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Effect of extended maceration and ethanol concentration on the extraction

and evolution of phenolics, colour components and sensory attributes of
Merlot wines

Article  in  Australian Journal of Grape and Wine Research · December 2012

DOI: 10.1111/ajgw.12009

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Casassa et al. Extended maceration and ethanol concentration 25

Effect of extended maceration and ethanol concentration on

the extraction and evolution of phenolics, colour components
and sensory attributes of Merlot wines
L. FEDERICO CASASSA, C.W. BEAVER, M.S. MIRELES and J.F. HARBERTSON
School of Food Science. Irrigated Agricultural Research and Extension Center, Washington State University (WSU),
Prosser, WA 99350, USA
Correspondence author: Dr James F. Harbertson, email jfharbertson@wsu.edu

Abstract
Background and Aims: Anecdotal evidence suggests that there may be a synergistic effect of ethanol (EtOH) and
prolonged skin contact on the extraction of certain phenolics that may negatively impact wine sensory properties.
The combined effect of extended maceration (EM) and EtOH concentration was studied during winemaking and
bottle ageing.
Methods and Results: The evolution of phenolics and colour components was followed for up to 1 year of bottle
ageing. Harvest skins and seeds and those obtained from the pomace after maceration were also analysed. Sensory
attributes were studied by Quantitative Flavour Profiling. The relationship between the chemical and sensory data
was explored with Partial Least Square Regression. EtOH concentration differing by 1.2% v/v had no effect on tannin
and anthocyanin extraction, colour, tannin mean degree of polymerisation, polymeric pigment formation and
recovery of anthocyanins and tannins in the pomace after maceration. The maceration length defined the chemical
and sensory profile of the wines. The tannin content of wines produced with EM was mainly derived from seed
tannins, whereas control wines had a balanced proportion of seed and skin tannins. The anthocyanin concentration
was lower in EM wines, whereas polymeric pigments and tannins were predictors of astringency. In control wines,
perceived red colour was associated with anthocyanins, vitisins, a* (red component), and small polymeric pigments.
Significance of the Study: Evidence of the nature and interrelation of the chemical and sensory composition of
wines obtained with EM is provided. Major chemical features responsible for the sensory properties of wines
produced by two contrasting skin contact regimes are identified.

Keywords: colour, ethanol, extended maceration, phenolic, sensory analysis

Introduction reported to decrease with ripening (del Llaudy et al. 2008,

Extended maceration (EM) prolongs skin and seed contact after
the must has fermented to dryness (Sacchi et al. 2005). This
practice has gathered attention because of its potential to
enhance phenolic extraction (Auw et al. 1996, Canals et al.
2005), stabilise wine colour (Auw et al. 1996, Puertas et al.
2008) and alter mouthfeel properties (Schmidt and Noble 1983,
Joscelyne 2009). Because of the anecdotal belief that full-bodied
wines can be obtained only from fruit that undergoes extended
ripening, there is a concern about the synergistic effect of
ethanol (EtOH), resulting from high °Brix fruit, and prolonged
skin contact on the extraction of certain phenolics that may
impart negative sensory properties. Therefore, it is of interest to
document the chemical and sensory characteristics derived from
the interaction between EM and EtOH concentration during
winemaking.
Fruit maturity plays a critical role in anthocyanin and tannin
extraction, polymeric pigments’ formation and modulation of
astringency (Canals et al. 2005, del Llaudy et al. 2008, Harbert-
son et al. 2009). Anthocyanin extractability into wine progres-
sively increased from ~14°Brix to 26.7°Brix (Canals et al. 2005,
Fournand et al. 2006, del Llaudy et al. 2008). Conversely,
tannin extraction into wine, particularly from seeds, has been
Bautista-Ortín et al. 2012). Elevated EtOH concentration,
however, resulting from high °Brix fruit may increase the
intrinsically lower extractability of seed tannins, reportedly
because of the dissolutive effect of EtOH on the lipidic outer coat
of the seeds (Glories and Saucier 2000). In turn, enhanced
tannin extraction from seeds during maceration increased per-
ceived astringency of wine (Harbertson et al. 2009).
Seed tannins are compartmentalised in thin-walled paren-
chyma cells located between the cuticle and the inner lignified
layers of the seeds (Thorngate and Singleton 1994, Adams
2006). Seed tannins are composed by catechin, epicatechin and
epicatechin-3-O-gallate (Prieur et al. 1994), which are present
mainly as monomers, but oligomers and polymers also exist.
Moreover, the mean degree of polymerisation (mDP) of seed
tannins is lower than that of skin tannins (Prieur et al. 1994,
Souquet et al. 1996). Generally, longer maceration times
increase the contribution of seed tannins (Vrhovsek et al. 2002,
del Llaudy et al. 2008). In model wines, seed tannins contrib-
uted 90% of the wine tannin content after 3 weeks of macera-
tion (González-Manzano et al. 2004) and similar results were
found on an industrial scale (Harbertson et al. 2009). Conflict-
ing results however, have been reported for the effect of variable

doi: 10.1111/ajgw.12009
© 2013 Australian Society of Viticulture and Oenology Inc.
26 Extended maceration and ethanol concentration
EtOH concentration on seed extraction. In model wines, Single-
ton and Draper (1964) found that 14% EtOH extended the lag
phase of tannin extraction compared with that of 11% EtOH,
which was attributed to a tissue toughening effect by EtOH. Yet,
at longer contact times, higher EtOH increased extraction (Sin-
gleton and Draper 1964). In another report, an EtOH content
ranging from 0 to 10% v/v had a marginal effect on the extrac-
tion of catechin, epicatechin and proanthocyanidins dimers B1,
B2 and B4 over a 96-h extraction period (Oszmianski et al.
1986) although no further analysis was done beyond this point.
Skin proanthocyanidins are located in the vacuoles of thick-
walled hypodermal cells (Adams 2006), with those associated
with cell wall polysaccharides having a higher degree of polym-
erisation (Gagné et al. 2006). Extraction of monomeric and
oligomeric tannins from skins occurs early during maceration
(Koyama et al. 2007). Relative to seeds, however, the extraction
pattern of skin tannins of high molecular weight follow an
erratic behaviour because of the subsequent non-covalent
rebinding by cell wall material (Bindon et al. 2010, Hanlin et al.
2010). As in seeds, the effect of EtOH concentration on tannin
extraction from skins has proven to be inconsistent. In model
wines, increasing the EtOH concentration from 0 to 13% v/v
increased extraction of skin tannins in unripe fruit (Canals et al.
2005), but no differences were found when comparing extrac-
tion solutions at 11 and 13% v/v EtOH applied to fruit at
technological maturity (Gambuti et al. 2009).
Although the loss of anthocyanins during EM is well docu-
mented (Scudamore-Smith et al. 1990, Kelebek et al. 2006), the
effect of variable EtOH concentration on anthocyanin extraction
remains unclear. In addition to their direct role on wine colour,
the presence of anthocyanins during maceration increases the
solubility and retention of tannins via the formation of poly-
meric pigments (Kantz and Singleton 1991, Singleton and
Trousdale 1992). Polymeric pigments encompass a heterogene-
ous array of compounds, including pigmented tannins and
acetaldehyde cross-linked products (Somers 1971, Kennedy and
Hayasaka 2004). These pigments differ from intact anthocyanins
in that they are partially resistant to bisulfite bleaching and
more resilient to pH change (Somers and Evans 1977). There
appears to be, however, a complex relationship between tannin
content, anthocyanin extraction (or loss) and polymeric
pigment formation during maceration.
Quantitative Flavour Profiling (QFP) is a sensory technique
where trained panellists evaluate a list of descriptive terms pre-
viously selected by a group of experienced tasters (Stampanoni
1993). The QFP approach has the advantage over Quantitative
Descriptive Analysis in that the panellists do not have any
preconceived ideas because they are not involved in the devel-
opment of the descriptors (Stampanoni 1994).
Here we report the combined effect of EM and EtOH con-
centration on the extraction and evolution of anthocyanins,
tannins and colour components during and post-maceration,
and up to 1 year of bottle ageing. Sensory properties were
studied with QFP.
Materials and methods
Winemaking
Merlot grapes (clone 3) were harvested on 23 September 2010
from Goose Ridge vineyard in Paterson, WA, USA, which is
located in the American Viticultural Area-designated Columbia
Valley. Two adjacent vineyard blocks differing by ~1°Brix, to
target two levels of final EtOH concentration, were harvested
separately into four 500-kg capacity bins. Two skin contact
treatments were established in triplicate for each block: control
Australian Journal of Grape and Wine Research 19, 25–39, 2013
wines, with a 10-day skin contact period, and extended mac-
eration wines (EM) with a 30-day skin contact period, affording
12 wines. The fruit was crushed and destemmed using a
Mearelli crusher (Cinquemiglia, Città di Castello, Italy);
50 mg/L of sulfur dioxide (SO2) was added. The must was fer-
mented in 300 L stainless steel jacketed fermentors with mobile
lids (Ghidi Metalli, Buggianao, Italy) filled with ~120 kg of
must. Musts were inoculated 4 h after crushing with dry yeast
(Lalvin EC-1118, Lallemand, Montreal, Canada) at a rate of
250 mg/L. Malolactic bacteria (Lalvin VP41, Lallemand) were
added 48 h after yeast inoculation at a rate of 10 mg/L. Diam-
monium phosphate was added to raise the yeast assimilable
nitrogen to 225 mg/L prior to fermentation. Sugar consumption
during fermentation was monitored with a handheld densito-
meter (DMA 35N, Anton Paar, Graz, Austria) and tank tempera-
ture was maintained at 26 ⫾ 2°C using a web-based
fermentation system (TankNet, Acrolon Technologies, Sonoma,
CA, USA). Reducing sugar was measured by the Rebelein
method (Iland et al. 2004). Cap management consisted of a
whole-volume tank pump-over followed by a 2-min punch
down twice a day during fermentation. Alcoholic fermentation
was completed (reducing sugars <2 g/L) after 9 days. During
post-fermentation, EM wines received one 2-min punch down
per day, after which the tanks were sealed and purged under lid
with N2 (30 L/min ¥ 3 min). After completion of the skin
contact time, free run wines were transferred to 19 L glass
carboys fitted with airlocks and stored at 22 ⫾ 2°C. Malolactic
fermentation was monitored by enzymatic analysis of L-malic
acid (Unitech Scientific, Hawaiian Gardens, CA, USA); after
completion of malolactic fermentation (MLF), the wines were
racked, cold-stabilised (90 days at 2 ⫾ 2°C) and adjusted to
35 mg/L free SO2. The wines were bottled in February 2011 in
750 mL bottles sealed with screw caps, leaving a 16 mL head-
space using a capper machine (Technovin TVLV, Saxon, Swit-
zerland) and stored at 10 ⫾ 2°C.
Chemical analysis reagents
Reagents for spectrophotometric analyses are reported in Har-
bertson et al. (2009). Phloroglucinol, L-ascorbic acid, L-tartaric
acid (99%), L-malic acid (97%) and L-lactic acid (85%,
13 mol/L) were purchased from Sigma-Aldrich (St. Louis, MO,
USA). Malvidin-3-O-glucoside was purchased from Polyphenols
Laboratories (Sandnes, Norway). Acetic acid (99.7%, 17 mol/L)
and high-performance liquid chromatography (HPLC)-grade
solvents were obtained from Merck (Darmstadt, Germany).
Fruit and wine basic analysis
For fruit analysis, three 15-cluster replicates were randomly
selected from each vineyard block. For each replicate, berries
were separated from the clusters and placed onto a table where
two sets of 30 berries were selected at random. The juice was
extracted using an IKA™ A11 analytical mill (Fisher Scientific,
Waltham, MA, USA) and the pulp solids and liquid were trans-
ferred to 50-mL tubes and centrifuged (5000 g ¥ 6 min at 5°C),
and the supernatant was analysed as follows. Total soluble solids
(°Brix) were determined by a temperature-compensating
refractometer (Atago RX-5000, Tokyo, Japan). Titratable acidity
(pH 8.2 end-point) was measured with an automatic titrator
(Mettler-Toledo, Columbus, OH, USA), and pH was obtained
using a digital pH meter (Fisher Scientific). Phenolics in the fruit
were analysed as described (Harbertson et al. 2002, 2003), with
results expressed on a fresh weight (FW) basis. EtOH concen-
tration was measured with a digital infrared spectrophotometer
© 2013 Australian Society of Viticulture and Oenology Inc.
Casassa et al.
(Anton Paar). Free and total SO2 were measured by the aspira-
tion method (Iland et al. 2004) throughout the study.
Spectrophotometric analysis
Must and wine samples were treated with 1 mmol/L sodium
azide to inhibit microbial activity, centrifuged (5000 g ¥ 5 min)
and filtered through 0.22 mm filters (Fisher Scientific, Westboro,
MA, USA) prior to analysis. Spectrophotometric measurements
were carried out with an Agilent 8453 ultraviolet (UV)-visible
spectrophotometer (Agilent Technologies, Santa Clara, CA,
USA). Anthocyanins, non-tannin phenolics, small polymeric
pigments (SPPs) and large polymeric pigments (LPPs) were
measured as detailed (Harbertson et al. 2003). Tannins in the
fruit, pomace and wines were analysed by protein precipitation
(Harbertson et al. 2002). A previously described protocol (Har-
bertson et al. 2009) was used to measure tannins and anthocy-
anins recovered in the skins and seeds collected from the
pomace of each replicate.
The Cie-Lab coordinates L* (lightness), C* (saturation),
H* (hue angle), a* (green/red component) and b* (blue/yellow
component) were calculated as reported in Casassa et al.
(2012).
Analysis of wines with HPLC-diode-array detection
(DAD)-electrospray ionisation-mass spectrometry (ESI-MS)
An Agilent 1100 series HPLC-DAD system coupled with a 6410
Triple Quadrupole ESI-MS instrument (Agilent Technologies)
were used for the chromatographic separations and confirma-
tion of peak identity, respectively. The MassHunter software
version B.04.00 (Agilent Technologies) was used for MS data
analysis.
Organic acids
Malic, lactic and acetic acids were measured by reversed-phase
HPLC (Pereira et al. 2010).
Anthocyanins
An HPLC method for separation of anthocyanins (Downey and
Rochfort 2008) was applied to grapes, wines and pomace
samples. Filtered samples were transferred to brown vials and
the anthocyanins were separated on a Unison UK C-18 column
(150 ¥ 4.6 mm, 3-mm particle size (Imtakt USA, Philadelphia,
PA, USA)) thermostated at 40°C and protected by a guard
column of the same packing material. Anthocyanins were iden-
tified by ESI-MS and quantified as equivalents of malvidin-3-
O-glucoside. Mass scans were performed from m/z 100 up to m/z
1000 in the positive ionisation mode.
Phloroglucinolysis
Grapes, wines and pomace samples were subjected to phloro-
glucinolysis as described (Kennedy and Jones 2001) with
minor modifications. Proanthocyanidin terminal and extension
units were separated on an Atlantis C18 column (250 ¥
4.6 mm, 5-mm particle size (Waters, Mildford, MA, USA)) pro-
tected by a guard column of the same material. The com-
pounds were identified according to relative retention times
and molecular ions (M–H)-, which were m/z 289.0 for catechin
(C) and epicatechin (EP); m/z 441.0 for epicatechin-3-O-gallate
(ECG); m/z 412.9 for C and EP-phloroglucinol adducts; m/z
428.9 for epigallocatechin-phloroglucinol adducts and m/z
565.0 for ECG-phloroglucinol adducts. Mass scans were run
© 2013 Australian Society of Viticulture and Oenology Inc.
Extended maceration and ethanol concentration 27
from m/z 100 to 1000 in the negative ionisation mode. The
mDP was calculated as reported (Kennedy and Jones 2001).
Polymeric pigments and vitisins
Polymeric pigments, vitisin A and vitisin B were analysed as
described by Peng et al. (2002) using a polystyrene divinyl-
benzene column (250 ¥ 4.6 mm, 5-mm particle size (Agilent
Technologies)) thermostated at 40°C and protected by a
PLRP-S guard column of the same packing material. The com-
pounds were identified by their UV spectrum and retention
time and were quantified as mg/L of malvidin-3-O-glucoside
equivalents.
Sampling protocol
Beginning at day 4 after crush, anthocyanins were determined
by spectrophotometry and by HPLC every 2 days during skin
contact (with samples taken immediately after the first pump-
over), at pressing (day 10 and 30) and at day 50, 100, 300 and
540 (1 year of bottle ageing) from pressing. Polymeric pig-
ments, vitisins and phloroglucinol adducts in wines were ana-
lysed at day 4, 30, 50, 100, 300 and 540. Values are reported
as an average (n = 3) followed by the standard error of the
mean.
Experimental design
The experiment was a full factorial design with two factors (skin
contact time and EtOH concentration) and two levels of each
factor (skin contact time: 10 and 30 days; EtOH concentration:
12 and 13% v/v). The harvest fruit comparison was carried out
by a two-sample Student’s t-test for independent samples
(degrees of freedom (d.f.) = 10, P < 0.05). A fixed-effect two-
way analysis of variance (ANOVA) (d.f. = 11) with a 5% level
for rejection of the null hypothesis was used to analyse the
effects of skin contact, EtOH concentration and their interaction
for all the chemical parameters measured. Fisher’s LSD test was
used as a post-hoc comparison of means. Data were analysed
with XLSTAT v. 2009 (Addinsoft, Paris, France). Regression
analysis, significance and correlation coefficients were calcu-
lated using the SigmaPlot (v. 10.0) package (Systat Inc.,
Chicago, IL, USA).
Sensory evaluation
Materials. Raspberry and cherry jams (Safeway Select,
Grandview, WA, USA), and 100° proof vodka (Smirnoff,
Diageo, London, UK) were obtained from local stores, and 6-n-
propylthiouracil (PROP) was obtained from Sigma-Aldrich.
Reference standards for aroma and colour were prepared in a
base wine (2008 Merlot) previously stripped of aroma com-
pounds under reduced pressure (30°C ¥ 45 min) using a Büchi
Syncore Polyvap (Flawil, Switzerland) to a final EtOH content
of 10.3% v/v (Table 1). For the ‘oxidised fruit’ standard, the
jams were cooked (80°C ¥ 10 min) and dissolved in base wine.
For colour standards, wines were obtained by varying the pH
and/or by addition of acetaldehyde (Sigma-Aldrich), hydrogen
peroxide (H2O2) and sulfur dioxide (SO2) (J.T. Baker, Phil-
lipsburg, NJ, USA) with specifications reported in Table 1 as
Cie-Lab coordinates. Unsalted crackers and deionised filtered
water (Easy Pure II, Thermo Scientific, Dubuque, IA, USA)
were provided for palate cleansing. Aroma attributes and
astringency were evaluated in tulip-shaped cobalt black glasses
(Libbey, Toledo, OH, USA) to avoid perceptual bias because of
colour, (Ross et al. 2008). Clear ISO wine glasses (ISO
3591:1977) were used only for colour evaluation.
28 Extended maceration and ethanol concentration
Table 1. Ingredients, specifications and lexicon of sensory analysis standards.
Reference
standard
Low
Aroma, flavour
Red fruit character 5 g each cherry and raspberry jam
Oxidised fruit 3 g each cooked cherry and raspberry
jam‡
Alcohol/hot Stripped wine§, EtOH: 10.3% v/v
Colour
Purple component NA
Red component NA
Brown component NA
Saturation C* = 49.53
Mouthfeel
Astringency PPT = 358 mg/L
†All standards were prepared at three levels and dissolved in 750 mL of base wine. ‡Jams (50 g) were cooked (80°C ¥ 10 min) and then dissolved in base wine. §Base
wine: 2008 Merlot stripped of aroma compounds under reduced pressure (30°C ¥ 45 min) to a final ethanol content of 10.3% v/v. a*, red component; b*, yellow
component; C*, saturation or chroma; EtOH, ethanol; H*, hue; L*,: lightness; NA, not applicable; PPT, protein precipitable tannins.
QFP
The wines were subjected to sensory analysis approximately 9
months after crushing. All individuals involved in the study
were previously screened for bitterness sensitivity to PROP
(Tepper et al. 2002) and for visual disorders using Ishihara
images (Legrand et al. 1945). None of the panellists had colour
deficiencies and 31% of the panellists were insensitive to PROP
(Pickering et al. 2004).
An assessment of the wines by three experienced wine
tasters (two super-tasters and one medium-taster) revealed the
absence of off-flavours and green aromas. This assessment also
served to select relevant sensory attributes that were deemed to
differ in intensity among the wines. Three aroma attributes
(hot/EtOH, red fruit and oxidised fruit), four colour descriptors
(purple, red, brown component and saturation) and one
mouthfeel attribute (astringency) were retained by consensus.
A trained panel (n = 15, eight males and seven females, ages
ranging from 23 to 62 years old) was recruited from the WSU
Prosser community and the professional staff of two wineries.
Panellists received minimal information about the nature of the
study and signed an informed consent form approved by the
WSU Review Board for human subject participation.
Panellists were trained over seven training sessions. Astrin-
gency was defined as a summation of two sensations, surface
texture and drying (Gawel et al. 2000). During the training and
evaluation sessions, a 15-cm line scale was used, labelled with
terms ‘low’ and ‘high’, at the 1 cm and 14 cm mark from the left
end of the scale, respectively. Except for the purple, red and
brown colour components, the standards were prepared at
‘low’, ‘medium’ and ‘high’ levels (Table 1), representing
anchors located at 1 cm, 7.5 cm and 14 cm from the left end in
Australian Journal of Grape and Wine Research 19, 25–39, 2013
Levels†
Medium High
15 g each cherry and raspberry jam 37.5 g each cherry and raspberry jam
7 g each cooked cherry and raspberry 15 g each cooked cherry and
jam raspberry jam
75 mL 100° proof vodka, EtOH: 125 mL 100° proof vodka, EtOH:
13.9% v/v 16.3% v/v
NA Cie-Lab parameters: L* = 29.8;
C* = 76.74; H* = 36.29; a* = 61.86;
b* = 45.43
NA Cie-Lab parameters: L* = 38.4;
C* = 56.59; H* = 17.95; a* = 53.84;
b* = 17.45
NA Cie-Lab parameters: L* = 45.3;
C* = 49.04; H* = 26.39; a* = 43.93;
b* = 21.8
C* = 64.48 C* = 83.77
PPT = 621 mg/L PPT = 1561 mg/L
the 15-cm line scale. Standards were available to panellists until
the beginning of the formal sessions.
The three replicates of each treatment were assessed during
four evaluation sessions in individual booths (20 ⫾ 2°C) lighted
with Lumichrome full spectrum lamps (6500 K) (Lumiram Elec-
tric Corporation, Larchmont, NY, USA). Aliquots (25 mL) of
wine at room temperature were poured into wine glasses coded
with three-digit random numbers and covered with aluminum
lids to trap volatiles. Wines were presented under a randomised
balanced block design. Results were collected in ballots with
responses decoded in cm. Individual performances were
assessed by checking the correlation of each panellist ratings
with the panel mean and by their contribution to the
panellist ¥ wine interaction for each attribute (Lattey et al.
2010). Based on these analyses, it was decided to remove data
from one panellist (final n = 14).
Data analysis and Partial Least Square Regression (PLSR)
The trained panel data were analysed by a three-way mixed-
effects ANOVA with replication considering panellist as a
random effect and treatment and replicate as fixed effects, with
their interactions. Separation of means was accomplished using
Fisher’s LSD with significance established as P ⱕ 0.05. The
XLSTAT v. 2009 package (Addinsoft) was used for ANOVA and
mean separation. The relationship between the chemical data
(as the X matrix, or predictors) and the sensory data (as the Y
matrix, or responses) at the two EtOH concentrations was
explored with PLSR. Only significant chemical parameters and
sensory attributes (P < 0.05) were included in the model. The
analysis was performed with X and Y weights normalised and
© 2013 Australian Society of Viticulture and Oenology Inc.
Casassa et al. Extended maceration and ethanol concentration 29

Table 2. Total soluble solids, titratable acidity and pH of the fruit from the two Merlot vineyard
blocks at harvest. Average values followed by SEM (n = 6).

Vineyard block Total soluble solids Titratable acidity pH

(°Brix) (g/L tartaric acid)

12% EtOH† fruit 23.4 ⫾ 0.4 7.2 ⫾ 0.01 3.59 ⫾ 0.04

13% EtOH fruit 24.3 ⫾ 0.6 7.1 ⫾ 0.01 3.65 ⫾ 0.04

Two-tailed Student’s t-test for independent samples to compare data. †EtOH: approximate potential ethanol content (% v/v) of the fruit
of each vineyard block. EtOH, ethanol; SEM, standard error of the mean.

Table 3. Berry weight and phenolic composition of the fruit from the two Merlot vineyard blocks at harvest. Average
values followed by SEM (n = 6).

Vineyard block Berry weight Skin anthocyanins‡ Skin tannins§ Skins mDP‡ Seed tannins§ Seeds mDP‡
(g) (mg/g FW) (mg/g FW) (mg/g FW)

12% EtOH† fruit 1.08 ⫾ 0.03a 0.57 ⫾ 0.02b 0.35 ⫾ 0.01b 14.82 ⫾ 0.32 3.26 ⫾ 0.16b 5.31 ⫾ 0.39
13% EtOH fruit 0.97 ⫾ 0.02b 0.73 ⫾ 0.02a 0.44 ⫾ 0.03a 15.45 ⫾ 0.52 3.97 ⫾ 0.11a 5.05 ⫾ 0.17

Two-tailed Student’s t-test for independent samples to compare data: different letters within a column indicate significant differences at P < 0.05. †EtOH: approximate
potential ethanol content (% v/v) of each vineyard block at harvest. ‡Determined by high-performance liquid chromatography-diode-array detection-electrospray
ionisation-mass spectrometry. §Determined by the Adams-Harbertson Assay (Harbertson et al. 2003). EtOH, ethanol; FW, fresh weight; mDP, mean degree of
polymerisation; SEM, standard error of the mean.

Table 4. Basic chemical analyses of the finished Merlot red wines at bottling. Average values followed by SEM (n = 3).

EtOH (%)† Skin Ethanol pH Titratable acidity Malic acid Lactic acid Acetic acid Free SO2
contact (% v/v) (g/L tartaric acid) (g/L) (g/L) (g/L) (mg/L)

12 Control 11.77 ⫾ 0.08b 3.76 ⫾ 0.03b 5.5 ⫾ 0.01 0.10 ⫾ 0.03 1.53 ⫾ 0.02a 0.49 ⫾ 0.06b 31 ⫾ 1
EM 12.11 ⫾ 0.16b 3.82 ⫾ 0.01a 5.4 ⫾ 0.01 0.13 ⫾ 0.01 1.33 ⫾ 0.06b 0.72 ⫾ 0.03a 28 ⫾ 1
13 Control 13.29 ⫾ 0.14a 3.78 ⫾ 0.01ab 5.4 ⫾ 0.01 0.10 ⫾ 0.02 1.47 ⫾ 0.03a 0.41 ⫾ 0.02b 30 ⫾ 2
EM 13.09 ⫾ 0.15a 3.79 ⫾ 0.01ab 5.6 ⫾ 0.02 0.13 ⫾ 0.01 1.26 ⫾ 0.01b 0.74 ⫾ 0.01a 29 ⫾ 1
Significance (P-values)
EtOH <0.0001 0.523 0.340 0.923 0.095 0.343 0.875
Skin contact 0.879 0.080 0.829 0.059 0.846 <0.0001 0.248
EtOH ¥ skin contact 0.053 0.191 0.371 0.763 <0.0001 0.135 0.182

Analysis of variance to compare data: different letters within a column indicate significant differences for Fisher’s LSD at P < 0.05. Control: 10-day skin contact;
extended maceration (EM): 30-day skin contact. †Approximate final ethanol content (% v/v) of each set of wines. EtOH, ethanol; SEM, standard error of the mean.

full cross-validation using the NIPALS algorithm (Unscrambler
version 10.1, Camo Software, Oslo, Norway).
Results and discussion
Fruit chemistry and basic wine composition
The present study evaluated the impact of the length of macera-
tion (10 and 30 days) and EtOH concentration (12 and 13%
v/v) on the extraction and evolution of colour and phenolics.
We selected two blocks of a Merlot single vineyard differing in
initial °Brix and therefore potential EtOH concentration. No
difference in the basic fruit chemistry, however, was found
between the 12% EtOH and the 13% EtOH fruit (Table 2).
Likewise, the mDP of skin and seeds of both vineyard blocks
were found at a comparable level (Table 3). Anthocyanins, skin
and seed tannins on an FW basis showed a 28, 25 and 18%
increase, respectively, in the 13% EtOH fruit over that of the
12% EtOH fruit, although these differences did not translate
into the wines, as it will be further commented.
Wines were analysed for basic composition after bottling,
which occurred 150 days after crush (Table 4). The difference in
the EtOH content of the wines of both vineyard blocks averaged
1.2% (v/v). While post-fermentation oxygen exposure during
EM was minimised by N2 purging under lids, EM wines showed
a 47% increase (12% EtOH wines) and an 80% increase (13%
EtOH wines) in the content of acetic acid. The latter can be
attributed to the oxidative metabolism of Acetobacter pasteurianus
and Acetobacter aceti, as reported in wines undergoing EM (Drys-
dale and Fleet 1988).
Phenolic extraction and colour evolution during winemaking
and ageing
Protein precipitable tannins were followed at 2-day intervals
during maceration (Figure 1) to gain insight into the kinetics of
extraction of these compounds as affected by maceration length.
Tannins peaked at the end of the maceration period of each
treatment. For EM wines, tannin extraction reached a plateau,

© 2013 Australian Society of Viticulture and Oenology Inc.

30 Extended maceration and ethanol concentration Australian Journal of Grape and Wine Research 19, 25–39, 2013

Figure 1. Extraction of protein precipitable tannins during fermentation, post-fermentation and bottle ageing (n = 3) of Merlot wines having
an approximate final ethanol content of (a) 12% and (b) 13%. ( ) Control: 10-day skin contact; ( ) extended maceration (EM): 30-day skin
contact. Black and grey arrows indicate pressing time in control and EM wines, respectively. CE, catechin equivalents. Different letters indicate
significant differences for Fisher’s LSD at P < 0.05. If not shown, error bars are obscured by treatment symbol.

Table 5. Tannin content and mean degree of polymerisation (mDP) observed in skins and seeds in the pomace, and
estimated proportion of skin and seed tannin extracted into Merlot wine. Averages values followed by SEM (n = 3).

Treatment Pomace Estimated extracted

proportion (%)

EtOH (%)† Skin Skin tannins‡ Skins mDP§ Seed tannins‡ Seeds mDP§ Skin tannin Seed tannin
contact (mg/g FW) (mg/g FW)

12 Control 0.14 ⫾ 0.04ab 5.42 ⫾ 0.14 2.96 ⫾ 0.08b 6.87 ⫾ 0.44 48a 52b
EM 0.11 ⫾ 0.01b 5.25 ⫾ 0.35 2.30 ⫾ 0.18c 7.23 ⫾ 0.26 22b 73a
13 Control 0.21 ⫾ 0.00a 5.16 ⫾ 0.31 3.62 ⫾ 0.25a 7.51 ⫾ 0.62 42a 58b
EM 0.14 ⫾ 0.01ab 5.16 ⫾ 0.21 2.66 ⫾ 0.16b 7.85 ⫾ 0.22 19b 80a
Significance (P-values)
EtOH 0.124 0.401 0.021 0.215 0.502 0.502
Skin contact 0.091 0.095 0.002 0.741 0.004 0.004
EtOH ¥ skin contact 0.403 0.103 0.429 0.105 0.754 0.754

Analysis of variance to compare data: different letters within a column indicate significant differences for Fisher’s LSD at P < 0.05. Control: 10-day skin contact;
extended maceration (EM): 30-day skin contact. †Approximate final ethanol content (% v/v) of each set of wines. ‡Determined by the Adams-Harbertson Assay
(Harbertson et al. 2003). §Determined by high-performance liquid chromatography-diode-array detection-electrospray ionisation-mass spectrometry. EtOH, ethanol;
SEM, standard error of the mean.

which occurred between day 10 and 22, and was followed by
an almost linear increase up to the peak of extraction. This
linear increase is consistent with the extraction of seed tannins
(Hernández-Jiménez et al. 2012). It has also been suggested that
post-fermentation extraction of tannins could be the result of a
desorption mechanism mediated by EtOH, which leads to the
disruption of the non-covalent interactions of the previously
extracted tannins that were bound to cell wall material (Hanlin
et al. 2010). Whether this reported linear increase is the result
of one or a combination of both mechanisms remains to be
clarified. By the end of the study (day 540), overall tannin
extraction was enhanced by 80% (12% EtOH) and by 50%
(13% EtOH) in EM wines relative to that of the controls.
In an effort to determine the origin of the tannins extracted
in control and EM wines, tannins in skin and seed samples
recovered after maceration were also analysed by protein pre-
cipitation and phloroglucinolysis, with results summarised in
Table 5. The extracted proportion of skin or seed tannins was
determined by the difference between what was found in either
the skin or seed at harvest and the amount left in the pomace
and then dividing by the total amount of tannin extracted
(Harbertson et al. 2009). Tannins recovered in seed pomace
were on average 22% (12% EtOH) and 37% (13% EtOH) lower
in the EM treatments, implying more tannin extraction from the
seeds. Indeed, tannins of seed origin accounted for 73 and 80%
of the wine tannin content at pressing in the 12% EtOH and
13% EtOH wines, respectively. In control wines, which were
pressed after 10 days of skin contact, the tannin concentration
recovered in the seeds after maceration was consistently higher
relative to that of the EM pomace, implying comparatively less
extraction from the seeds. Accordingly, skin tannins accounted
for 48% (12% EtOH) and 42% (13% EtOH) of the extracted

© 2013 Australian Society of Viticulture and Oenology Inc.

Casassa et al.
Table 6. Mass balance indicating the proportion of anthocyanins and total tannins extracted from Merlot fruit,
recovered in the pomace and unaccounted for.
Treatment Anthocyanin proportion (%)‡
EtOH (%)† Skin contact Extracted Recovered in
pomace
12 Control 45a 30a
EM 25b 6b
13 Control 36a 26a
EM 21b 8b
Significance (P-values)
EtOH 0.420 0.420
Skin contact <0.0001 <0.0001
EtOH ¥ skin contact 0.059 0.059
Analysis of variance to compare data: different letters within a column indicate significant differences for Fisher’s LSD at P < 0.05. Control: 10-day skin contact;
extended maceration (EM): 30-day skin contact. †Approximate final ethanol content (% v/v) of each set of wines. ‡Proportion was calculated on the basis of the
concentration of each phenolic class at the end of the skin contact time for each treatment with data analysed on a mg/g berry fresh weight basis. EtOH, ethanol.
tannins in control wines, indicating a balanced extraction from
both skins and seeds. As reflected by the P-values, it was the
skin contact treatment, but not the EtOH content, which deter-
mined the proportion of extracted seed and skin tannins.
In spite of some differences in the tannin content of seeds
and skins between both vineyard blocks, the difference in EtOH
content of 1.2% (v/v) on average between the two sets of wines
had no effect on tannin extraction (Figure 1). It has been sug-
gested that extraction of seed tannins can occur only after
hydration of the seeds is complete, which often occurs after the
seeds have absorbed 50% of their weight in water (Oszmianski
et al. 1986, Hernández-Jiménez et al. 2012). The leakiness of
the parenchyma cell outside the true seedcoat, however,
appears not to be function of the EtOH content (Adams and
Scholz 2008, Hernández-Jiménez et al. 2012), and in fact, under
our experimental conditions, it seems to be affected more by the
amount of time it takes to reach maximal seed hydration. This
hypothesis is supported by the higher proportion of tannin
extraction from seeds in EM wines, which was exclusively
affected by the skin contact time.
Interestingly, tannins recovered in the skins after macera-
tion had a significantly lower mDP than that found in the
skins of the fruit (Table 3). Conversely, the mDP of the tannins
recovered in the seeds from the pomace was higher than that
previously measured in the seeds at harvest. In the skin
tannins from pomace, the lower mDP resulted from the molar
contribution of epicatechin-3-O-gallate as terminal subunit
(21% on average), which was not previously detected on the
skin samples from the grapes (Supporting Figure S3). Because
epicatechin-3-O-gallate is proportionally higher in seeds but
virtually absent in skins (Prieur et al. 1994, Cortell and
Kennedy 2006), one plausible explanation for this result is a
rebinding of seed tannins onto the skin surface in the pomace,
a phenomenon not previously reported. Alternatively, the
lower mDP in the skins recovered in the pomace after mac-
eration suggests that the extraction of higher molecular weight
tannins from skins effectively took place, although these were
not recovered in the wines, as previously shown (Hanlin et al.
2011). On the other hand, no epigallocatechin was detected
on the seeds recovered from the pomace (Supporting Fig-
ure S4), suggesting that no incorporation of skin tannins on
seed pomace occurred.
© 2013 Australian Society of Viticulture and Oenology Inc.
Extended maceration and ethanol concentration 31
Total tannin (seeds + skins) proportion (%)‡
Unaccounted Extracted Recovered in Unaccounted
for pomace for
25b 11b 86a 3b
69a 19a 67b 15a
38b 11b 86a 3b
71a 17a 64b 19a
0.420 0.401 0.401 0.401
<0.0001 0.003 0.003 0.003
0.059 0.733 0.733 0.733
A mass balance analysis, assuming an average of 1 g per
berry on the grapes, was conducted to estimate the proportion
of both anthocyanins and tannins extracted from the original
fruit as well as the proportion recovered from the pomace
(Table 6). The proportion of tannins extracted was significantly
lower than that of the anthocyanins, with the vast majority of
them recovered in the pomace. This was particularly evident for
control wines, in which 89% of the fruit tannins on average
were recovered in the pomace after maceration.
The evolution of the mDP and the percentage of recoveries
were followed at critical points during winemaking to gain
qualitative and quantitative information on the average
polymer length (Figure 2). Beginning at day 30, EM wines at
both EtOH levels consistently showed an mDP lower than that
of the controls. This is consistent with a higher extracted pro-
portion of seed tannins, which had a comparatively much lower
mDP relative to that of skins (Table 3). From day 300 to day 540,
the mDP increased by 50 and 27% in the controls and by 42 and
24% in EM wines of the 12% EtOH and 13% EtOH treatments,
respectively, but this occurred along with a progressive decrease
of the recovery percentage from day 4 to day 540. The decrease
in the proportion of tannin recovered after phloroglucinolysis
during wine ageing suggests that wine tannins progressively
increase their compositional variability, leading to the adsorp-
tion of some tannin structures during the chromatographic
purification (Kantz and Singleton 1991, Kennedy and Hayasaka
2004) or yielding new structures that are unquantifiable using
the phloroglucinolysis method. The final result is a decrease of
the analysable tannin pool. In this context, the mDP as a sole
parameter of wine proanthocyanidin length should be taken
cautiously. Instead, an account of the distribution of polymer
lengths and proanthocyanidin concentration of each polymer
length in the final wine (Hanlin et al. 2011) could be followed.
The extraction and evolution of different anthocyanin
derivatives grouped as a function of the aglycone moiety are
shown in Figure 3. Generally, anthocyanin extraction into wine
was not affected by the two EtOH concentrations. ANOVA
showed no differences within the controls of both EtOH levels
for malvidin, petunidin, delphinidin and peonidin derivatives.
For EM wines, malvidin derivatives were higher in the 12%
EtOH wines, but the opposite was found in the 13% EtOH wines
for petunidin, delphinidin and peonidin derivatives.
32 Extended maceration and ethanol concentration
Figure 2. The mean degree of polymerisation (mDP, represented in lines) and conversion yield (represented in bars) at selected time points
during the winemaking process (n = 3) of Merlot wines having an approximate final ethanol content of (a) 12% and (b) 13%. ( , black bars)
Control: 10-day skin contact; ( , white bars) extended maceration (EM): 30-day skin contact. If not shown, error bars are obscured by
treatment symbol.
Irrespective of the aglycone, anthocyanins peaked between
day 4 and 6 of maceration. Subsequently, a variable loss contin-
gent upon the skin contact treatment was observed. Relative to
the controls, a dramatic loss of anthocyanins was observed
during the post-fermentation phase in EM wines, as previously
reported (Scudamore-Smith et al. 1990, Harbertson et al. 2009).
Loss of anthocyanin during prolonged skin contact can be attrib-
uted to a variety of factors including ionic adsorption by yeast
cell walls (Vasserot et al. 1997), adsorption onto bitartrate crys-
tals and other solids (Somers and Evans 1977, Cheynier et al.
2006), incorporation into polymeric pigments (Adams et al.
2004), and formation of pyranoanthocyanins and acetaldehyde
cross-linked products (Drinkine et al. 2007, Rentzsch et al.
2007). In the present study, re-adsorption onto grape pomace
can be ruled out, as only a marginal 6–8% of the initial anthocy-
anin content was recovered in the skin pomace of EM wines
(Table 6). Formation of pyranoanthocyanins proved to be a
minor factor, as the concentration of both vitisins A and B
combined ranged from 2.9 mg/L in EM wines to 3.5 mg/L in
control wines at day 540 (data not shown). Therefore, polymeric
pigment formation emerged as especially relevant for this study.
The formation of polymeric pigments during maceration
and ageing is shown in Figure 4a. EM wines increased their
polymeric pigment content by 98% (12% EtOH wines) and by
132% (13% EtOH wines) in the period spanning from day 4 to
pressing (day 30), whereas for control wines, the increase
during this same time period varied between 38 to 40%. Previ-
ously, Harbertson et al. (2009) reported that in EM wines (20
days of skin contact), LPPs increased three-fold relative to that
of a control (7 days of skin contact). In the present study, the
two-fold increase in the polymeric pigment content of EM wines
was consistent with the major registered losses of anthocyanin
derivatives (Figure 3). Because the anthocyanins recovered in
the skin pomace of EM wines were found in lower concentra-
tion relative to that of the controls (Table 6), it is tempting to
hypothesise that these missing anthocyanins could have been
incorporated into polymeric pigments. It appears, however, that
the formation of polymeric pigments alone can only be partially
responsible for the observed anthocyanin loss. Evidence of this
is found in that an increase in the polymeric pigment content of
12 mg/L (12% EtOH) and 14 mg/L (13% EtOH) from day 4 to
Australian Journal of Grape and Wine Research 19, 25–39, 2013
day 30 in EM wines occurred along with a drop of total anthocy-
anins of 202 mg/L (12% EtOH) and 261 mg/L (13% EtOH) in
this same period (data not shown). Control wines, in contrast,
mostly increased their polymeric pigment content in the last
stages of bottle ageing with major synthesis taking place
between days 300 and 540 (Figure 4a). The net result was an
equivalent concentration of polymeric pigments in all wines
after 540 days.
Results were also expressed as a proportion of polymeric
pigmented material to total pigment content (Figure 4b). In
addition to polymeric pigments, total pigment content included
monomeric anthocyanins, vitisin A and vitisin B (Alcalde-Eón
et al. 2006). At day 4, polymeric pigments contributed less than
5% to the total pigment content of the wines. From day 30
onwards, the contribution of polymeric pigments increased pro-
gressively and was determined by the skin contact treatment
(P < 0.0001). By day 540, polymeric pigments contributed 18%
of the total pigment content in control wines compared with 30%
for EM at both EtOH concentrations. It should be noted,
however, that in EM wines, the proportion of the contribution of
polymeric pigments to total pigments was higher not because the
concentration of polymeric pigments was higher but rather
because the actual concentration of anthocyanins was lower. For
control wines, a drastically less pronounced drop of anthocyanins
was observed thus resulting in a lower proportion of the contri-
bution of polymeric pigments to total pigments (Figure 4b).
Because skin contact time was a determining factor on the
tannin, anthocyanin and polymeric pigment content of the fin-
ished wines, the relationship between anthocyanins and SPPs
(i.e. pigments that do not precipitate with the bovine serum
albumin (BSA) protein) and LPPs (i.e. pigments that precipitate
with the BSA protein) was explored by regressing both of these
parameters against the anthocyanin content. Data were ana-
lysed on the basis of the skin contact treatment and over time to
demonstrate the progressive nature of this relationship. No rela-
tionship over time was observed between anthocyanins and
SPPs (control wines: r = 0.18, P = 0.34; EM wines: r = 0.22,
P = 0.22). A negative correlation was found when the anthocy-
anin content was regressed against LPPs, indicating that the
decrease in anthocyanins during winemaking occurred along
with the progressive and preferential formation of LPPs
© 2013 Australian Society of Viticulture and Oenology Inc.
Casassa et al. Extended maceration and ethanol concentration 33

Figure 3. Extraction and evolution of: (a) malvidin derivatives, (b) petunidin derivatives, (c) delphinidin derivatives, (d) peonidin derivatives,
and (e) cyanidin derivatives during fermentation, post-fermentation and bottle ageing (n = 3) of Merlot wines having an approximate final
ethanol content of 12 and 13%. ( ) Control: 10-day skin contact; ( ) extended maceration (EM): 30-day skin contact. Black and grey
arrows indicate pressing time in control and EM wines, respectively. Different letters indicate significant differences for Fisher’s LSD at
P < 0.05. If not shown, error bars are obscured by treatment symbol.

© 2013 Australian Society of Viticulture and Oenology Inc.

34 Extended maceration and ethanol concentration Australian Journal of Grape and Wine Research 19, 25–39, 2013

Figure 4. Evolution of:

(a) polymeric pigments
(mg/L malvidin-3-glucoside
equivalents), and (b) polymeric
pigment as a proportion of the
total pigment content at day 4,
30, 50, 100, 300 and 540
(n = 3) of Merlot wines having
an approximate final ethanol
content of 12 and 13%.
Control: 10-day skin contact;
extended maceration (EM):
30-day skin contact.

Table 7. Means separation for the sensory attributes of Merlot wines assessed by a trained panel (n = 14).

Treatment Wine attributes‡

Colour component Aroma, flavour Mouthfeel

EtOH (%)† Skin contact Purple Red Brown Saturation Red fruit Oxidised fruit Hot/alcohol Astringency

12 Control 8.04ab 7.16a 1.59b 9.07b 4.61c 2.63 5.20b 6.53c

EM 8.59b 5.91b 1.91ab 9.62a 6.71b 2.43 5.96b 8.77b
13 Control 7.41a 7.74a 1.62b 9.38ab 7.90a 2.78 7.44a 8.09b
EM 7.78a 6.95b 2.04a 9.92a 5.85b 3.44 7.94a 10.48a

Analysis of variance to compare data: different letters within a column indicate significant differences for Fisher’s LSD at P < 0.05. Control: 10-day skin contact;
extended maceration (EM): 30-day skin contact. †Approximate final ethanol content (% v/v) of each set of wines. ‡Evaluations were made along a 15-cm line scale.
EtOH, ethanol.

(Figure 5). The coefficients of determination (r2) indicated that
66% of the variation in polymeric pigment formation was
explained by the observed decrease in anthocyanins in EM
wines, whereas for control wines, 56% of this variation was
explained.
The evolution of the Cie-Lab colour parameters during mac-
eration, post-maceration and ageing is shown in Figure 6. Light-
ness (L*) was higher in the 12% EtOH wines but no major
difference was found in the other Cie-Lab parameters between
the wines of both EtOH concentrations. A difference between
both skin contact treatments, however, was observed for L*,
C* (saturation) and a* (red component). EM increased L*
decreased C* and a* relative to that of the controls from day 10
to 50 (Figure 6a,b,d), but after 1 year of bottle ageing, only the
EM wines of the 12% EtOH treatment had a lower value of C*
and a*. The hue angle (H*) and the yellow component (positive

© 2013 Australian Society of Viticulture and Oenology Inc.

Casassa et al.
Figure 5. Three-dimensional scatter plot of the relationship
between anthocyanins and large polymeric pigments (LPPs) as a
function of time of Merlot wines having an approximate final ethanol
content of 12 and 13%. Data points represent wines of the three
replicates at day 30, 50, 100, 300 and 540 after crushing. ( )
Control: 10-day skin contact; ( ) extended maceration (EM):
30-day skin contact. Control and EM wines from both ethanol levels
were pooled together for the analysis. Linear correlations of anthocy-
anins versus LPPs: control wines: r = 0.75, P < 0.0001; EM wines:
r = 0.81, P < 0.0001. A520: absorbance units at 520 nm.
b* value) were higher for EM wines at the 12% EtOH concen-
tration, but no differences were observed in these two param-
eters for the 13% EtOH wines.
The most dynamic changes in anthocyanins and polymeric
pigments were also reflected in the Cie-Lab colour parameters.
For EM wines, a rapid increase in polymeric pigments from day
4 to 30 occurred along with a decrease in a* and C* and an
increase in L*, thus resulting in a significant colour difference
(P < 0.05) relative to that of the control wines at this point (data
not shown). For control wines, polymeric pigment formation
was favoured from day 300 to 540, which consequently
increased L* and decreased C* (Figure 6a,b, respectively).
Sensory analysis by QFP
Wines were analysed for selected sensory attributes (Table 1) by
a trained panel using the QFP approach. For the 13% EtOH
wines, EM wines were perceived to have a more pronounced
brown colour component (Table 7), which was also reflected in
the higher H* value (Figure 6c), in the positive b* value (i.e.
yellow tones, Figure 6e) and in the polymeric pigments content
at day 300 (Figure 4a) of these wines. Yokotsuka et al. (2000)
attribute a decrease in red colour and an increase in the brown
component in EM wines to the oxidative polymerisation of
anthocyanins with other phenols to produce polymeric pig-
ments. As evidenced also by the acetic acid content, the oxida-
tive process on EM wines was clearly reflected in the colour
measurements and also in the perceived colour.
EM has been reported to enhance bitterness because of the
contribution of low molecular weight tannins from seeds (Yoko-
tsuka et al. 2000, Joscelyne 2009). In the present study, assess-
ment of the wines by three bitter-sensitive tasters (Pickering
© 2013 Australian Society of Viticulture and Oenology Inc.
Extended maceration and ethanol concentration 35
et al. 2004) revealed no difference in bitterness, thus this
attribute was excluded from those that can potentially differen-
tiate the wines. Among these potential attributes, astringency
was rated 34 and 30% higher in the EM wines of the 12 and
13% EtOH treatments, respectively. In Cabernet Sauvignon
(Scudamore-Smith et al. 1990) and Merlot wines (Yokotsuka
et al. 2000) produced with an EM of 42 and 64 days, respec-
tively, the astringency rating was 22 and 24% higher relative to
that of the controls. In the present study, the higher astringency
rating in EM wines is consistent with the relatively high tannin
content of the seeds (Harbertson et al. 2002), which were the
primary source of tannins for these wines (Table 5). It is also
possible that the higher polymeric pigment content at day 300
in the EM wines (Figure 4) could have contributed to the rela-
tive higher perceived astringency as well.
Control wines had a higher predominance of the red com-
ponent, lower brown component and lower astringency ratings.
The red fruit character received the lowest rating for the 12%
EtOH control wines, but the opposite was found for the 13%
EtOH control wines; EM wines showed intermediate values for
this attribute. The EtOH concentration had no effect on any
sensory attribute, with the exception of the hot/alcohol sensa-
tion in itself: the 13% EtOH wines were rated higher in hot/
alcohol than the 12% EtOH wines.
PLSR
To gain insight into the chemical features that were responsible
for relevant sensory attributes in the wines, PLSR was applied
using the chemical variables as predictors of the sensory data at
a discrete point, corresponding to 3 months of bottle ageing.
Using two latent factors, PLSR scores separated the wines into
four quadrants, with factor 1 discriminating the wines according
to the skin contact treatment and factor 2 separating the wines
as a function of the EtOH concentration (Figure 7a).
PLSR loadings (Figure 7b) defined EM wines with a positive
correlation between the chemical attributes tannins, polymeric
pigments (HPLC), LPPs, b* (yellow component) and the sensory
attributes brown component and astringency. Similarly, LPPs
and tannins clustered in close proximity in young Merlot wines
also subjected to PLSR (Jensen et al. 2008); Villamor et al.
(2009) report a correlation coefficient of 0.677 between per-
ceived astringency and LPPs, consistent with the clustering of
LPPs and astringency in the present study. The descriptor hot/
EtOH was located close to its chemical counterpart for the 13%
EtOH wines and the brown colour component clustered near
the non-tannin phenolic and acetic acid content. The remaining
sensory attributes and the chemical variables pH, lactic acid and
H* were located within the inner annulus of the PLSR loadings
plot, implying that these variables were not relevant to the PLSR
model.
For control wines, PLSR loadings revealed a strong correla-
tion between the chemical attributes anthocyanin derivatives,
SPPs, S vitisins (visitin A + vitisin B), mDP, a*, C* and the sensory
red colour component, thus suggesting that red colour is associ-
ated with anthocyanins, pyranoanthocyanins and miscellaneous
cycloaddition products or pigmented oligomers. SPPs are com-
posed by tannin–anthocyanin dimers either of direct condensa-
tion or mediated by acetaldehyde (Adams et al. 2004). Among
them, the dimer catechin-(4a→b8)-malvidin 3-O-glucoside
presents a wavelength of maximum absorption at 520 nm at
wine pH which corresponds to red colour (Salas et al. 2004). This
is consistent with the clustering of SPPs around the red compo-
nent. SPPs may also contain cycloaddition products (Adams et al.
2004), which explains the close proximity of SPPs and S vitisins.
36 Extended maceration and ethanol concentration Australian Journal of Grape and Wine Research 19, 25–39, 2013

Figure 6. Evolution of: (a) lightness (L*), (b) saturation (C*), (c) hue angle (H*), (d) a* parameter and (e) b* parameter during fermentation,
post-fermentation and bottle ageing (n = 3) of Merlot wines having an approximate final ethanol content of 12 and 13%. ( ) Control: 10-day
skin contact; ( ) extended maceration (EM): 30-day skin contact. Black and grey arrows indicate pressing time in control and EM wines,
respectively. Different letters indicate significant differences for Fisher’s LSD at P < 0.05. If not shown, error bars are obscured by treatment
symbol.

© 2013 Australian Society of Viticulture and Oenology Inc.

Casassa et al.
Conclusions
In this experiment, the maceration length defined the chemical
and sensory profile of the wines. A difference of 1.2% v/v EtOH
had no major effect on tannin and anthocyanin extraction,
mDP, polymeric pigment formation and pomace recovery of
anthocyanins and tannins from skins. Further, at the EtOH
concentrations and maceration lengths studied here, extraction
of tannins from skins was not significantly affected. Conversely,
extraction of tannins from seeds was contingent upon macera-
tion length and occurred under EtOH concentrations that can be
considered at the low end of those commercially relevant, sug-
gesting that even in low EtOH conditions, extraction of tannins
from seeds readily occurs and is mainly driven by maceration
length. Future research may be directed to evaluate a difference
in EtOH content greater than 2% EtOH (e.g. achieved by chap-
talisation of grapes at the same maturity level) to further
explore the effects of EtOH.
Qualitative analysis of tannins in grapes and seed/skin
pomace samples recovered after maceration suggested a rebind-
ing or incorporation of previously extracted seed tannins onto
the skins during maceration, although the reciprocal process,
i.e. the incorporation of skin tannins onto the seeds, was not
observed. Also, this phenomenon was independent of the mac-
eration length and the EtOH concentration. In light of the rather
low recovery percentages after phloroglucinolysis obtained in
the pomace and in aged wine samples, a combination of differ-
ent analytical approaches, e.g. fractionation followed by acid-
catalysis of the individual fractions, should help to gain a more
comprehensive understanding of these phenomena.
Consistent with an astringency rating 32% higher relative to
that of the controls, the contribution of seed-derived tannins in
EM wines varied from 73 to 80%, whereas control wines had a
© 2013 Australian Society of Viticulture and Oenology Inc.
Extended maceration and ethanol concentration 37
Figure 7. Partial Least
Squares Regression analysis:
(a) wine scores, and (b)
correlation loadings of the
relationship between chemical
analysis (X matrix, in blue
fonts) and sensory attributes
(Y matrix, in red underlined
fonts). Factor 1 (horizontal)
explained 87% of wine
chemical parameters and 27%
of wine sensory attributes,
while factor 2 (vertical)
explained 6 and 22%,
respectively. For conciseness,
only significant chemical and
sensory attributes (P < 0.05)
were included in the model.
Control (C): 10-day skin
contact; extended maceration
(EM): 30-day skin contact.
balanced proportion of seed and skin tannins. In EM wines,
major losses of anthocyanins occurred during post-maceration
along with the synthesis of LPPs, suggesting that the post-
fermentation stage has a larger influence on the formation of
polymeric pigments relative to other winemaking steps. For
control wines, major synthesis of polymeric pigments occurred
towards the end of bottle ageing, but the contribution of poly-
meric pigments to total pigments remained lower relative to
that of the EM wines.
Sensory analysis by QFP coupled with PLSR revealed
that polymeric pigments, LPPs and tannins clustered around
astringency in EM wines. From this perspective, maceration
techniques that favour seed tannin extraction and LPPs for-
mation should result in more astringent wines. For con-
trol wines, anthocyanin derivatives, S vitisins, mDP and SPPs
clustered around the sensory descriptor red component,
suggesting that perceived red colour is associated with mono-
meric anthocyanins, cycloaddition products and pigmented
oligomers.
Acknowledgements
The Washington Wine Advisory Committee, the Fulbright Com-
mission and the Walter Clore scholarship are thanked for finan-
cial support. Goose Ridge vineyard (Washington State, USA) is
thanked for generous donation of the grapes for this research.
We also extend our gratitude to Dr Richard Larsen (WSU
Prosser) and Dr John Thorngate (Constellation Wines, USA) for
critical review of the manuscript. Panellist members from the
WSU Prosser community and Chateau-Saint Michelle Wine
Estates are acknowledged for outstanding commitment to
this study.
38 Extended maceration and ethanol concentration
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Supporting Information
Additional Supporting Information may be found in the online
version of this article: http://onlinelibrary.wiley.com/doi/10.
1111/ajgw.12009/abstract
Figure S1. Evolution of the percentage mol extension subunits
of: (a) epigallocatechin, (b) catechin, (c) epicatechin and (d)
epicatechin-3-O-gallate, at day 4, 7, 30, 50, 100, 300 and 540
(n = 3). Control: 10-day skin contact; extended maceration
(EM): 30-day skin contact. †Approximate final ethanol content
(% v/v) of each set of wines.
Figure S2. Evolution of the percentage mol terminal subunits
of: (a) catechin, (b) epicatechin and (c) epicatechin-3-O-gallate,
at day 4, 7, 30, 50, 100, 300 and 540 (n = 3). Control: 10-day
skin contact; extended maceration (EM): 30-day skin contact.
†Approximate final ethanol content (% v/v) of each set of
wines.
Figure S3. Percentage mol extension and terminal subunits of
skins and seed of the grapes at harvest (n = 3). †EtOH: approxi-
mate potential ethanol content (% v/v) of each vineyard block.
Figure S4. Percentage mol extension and terminal subunits of
skins and seed recovered in the pomace after maceration
(n = 3). †Approximate final ethanol content (% v/v) of each set
of wines.
Figure S5. HPLC-DAD chromatograms registered at 280 nm
after acid catalysis in excess of phloroglucinol on a) seeds at
harvest, b) skins at harvest, c) wine at day 540, d) seed pomace
and e) skin pomace. Peak assignment: (1) epigallocatechin-
phloroglucinol adduct, (2) catechin-phloroglucinol adduct,
(3) epicatechin-phloroglucinol adduct, (4) catechin, (5)
epicatechin-gallate-phloroglucinol adduct, (6) epicatechin, (7)
epicatechin-gallate.
Table S1. Degrees of freedom (d.f.) and F ratios from analysis of
variance of trained panel evaluation (n = 14) of Merlot wine
sensory attributes.

Manuscript received: 19 July 2012

Revised manuscript received: 9 October 2012
Accepted: 4 November 2012

© 2013 Australian Society of Viticulture and Oenology Inc.

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