposure (TOE) in the bottle for each wine are the sum of closure contribution and TPO.

Closure contribution is intended as the combination of oxygen ingress through the closure (OTR) and the amount of oxygen released from the closure following insertion into the bottleneck. These values were provided by the manufacturer and were calculated using the procedure described by J.B. Dieval et al.39 A slightly lower free SO2 level was detected in W1high and W2high 10 months after bottling.

Standard chemical analyses and spectrophotometric measurements

Standard chemical analyses (alcoholic strength by volume, titratable acidity, pH, volatile acidity, free and total SO2 and total polyphenols (Folin-Ciocalteu) were measured according to the OIV Compendium of International Methods of Wine and Must Analysis.40

Condensed tannins (proanthocyanidins, or PAs) were evaluated,41 and total anthocyanins and SO2 bleaching anthocyanins were determined according to P. Ribéreau-Gayon and E. Stone­street.42 Vanillin reactive flavans (VRF) were determined according to R. Di Stefano and S. Guidoni.43

Color intensity and hue were evaluated according to Y. Glories methods.44 All analyses were performed in triplicate.

HPLC analysis of anthocyanins
HPLC separation of monomeric anthocyanins also was performed according to the OIV Compendium of International Methods of Wine and Must Analysis.40 Twenty µL of wine or calibration standards were injected into the column. All samples were filtered through 0.45 µm membrane filters into glass vials and immediately injected into the HPLC system.

Human saliva
Whole saliva was obtained by mixing saliva samples collected from six non-smoking volunteers (three males and three females). Electrophoresis analyses were performed on the resulting supernatant.

Saliva precipitation index determination
The SPI was determined as reported by A. Rinaldi et al.38 The calibration curve was obtained by the density reduction of two protein bands selected from the pool of salivary proteins that were better correlated with sensory analysis. The densitometric analysis of proteins was performed before and after the interaction of saliva with five standard solutions containing tannic acid (2 to 10 g/L in water).

Sensory analysis
Selection and training sessions:
Twenty-four subjects were recruited from a viticulture and enology class at the University of Naples to participate in the sensory sessions. All had experience as wine tasters, but with different backgrounds: six aroma researchers, seven winemakers and 11 enology students. Panelists were trained to differentiate astringency from bitterness and sourness using 3.0 g/L tannic acid, 0.25 g/L caffeine-monohydrate and 4.0 g/L tartaric acid as examples of astringency, bitterness and sourness, respectively.

Eighteen panelists indicated an ability to discriminate among these taste stimuli. Selected panelists trained with astringency rating evaluated overall astringency of different concentrations (from 0.1 to 5.0 g/L) of commercial tannin on a nine-point scale (absent, very weak, weak, weak-moderate, moderate, moderate-strong, strong, very strong or extremely strong) first in water and then in wine solution.

In each session five unknown samples (10 ml were presented in balanced random order at room temperature [18° ± 2° C] in black tulip-shaped glasses). The panelists were instructed to pour the whole sample in their mouth, hold it for eight seconds, expectorate and rate the perceived overall astringency using the nine-point scale. Panelists waited four minutes before rinsing with de-ionized water for 10 seconds twice and then waited at least 30 seconds before the next sample. Each sample was evaluated within five minutes.

Astringency was expressed as the maximum intensity perceived. The data obtained were used for assessing the reliability and consistency of the panelists, which were considered acceptable (P < 0.05 for reproducibility of scores of replicate samples). The accuracy of rating was monitored with use of standards during each tasting session, consisting of three commercial tannin wine solutions (very weak astringency = 0.1 g/L; moderate astringency = 2.5 g/L; and extremely strong astringency = 5.0 g/L) to provide reference for three points on the nine-point scale.

Evaluations of panel performance were based on a one-way random model under the assumption that panelists are homogeneous. The tendency toward consistency in the repeated measurements of the sample was referred to as the reliability. The reliability coefficient was used for assessing performance of the panel.45

Sensory evaluation sessions: At the beginning of each session, panelists tasted three solutions for astringency. The same procedure and conditions were applied for red wine evaluation. During the eight tasting sessions, three experimental wines were evaluated in duplicate.

Statistical analysis
All of the data are expressed as the arithmetic average ± standard deviation of three replicates. Analysis of variance was performed on phenolic compound and sensory data. Fisher’s Least Significant Differences procedure was used to discriminate among the means of the variables.

Effect of MOx on phenolics and astringency of bottle-aged red wines

What is the effect of micro-oxygenation on changes in phenolic composition, color, SPI and astringency during aging? An increase of color intensity and total anthocyanin content was observed in both wines three months after the MO1 treatment.

A significant increase previously has been reported in the color intensity just after the micro-oxygenation treatment,18,46,47 which was due to the formation of new pigments such as those derived from the combination of anthocyanins and flavanols via the formation of ethyl bridges.48 The higher values of total anthocyanins in MOx wines confirmed this hypothesis.

Increased levels of micro-oxygenation to treatment MO2 resulted in a significant decrease of SO2 decolorable anthocyanins. Therefore, the higher levels of oxygen, the higher the formation of new anthocyanin-derived pigments stable to pH changes and bisulfite bleaching.14

After 42 months, all wines showed an increase of hue and a decrease of total and SO2 decolorable anthocyanins. However, the effect of micro-oxygenation on color intensity and hue for both wines and for total and SO2 decolorable anthocyanins for Wine A was no longer detected.

Several authors have observed that, during aging in barrel and/or bottle for several months, the differences between MOx wines and respective control wines were minimized.22,46 Our results suggest that, with time, this trend is enhanced to such an extent as to cancel the differences.

A decrease of native monomeric anthocyanins with MOx was detected in agreement with reported results18,22,47 and can be related to the involvement of these molecules in the oxygen-activated reactions between anthocyanins and flavanols. These reactions determine the formation of anthocyanin-ethyl flavanol compounds, which are unstable and may undergo cleavage of the ethyl bridge with consequent liberation of monomeric anthocyanins.14 This latter phenomenon could explain the increase of several monomeric anthocyanins observed in previous studies.17 In agreement with literature, the content of total monomeric anthocyanins in bottle decreased over time.49,50

Three months after the treatments, the content of total phenolics increased with MOx treatment for both wines. Since different phenolic classes possess slightly different chemical properties, the data observed may be due to the formation of phenolics with a higher reactivity toward the Folin-Ciocalteu reagent used for the analysis.51 In contrast, the aged MOx wines showed a lower content of total phenolics conpared to control wines, indicating that phenolic compounds changed or rearranged over time, giving less reactive compounds.

In Wine A, no statistically significant difference in PAs was detected at both dates of sampling. According to U. Vrhovsek et al.,52 the Bate-Smith reaction used to determine the tannins provides an estimation of high proanthocyanidins corresponding to more than five units, therefore no variation in this kind of molecule was observed.

In spite of the same initial content of PAs of the two wines, Wine B showed a decrease of PAs when the higher level of micro-oxygenation was applied. This might be due to the fact that PAs can have different reactivity toward oxygen and oxygen-derived compounds, depending on the monomers constituting the polymers as well as the polymerization degree.53

Variations in the degree of polymerization of tannic molecules are also suggested by the finding that changes in VRFs (corresponding to phenolic polymers of two to four units) are detected with MOx and time for both wines. For Wine A, a decrease was always observed when MO2 level was applied; for Wine B, a decrease of VRF always occurred when the MO1 level was applied.

Because vanillin reacts only with terminal units of tannic molecules, its decrease may be caused either by precipitation of tannins or by an increase of their polymerization degree. Concerning the degree of polymerization, different behaviors were reported. For some researchers it was thought that MOx induced the polymerization of PAs,19 but recently no variation of degree of polymerization of proanthocyanidins was detected.22

It is not very clear if the changes of the polymerization degree of proanthocyanidins1,9—or the combination of proanthocyanidins and anthocyanidins—are responsible for the decrease of astringency of wine. Therefore, only on the basis of these analyses, the effect of micro-oxygenation and bottle aging on wine astringency is not predictable.

In this study concerning sensory analysis, the variation occurring in phenolic composition three months after MOx is not enough to cause a significant variation of the astringency (see “Evolution of Astringency of Wine A” and “Evolution of Astringency of Wine B” above).

For both wines, a decrease of astringency was detected after 42 months of aging in bottle. No effect of MOx was observed in Wine A while, for Wine B, a significant decrease of astringency was detected with increasing MOx levels. This result is confirmed by data on SPI (see “Impact of Micro-Oxygenation on SPI” above), which gives a direct measure of the reactivity of whole wine phenolics with salivary proteins.

In the literature, both a significant decrease17 and no effect21 on the astringency with micro-oxygenation of wines have been reported. This is the first time that a significant effect was observed with time, and it occurs only in the lower pH wine. Therefore, in agreement with recent findings22 showing that pH exerts a major effect on the evolution of phenolic compounds during aging, our data seem to show that this effect can result in a variation of wine reactivity toward salivary proteins and, then, in wine astringency.

However, this result can be also due to: a) differences in phenolic composition between the two wines; b) the direct effect of wine pH on astringency perception34 and, c) the lower content of molecular SO2 protecting wine components from oxidation.

Effect of OTR on changes in phenolic composition, color, SPI and astringency

To evaluate the effect of nano-oxygenation, two red wines with a different level of TPO at bottling (9.8 mg/L = W1, and 6.5 mg/L = W2) were sealed with different closures and analyzed after 10 months of storage in bottle. Three increasing OTR conditions (Wlow, Wmedium and Whigh), ensured by using synthetic closures with controlled oxygen permeability, were compared.

Color intensity of W1low did not differ between W1high and W1medium, while W1medium showed slightly higher color intensity than W1high. This result contrasts with data reported by S. Caillé et al.,21 which showed a positive correlation between color intensity of red Grenache wine and OTR. The different trend may depend on the differences in TPO of closures used in the two studies. However, wine type can also affect the evolution of color intensity during bottle aging.

For W1, a significant loss of total monomeric anthocyanins of W1medium and W1high with respect to W1low was observed. The last two samples were similar to each other. The highest percentage of loss of monomeric anthocyanins (comprised between 38% and 44% of total monomeric anthocyanins) with respect to that observed in literature26,27 can be related to the highest oxygen exposure of W1. Further studies aimed to determine the relationship between OTR, the antioxidant power of wine and time of storage can better explain these phenomena.

No differences among closures for color intensity and total monomeric anthocyanins were detected for W2 wines. These wines were characterized by lower total oxygen exposure (6.5 mg/L compared to 9.8 mg/L for W1 wines), and higher content of phenolic compounds (3,400 mg/L compared to 2,300 mg/L for W1 wines), acting as oxygen-quenching compounds could explain this result.

OTR was observed to have a significant effect on total phenolic content of W1 wines, while no differences were observed among W2 wines. Each phenolic compound had different reactivity toward the Folin-Ciocalteu reagent.51 The results indicate that the OTR of closures can determine a variation into the chemical nature of wine phenolics. While no variation in PAs was detected, a significant loss of VRF of W1high and W1medium was detected compared to W1low.

Since vanillin reacts with free carbons C6 and C8 of the A ring of flavanols, the decrease of low molecular weight proanthocyanidins reactive toward vanillin is consistent with the minor presence of nucleophile sites on flavanol molecules due to the significant effect of OTR of closures. The fact that this phenomenon occurs only for W1 and not for W2 indicates that both TOE and the native composition in phenolics of red wine are determining factors in development of condensation and polymerization reactions of tannins.

The SDS-PAGE analysis of human saliva after the interaction with experimental wines was performed to determine the SPI values of bottled wines. For W1 wine, the SPI was significantly lower when the bottles were sealed with closures providing high OTR.

A significant decrease in SPI was detected for W2 wine. The loss of phenolics reactive toward salivary proteins positively correlated with OTR. These results are partially confirmed by the sensory rating of astringency, which is in agreement with SPI data. W1high was less astringent than W1low while in contrast with SPI data; W1medium did not differ from W1high (see “Influence of Closure on Astringency of Wines” above). For W2, no differences among wines were detected.

The discrepancy between sensory analysis and SPI can be attributed to different reasons such as the interference of wine components, changes occurring in phenolic stimuli and sensitivity of the two analytical methods used.

Regarding the interference of wine components such as tartaric acid, ethanol, fructose and mannoproteins, a recent study showed that they affect both sensory perception of astringency and SPI, and this effect depends on wine phenolics.34

However, the entity of the effects detected were not the same for the two methods considered. It is therefore likely that oxygen exposure can determine changes in wine phenolics detectable only by means of SPI because this analysis is more sensitive than sensory analysis to little changes in binding reactivity of tannins.

Pre- and post-bottling O2 exposure influences sensory-active phenolics of red wines
Oxygen exposure to red wine before and after bottling affects the evolution of phenolics and astringency during bottle aging. This is the first time that a direct effect of a) addition of micro quantities of oxygen to a wine before bottling and, b) oxygen permeating toward closures on the reactivity of wine phenolics toward salivary proteins has been demonstrated. These effects are the function of aging time, wine initial composition (pH and phenolic composition) and dissolved oxygen level.

Before applying micro-oxygenation and choosing a bottle closure, a winemaker must consider the expected consumption date of a bottled wine, the total package oxygen present at bottling and wine composition.

Further studies about the influence of each wine compound on the evolution of wine astringency and reactivity toward salivary proteins during bottle aging are needed to improve the use of both micro-oxygenation and closures at specific OTRs in the wine industry.


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