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Micro-oxygenation impacts on wine colour
Sierra Rayne

Ph.D., P.Chem.1,2

Chemistry, Earth and Environmental Sciences
The University of British Columbia
Okanagan, Kelowna
British Columbia, Canada
Ecologica Environmental Consulting
British Columbia, Canada


Despite the well-known relationship between oxygen exposure
and colour development in wines, relatively few openly available
studies have investigated the potential impacts of micro-oxygenation.
The current report describes the temporal evolution in the optical
properties of replicate full-scale commercial micro-oxygenated trials
and control wines from two varieties, Vitis vinifera L. cv. Merlot and
Cabernet Sauvignon. Wines were tracked from pressing through a
stainless steel tank-aging period, and into the oak barrel aging.

Descriptions of Colour Parameters. Colour intensity (CI) was
determined as the sum of absorbances at 420nm (A420), 520nm (A520),
and 620nm (A620) using a 1mm pathlength cell. Colour composition
was calculated as the contribution of each of the three components
of the overall colour: percentage of yellow=A420/CI3100; percentage
of red=A520/CI3100; percentage of blue=A620/CI3100. Tint was
calculated as the ratio of the absorbances at 420nm and 520nm using
a 1mm pathlength cell: tint=A420A520. Brilliance was calculated using
the formula (1-[(A420+A620)/(23A520)])×100.
The total colour of pigments (TCP) was determined by diluting
a 0.5mL wine sample in 20mL of 0.1M HCl. Absorbance at 520nm
was measured in a 10mm pathlength cell after 30 minutes to ensure
complete conversion of anthocyanins into the flavylium form, and the
reading corrected for dilution. Wine colour (WC) was determined by
adding 20μL of 10% (v/v) acetaldehyde in ethanol to 2mL of wine.
After 45 minutes, the absorbance at 520nm was measured in a 1mm
pathlength cell. Colour due to derivatives resistant to sulfur dioxide
bleaching (CDRSO2) was determined by adding 75μL of a 20%
(w/v) sodium metabisulfite solution to 5mL of wine and measuring
the absorbance at 520nm after 10 minutes in a 1mm pathlength cell.
The colour due to pigments not resistant to sulphur dioxide bleaching
(CDNRSO2) was calculated as TCP minus CDRSO2.
Wine aging (WA) was calculated as CDRSO2 divided by TCP. The
chemical age of the wine (CAW) was calculated as 100%3CDRSO2
divided by WC. Phenol content (I280) was estimated by diluting
a 1mL sample of wine 100-fold in distilled water, measuring the
absorbance at 280nm in a 10mm pathlength cell, and multiplying by
100 (to account for dilution) to obtain the I280 value. Total monomeric
anthocyanin (TMA) content (mg/L) was estimated using the following
formula: 20×(TCP-[5/33CDRSO2]). Coloured monomeric anthocyanin

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October 2007


The Australian & New Zealand Grapegrower & Winemaker 75

High-quality wines are traditionally stored in oak barrels for
periods in the range of months to years to improve their sensorial
attributes. Contact between the oak wood and wine allows a number
of compounds to diffuse out of the oak into the wine, and these
substances are known to improve the intensity and complexity of wine
flavor (1). Additional benefits of oak aging include colour stabilisation,
lower astringency and bitterness, and the disappearance of excessive
vegetative/herbaceous aromas. Previous work has associated these
changes to the small amounts of molecular oxygen (i.e., O2) that
diffuse through the oak wood and between the stave interstices,
and also enter through the bunghole during topping-up (2,3).
But oak barreling is an expensive and laborious task, and
can introduce the risks of microbiological contamination (e.g.,
Brettanomyces) due to the nutrients provided by the oak, coupled with
the attractive wood-wine interface for biofilm growth and difficulty
in sanitising the highly-porous wood before and between uses. For
these reasons, micro-oxygenation has been proposed for attempting
to reproduce, and even accelerating, the positive oxidative wine
transformations that take place during oak barrel aging (4). Microoxygenation is a process where wines are continuously exposed to
oxygen. Oxygen is supplied in the form of the compressed molecular
gas via a micron-sized diffuser positioned close to the bottom of
a stainless steel tank or oak/stainless steel barrel. Flow rates are
controlled by a pressure release chamber, and typically range from
0.25 to 100mL/L/month (5).
The objectives of the micro-oxygenation process include improved
palatability, enhanced colour stability and intensity, increased
oxidative stability, decreased reductive character, and decreased
vegetative aroma (6). The process of micro-oxygenation begins with
an assessment of the wine and a review of the winemaker’s objectives.
From this information, a micro-oxygenation program can be developed.
The technique of micro-oxygenation can be started at any stage during
the winemaking process but has been reported to be typically, and
most effectively, begun at the end of alcoholic fermentation and prior
to malolactic fermentation (4). However, several important unresolved
questions remain regarding micro-oxygenation, such as the long-term
aging effects on wine quality, risks of promoting Brettanomyces and
other microbial growth during tank or barrel aging, the formation of
sulphide dimers, and pyrazine/thiol degradation (4,6).

(CMA) content (mg/L) was estimated using
the following formula: 203(A520 -CDRSO2).
Percent of coloured monomeric anthocyanins
out of the total monomeric anthocyanins
(CMATMA) was calculated by dividing CMA
by TMA.
Winemaking Details. A summary of
the vinification protocols for the Merlot and
Cabernet Sauvignon wines is given in Table 1.
The full-scale commercial micro-oxygenation
treatments and control wines for each variety
were harvested, crushed, pressed, tanked, and
barrelled on the same dates, and were treated
equivalently in all regards with the exception
of exposure to micro-oxygenation.
In brief, Vitis vinifera L. cv. Merlot grapes
were harvested by hand-picking between 2630 September, 2006, were crushed/destemmed
60% whole on 3 October, and fermented for 14
days until pressing of the grapes on 17 October.
The control Merlot wine was then placed in a
stainless steel tank until 8 November (a 22 day
period), at which point it was barrelled. The
treatment Merlot wine was placed in a 4550L
stainless steel tank on 17 October and exposed
to a micro-oxygenation regime at dosing rates
between 24 and 34mL/L/month for a 15 day
period until 1 November, after which the
treatment wine was held in the stainless steel
tank until barrelling on 8 November.
Similarly, Vitis vinifera L. cv. Cabernet
Sauvignon grapes were harvested by handpicking on 26-27 October, 2006, were
immediately crushed/destemmed 60% whole,
and fermented for 12 days until pressing of the
grapes on 8 November. The control Cabernet
Sauvignon wine was then placed in a stainless
steel tank until 4 December (a 26 day period),
at which point it was barrelled. The treatment
Cabernet Sauvignon wine was placed in a


Micro-oxygenation of the two red wine
varieties (Merlot and Cabernet Sauvignon)
resulted in different colour characteristics
compared to the control wines (Figure 1). In
general, the Cabernet Sauvignon wines were
slower to respond to micro-oxygenation than
their Merlot counterparts, requiring between
10 and 30 days longer to show the effects of
After 181 days following the press, the
micro-oxygenated Merlot trials had higher CI,
CMA, and CMATMA, and lower percentage
of yellow relative to the non-micro-oxygenated
control wines. There was no significant
difference in the percentage of red or blue, tint,
brilliance, I280, and TMA between treatment
and control wines after the 181 day aging period.
The results suggested that micro-oxygenation
does not affect the total quantity of phenolics
in wine (as inferred by the I280 value), but
alters the partitioning of these compounds
between more polymerised, stable, or coloured
forms. The 181 day aged micro-oxygenated and
control Merlot wines had lower CI, percentage
of red, brilliance, TCP, WC, CDNRSO2, TMA,
CMA, and CMATMA, and higher percentage
of yellow and blue, tint, CAW, WA, and I280
relative to samples collected immediately after
the press. The micro-oxygenated Merlot wine
had a higher CDRSO2 than the aged control
and the just-pressed samples, and there was
no significant difference in CDRSO2 between
the aged control and just-pressed samples.
Added colour stability is a perceived benefit
of micro-oxygenation, and is supported by the
Merlot trials.
At 159 days following the press, the microoxygenated Cabernet Sauvignon trials had
higher CI, % of yellow and blue, tint, CDRSO2,
CAW, and lower percentage of red, brilliance,
and CMA relative to the control wines.
There was no difference in the WC, TCP,

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MOX (day 181)



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NOX (day 181)

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MOX (day 159)



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between the micro-oxygenated and control
Cabernet Sauvignon wines. The 159 day
aged micro-oxygenated and control Cabernet
Sauvignon wines had lower CI, percentage of
red, brilliance, WC, TMA, CMA, CMATMA,
and higher percentage of yellow and blue,
tint, CDRSO2, and CAW relative to samples
collected immediately after the press. There
was no significant difference in TCP, I280,
or CMATMA between either the microoxygenated or control wines after 159 days of
aging and samples collected immediately after
the press. The micro-oxygenated Cabernet
Sauvignon wine had a lower CDNRSO2 and
TMA, and a higher WA, than the just-pressed
sample, and there was no difference in these
variables between the aged treatments and
Although the Merlot and Cabernet wines
(both micro-oxygenated and controls) had
similar changes in their colour properties
over the six-month aging period, there were
several notable different effects due to microoxygenation between the two varieties. Microoxygenated Merlot wines had a lower percentage
of yellow and no significant difference in
percentage of blue relative to aged controls.
In contrast, the micro-oxygenated Cabernet
Sauvignon had a higher percentage of yellow
and blue, and a lower percentage of red,
relative to the control wines. These differences
led to micro-oxygenation increasing the tint
and decreasing the brilliance of the Cabernet
Sauvignon wines, whereas no difference
was observed for these variables between
treatments and controls in the Merlot wines.
Micro-oxygenation led to an increase in the
CMA in the Merlot wines, and the opposite
influence in the Cabernet Sauvignon. Microoxygenation increased the TCP and CMATMA
relative to the controls for the Merlot wines,
but no significant treatment effects on these
variables were observed for the Cabernet
Sauvignon trials. Significant varietal effects on
all variables were observed, with the exception
of percentage of blue and WA.
A multivariate technique (principal
components analysis, or PCA) was also applied
to the colour dataset on the MOX and control
wines to further distinguish the treatment
effects within and between varieties. PCA of
the colour characteristics explained 84.5%
of the variation in the data in the first two

(a) Merlot


yo u wa

wa t e







7100L stainless steel tank on 8 November
and exposed to a micro-oxygenation regime
at dosing rates between two and 44mL/L/
month for a 14 day period until 22 November,
after which the treatment wine was held in
the stainless steel tank until barrelling on 4



NOX (day 159)









Wavelength (nm)

Fig. 1. Ultraviolet-visible spectra for must immediately after pressing and the micro-oxygenated (MOX)
and non-micro-oxygenated controls (NOX) for the Merlot (a) and Cabernet Sauvignon (b) wines at 181
days and 159 days, respectively.

76 The Australian & New Zealand Grapegrower & Winemaker


October 2007

It stacks up

Table 1. Details on the vinification processes for the Merlot and Cabernet
Sauvignon wines.


Cabernet Sauvignon

Harvest date

Sept. 26-30, 2006

Oct. 26-27, 2006





60% whole

60% whole

Fermentation time

14 days

12 days

Tons processed



Wine volume obtained (L) 5530


Harvest Brix



Finished alcohol (v/v)




Harvest must TA (mg/L) 4.9


Finished wine TA (mg/L)



Harvest must pH



Finished wine pH



Press date

Oct. 17

Nov. 8

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Micro-oxygenation regime
Tank volume (L)



Dosing rates (mL/L/month) 30 (Oct. 17-19; days 0-2) 40 (Nov. 10-12; days 2-4)
24 (Oct. 20-24; days 3-7) 44 (Nov. 13-18; days 5-10)

Barrelling date

34 (Oct. 25-30; days 8-13)

20 (Nov. 19; day 11)

34 (Oct. 31; day 14)

4 (Nov. 20-21; days 12-13)

34 (Nov. 1; day 15)

2 (Nov. 22; day 14)

Nov. 8 (day 22)

Dec. 4 (day 26)

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Titratable acidity

October 2007



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dimensions, with 68.5% and 16.0% explained by factor 1 and factor 2,
respectively (Figure 2). Most of the separation occurred within factor
1 which was positively loaded with the majority of the variables,
and negatively loaded with percentage of yellow and blue, tint, WA,
and CAW. The positive factor 1 variables were split into those which
loaded positively on factor 2 (CI, WC, TCP, CDNRSO2, TMA, CMA,
and I280), and those loaded negatively on factor 2 (percentage of red,
brilliance, and CMATMA). Negative factor 1 variables all displayed
positive factor 2 loadings, and included a percentage of yellow and
blue, tint, WA, and CAW.
Variables with negative factor 1 loadings all describe aged wines
with increasing values of the descriptors. WA and CAW are both
descriptors whose calculated values represent the state of wine aging.
The percentage of yellow and blue are also indicators of wine aging. As
a wine ages, the dominantly red monomeric anthocyanins polymerize
via different mechanisms (described below) that generally lead to
increases in the % of yellow and blue. With the exception of CDRSO2,
all positive factor 1 loadings describe young wines with increasing
values of the descriptors. For example, young wines generally have
higher CI, WC, CDNRSO2, CMA, TMA, brilliance, and % of red,
characteristic of higher levels of the monomeric anthocyanins.
The PCA also separated the wines by variety and treatment, as well
as the course of the aging. Cabernet Sauvignon controls and treatments
grouped together during the early stages of aging (up to 14 days postpress), after which the treatment effects began to discriminate the
samples. The Merlot wines were immediately discriminated following
the press, and grouped separately from the Cabernet Sauvignon
samples. By the later stages of aging (>83 days for the Merlot and >61
days for the Cabernet Sauvignon), both varieties exhibited discrete
groupings for the treatments and controls. Sample positions after this
time did not change much within each group, indicating the wines
were beginning to stabilise.
Few previous studies have investigated the potential impacts
of micro-oxygenation on wine colour, and some of the results are
conflicting. One finding reported a decrease in TMA of micro-


The Australian & New Zealand Grapegrower & Winemaker 77





% of blue



I2 8 0







% of yellow




Component 2 (16.0%)


% of red









Cab. Sauvignon Treatment
(days 61 to 159)

Merlot Treatment
(days 83 to 181)




Cab.Sauvignon Control
(days 61 to 159)




Cab.Sauvignon Treatment & Control
(days 0 to 14)
Merlot Control
(days 83 to 181)

Merlot Treatment & Control
(days 0 to 49)






Component 1 (68.5%)
Fig. 2. Principal components analysis of (a) scores (variables) and (b)
loadings (samples) and for the micro-oxygenated and control Merlot and
Cabernet Sauvignon wines.

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78 The Australian & New Zealand Grapegrower & Winemaker

oxygenated wines relative to control wines (7), in contrast to another
work (8) and our results, where both varieties had no change. Both
increases (7,9,10) and decreases (8) in CI of micro-oxygenated wines
relative to control wines have been observed. We found an increase
in CI due to micro-oxygenation. Two previous works (8,9) found
more stable colour in micro-oxygenated wines, similar to the higher
CDRSO2 values for micro-oxygenated wines reported here. One study
has reported that micro-oxygenation before oak aging produces wines
evolved to yellowish tones (9), while another investigation showed an
increase in percentage of blue of micro-oxygenated wines (10). We
found an increase in the percentage of yellow and blue of our microoxygenated Cabernet Sauvignon wine, but the Merlot wine had a
lower percentage of yellow, and did not have a significantly different
percentage of red or blue, relative to the control.
Methods to characterise polymeric phenols, such as the mean
degree of polymerisation or HCl index have either shown no
differences (7) or an increase in the average molecular weight of
polyphenolic compounds occurring with micro-oxygenation (7,10,11).
Structural studies suggest that micro-oxygenation favors reactions
that lead to a greater formation of pyranoanthocyanins and ethyllinked compounds (8,9), but does not change the overall quantity of
phenolics in the wine. Our results, using the spectral I280 variable as
a proxy for phenolic content, support these previous findings. These
structural changes in the tannin framework are consistent with current
mechanistic understandings of polyphenol oxidation in wine, where
molecular oxygen in the presence of ortho-diphenols (e.g., catechins,
anthocyanins) produces hydrogen peroxide, which can oxidise ethanol
to acetaldehyde (12). The acetaldehyde produced in this manner may
react directly with anthocyanins to form both pyranoanthocyanins and
ethyl-linked products.


October 2007

The distinct responses in the magnitude and direction of wine
colour descriptors among the two varieties exposed to microoxygenation suggests different net chemical processes are occurring
in these wines. No universal approach, as yet, appears to exist for
predicting the effects of micro-oxygenation on red wine colour.
This may be due to different relative contributions of the various
polyphenolics occurring in the wines between varieties (and interannual and geographic variations within a variety based on climate,
soil, and vineyard management practices). The relative importance
and the structure of the end products depend not only on the initial
wine composition, but also on the presence of yeast metabolites, pH,
temperature, and oxygen exposure (13-15).
Anthocyanins are the most significant components responsible
for the purple-red colour of young wines (14). These compounds
are unstable and participate in reactions during fermentation and
maturation to form more complex pigments, which mainly arise from
the interaction between anthocyanins and other phenolic compounds,
especially the flavanols (16).
Micro-oxygenation is intended to promote the more rapid
polymerisation of these pigments via oxidative processes, similar to
aging in oak barrels. Several mechanisms have been confirmed for the
formation of these new pigments:
l Direct reactions between anthocyanins and flavanols, having a
lmax~520nm (515 to 526nm) and a dominant % of red (15,18).
l Reactions involving acetaldehyde, with the formation of
anthocyanin-tannin adducts linked by an ethyl bridge, typically
having a lmax~528-540nm and shifting the wine colour towards
increased percentage of blue (19).
l Formation of pyranoanthocyanins through the reaction between
anthocyanins and other compounds such as vinylphenols,
acetaldehyde, or pyruvic acid, among others, typically having
a lmax~480 to 510nm and shifting the wine colour towards an
increased percentage of yellow (17,20,21).
Collectively, these mechanisms lead to the formation of higher
molecular weight compounds that stabilise wine colour, partly
resisting decolourisation by sulphur dioxide and providing better
colour stability at wine pH. Observations of colour derivatives more
resistant to sulphur dioxide bleaching in the treatments for both
varieties is consistent with micro-oxygenation promoting mechanisms
(1), (2), and (3). Stable percentage of red in the micro-oxygenated
Merlot wines relative to controls suggests that mechanism (1) may be
dominant for this variety. The increase in yellow and blue tonalities
for the micro-oxygenated Cabernet Sauvignon wines relative to
controls suggests that mechanisms (2) and (3) may be most important

in this variety. Cabernet Sauvignon wines generally have higher
anthocyanin and flavonol concentrations compared to Merlot (22).
The ortho-diphenolic groups on anthocyanins and higher polyphenols
are hypothesised to react with molecular oxygen to give hydrogen
peroxide, which then oxidises ethanol to acetaldehyde (12). The
acetaldehyde produced in this manner then reacts with anthocyanins
and other pigments under mechanisms (2) and (3) to yield more stable
ethyl-bridged and pyrano-complexes.
Higher anthocyanin levels in Cabernet Sauvignon wines would
thus be expected to lead to higher acetaldehyde concentrations
following oxygen exposure, favoring mechanisms (2) and (3) as was
observed. Conversely, lower anthocyanin levels in Merlot wines would
result in lower acetaldehyde concentrations, which appears to favour
direct reactions between anthocyanins and flavanols. Compositional
differences in the types of anthocyanins and other polyphenols
between the varieties is also a factor governing the relative magnitudes
of the various pathways. The longer time for Cabernet Sauvignon
wines to show an impact of micro-oxygenation also suggests that the
kinetics for mechanisms (2) and (3) are slower than mechanism (1),
but all these influences are poorly understood at present and warrant
continued research.

Puech, J.L., F. Feulliat, and J.R. Mosedale. 1999. Am J Enol Vitic 50: 469.
Vivas, N., Y. Glories, and P. Raymond. 1997. Rev Fr Oenol 166: 37.
Singleton, V.L. 1995. Am J Enol Vitic 46: 98.
Parish, M., D. Wollan, and R. Paul. 2000. Australian & New Zealand Grapegrower Winemaker Annual
Technical Issue: 47.
Dempsey, C. 2001. Wine Business Online June.
Zoecklein, B.W., R. Carey, and P. Sullivan. 2003. Wine East 31: 28.
Atanasova, V., H. Fulcrand, V. Cheynier, and M. Moutounet. 2002. Anal Chim Acta 458: 15.
del Carmen Llaudy, M., R. Canals, S. Gonzalez-Manzano, J. Miquel Canals, C. Santos-Buelga, and F.
Zamora. 2006. J Agric Food Chem 54: 4246.
Cano-Lopez, M., F. Pardo-Minguez, J.M. Lopez-Roca, and E. Gomez-Plaza. 2006. Am J Enol Vitic
57: 325.
Perez-Magarino, S., M. Sanchez-Iglesias, M. Ortega-Heras, C. Gonzales-Huerta, and M.L. GonzalesSanjose. 2006. Food Chem 101: 881.
Jones, P.R., M.J. Kwiatkowski, G.K. Skouroumounis, I.L. Francis, K.A. Lattey, E.J. Waters, I.S.
Pretorius, and P.B. Hoj. 2004. Wine Ind J 19: 17.
Wildenradt, H.L., and V.L. Singleton. 1974. Am J Enol Vitic 25: 127.
Gao, L., B. Girard, G. Mazza, and A.G. Reynolds. 1997. J Agric Food Chem 45: 2003.
Mazza, G. 1995. Crit Rev Food Sci Nutr 35: 341.
Fulcrand, H., P.J. Cameira dos Santos, P. Sarni-Manchado, V. Cheynier, and J. Favre-Bonvin. 1996. J
Chem Soc, Perkin Trans 1 7: 735.
H. Liao, Y. Cai, and E. Haslam. 1992. J Sci Food Agric 59: 299.
Romero, C., and J. Bakker. 2000. Int J Food Sci Technol 35: 129.
Remy, S., H. Fulcrand, B. Labarbe, V. Cheynier, and M. Moutounet. 2000. J Sci Food Agric 80: 745.
Saucier, C., D. Little, and Y. Glories. 1997. Am J Enol Vitic 48: 370.
Fulcrand, H., P. Cameira dos Santos, P. Sarni Manchado, V. Cheynier, and J. Favre Bonvin. 1996. J
Chem Soc, Perkin Trans 1 7: 735.
Romero, C., and J. Bakker. 2000. J Sci Food Agric 81: 252.
Mazza, G., L. Fukumoto, P. Delaquis, B. Girard, and B. Ewert. 1999. J Agric Food Chem 47: 4009. ■

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• Increased yields of premium wine
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October 2007

Tel: 03 9463 1999


The Australian & New Zealand Grapegrower & Winemaker 79

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