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The impact of wine components on


fractionation of Cu and Fe in model wine
systems: Macromolecules...

Article November 2016


DOI: 10.1016/j.foodres.2016.11.017

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

The impact of wine components on fractionation of Cu and Fe in model


wine systems: Macromolecules, phenolic and sulfur compounds
Nikolaos Kontoudakis a,b,, Mark Smith c, Anque Guo a,d, Paul A. Smith c, Geoffrey R. Scollary a,e,
Eric N. Wilkes c, Andrew C. Clark a,b
a
National Wine and Grape Industry Centre, Mambarra Drive, Wagga Wagga, NSW 2678, Australia
b
School of Agricultural and Wine Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, NSW 2678, Australia
c
Australian Wine Research Institute, Glen Osmond, SA 5064, Australia
d
College of Enology, Northwest A&F University, Yangling, Shaanxi 712100, PR China
e
School of Chemistry, The University of Melbourne, Vic. 3010, Australia

a r t i c l e i n f o a b s t r a c t

Article history: A variety of techniques have been developed with the ability to measure different forms of metals in wine with
Received 28 September 2016 the ultimate aim of providing a more accurate indicator of metal induced spoilage of wine. This study was con-
Received in revised form 9 November 2016 ducted in order to identify which wine components inuence the measurement of Cu and Fe in their fractionated
Accepted 15 November 2016
and/or electrochemically active forms. The measurement techniques involved detection of labile Cu by stripping
Available online xxxx
potentiometry and fractionation of Cu and Fe by sequential solid phase extraction, with detection by inductively
Keywords:
coupled plasma-optical emission spectroscopy. The wine components assessed included those extracted from
Copper wine (red wine tannin, white wine protein, white wine polysaccharide, red wine polyphenol, white wine poly-
Iron phenol), and commercially available monomeric compounds, including phenolic compounds and sulfur-contain-
Wine fractionation ing compounds. For Cu, only hydrogen sulde, which is known to induce the formation of Cu(I) sulde, showed
Speciation any appreciable inuence on the fractionation and electrochemical detection of Cu. This form of Cu was also iden-
Stripping potentiometry tied as the major component of red and white wines. For Fe, the fractionation was different for red versus white
Labile Cu wine, and inuenced signicantly by extracted red wine polyphenol, ()-epicatechin, gallic acid and tartaric
Macromolecules
acid. The wine components showed more inuence on Fe at pH 4.00 compared to pH 3.25. These results enable
Hydrogen sulde
a targeted use of these techniques in the assessment of metal-induced spoilage of wine.
2016 Elsevier Ltd. All rights reserved.

1. Introduction the accumulation of certain sulfur compounds (hydrogen sulde,


methanethiol), when wine is aged in low oxygen conditions (Ugliano,
Copper (Cu) and iron (Fe) in wine are linked to both the reductive and 2013; Viviers et al., 2013). These compounds can be responsible for unde-
oxidative development of wine (Clark, Wilkes, & Scollary, 2015; sirable aromas and the repression of desirable characters of the wine.
Ribreau-Gayon, Glories, Maujean, & Dubourdieu, 2006; Ugliano, 2013; Given the potential inuence that Cu and Fe can exert on wine devel-
Viviers, Smith, Wilkes, & Smith, 2013). Their contribution to oxidation is opment, a variety of instrumental techniques have been developed to
primarily through the catalysis of reactions induced by molecular oxygen, allow measurement of both the total concentrations and different
ultimately contributing to the oxidation of wine components such as forms of the metals in wine (Clark et al., 2015). The latter types of tech-
sulfur dioxide, phenolic compounds and ethanol (Clark et al., 2015; niques either classify the metal in broad terms (i.e., fractionation) or as
Danilewicz & Wallbridge, 2010; Li, Guo, & Wang, 2008). In some cases, specic ligand metal chelates (i.e., speciation) (Clark, Kontoudakis,
the oxidation reactions can be desirable when they occur in a controlled Barril, Schmidtke, & Scollary, 2016; Danilewicz, 2014; Karadjova, Izgi,
manner, such as during the barrel aging of red wine, but often they are & Gucer, 2002; Pohl & Prusisz, 2009; Pohl & Sergiel, 2009; Wiese &
detrimental to the sensory features of the wine (Avizcuri, Schwedt, 1997). The fractionation and speciation measurements have
Senz-Navajas, Echvarri, Ferreira, & Fernndez-Zurbano, 2016; Culler, been developed with the aim of measuring a form of the total metal con-
Cacho, & Ferreira, 2007; Escudero, Herndez-Orte, Cacho, & Ferreira, centrations that will be a better measure of metal activity in wine than
2000). In terms of promoting reduction in wine, Cu and Fe are linked to the total concentration. However, in the scientic literature there has
been more emphasis on the development of methods rather than stud-
Corresponding author at: National Wine and Grape Industry Centre, Mambarra Drive,
ies linking the measurement to the role of Cu and Fe in oxidative and
Wagga Wagga, NSW 2678, Australia. reductive development of wine. An example of the latter was the use
E-mail address: nkodoudakis@csu.edu.au (N. Kontoudakis). of spectrometric measures of Fe(III) tartrate in model wine systems to

http://dx.doi.org/10.1016/j.foodres.2016.11.017
0963-9969/ 2016 Elsevier Ltd. All rights reserved.

Please cite this article as: Kontoudakis, N., et al., The impact of wine components on fractionation of Cu and Fe in model wine systems:
Macromolecules, phenolic and sulfur compounds, Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.11.017
2 N. Kontoudakis et al. / Food Research International xxx (2016) xxxxxx

follow the redox equilibrium reached between Fe(II) and Fe(III) in oxi- and the metal ions. Karadjova et al. (2002) utilised general precipitation
dizing model wine systems (Danilewicz, 2014). Further work procedures for polysaccharides and proteins in wine to show signicant
established a measure of Fe(II) in white wine utilising a colorimetric concentrations of Cu and Fe in both types of precipitate, inferring asso-
ferrozine measure that accounted for bias towards Fe(II) during the ciation of metals by the macromolecules. Furthermore, complexation
measurement (Danilewicz, 2016). The results showed wine exposed capacity studies have been conducted in red wine, using electrochemi-
to oxygen exhibited a decrease in the Fe(II):Fe(III) ratio and that the cal techniques, and have shown complexing ligand concentrations and
nal ratio reached was dependent on the composition of wine. The frac- stability constants of ligands (Vasconcelos, Azenha, & Freitas, 1999).
tionation of Cu and Fe by solid phase extraction and analysis by induc- This study was conducted in order to identify which wine compo-
tively coupled plasma with optical emission spectroscopy detection nents inuence the measurement of Cu and Fe in their fractionated/
(SPE ICP-OES) has also been used to investigate the fractions of Cu and electrochemical forms and to enable an improved understanding for
Fe more relevant to oxygen consumption rates in wine with ascorbic the use of such measurements in following metal activity in wine. The
acid added (Rousseva, Kontoudakis, Schmidtke, Scollary, & Clark, measurement techniques involved SPE ICP-OES fractionation for both
2016). In this study, it was found that the fractions of Cu that were Cu and Fe, and stripping potentiometry for Cu. The impact of pH and dif-
able to elute through a C18 and cation exchange SPE cartridge were cor- ferent metal ratios was also to be assessed for various wine components.
related with the highest oxygen consumption rates. The Cu that was
trapped on a cation exchange column also showed reasonable correla- 2. Materials and methods
tion with oxygen consumption rates whilst the Cu retained on the C18
cartridge showed little relationship to oxygen consumption rates. The 2.1. Chemicals, samples and equipment
total Fe concentration and corresponding fractions had poor correlation
with the oxygen consumption rates in the wine. Finally, the electro- All glassware was soaked overnight in 10% (v/v) nitric acid (VWR,
chemical measurement of labile and non-labile Cu, meaning Cu able to Radnor, PA, USA) and then rinsed with copious amounts of 18.2 M
be detected and not detected by the technique, respectively, showed water prior to usage (Millipore Milli-Q Plus, Billerica, MA, USA). Solu-
an ability to monitor the conversion of Cu(II) to Cu(I) sulde in wine tions and dilutions were prepared using the same Type 1 quality water.
(Clark et al., 2016). This technique showed a signicant drop in the la- For the sample preparation nitric acid 70%, hydrochloric acid 34
bile Cu measured in a wine when hydrogen sulde was added to a 37%, sodium hydroxide and ammonium iron(II) sulfate hexahydrate
wine (in the form of sodium sulde) and Cu(I) sulde was generated. of trace metal grade were used (Sigma-Aldrich, St. Louis, MO, USA).
Conversely, in model wine with Cu(I) sulde present, the loss of volatile ()-Epicatechin ( 90%), gallic acid, quercetin 3-glucoside ( 98%),
hydrogen sulde induced an increase in labile Cu that was attributed to caffeic acid ( 98%), ellagic acid ( 95%), sodium sulde, sodium
disassociation of Cu(I) sulde. methanethiolate (90%), sodium ethanethiolate (90%), carbon disul-
However, it is knowledge of the wine components that inuence de ( 99%), dimethyl sulde ( 98%), L-cysteine (97%), L-glutathione
fractionation of the metal within wine that is most lacking from frac- reduced (98.0%), 2-furanmethanethiol (98%), 4-mercapto-4-methyl-
tionation methods. For example, in the case of the electrochemical mea- 2-pentanone, L-(+)-tartaric acid (99.5%), potassium hydrogen tartrate
surement of labile and non-labile Cu (Clark et al., 2016), it is known that (99%), potassium nitrate (99.0%), ammonium acetate (98%) and iron,
Cu(II) tartrate is measured as labile and Cu(I) sulde is measured as copper and lutetium standards for ICP were purchased from Sigma-Al-
non-labile, but the classication of other Cu complexes is not known. drich. Absolute ethanol, acetic acid (N99.7%) and copper(II) sulfate
Similarly, for the fractionation of Cu and Fe by SPE ICP-OES, little is pentahydrate (N99%) were obtained from VWR and mercury chloride
known about the inuence of major wine components on the fraction- (N 99.5%) from Ajax (Taren Point, Australia).
ation (Pohl & Prusisz, 2009; Pohl & Sergiel, 2009; Rousseva et al., 2016). Ten white and ten red wines were selected for this study and all
Although the residual and cationic forms of Cu were shown to correlate were commercially available. All wines were sealed under screw cap
best with oxygen consumption in a group of Chardonnay wines and analysed immediately after opening of the wine bottles.
(Rousseva et al., 2016), not much is known about the identity of these The concentrations of Cu and Fe in wines and model wine solutions
fractions of Cu other than they can constitute Cu(II) organic acid com- were determined by inductively coupled plasma optical emission
plexes (Pohl & Sergiel, 2009; Rousseva et al., 2016). spectroscopy (Varian 710 ICP-OES, California, U.S.A.) with utilisation of
The components of wine that can interact with Cu(II), Fe(II) and a High Salts Nebuliser/Spray Chamber. Cu was monitored at
Fe(III) are numerous and vary widely in general type. They can consist 324.754 nm and Fe at 259.940 nm. The instrumental conditions
of the wine macromolecules, including proteins, polysaccharides, tan- were: plasma power 1.30 kW, plasma ow 15.0 L/min, auxiliary ow
nins and their aggregate combinations, as well as monomeric species, 1.5 L/min, nebuliser pressure 200 kPa, stabilization time 15 s, sample
including organic acids, phenolic compounds, and sulfur-containing uptake 60 s, rinse time 60 s, and pump rate 15 rpm. The fast rinse
compounds (Danilewicz, 2016; Karadjova et al., 2002; Kreitman, pump was disabled as rapid sample introduction to the plasma led
Danilewicz, Jeffrey, & Elias, 2016a, 2016b). Although many studies to it being extinguished. Quantication was conducted via the use of
have been conducted on the complexation of metal ions to these com- Lu (261.541 nm) as an internal standard and standards were prepared
ponents in non-wine-like conditions (higher pH, ethanol-free or with- in a model wine system and diluted 4-fold with 5% (v/v) HNO3.
out organic acids), the specic conditions of wine are likely to be Electrochemical experiments were performed as described by
critical in indicating the dominant binders of metals. For example, al- Clark et al. (2016) with a 797VA Computrace (Metrohm, Herisau,
though phenolic compounds are known to be signicant binders of Fe Switzerland) and managed with the VA Computrace 797 PC Software
in aqueous conditions (typically at pH N 4), Danilewicz showed with (v 1.3). A DropSens 110 screen printed carbon electrode (DropSens,
spectroscopic evidence that little chelation occurred between Fe and Llanera, Spain) was utilised, which consisted of a 4 mm carbon work-
monomeric phenolic compounds at pH 3.6 when 5 g/L tartaric acid ing electrode, carbon counter electrode and silver reference elec-
was present (Danilewicz, 2014; Danilewicz, 2016). Consequently, al- trode, and the electrode was connected to the 797 VAComputrace
though Fe is known to interact with phenolic compounds in wine con- system via a CAC connector cable (DropSens). Sample ow (2 mL/
ditions via the transfer of electrons, the interaction was not sufcient min) into a cell methacylate wall-jet ow cell (DropSens) was con-
to displace the predominant binding between tartaric acid and Fe. Alter- trolled with a Waters Reagent Manager pump (Waters, Milford,
natively, Pohl and Prusisz (2009) showed that the presence of 500 mg/L USA), where by the restrictor coil was 120 mm in length to provide
of tannic acid with Fe(III) could induce different SPE ICP-OES fraction- a back pressure of 100200 psi. Stripping solutions were controlled
ation of Fe compared to a control (Fe(III) without tannic acid present). with an Ismatec peristaltic pump (Zurich, Switzerland), using PVC
Other studies, have shown interactions between wine macromolecules tubing (2.05 mm i.d.) enabling a ow rate of 1.5 mL/min, and the

Please cite this article as: Kontoudakis, N., et al., The impact of wine components on fractionation of Cu and Fe in model wine systems:
Macromolecules, phenolic and sulfur compounds, Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.11.017
N. Kontoudakis et al. / Food Research International xxx (2016) xxxxxx 3

stripping solutions were passed through an on-line Alltech Elite Table 1


Degasser. A six channel Rheodyne TPMV valve was used to switch General information of white wine polysaccharide, red wine polyphenol and red wine tan-
nin extracts.
between sample and washing/stripping solutions. A second six chan-
nel Rheodyne TPMV valve was used to switch between the 60% (v/v) Carbohydrates Mole % g/mL
aqueous ethanol wash solution and the 4.0 M ammonium acetate Mannose 45.9 331.2
stripping solution. Stripping potentiograms were plotted as dt/dU Rhamnose 5.0 36.6
vs U, by the VA Computrace 797 PCSoftware (v 1.3), where U repre- Glucuronic acid 4.0 31.3
Galacturonic acid 3.9 33.4
sents potential (V) and t represents time (s).
Glucose 5.0 36
Galactose 26.2 188.8
2.2. Extraction of wine macromolecules Xylose 1.1 6.4
Arabinose 7.4 44.8
2.2.1. White wine protein Fructose 1.5 10.1

The protein fraction was isolated according to Van Sluyter et al. Red wine polyphenol information
(2009) and has a protein concentration of 1 g/L containing mostly Total tannin (mg/L epicatechin equivalents) 2477.5
three thaumatin-like proteins in these ratios; A 77.3%, B 17.6%, C 5.1%. Total anthocyanins (mg/L malvidin 3-glucoside equivalents) 320.6
The composition and concentration of haze-forming proteins in must Total phenolics (au at 280 nm) 66.9
and wines were assessed in solution using high performance liquid Non-bleachable pigment (au at 520 nm post SO2 treatment) 4.6

chromatography (HPLC). HPLC analysis was performed as previously Red wine tannin information
described with some modications (Van Sluyter et al., 2009). Separation Tannin concentration (mg/L epicatechin equivalents) 1290
was achieved using Agilent 1260 UHPLC with Prozap Expedite C18 % Yield 37.6
column (10 mm 2.1 mm i.d., Grace technologies), a solvent system % ()-Epigallocatechin subunits 26.8
% ()-Epicatechin-3-O-gallate subunits 6.1
of 0.1% TFA/H2O (Solvent A) and 0.1% TFA/ACN (Solvent B), at 0.75
Mean degree of polymerisation (mDp) 9.1
mL/min and 35 C column temperature. The mobile phase gradient MM (g/mol) 2598
was: 01 min 1020% Solvent B, 14 min 2040% B, 46 min 4080% % Coloured (520 nm:280 nm) 7.6
B, 67 min 80% B, 7.01 min 10% B, and 710 min 10% B. Detection of pro-
teins was achieved by Diode Array monitoring at 210 nm. Identication
of TLPs and chitinases was achieved by comparing the retention times of wine. The column was washed with H2O/0.1% (v/v) formic acid (2 L)
sample peaks with isolated standards and quantitation was achieved by and the polyphenols eluted using 2:1 acetone:H2O (2 L). The acetone
comparing the peak areas with those of a standard curve of thaumatin was removed under vacuum using a rotary evaporator at 30 C and
(15 mg/L to 500 mg/L) (Sigma-Aldrich). Retention times of A, B and C: the total volume reduced to ~ 100 mL. Compositional information for
2.0 min, 2.2 min, 2.3 min, respectively. this extract are presented in Table 1 and were obtained using previously
described methods (Mercurio, Dambergs, Herderich, & Smith, 2007).
2.2.2. White wine polysaccharide
A 20 L volume of 2010 Chardonnay wine from the Barossa Valley
2.2.5. Red wine tannin
region was stripped of proteins using a strong cation exchange resin
A modied version of the published method (McRae, Kassara,
(Macro-Prep High S resin, Bio-Rad, Hercules, CA, USA) and phenolics
Kennedy, Waters, & Smith, 2013) was used. Briey, Toyopearl HW-
(Amberlite FPX-66 resin, Rhom and Hass) as described by Van
40F size exclusion medium (Optigen Scientic Pty Ltd., Port Adelaide,
Sluyter et al. (2009). The wine was evaporated by 75% under vacuum
Australia) in a glass column (50 450 mm) was equilibrated with
at b 40 C. Four volumes of cold ethanol (96% (v/v), Tarac Industries,
H2O/0.1% (v/v) formic acid wine prior to loading wine (600 mL). The
Grifth, Australia) were added and after 16 h at 4 C the precipitate
column was then washed with H2O/0.1% (v/v) formic acid (2 L) follow-
was removed by centrifugation. The precipitates were washed
ed by 1:1 MeOH/ H2O with 0.1% (v/v) formic acid (approximately 6 L).
twice with 96% (v/v) ethanol before being recovered by centrifuga-
Tannin was eluted with 2:1 acetone/H2O with 0.1% (v/v) formic acid
tion, and extensively dialysed against water (3.5 kDa MW cutoff,
(1 L). The solvent was removed by rotary evaporator (30 C) followed
Thermoscientic, Rockford, IL, USA) for 36 h at 22 C before being
by freeze drying. The composition of the isolated tannin is shown in
freeze dried. (Gawel, Smith, & Waters, 2016). The carbohydrate dis-
Table 1 using previously described methods (Cozzolino et al., 2004;
tribution of the polysaccharide isolate is shown in Table 1.
McRae et al., 2012; Mercurio et al., 2007).
2.2.3. White wine polyphenol
White wine polyphenols were extracted as per Gawel, Van Sluyter, 2.3. Preparation of model wine samples with wine components and metals
Smith, and Waters (2013). Four litres of wine (2012 Lafazanis
Moschollero, Peloponnese, Greece) was slowly introduced to the top All model wine samples were prepared in triplicate in a tartrate-
reservoir of a 1 m 2.5 cm column packed with Amberlite FPX-66 based wine system consisting of 12% (v/v) ethanol, 0.011 M potassium
resin (Rhom and Haas, Midland, MI, USA) and allowed to ow through hydrogen tartrate, and 0.007 M tartaric acid. The pH was 3.25 but was
under gravity. The resin was washed with two column volumes of ultra- adjusted to pH 4.00 when required with the addition of 10% (w/v)
pure water before being exhaustively eluted with 96% (v/v) ethanol. NaOH. Prior to sample preparation, the oxygen concentration was de-
The ethanol was reduced under vacuum at 30 C to provide a nal vol- creased to b0.1 mg/L by sparging solutions with argon (High purity
ume of ~100 mL of the extracted white wine polyphenols. The extract grade, BOC, Sydney, Australia). A Fibox 3 LCD trace, manufactured by
was analysed via UVVis (Cary 60, Agilent Technologies) in 2 mm Precision Sensing GmbH (Regensburg, Germany), was utilised for mea-
quartz cells, values are corrected for path length. A280 4.04au, A320 surement of dissolved oxygen concentrations in conjunction with sen-
3.93au, A370 0.45au. sors of the type PSt3 (detection limit 0.015 mg/L, 0100% oxygen).
The sensors came pre-calibrated from the manufacturer.
2.2.4. Red wine polyphenol At pH 3.25, the metal treatments included 0.4 mg/L Cu, 3.0 mg/L Fe,
A modication of the published method was used for red wine poly- 3.0 mg/L Fe + 0.2 mg/L Cu and 3.0 mg/L Fe + 0.4 mg/L Cu. Cu was added
phenol isolation (McRae, Falconer, & Kennedy, 2010). Briey, a glass col- as copper(II) sulfate pentahydrate and Fe was added as ammonium
umn packed with 600 mL of Toyopearl HW-40C (Sigma-Aldrich) was iron(II) sulfate hexahydrate. The Fe(II) redox form of Fe was chosen
equilibrated with H2O/0.1% (v/v) formic acid prior to loading 2 L of over Fe(III) as this form of Fe typically dominates in wines sealed

Please cite this article as: Kontoudakis, N., et al., The impact of wine components on fractionation of Cu and Fe in model wine systems:
Macromolecules, phenolic and sulfur compounds, Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.11.017
4 N. Kontoudakis et al. / Food Research International xxx (2016) xxxxxx

under screw cap. The concentrations of Fe and Cu adopted were typical sulfur compounds were analysed on the same day of preparation rather
of that observed in white and red wines (Fig. 1). than after seven days.
The wine components to be assessed were added at typical wine-
like concentrations (Ferreira, Piarra-Pereira, Monteiro, Loureiro, & 2.4. Solid phase extraction scheme
Teixeira, 2001; Mercurio, Dambergs, Cozzolino, Herderich, & Smith,
2010), apart from the sulfur-containing compounds which were A modied approach for the fractionation of Cu and Fe was applied
added at a mole ratio of 2:1 for sulfur compound to metal ion. For (Pohl & Prusisz, 2009; Pohl & Sergiel, 2009; Rousseva et al., 2016) (Sup-
each metal treatment, the following wine components were added: plementary Fig. 1). DVB non-polar cartridges (Strata SDBL 100 m Sty-
control (no further addition), white wine protein (50 mg/L), white rene-divinylbenzene, 1 g/6 mL, Phenomenex, Torrance, CA, U.S. A) and
wine polysaccharide (200 mg/L), white wine polyphenol (200 mg/L), SCX cation exchange cartridges (Strata SCX 55 m, 70 , 1 g/6 mL)
red wine polyphenol (2000 mg/L), red wine tannin (1000 mg/L) and hy- were used in series. The DVB cartridge was conditioned with 20 mL
drogen sulde (two-fold mole excess over metals ions, 2:1). The sam- methanol, followed by 40 mL water, 10 mL 1 M HCl, and nally with
ples containing Cu were placed in 50 mL Schott glass bottles and 40 mL of water. The cartridge was then dried under vacuum prior to
closed with silicone lined screw caps (Duran Group GmbH, Wertheim/ usage. The SCX cartridge was conditioned with the following solutions
Main, Germany) to avoid head-space or bubbles (~68 mL sample vol- in sequence: 40 mL of water, 10 mL of 1.0 M HCl, 20 mL of water,
ume). The remaining metal treatment samples were prepared in 10 mL of 1.0 M NaOH and nally with 40 mL water. The cartridge was
20 mL standard GC headspace vial tightly sealed with a PTFE/silicone then also dried under vacuum.
lined screw cap (CrossLab, Agilent, Santa Clara, CA, USA), again with The conditioned cartridges were then mounted on a peristaltic
no head-space volume or bubbles present. In all samples, the oxygen pump (Gilson minipuls 3, Middleton, WI, U.S.A.) and 15 mL of each sam-
concentration was b0.5 mg/L. The samples were stored for seven days ple was passed through the DVB cartridge at a rate of 1 mL/min. A 5 mL
in anaerobic jars sparged with Ar whereby the oxygen concentration aliquot of the eluent was used for measurement of the metal ions not
was maintained to b 0.1 mg/L (or 1% oxygen saturation). retained by the DVB cartridge, allowing calculation of the hydrophobic
At pH 4.00, two metal treatments were applied: 0.4 mg/L Cu and metal fraction, being equivalent to the total metal concentration in the
3.0 mg/L Fe. The following components were added: red wine polyphe- wine minus the metal not retained by the DVB cartridge (non-hydro-
nol (2000 mg/L), red wine tannin (1000 mg/L), ()-epicatechin (1000 phobic). The remaining 10 mL aliquot of the solution already eluted
mg/L), gallic acid (100 mg/L), caffeic acid (100 mg/L), quercetin 3-glu- through the DVB column was then passed through the second column
coside (15 mg/L), ellagic acid (10 mg/L), hydrogen sulde, ethanethiol, (SCX) at a rate of 1 mL/min. The eluent from this second column was
methanethiol, carbon disulde, dimethyl sulde, L-cysteine, L-glutathi- measured for the residual fraction of metal ions. The cationic fraction
one, 2-furanmethanethiol (2-FMT) and 4-mercapto-4-methyl-2- was calculated as the non-hydrophobic minus the residual fraction.
pentanone (4-MMP). All sulfur compounds were added in a two-fold Thus, three metal fractions were obtained hydrophobic, residual and
mole excess over the metals ion concentration. Sample preparation cationic. Blank samples were performed using a model wine system
and storage were conducted as described for pH 3.25, although the (12% aqueous ethanol, 0.011 M potassium hydrogen tartrate, 0.007 M
tartaric acid, pH 3.25), which identied any background Cu and Fe con-
tamination stemming from the non-polar and cation exchange
0.9 cartridges.
a Cationic Residual Hydrophobic
Prior to ICP-OES analysis, the primary eluents from the DVB and SCX
0.8
columns, and also the untreated samples (for determination of total
Cu concentration (mg/L)

0.7 metal ion concentration), were diluted 4-fold with 5.0% (v/v) HNO3.
0.6

0.5
2.5. Measurement of labile copper

0.4
Labile copper was measured in wine and model wine samples by
0.3 medium exchange stripping potentiometry utilising screen printed car-
0.2 bon electrodes (Clark et al., 2016).

0.1
3. Results and discussion
0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
3.1. Analysis of wines
White wine Red wine

5.0 Ten white and ten red wines, encompassing a large range of total Cu
b Cationic Residual Hydrophobic
and Fe concentrations (Fig. 1), were analysed via SPE ICP-OES to quan-
4.0 tify Cu and Fe in terms of hydrophobic, residual and cationic fractions.
Fe concentration (mg/L)

In this study, SPE cartridges rather than resins were utilised, as per
Rousseva et al. (2016), although the SPE cartridge adopted to retain hy-
3.0
drophobic wine components was divinylbenzene-based composition
rather than C18. Hence the material adopted was more like the
2.0 Dowex 50 W resin utilised by Pohl and Prusisz (2009) and Pohl and
Sergiel (2009) to retain hydrophobic Cu and Fe complexes in beer and
1.0 wine samples. The divinylbenzene SPE cartridge was chosen for this
study rather than C18 material as the former is known to retain a larger
amount of phenolic compounds, including phenolic acids, when they
0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 are extracted from acidic aqueous conditions (Michalkiewicz, Biesaga,
White wine Red wine
& Pyrzynska, 2008). Given the modication of the technique compared
to past studies (Pohl & Prusisz, 2009; Pohl & Sergiel, 2009; Rousseva
Fig. 1. The concentrations of hydrophobic, residual and cationic forms of (a) Cu and (b) Fe et al., 2016), the fractionation of the 20 wines shown in Fig. 1 warrants
in red and white wines. The error bars are the st.dev. (n = 3). specic discussion.

Please cite this article as: Kontoudakis, N., et al., The impact of wine components on fractionation of Cu and Fe in model wine systems:
Macromolecules, phenolic and sulfur compounds, Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.11.017
N. Kontoudakis et al. / Food Research International xxx (2016) xxxxxx 5

Fig. 1a shows the concentrations for the different Cu metal ion frac- able to be deposited onto the electrode as reported previously. This sug-
tions obtained by SPE ICP-OES analysis. The hydrophobic fraction was gested a component in wine that was sufciently strong to avoid disso-
the dominant form of Cu for the majority of the wines, with the average ciation in the diffusion layer of the electrode. The Cu(II) organic acid
percentage of hydrophobic Cu as a function of total Cu being 78%. The complexes (tartaric acid, malic acid and citric acid) are all classied as
proportion of hydrophobic Cu ranged from 40 to 100% of total Cu. labile Cu forms with the electrochemical technique (Clark et al., 2016).
There was no obvious trend evident between red and white wines for This means that an alternate wine component is binding with Cu suf-
Cu fractionation. The hydrophobic fraction was proposed to consist of ciently strongly as to be classied as non-labile Cu. From Figs. 1a and
strong complexes between Cu and various avonoids and other poly- 2, it is evident that the hydrophobic and non-labile forms of Cu are
phenols (Pohl & Sergiel, 2009). It is of interest to note that the hydro- most likely due to the interaction of Cu with the same binding agent.
phobic fraction of total Cu dominates in white wine as much as it does For further insights into the wine components imparting signicant in-
in red wine despite the phenolic concentration being much lower in uence on metal ion fractionation in wine, the SPE ICP-OES and electro-
white wines compared to red wines. chemical technique were used to analyse metal ions in a variety of
The residual fraction was the second most abundant fraction for Cu model wine systems.
which ranged from 0 to 50% of total Cu (Fig. 1a). This fraction, which
consisted of metal ions passing through both divinylbenzene and cation
exchange cartridges, was suggested in similar studies to be due to neg- 3.2. Wine components and the electrochemical assessment of Cu
atively-charged or neutral complexes of the metal ions (Pohl & Sergiel,
2009; Rousseva et al., 2016). These complexes were also not sufciently The results from the analysis of the wine components in model wine
hydrophobic to be retained by the hydrophobic column. According to systems at pH 3.25 and 4.00 are shown in Table 2. The wine components
Pohl and Sergiel (2009), the majority of the Cu(II) tartrate complexes included those extracted from wines (red wine polyphenol, red wine
were measured in the residual fraction (68.2% of total Cu at pH 3.5) tannin, white wine polysaccharide, white wine protein and white
with the remainder being cationic. The cationic fraction was attributed wine polyphenol) as well as commercially available standards of a vari-
to uncomplexed Cu(II) ions, loosely bound Cu or positively charged ety of monomeric compounds found in wine (phenolic compounds and
complexes that could elute from the hydrophobic column and dissoci- sulfur-containing compounds). The commercial standards, red wine
ate in the cation exchange column (Pohl & Sergiel, 2009; Rousseva et polyphenol and red wine tannin were assessed at pH 4.00, a relatively
al., 2016). It is more likely that the overall equilibrium between the dif- high pH but still relevant to red wine, and more likely to induce binding
ferent complexes of Cu(II), and their speed of re-equilibration during of Cu to wine components. The components specic to white wine
passage through the SPE cartridges, inuences the overall partitioning (white wine polysaccharide, white wine protein and white wine poly-
of the Cu. The results of Fig. 1a differ from those described by Pohl and phenol) were assessed at a lower pH more akin to conditions encoun-
Sergiel (2009), given that the residual Cu for the wines in Fig. 1a were tered in white wines. To allow direct comparison to the white wine
generally more abundant than the cationic Cu. However, Pohl and macromolecules, the red wine polyphenol and red wine tannin were
Sergiel (2009) did use a different form of cationic exchange material also assessed at pH 3.25. Hydrogen sulde was also examined at both
(i.e., resin versus packed cartridges), and also only analysed four red pH values given its known ability to induce the formation of non-labile
wines. Cu at pH 3.25.
Fe showed different fractionation compared to Cu, including a sub- From the results in Table 2, it is evident that only the addition of hy-
stantial difference between the fractionation in white and red wines drogen sulde to the model wine system afforded a signicant change
(Fig. 1b). The hydrophobic form was the least abundant Fe fraction for in the classication of Cu by the electrochemical technique. For all
most of the wines (b 20% of total Fe), apart from one wine (see wine 8, other model wine systems the predominant form of Cu was labile Cu,
Fig. 1b). This suggested that for the majority of the wines, the proportion identical to the classication of Cu in the model wine without any bind-
of Fe in complexes able to be retained by the divinylbenzene SPE car- ing component added, that is, with Cu present as Cu(II) tartrate. Howev-
tridge was low, regardless of wine colour. For white wines the main Fe er, in the case of hydrogen sulde, all the Cu was classied as non-labile
fraction was generally the cationic form (3380%), while for red wines as reported earlier, regardless of the sample pH. The formation of Cu(I)
it was the residual form (4973%). As was the case for cationic Cu in sulde (Kreitman et al., 2016a) in the model wine system is responsible
these wines, the cationic Fe fraction was proposed to contain for the non-labile Cu, some of which would be removed as particulate
uncomplexed Fe ions (Fe(II) and/or Fe(III)) and loosely bound Fe spe- Cu(I) sulde by the ltration step prior to electrochemical analysis of
cies able to be dissociated in the cation exchange column (Pohl & the sample (Clark, Grant-Preece, Cleghorn, & Scollary, 2015). Part of
Prusisz, 2009). The residual Fe was suggested to be neutral or anionic the non-labile Cu is also suspended Cu(I) sulde particles that are not
complexes of Fe, and Pohl and Prusisz (2009) showed signicant detected by the electrochemical sensor (Clark et al., 2016).
amounts of residual Fe (7375% of total Fe) induced by the presence
of citric acid or tannic acid at pH 3.00. As for Cu, it is more likely that 0.9 Non-labile Labile
the overall equilibrium between the different complexes of Fe(II), and
0.8
their speed of re-equilibration during passage through the SPE car-
tridges, also inuences the overall partitioning of the Fe. 0.7
Cu concentration (mg/L)

Cu was measured in wines by medium exchange stripping 0.6


potentiometry utilising screen printed carbon electrodes (Clark et al., 0.5
2016) to classify Cu as either labile or non-labile. An advantage of this
0.4
technique is that the wine can be analysed without any pre-treatment
other than ltration, and hence the distribution of the different Cu 0.3
forms is not inuenced by wine preparations prior to the measurement. 0.2
The labile Cu consisted of Cu in the wine that could be deposited onto a
0.1
mercury electrode, through the application of a 900 mV potential,
whilst non-labile Cu was Cu that could not be deposited. It was expected 0.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
that free Cu ions or loosely bound Cu, that dissociated in the diffusion White wine Red wine
layer of the electrode allowing deposition of Cu onto the electrode,
would be classied as labile Cu. Fig. 2 demonstrates that most of the Fig. 2. The labile and non-labile Cu concentrations as measured by ow medium exchange
Cu in both white and red wine is in the non-labile form, hence not constant current stripping potentiometry. The error bars are the st.dev. (n = 3).

Please cite this article as: Kontoudakis, N., et al., The impact of wine components on fractionation of Cu and Fe in model wine systems:
Macromolecules, phenolic and sulfur compounds, Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.11.017
6 N. Kontoudakis et al. / Food Research International xxx (2016) xxxxxx

Table 2 For Fe the cationic and residual fractionated forms are the most
Total Cu measured by ICP-OES and labile/non-labile Cu measured by ow medium ex- abundant. The red wine polyphenol, epicatechin and gallic acid have
change constant current stripping potentiometry in model wine systems containing ex-
tracted red wine macromolecules, monomeric phenolic and sulde-containing
higher concentration of residual Fe than the control while red wine tan-
compounds at pH 4.00 and in red and white wine macromolecules and hydrogen sulde nin, caffeic acid, quercetin-3-glucoside and ellagic acid are generally
at pH 3.25. The error bars are the st.dev. (n = 3). similar to the control. Interestingly, the Fe(II) in the tartrate-containing
control has a higher concentration of residual Fe at pH 4.00 than at
Treatment Total Labile Non-labile
pH 3.25 (Fig. 3b versus Fig. 5b), which is consistent with the results re-
pH 4.00 Control 0.39 0.01 0.40 0.01 n.d.
ported by Pohl and Prusisz (2009). The latter study observed an increase
Red Wine Polyphenol 0.41 0.01 0.42 0.01 n.d.
Red Wine Tannin 0.45 0.02 0.38 0.01 0.07 0.01 in residual Fe in the presence of citrate as the pH of the solution was in-
Epicatechin 0.41 0.01 0.42 0.01 n.d. creased, and it is expected that a similar effect would occur for tartrate.
Gallic acid 0.39 0.01 0.41 0.01 n.d. The hydrophobic form is the least abundant Fe fraction, similar to the
Caffeic acid 0.38 0.01 0.43 0.01 n.d. wines analysed (Fig. 1b), however, it was present in trace amounts in
Quercetin 3-glucoside 0.39 0.01 0.45 0.01 n.d.
Ellagic acid 0.43 0.01 0.41 0.01 0.03 0.01
all samples other than quercetin-3-glucoside. Its higher concentration
H2S 0.43 0.02 0.01 0.01 0.42 0.03 than the control indicated some minor retention of Fe in the presence
MeSH 0.39 0.01 0.45 0.01 n.d. of phenolic compounds at pH 4.00 by the divinylbenzene SPE cartridge.
EtSH 0.40 0.01 0.43 0.01 n.d. The SPE ICP-OES analysis of the pH 4.00 model wine systems con-
CS2 0.42 0.01 0.39 0.01 0.03 0.01
taining Cu and the sulfur compounds (Fig. 4a) showed the hydrogen
DMS 0.41 0.01 0.38 0.01 0.03 0.01
Cysteine 0.39 0.01 0.39 0.01 n.d. sulde provided a contrasting result compared to the other sulfur com-
Glutathione 0.37 0.01 0.45 0.01 n.d. pounds, as was the case for the electrochemical study (Table 2). All the
2-FMT 0.39 0.01 0.41 0.01 n.d. Cu was detected as hydrophobic Cu in the presence of hydrogen sulde,
4-MMP 0.30 0.01 0.46 0.01 n.d. conrming the similarity between the electrochemical (non-labile) and
pH 3.25 Control 0.39 0.01 0.43 0.01 n.d.
SPE ICP-OES (hydrophobic) measures of Fig. 1a and Fig. 2. For the other
White wine protein 0.38 0.01 0.38 0.01 n.d.
White wine polysaccharide 0.39 0.01 0.40 0.01 n.d. sulde-containing compounds, the Cu fractionation is similar to the
White wine polyphenol 0.42 0.01 0.39 0.01 0.03 0.01 control, with mainly cationic Cu and no residual Cu. Regarding the Fe
Red wine polyphenol 0.41 0.01 0.44 0.01 n.d. fractionation, none of the sulde-containing compounds demonstrated
Red wine tannin 0.40 0.01 0.38 0.01 0.02 0.01
any capacity to inuence Fe fractionation, with the major Fe fraction
H2S 0.40 0.01 0.01 0.01 0.39 0.01
being cationic in all cases (Fig. 4b). A larger amount of residual Fe in
the control compared to sulfur compound-containing samples was evi-
Such a result demonstrates that the electrochemical technique is rel- dent, suggesting an increased inuence of anionic or neutral Fe tartrate
atively specic towards the binding of Cu by hydrogen sulde based on complexes on the elution of Fe from the cation exchange cartridge in the
the wine components assayed in this study. Furthermore, given the re- absence of sulfur compounds. It is not certain why this was the case, but
sults from the wine analysis (Fig. 2) it is likely that the majority of Cu in potentially the sulfur compounds may have ensured that Fe remained in
wine is in the form of Cu(I) sulde, regardless of the wine being white or the reduced Fe(II) form which complexes tartrate with lower stability
red. Franco-Luesma and Ferreira (2014) have also reported that bound than Fe(III). Kreitman et al. (2016b) have recently described the gradual
measures of hydrogen sulde in wine are correlated to the wine Cu reduction of Fe(III) to Fe(II) in wine conditions by hydrogen sulde or
concentration. cysteine.
It is possible that other wine components not assayed (i.e., different At pH 3.25, the wine macromolecules and hydrogen sulde were
types of polysaccharides with increasing acid functionality), or combi- assessed for their binding ability with Cu and Fe with the use of SPE
nations of different components, might be able to induce signicant ICP-OES. The metal treatments 0.4 mg/L Cu and 3.0 mg/L Fe are present-
binding of Cu. However, the absence of even a minor impact by the ed in Fig. 5a and b. Given the lack of any differences in results for sam-
non-hydrogen sulde wine components on the electrochemical classi- ples with combined or individual metal ions, the treatment 3.0 mg/L
cation of Cu strengthens the likelihood of hydrogen sulde being re- Fe + 0.2 mg/L Cu and 3.0 mg/L Fe + 0.4 mg/L Cu are shown in Supple-
sponsible for the non-labile fraction of Cu in red and white wines. mentary Table 2. Similar to the results at the pH 4.00, it is evident that
Previous studies have shown the ability of the non-labile Cu portion to for Cu fractionation only hydrogen sulde shows fractionation different
decrease signicantly, and the labile Cu to reach total Cu concentrations, to the control. For all the wine macromolecules the cationic Cu is the
when wine is left in open containers for extended periods of time (Clark major Cu form while for hydrogen sulde all the Cu is hydrophobic.
et al., 2016). Such a result is more consistent with the volatile loss of hy- For Fe, none of the wine macromolecule components or hydrogen sul-
drogen sulde and the consequent dissociation of Cu(I) sulde. Al- de showed any difference to the control. Furthermore, at pH 3.25 the
though, it is possible that the oxidation of non-volatile complexing residual Fe for the red wine polyphenols and tannin are much lower
agents could also afford the release of Cu from non-labile forms. than observed at pH 4.00. This was also the case for the control, but
the reduction in residual Fe was larger for the red wine components.
3.3. Wine components versus SPE fractionation This suggests that both Fe(II) tartrate and Fe(II) phenolic complexes
contribute more to residual Fe at higher pH, a result similar to that ob-
SPE ICP-OES was used to study the interaction of both Cu and Fe with served by Pohl and Prusisz (2009) for Fe(III) citrate and Fe(III) tannic
wine components in model wine systems at pH 3.25 and 4.00. Fig. 3a acid. For the other two metal ion treatments (3.0 mg/L Fe + 0.2 mg/L
and b shows the hydrophobic, residual and cationic fractions for Cu Cu and 3.0 mg/L Fe + 0.4 mg/L Cu) no differences were found in com-
and Fe respectively, with red wine polyphenol, red wine tannin and parison with the results mentioned for the samples with individual
commercial monomeric representative phenolic compounds at metals present (0.4 mg/L Cu and 3.0 mg/L Fe) (Supplementary Tables
pH 4.00. For Cu the predominant fraction was cationic Cu in all cases. 1 and 2).
A trace amount of residual Cu was present only in the samples with
red wine tannin and ellagic acid. The hydrophobic Cu concentration 4. Conclusion
was generally low (b0.15 mg/L) and comparable to the control indicat-
ing that weak or no complexes had formed with Cu and the phenolic The results demonstrate that hydrophobic Cu and non-labile Cu, as
wine components. There was a slight increase in the hydrophobic Cu measured by SPE ICP-OES and an electrochemical technique, respective-
for the red wine tannin sample, but this increase was not statistically ly, are the major forms of Cu in both red and white wines. Given the
signicant (P N 0.05). components assessed in the model wine systems at pH 3.25 and/or

Please cite this article as: Kontoudakis, N., et al., The impact of wine components on fractionation of Cu and Fe in model wine systems:
Macromolecules, phenolic and sulfur compounds, Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.11.017
N. Kontoudakis et al. / Food Research International xxx (2016) xxxxxx 7

0.5
a Cationic Residual Hydrophobic

Cu concentration (mg/L)
0.4

0.3

0.2

0.1

0.0
Control Red Wine Red Wine Epicatechin Gallic acid Caffeic acid Quercetin 3- Ellagic acid
Polyphenol Tannin glucoside

Cationic Residual Hydrophobic


4.0
b

3.0
Fe concentration (mg/L)

2.0

1.0

0.0
Control Red Wine Red Wine Epicatechin Gallic acid Caffeic acid Quercetin 3- Ellagic acid
Polyphenol Tannin glucoside

Fig. 3. The concentrations of hydrophobic, residual and cationic forms of (a) Cu and (b) Fe in model wine systems with extracted wine macromolecules and monomeric phenolic
compounds at pH 4.00. The error bars are the st.dev. (n = 3).

0.6 Cationic Residual Hydrophobic


a 4.00, it is most likely that these fractions are due to the presence of
copper(I) sulde in wine, rather than as complexes with hydrophobic
0.5
phenolic compounds as suggested by previous studies (Pohl & Sergiel,
Cu concentration (mg/L)

2009; Rousseva et al., 2016). It is possible that combinations of some


0.4
of the wine components may contribute to some synergistic effects on
Cu fractionation. However, the wine phenolic extracts, which would
0.3
contain a variety of different phenolic compounds, did not show any
0.2
such synergistic effects for the fractionation of Cu. Furthermore, many
of the sulfur compounds were utilised at concentrations much higher
0.1 than present in wine (i.e., MMP, cysteine, methanethiol) and hence
would be less likely to show impacts on Cu fractionation at lower con-
0.0 centrations. It is possible that other unidentied components of wine
Control H2S MeSH EtSH CS2 DMS Cysteine Glut. 2-FMT 4-MMP may also induce signicant binding of Cu to provide hydrophobic or
5.0 Cationic Residual Hydrophobic non-labile Cu. However, the conversion of non-labile Cu to labile Cu
b by heating and/or storage of wine in open containers is consistent
with the assignment of this fraction of Cu being Cu(I) sulde and in
4.0
equilibrium with volatile hydrogen sulde. It is interesting that this
Fe concentration (mg/L)

major form of Cu in wine (hydrophobic) was the form least correlated


3.0 with the rate of oxygen consumption in Chardonnay wine (Rousseva
et al., 2016). Given the link between hydrophobic Cu, non-labile Cu
2.0 and Cu(I) sulde it follows that the former two would provide measures
of Cu that are less able to catalyse oxygen consumption. Alternatively,
labile Cu, cationic Cu and residual Cu would be expected to be more ac-
1.0
tive in catalyzing oxygen consumption in wine.
For Fe, there is an obvious contrast between red and white wines,
0.0 whereby the residual form of Fe dominates in red wines, whilst the cat-
Control H2S MeSH EtSH CS2 DMS Cysteine Glut. 2-FMT 4-MMP
ionic form is more prevalent in white wines. As expected, the increased
residual fraction was linked to phenolic compounds, however, it was
Fig. 4. The concentrations of hydrophobic, residual and cationic forms of (a) Cu and (b) Fe
in model wine systems with sulfur-containing compounds at pH 4.00. The error bars are only a select few phenolic compounds (red wine polyphenol, epicate-
the st.dev. (n = 3). chin and gallic acid) that provided signicant amounts of the residual

Please cite this article as: Kontoudakis, N., et al., The impact of wine components on fractionation of Cu and Fe in model wine systems:
Macromolecules, phenolic and sulfur compounds, Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.11.017
8 N. Kontoudakis et al. / Food Research International xxx (2016) xxxxxx

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Please cite this article as: Kontoudakis, N., et al., The impact of wine components on fractionation of Cu and Fe in model wine systems:
Macromolecules, phenolic and sulfur compounds, Food Research International (2016), http://dx.doi.org/10.1016/j.foodres.2016.11.017
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