COPPER AND IRON SPECIATION IN WHITE WINE: IMPACT ON

Transcription

Master international Vintage
Groupe École Supérieure d'Agriculture
d'Angers
National Wine and Grape Industry Centre
McKeown Drive
Wagga Wagga NSW 2678
55 rue Rabelais
Australia
49007 ANGERS
Supervisor: Dr. CLARK Andrew
France
COPPER AND IRON SPECIATION IN WHITE WINE:
IMPACT ON WINE OXIDATION AND INFLUENCE OF PROTEIN FINING
AND INITIAL COPPER AND IRON JUICE CONCENTRATIONS
Master’s Thesis
Promotion: 11
Date: 9th September 2014
ROUSSEVA Michaela
International Vintage Master student, ESA
Coordinatior: MAURY Chantal
BIBLIOGRAPHIC NOTICE
AUTHOR: ROUSSEVA Michaela
Promotion: 11
Coordinator: MAURYChantal
Title of the thesis: ”Copper and iron speciation in white wine: Impact on wine oxidation and
influence of bentonite fining and initial copper and iron juice concentrations”;
Pages: 74; Tables: 13; Figures: 16; Bibliography: 103; Annexes: 7.
Keywords:...white wine, copper, iron, speciation, bentonite, oxidation
AUTHOR’S SUMMARY
PLAN & AIM
OF
THE
STUDY
The aim of this project is to carry out a detailed study of copper (Cu) and
iron (Fe) distribution and concentrations in wine and their effect on the wine’s
quality and characteristics throughout different stages of the winemaking
process. The main focus is on the analysis of specific species of Cu and Fe,
and not only as usual on their total concentrations.
First of all, the wines used in the experiment were specifically produced
for this project. A total of six different treatments, with three repetitions per
METHODS &
treatment, were prepared in order to compare the influence of different copper
TECHNIQUES
and iron concentrations in juice on the final concentrations in wine. Samples
were taken to analyse and thus compare the following stages: from the juice
before alcoholic fermentation, from the wine at the end of alcoholic
fermentation, from the wine before and after bentonite fining and from the
already bottled wine to measure the rate of oxidation.
A solid phase extraction (SPE) method enabled the separation of both
copper and iron species into 3 groups – hydrophobic, residual and cationic.
These wine samples were then analysed by inductively coupled plasma optical
emission spectroscopy (ICP-OES). This approach provided detailed
information about how a different distribution of copper and iron species in
the wine could be impacted by wine production processes and how they could
impact the development of the wine.
Some interesting results were obtained which showed that, for example,
RESULTS
the alcoholic fermentation was completed quicker in samples where there
CONCLUSION
initially was a higher copper concentration. It was also established that
generally the most significant portion of the copper which is found in the wine
after alcoholic fermentation is in the cationic fraction and that this outcome
doesn’t seem to be affected by the initial copper concentration in the juice. For
iron, it could be pointed out that the cationic species are the most predominant
form for all wine samples, accounting for more than 50% of the total
concentration. The application of calcium bentonite was reported to have a
significant impact on the total copper and iron concentrations and at the same
time was found to also alter the distribution of the metals amongst the
different species. In regards to the oxidation process, a good correlation was
discovered between the residual fractions of both copper and iron and the
oxidation rate, which suggests this form of the metals warrants further
investigation as a potentially potent oxidative catalyst in wine.
The results obtained in this research reveal remarkable new aspects about
Cu and Fe speciation in white wine. They open new opportunities for further
research on the influence of copper and iron speciation on winemaking.
NOTICE BIBLIOGRAPHIQUE
AUTEUR : ROUSSEVA Michaela
Promotion : 11
Patron de mémoire : MAURY Chantal
Signalement du mémoire : « Spéciation du cuivre et du fer dans le vin blanc : influence de la
teneur initiale en cuivre et en fer dans le jus, de l’impact du collage protéique et impact sur
l’oxydation du vin »; Pages : 74; Tableaux : 13; Figures : 16; Bibliographie : 103;
Annexes : 7.
Mots-clés : vin blanc, cuivre, fer, spéciation, collage, oxydation.
RÉSUMÉ D'AUTEUR
L'objectif de ce projet est de mener une étude détaillée sur les
concentrations et la distribution de cuivre (Cu) et de fer (Fe) dans le vin et leur
effet sur ses qualités et caractéristiques pendant différentes étapes de la
BUTS DE
vinification. L'approche originale qui a été choisie pour cette étude, a portée
L'ETUDE
essentiellement sur l’analyse des espèces spécifiques de Cu et de Fe, et pas
seulement sur leurs concentrations totales, ce qui est étudié généralement.
Tout d'abord, les vins de Chardonnay utilisés ont été spécialement élaborés
METHODES &
pour
cette étude. Les essais ont consistés à la réalisation de 6 traitements
TECHNIQUES
différents, avec 3 répétitions par traitement, afin de comparer l'influence de
différentes concentrations de Cu et de Fe dans le vin. Des échantillons étaient
prélevés pour analyser et comparer les phases suivantes : le jus avant la
fermentation alcoolique, le vin à la fin de la fermentation alcoolique, le vin
avant et après le collage protéique et le vin embouteillé pour mesurer le taux
d’oxydation.
L’extraction en phase solide (SPE) a permis la séparation des espèces du
cuivre et du fer en 3 groupes - hydrophobe, résiduelles et cationiques. Ces
échantillons de vin ont ensuite été analysés par une spectrométrie
d'émission optique (ICP-OES). Cette approche a fourni des informations
détaillées sur comment une distribution différente de ces espèces dans le vin
pourrait influer sur son développement.
RESULTATS
Des résultats intéressants ont été obtenus qui ont montré que, par exemple,
CONCLUSIONS la fermentation alcoolique a été achevée plus rapidement dans les échantillons
contenant initialement plus de cuivre. Il a également été établi que la teneur la
plus importante de cuivre qui se trouve dans le vin après fermentation
alcoolique est sous la forme cationique et cela ne semble pas être affecté par la
concentration initiale de cuivre dans le jus. Pour le fer, il peut être indiqué que
les espèces cationiques sont les plus répandues dans tous les échantillons de
vin, et qu’elles représentent plus de 50% de la concentration totale. En plus,
l'application de la bentonite de calcium a révélé un impact significatif sur les
concentrations totales de cuivre et de fer et en même temps elle a modifié la
répartition des trois espèces de métaux. En ce qui concerne le processus
d'oxydation, une bonne corrélation a été découverte entre les fractions
résiduelles à la fois de cuivre et de fer et le taux d'oxydation, ce qui pourrait
indiquer que c'est probablement ces espèces de métaux qui sont responsables
de l'élaboration de ce processus.
Les résultats obtenus présentent des nouvelles possibilités d’étudier
l'influence de la spéciation de Cu et de Fe sur la vinification des vins blancs et
rouges.
PLAN
INDICATIF
Acknowledgements
First of all, I would like to thank Dr. Andrew Clark who has been my mentor, supervisor,
teacher, guide and friend from the first moment we started working together on this project. I
am forever grateful for his support and incredibly valuable advice on every step of the way.
I would also like to thank the National Wine and Grape Industry Centre for giving me the
opportunity to work besides their wonderful team, from whom I learned so much, and
especially Prof. Alain Deloire for making this possible.
To Campbell Meeks, whose assistance and great knowledge made the execution of the
project possible. Thank you for being my mentor into learning the tips and tricks of
Australian wines and winemaking.
To Dr. Leigh Schmidtke, whose expertise and devotion made the execution of this project
much easier and more enjoyable.
To everyone at the NWGIC, who accepted me as an equal from the first day and made my
stay so much more pleasant. Thank you for your kindness, hospitality and for always being
there for anything that I needed. You truly made me feel a part of your incredible team and
thus helped me develop new skills and expand my knowledge in a way I never thought
possible.
To all my friends in Australia, who quickly became a true family to me. Thank you for
accepting me as a friend and for introducing me to Australian culture and life, and for always
being there for me. This has been the most unique experience in my life so far and it wouldn’t
have been the same without you.
To Dr. Chantal Maury for her time, support and supervision, who made sure I stayed on
track every step of the way.
To my family who never stopped supporting me and who always believed in me, even in
those moments when everything went wrong. Knowing you are always there beside me made
me push myself even more and get the best out of every moment.
Table of contents
BIBLIOGRAPHIC NOTICE ..................................................................................... A
NOTICE BIBLIOGRAPHIQUE ............................................................................... B
Acknowledgements ...................................................................................................... C
Introduction .................................................................................................................. 1
The research centre .............................................................................................................. 1
Geographical situation ......................................................................................................... 2
NSW and its’ wine regions .................................................................................................. 3
Project background and missions ....................................................................................... 4
Problematic ........................................................................................................................... 7
Hypothesis ..................................................................................................................... 8
Main tasks ............................................................................................................................. 9
Metals in wine – a short review ................................................................................... 9
Introduction .......................................................................................................................... 9
Legal levels and limits .................................................................................................... 13
Copper in wine.................................................................................................................... 14
Iron in wine ......................................................................................................................... 17
Metal-induced spoilage ...................................................................................................... 19
Turbidity.......................................................................................................................... 19
Oxidation ......................................................................................................................... 21
Reductive process ........................................................................................................... 23
Bentonite ............................................................................................................................. 24
Metal removal ..................................................................................................................... 26
Methods for metal determination (Cu and Fe more specifically) in grape juice and
wine ...................................................................................................................................... 27
Fractionation and Speciation ......................................................................................... 30
Iron (Fe) speciation ..................................................................................................... 33
Copper (Cu) speciation ................................................................................................ 34
Materials and methods ............................................................................................... 36
Winemaking stage .............................................................................................................. 36
Analytical stage................................................................................................................... 38
Solid Phase Extraction (SPE) usage .............................................................................. 38
SPE column preparation ................................................................................................ 39
Instruments ......................................................................................................................... 41
DMA 35N Anton Paar GmbH portable density meter ............................................... 41
Robotic Sample Processor XL titrator and Flow Injection Analysis Sulfur Dioxide
Analyser ........................................................................................................................... 42
Flame Atomic Absorption Spectroscopy (FAAS) ........................................................ 42
Inductively coupled plasma optical emission spectrometry (ICP-OES).................... 42
High Performance Liquid Chromatography (HPLC) ................................................ 44
Konelab™ 20 Clinical Chemistry Analyzer .................................................................. 44
Oxygen decay analysis by luminescence analysis (Fibox 3 LCD trace) ..................... 44
Reagents and solutions ....................................................................................................... 45
Results and discussions .............................................................................................. 47
Wine production ................................................................................................................. 47
Grape juice – treatment and measurements ................................................................ 47
Alcoholic fermentation ................................................................................................... 48
Wine composition ........................................................................................................... 50
Copper and Iron speciation ............................................................................................... 51
Optimisation of methodology and approach ................................................................ 51
Metal concentration in grape juice ............................................................................... 52
Copper and iron speciation in wine .............................................................................. 54
Copper speciation ......................................................................................................... 55
Iron speciation.............................................................................................................. 58
Copper and iron speciation in wine before and after bentonite fining ......................... 60
Copper speciation and influence of bentonite fining ................................................... 61
Iron speciation and influence of bentonite fining ........................................................ 63
Impact of copper and iron speciation in wine on the oxygen decay rate ...................... 65
Influence of copper speciation on oxidation ................................................................. 67
Influence of iron speciation on oxidation ..................................................................... 68
Influence of bentonite addition on oxidation ................................................................... 70
Limitations .................................................................................................................. 72
Conclusion and future scope of work ....................................................................... 72
Conclusion ........................................................................................................................... 72
Future scope of work.......................................................................................................... 74
Bibliography ................................................................................................................ 75
Appendix A: Temperature and sugar concentrations during alcoholic
fermentation ................................................................................................................ 89
Appendix B: Measurements for all wine samples (T1-T6) by High performance
liquid chromatography (HPLC) ................................................................................ 91
Appendix C: Speciation Procedure Protocol ........................................................... 92
Appendix D: Work programme ................................................................................ 94
D.1: Winemaking................................................................................................................ 94
D.2: Part 1 ........................................................................................................................... 95
D.3: Part 2 ........................................................................................................................... 96
D.4: Part 3 ........................................................................................................................... 97
Figures
Figure 1: Scheme of the metal speciation procedure for both copper and iron
samples .................................................................................................................................... 41
Figure 2: Density measurements taken during alcoholic fermentation in ⁰Baumé. .. 49
Figure 3 : Calibration graph for iron in grape juice. .................................................... 52
Figure 4 : Calibration graph for copper in grape juice. ............................................... 53
Figure 5 : Copper and iron concentrations [mg/L] in wine after alcoholic
fermentation. Error bars indicate the standard deviation. ................................................ 54
Figure 6 : Copper and iron concentrations [mg/L] in grape juice after metal
additions. Error bars indicate the standard deviation. ...................................................... 54
Figure 7: Copper speciation concentrations [mg/L] for all samples (T1-T6). Error
bars indicate the standard deviation. ................................................................................... 56
Figure 8 : Copper fractions in wine as percentage of the total concentration. Error
bars indicate the standard deviation. ................................................................................... 57
Figure 9: Iron speciation concentrations [mg/L] for all samples (T1-T6). Error bars
indicate the standard deviation............................................................................................. 58
Figure 10: Iron fractions in wine as percentage of the total concentration. Error bars
indicate the standard deviation............................................................................................. 59
Figure 11: Copper speciation as percentage of total [%] before and after bentonite
addition. Error bars indicate the standard deviation. ........................................................ 62
Figure 12: Iron speciation as percentage of total [%] before and after bentonite
addition. Error bars indicate the standard deviation. ........................................................ 65
Figure 13: Correlation between the oxygen decay rate [1/min] and the total copper
concentrations [mg/L]. ........................................................................................................... 67
Figure 14: Correlation between the oxygen decay rate [first order] and the
hydrophobic, residual and cationic copper fractions [mg/L] in the wine. ........................ 68
Figure 15: Correlation between the oxygen decay rate [first order] and the total iron
concentration [mg/L]. ............................................................................................................ 69
Figure 16: Correlation between the oxygen decay rate [first order] and the
hydrophobic, residual and cationic iron fractions [mg/L] in the wine.............................. 69
Tables
Table 1 Annual summary of climate components for Wagga Wagga in 2011 ............. 2
Table 2: Maximum acceptable limits for metal ions, according to OIV regulation... 14
Table 3: Chemical elements and their wavelength [nm] determined by the 710-ES
ICP-OES ................................................................................................................................. 43
Table 4: Copper concentrations in grape juice on average for each treatment before
and after metal additions [mg/L]. The uncertainty indicated is the standard deviation. 48
Table 5: Iron concentrations in grape juice on average for each treatment before and
after metal additions [mg/L]. The uncertainty indicated is the standard deviation. ....... 48
Table 6: Sugar measurements for fructose and glucose in wine samples [mg/L]. The
uncertainty indicated is the standard deviation. ................................................................. 50
Table 7: Titratable acidity (TA), pH, free SO2 (FSO2) and total SO2 (TSO2)
measurements for all wine samples after the end of the alcoholic fermentation. The
uncertainty indicated is the standard deviation. ................................................................. 51
Table 8: Total and speciation copper concentrations [mg/L] in wine all samples (T1T6). The uncertainty indicated is the standard deviation. Means with the same letters
are not significantly different (SD) at p=0.05. ..................................................................... 55
Table 9: Total and speciation iron concentrations [mg/L] in wine all samples (T1T6). The uncertainty indicated is the standard deviation. Means with the same letters
are not significantly different (SD) at p=0.05. ..................................................................... 58
Table 10: Copper speciation concentrations [mg/L] before and after bentonite
addition. The uncertainty indicated is the standard deviation. ......................................... 61
Table 11: Iron speciation concentrations [mg/L] before and after bentonite addition.
The uncertainty indicated is the standard deviation .......................................................... 63
Table 12: The first order oxygen decay rates presented in [1/min] for all samples
(T1-T6). Means with the same letters are not significantly different (SD) at p=0.05. ..... 66
Table 13 : Comparison between oxygen decay rates [first order] of wines before and
after bentonite addition. Means with the same letters are not significantly different (SD)
at p=0.05. ................................................................................................................................. 70
Introduction
The research centre
The National Wine and Grape Industry Centre (NWGIC) is a research centre which
combines the efforts of three major Australian institutions – Charles Sturt University (CSU),
New South Wales Department of Primary Industries (DPI) and the New South Wales Wine
Industry Association (NSW WIA). It was created in 1997 and since then many experts from
different fields in the sector have joined the research team which is located in CSU’s campus
in Wagga Wagga, NSW.
Nowadays the team at NWGIC includes a number of well-known researchers who have
established themselves as leading researchers in Australia and Internationally in certain areas.
The Centre also has an ever-increasing group of young and promising researchers and PhD
students who contribute with their new ideas and high motivation to the constant
development and improvement of the work carried out in the Centre.
During the 17 years since its establishment, the NWGIC has increased significantly
parameters and themes of its work and it includes projects in the following fields:
1. Vine Health and Diseases
2. Wine Sciences (viticulture and oenology)
-
Vine physiology
-
Fruit and wine composition, chemistry
-
Wine making, wine styles and consumer preferences
-
Decision making tools (remote sensing, modelling, process)
3. Education, training and communication
The goal of the NWGIC is an integrated approach towards vineyard management and
wine making. This is to serve wine producers from the region in order to help them improve
the quality of their wines, so that they can enhance their positions in the increasingly
competitive Australian and international wine sector.
Geographical situation
Wagga Wagga is also referred to as “The Capital of Inland New South Wales" as it is the
largest populated centre in the interior of NSW and has become an important agricultural and
commercial centre in the Riverina region. The town is of significant strategic importance as it
is situated halfway between Sydney and Melbourne and at the same time the Murrumbidgee
River flows through it.
CSU has many different campuses and the one in Wagga Wagga is the largest covering
over 640 hectares of land. Diverse facilities have been built and are available to students and
staff, some of which include a campus farm, an equine centre, a vineyard, a winery and a
huge range of technical and industry standard facilities.
The climate is typically temperate. The summers are hot and dry with an average
temperature of 31.2oC in the hottest month of the year – January. In winter it is cold and the
minimum temperatures start from 16.2oC in February and drop down to 2.7oC in July. Frosts
during that season are a frequent sight, too. The region can be described as fairly dry with an
annual rainfall average of 580 mm, which is evenly distributed throughout the year at around
48 mm/month even during the summer (data from the Wagga Wagga Agricultural Institute1)
Table 1 Annual summary of climate components for Wagga Wagga in 20111
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Average/
Total
24
31
41
36
47
48
50
44
34
32
29
18
36
Max.
temp. [°C]
32.7
30
25.7
22
17
15
14
17
19
23
28.3
27.1
22.5
Min. temp. [°C]
17.4
17.2
13.9
9.2
4.3
3.5
3.3
5.2
4.9
8.7
13.1
12.8
9.5
Wind run
107
85
80
48
39
40
55
56
80
75
86
97
71
Pan
evaporation
[mm]
248
157
131
88
49
34
42
67
95
141
179
211
1441.1
Rainfall [mm]
44.4
158
51.4
27
27
26
32
41
25
19.8
148
65.2
664
2011, month
Humidity [%]
Source:
NSW
DPI
–
Primary
Industries
http://www.dpi.nsw.gov.au/research/centres/wagga/climate
1
Science
and
Research;
2
NSW and its’ wine regions
There are 14 separate wine regions in New South Wales. All of them are subject to very
particular climate and soil conditions setting them apart and giving the wines from each one
of them different and signature characteristics. Here we find one of the oldest wine regions in
Australia – Hunter valley – and at the same time a few more recently established cooler
climate regions – like Orange, for example, where the grapes for the current experiment come
from – which have been quickly gaining in popularity with their high quality wines.
Although we can find more than one grape varieties in every region, a typical variety has
been identified for each one of them which is said to have adapted best to the specific
conditions in the area. According to the New South Wales Wine Industry Association, the
most representative grapevine varieties for each of the regions are as follows:
1. Canberra District – Shiraz
2. Cowra – Chardonnay
3. Gundagai –Shiraz
4. Hastings River – Verdelho
5. Hilltops – Cabernet Sauvignon
6. Hunter Valley –Semillon
7. Mudgee – Cabernet Sauvignon
8. New England – Riesling
9. Orange – Sauvignon Blanc
Source:
NSWWIA’s
website
http://www.nswwine.com.au/pages/NSW-WineRegions.html
10. Perricoota – Shiraz
11. Riverina – Botrytis Semillon
12. Shoalhaven Coast – Chambourcin
13. Southern Highlands –Pinot Noir
14. Tumbarumba – Chardonnay
The number of wineries and grape growers have been steadily increasing in the region
over the last couple of year and the region is turning into one of the most dynamic in
3
Australia offering a wide range of good-quality wines. This is of course one of the main
reasons why there is a big demand for more knowledge about the wine-making process
amongst NSW’s wine producers who are continuously looking for ways to improve their
techniques and thus strengthen and improve their leading positions in Australia's fastdeveloping wine sector. With our work and research we are aiming to help them in their goals
and in this way support the regional wine industry.
Project background and missions
The focus of this project is the impact of several aspects of wine production on total metal
concentrations in wine, and also on the form in which the metal ions exist. The metals which
were focused upon were iron (Fe) and copper (Cu). This research was carried out on white
Chardonnay wines, produced specifically for the purposes of the study. Following and
controlling the development of the wine from the first until the last step of the production
process, allowed for a better and in-depth understanding of the final wine. This approach
enabled investigation of the different copper and iron fractions at several stages of the
production process, so as to assess how the metal fractions may be influenced by specific
winemaking techniques, i.e. protein stabilisation and fermentation. It also allowed an
assessment of the different metal forms on the rate of oxidation of the wines.
To begin with, it is important to note that the fast technological development in the wine
sector over the last 10-15 years has led to the use of a wide range of new equipment and
techniques both in the vineyard and in the cellars. Due to these innovations the risk of having
higher concentrations of Cu and Fe in wine due to contamination has decreased significantly.
However, increased knowledge on the roles of copper and iron in wine, even at lower
concentrations still necessitates their constant monitoring.
It has been reported on various occasions that metal ions present in wine can originate
from different sources such as the grapes, dust residues or contamination with winemaking
equipment or through winemaking additions (Ough and Amerine 1988; Laurie and
Waterhouse 2006; Ribéreau-Gayon, Glories et al. 2006). The copper content is reduced
significantly throughout the alcoholic fermentation, but depending on the initial metal
concentration this decrease may not be significant enough to protect the wine from problems
related to its stability. Metal ions can induce haze formation and changes in the wine’s
organoleptic properties (de Campos and Araújo 2001) and this is why it is important to
4
determine both the total concentration and the exact form in which the metals are present in
the wine.
The iron content, for example, may have a strong influence on the evolution of the wine
during fermentation. The significance of its impact may vary according to the level of
aeration of the ferments, the presence or absence of colouring agents, the extent to which the
iron interacts with the yeast, and the quantities which will attach to the sediments at the end
of the process (Olalla, González et al. 2000).
On the other hand, when copper is found in higher concentrations and especially in
combination with other metal ions which are normally present in the wine, such as iron,
manganese, zinc, nickel, lead and vanadium, it can provoke specific reactions leading to
considerable health risks for the consumer (Naughton and Petróczi 2008). What is more, it
has been established that copper plays a role in the accumulation of a wide range of
undesirable effects in wine and grape juice. Concentrations of around 5 mg/L or higher can
cause a metallic taste, haze, browning and other undesirable effects on wine (Li, Guo et al.
2008). Its presence in higher concentrations in the juice can even have an impact on the
alcoholic fermentation, as it has been reported on several occasions that it can inhibit the
growth and development of several groups of certain naturally-occurring microorganisms,
i.e.: Oenococcus oeni (5–10 mg/L) (Vidal, Poblet et al. 2001), Saccharomyces cerevisiae
(32–75 mg/L)(de Oliva-Neto and Yokoya 2001; Brandolini, Tedeschi et al. 2002),
Lactobacillus fermentum (75–300 mg/L) (de Oliva-Neto and Yokoya 2001) or Lactobacillus
mesenteroides (55–150 mg/L) (de Oliva-Neto and Yokoya 2001).
The investigation of the behaviour of copper during and after the fermentation process has
been of particular interest to researchers in the last years. Nonetheless, most studies have
focused on assessing the impact of different total concentrations of the metal. It is, however,
the chemical form, not the total concentration of the element, that will generally determine
and influence the spoilage potential of the wine. In their research Green, Clark and Scollary
(1997) have suggested that wine spoilage can occur even at very low copper concentrations.
Other sources also outline that copper can be stable (i.e. remain stable) in very acid aqueous
conditions – at pH=1 – and that the pH, age and the organic matter present in wine can also
make for a more stable wine (Arcos, Ancin et al. 1993). In addition, some other components
5
of the wine such as tannins, peptides and proteins take part in the formation of copper
complexes in white wine (Wiese and Schwedt 1997).
To sum up, all of the reactions stated above have been related to the increased levels of
iron and copper in wine and/ or grape juice. These types of spoilage are very common and
thus very often additional actions are required to be taken in order to protect the wine and
ensure the good quality and health safety of the final product. The goal of the current project
is to execute a profound research on how alcoholic fermentation and protein stabilisation
affects the presence of copper and iron, and how these metal ions impact the oxidation of
wine after bottling. What is more, the focus will be to look into not only the final and total
concentrations of the metals, but also certain forms in which they are found in the wine. This
is an important aspect of the study, as researchers have often speculated that certain
undesirable reactions in wine and juice may be caused by different forms of the same
chemical element, and thus alter the chemical and physical properties of the final product
(Wiese and Schwedt 1997).
The development of wine has been strongly linked to its copper and iron concentrations:
-
A major problem in the past was the formation of hazes when the metal
concentrations were particularly high – around 5 mg/L for copper (Li, Guo et al.
2008) and ≤4 mg/L for iron (Jackson 2008). The use of stainless steel equipment
and smaller amounts of chemical products for grapevine treatment has
significantly limited the occurrence of this spoilage.
-
In the presence of higher oxygen concentrations, both copper and iron play the
role of main catalysts for the consumption of oxygen and thus alter the wine’s
character through a process known as oxidation (Clark and Scollary 2002).
-
When the aeration of wine is limited and there are only low concentrations of
oxygen available during its storage especially after bottling, copper and iron can
catalyse the production of hydrogen sulfide (H2S) which is responsible for the
formation of a distinct rotten egg aroma in wine as well as the loss of varietal/fruit
aromas (Viviers, Smith et al. 2014).
These problems have been studied for total copper and iron concentrations. Little work
has been performed linking different forms of metal ions to these spoilage phenomena. By
6
identifying and quantifying the forms of copper and iron in relation to the winemaking
process and the properties of the final wines, valuable information will be obtained about the
possible spoilage that different forms of the metal ions may lead to in wine. A number of
studies have already been carried out on metal speciation, but there is still a need for further
knowledge about how and at what stage of the process do specific metal ion species interact
with the wine. This information will provide winemakers in Australia and internationally
with more knowledge about their wine product and thus give them the possibility of having
better control over the quality and organoleptic characteristics of the final product.
Problematic:
Do the alcoholic fermentation and bentonite
fining influence the speciation of iron and copper in
white wine and does this speciation have an impact
on the oxidation rate of wine after bottling?
7
Hypothesis
Copper and Iron Speciation in white wine:
Impact on wine oxidation and influence of protein fining and initial copper and iron juice concentrations
Hypothesis 1 (Part 1):
Do initial copper
and iron
concentrations in white
grape juice impact on
copper and iron
speciation?
Does protein fining
with calcium bentonite
impact on copper and
iron speciation in
white wine?
Does copper and
iron speciation impact
on the rate of white
wine oxidation?
Yes
No
Yes
No
Yes
No
8
Main tasks
1. Produce white Chardonnay wine, divided in 6 different treatments/samples. Monitor
the development of the wines through the alcoholic fermentation and account for any
possible changes and differences between treatments that might be due to the specific
copper and iron concentrations of each sample.
2. Perform protein stabilisation on half of the volume for all treatments and carry out
thorough analysis on wines before and after this procedure. Compare results in order
to establish any differences caused by the specific copper and iron speciation of the
wine samples.
3. Monitor and carefully measure the oxidation process and its rate in all wine samples
after bottling. Register and compared gathered data and relate the results to specific
the copper and iron speciation of the separate treatments.
Metals in wine – a short review
Introduction
Since early ancient times it has been recorded that fermented grape juice has been a major
component in the human diet. Its pleasant taste and beneficial health characteristics
contribute to the fact that it still is a preferred alcoholic beverage amongst an increasing
number of people all around the world (OIV 2012). This interest in wine has lead scientists
from different fields to investigate every aspect of wine – from the fresh grapes throughout
the wine-making process and all the way to the content and composition of the final product.
The aim is to become more familiar with the product, so as to be able to control and predict
the outcome and reach an envisioned and desired goal.
Nowadays consumers have become more demanding than ever when it comes to the
quality and taste of wines which makes it that much more important for producers to know
how their wines are developing during and after wine production and to make sure they meet
all the health and quality standards required. This is why there is a need for further and more
detailed information about what actions need to be taken to avoid any spoilage or undesirable
characteristics from developing in the wine. What is more, this information needs to be
9
presented to the producers in a clear and well-structured manner, so that they could easily
apply it whenever they need to.
The aim of this project is to carry out a detailed study of one group of components found
in wine – metals. The focus will be on copper (Cu) and iron (Fe) and their specific forms in
wine as they are usually found in significant concentrations in both the grape juice and the
wine and can influence the character and properties of the final product. These two elements
have been reported on various accounts to affect the wine’s quality and characteristics
throughout different stages of the wine-making process (de Oliva-Neto and Yokoya 2001;
Vidal, Poblet et al. 2001; Brandolini, Tedeschi et al. 2002), but also during its storage and
transportation (Danilewicz and Wallbridge 2010; Viviers, Smith et al. 2013).
It is considered that there are two possible origins of metals in wine – from natural
environmental sources, and from contamination due to the techniques and procedures applied
during the production process. Firstly, they are naturally accumulated by the plant from the
soil through the roots. The environmental conditions, as well as the grape variety, the
maturity of the grapes and their current condition also affect the concentration of these
elements in the grapes. This is considered to be a typical characteristic for each wine and has
been previously used to determine and differentiate wines from different locations (Gonzalez,
Gonzalez et al. 1987). Some metals, including heavy metals such as copper (Cu), manganese
(Mn), zinc (Zn) and others are indispensable for the healthy growth and development of both
plants and animals in small quantities. They are most often referred to as “trace elements”. If
found in higher doses, however, they become toxic and can cause severe health problems.
Another source of metals in the wine is the atmospheric deposition of airborne particulate
matter on grapes (Pyrzynska 2007; Woldemariam and Chandravanshi 2011). This however
accounts for only a small part of the total metal content. Taking into consideration the
technological innovations and constant improvements of the techniques applied by
winemakers we can assume that a significant part of the metals which can be found in the
grapes and thus in the grape juice and the wine come from the equipment utilised and the
addition of products during the entire process – from the grape-growing to the winemaking
and finally the bottling and storage.
Being aware of the variety and quantity of the metal ions present in the wine is an
important aspect of winemaking. Some of them can have important effects on the
10
organoleptic properties of the wine and certain play an essential role in oxidative–reductive
reactions resulting in wine browning, turbidity, cloudiness, and astringency (Pohl 2007;
Pyrzynska 2007; Li, Guo et al. 2008). On the one hand, elements such as aluminium (Al),
copper (Cu), iron (Fe), manganese (Mn), nickel (Ni) and zinc (Zn) have been thoroughly
studied and it has been shown that they can influence the wines by the formation of haze and/
or by inducing some undesirable changes of aroma, colour and taste through oxidationreductive aromas. On the other hand, some of the elements – copper (Cu) and zinc (Zn), for
example – are also of interest for numerous researchers regarding their toxicity in case of
excess concentrations in the wine. These elements are essential for certain physiological
processes in the human body and thus a requirement in a balanced diet, but at the same time
they are potentially toxic if consumed in higher concentrations (Pyrzynska 2007). There are
some other metals which don’t have any nutritional value, but have been found to be highly
toxic and have a serious effect on human health (Galani-Nikolakaki, Kallithrakas-Kontos et
al. 2002). In the case of wine some of those elements are arsenic (As), cadmium (Cd), lead
(Pb), etc.
In the long term high concentrations of heavy metals have a negative effect on the
environment as they can be responsible for polluting water sources and soils. In viticulture
there are a series of pesticides, fungicides and fertilizers which are widely used and contain
cadmium, copper, manganese, lead and zinc compounds. For copper in particular, the main
source of contamination, apart from the direct contact with some Cu-containing wine-making
equipment, are the chemical compounds used to protect the plants and later to treat the wine
(Tariba 2011). Copper sulfate (CuSO4) is one of the most common chemical products in
viticulture and it is widely used against downy mildew (Catarino, Pimentel et al. 2005).
However, copper is one of the trace elements which the yeast need in order to go through the
alcoholic fermentation. In fact, the major part of copper in juice will not make it to wine as
copper, in presence of yeast, sediments in the forms insoluble sulfides and is removed
together with the yeast lees (Kristl, Veber et al. 2003). After fermentation some residual
yeast-derived organic sulfur compounds may exist and can contribute to some undesirable
off-putting aromas in the wine. To eliminate these compounds copper in the form of copper
sulfate penthahydrate (CuSO4.5H2O) can be added to the wine to remove the sulfide aroma,
but again increases the total concentration of the element.
11
Chromium (Cr) is another element which has come to the attention of researchers in the
last years due to the fact that it was discovered that its levels increase with the ageing of the
wine. There are a few possible sources for chromium some of which include contamination
from the stainless steel equipment used for the last stages of the wine-making process before
bottling or even from some chromium oxides which are used to give some bottles colour
(Volpe, La Cara et al. 2009).
The iron (Fe) content in the grape juice is in a closely related to the specific pedoclimatic
conditions of their area of origin. It could be accumulated by the plant via its roots or it can
enter the berries if they are covered by iron dust. Wines with increased concentration of Fe
have been reported to be unstable in the long term and could result in certain problems during
storage after bottling (Galani-Nikolakaki, Kallithrakas-Kontos et al. 2002).
There are a few possible sources of lead (Pb) in the wine, including the natural Pb found
in the eco-system and some extracted during winemaking techniques or from enological
products. In some research the levels of Pb found in wine have been related to the use of
leaded gasoline, tin-lead capsules and brass alloys. It is of essential important to strictly
control the concentration of lead (Pb) in the wine because of its possible toxic effect on
human health (Teissedre, Cabanis et al. 1994; Kaufmann 1998).
The concentration of some metals depends on the specific environmental conditions to
which the grapevine is exposed during the last stages of the vegetative period. Potassium (K)
and calcium (Ca) are two of the most common elements from this group which can influence
the wine. K is a macroelement and thus is required in higher concentration by the plant
during its growing stages. If found in excess concentrations in the wine it can precipitate as
potassium hydrogen tartrate (KC4H5O6) in bottle forming insoluble crystals(Farkaš 1988).
Calcium can also precipitate in the form of calcium tartrate (CaC4H4O6) and cause spoilage of
the wine. This problem most often arises after the use of calcium carbonate (CaCO3) in
deacidification, but can also be a result from the use of cement cooperage, filter pads and
fining agents (Jackson 2008).
There are several different possible sources of aluminium (Al) in wine. Higher aluminium
concentrations can come from aluminium compounds which are present in the soil or can be
a result of excessive use of certain pesticides on the plants. Storing the wines in aluminium
tanks or using aluminium-based bentonite to stabilise them before bottling are some of the
12
most probable causes for higher levels of the metal in the final product (Larroque, Cabanis et
al. 1994). Concentrations above 3 mg/L have been reported to cause a cloudiness effect in the
wine which is referred to as “aluminium haze” (Kelly and Blaise 2006).
Manganese (Mn) is another metal ion which is said to play a role in the oxidation of wines
in combination with iron and copper. A few studies have discovered that Mn(II) ions provoke
the formation of acetaldehyde, which can polymerize phenolic materials and hence change
the sensory qualities of the (Cacho, Castells et al. 1995; Benítez, Castro et al. 2002).
Cadmium (Cd) is associated to the group of “heavy metals” and it is toxic to human
health even in low concentrations. The contamination comes from air and water, but its main
source is actually food, because significant doses can be accumulated in the food chain. The
effect it has in the organism is that it blocks some enzymes, which may lead to kidney and
metabolic malfunctions (Ribéreau-Gayon, Glories et al. 2006).
Zinc (Zn) is a vital element for plant health because it is involved in plant growth and
more specifically the auxin metabolism. A deficiency slows down the plant development and
can also affect the colour and distribution of the leaves. Elevated zinc concentrations in the
must may originate from specific dithiocarbamate-based fungicides applied in the vineyard.
In mechanically managed vineyards damaged iron wires can galvanize and thus account for
some zinc in the plants. The winemaking equipment is the other likely source of
contamination. A study on some Australian wines revealed base metal concentrations of zinc
of 1 mg/L in white Chardonnay wines and 1.2 mg/L in red wines from Shiraz (Viviers, Smith
et al. 2013). Along with some other metals – aluminium, manganese, copper, iron, etc. – zinc
also contributes to the formation of hazes in the wine and an alteration in its sensory
characteristics (Tariba 2011).
Legal levels and limits
Controlling the levels of all the compounds of wine, including metals, is an important part
of the production process. Many of them can be harmful for human health above certain
limits which are often referred to as “Maximum Residue Levels” (MRL) or “Maximum
Acceptable Levels” (MAL). For the moment there isn’t a universal regulation regarding the
acceptable levels of active substances found in wine which has been accepted and is
applicable worldwide. In most cases each country imposes rules and defines the maximum
13
concentrations for a number of elements and compounds in order to restrict their content in
the final product.
Office International de la Vigne et du Vin (OIV) which is an international research and
regulation centre based in France, has established a chart with the MAL in wine for a variety
of compounds. Some of the most important amongst the metallic ions are:
Table 2: Maximum acceptable limits for metal ions, according to OIV regulation
Element
Arsenic (As)
Cadmium (Cr)
Copper (Cu)
Lead (Pb)
Zinc (Zn)
Maximum Acceptable
Limit [mg/L]
0.2
0.1
1.0
2 mg/l for liqueur
wines produced from
unfermented or
slightly fermented
grape must
0.15
5
Source: OIV Code Sheet – Issue 2012/01; European Union Regulation, 1990
For the moment there is no MAL for the levels of iron in the wine, but there are strict
regulations as to the amount of iron products used in the vineyard and in the winery
throughout the winemaking process. The aim is to make sure the final iron content in the
wine remains in reasonable levels, so that the recommended daily intake of iron for humans
doesn’t exceed 14 mg, which is the amount stated in the European Regulation No 1169/2011
from 25 October 2011. To place this amount in context, in a research on the iron content in
Spanish wines and grape juice, Olalla et al. (2000) found that the concentration of the metal
varies from 3 ± 2 mg/L in the grape juice and 3 ± 1 mg/L in white wines(Olalla, González et
al. 2000).
Copper in wine
There are certain metals which are very important for humans and other animals, because
they play an essential role in diverse processes. Copper is one of those elements and is
reported to be the third most abundant metal element in living organisms. It is a component
of more than 30 enzymes in the human body – collagen synthesis, for example – and it is
involved in the metabolism of both iron and energy (Arredondo and Núñez 2005). Even
14
though copper is a major and much-needed component for humans, it can also become toxic
to living cells if present in higher concentrations. Research on the subject reveal that it can
bind to thiol moieties or catalyse a Fenton reaction resulting in the production of a hydroxyl
radical which can potentially cause cellular damage (Presta and Stillman 1997).
There are some agricultural techniques in the vineyard which include the use of copper
compounds, and can result in a higher level of copper residues in the grapes at harvest
(Wightwick, Mollah et al. 2008). This can affect the fermentation process by slowing down
or even completely stopping the yeast activity and thus leading to a stuck fermentation
(Brandolini, Tedeschi et al. 2002). Although higher copper concentrations in the soil result in
an increase of the copper levels in berries with a more significant accumulation being
observed from Chardonnay white grapes than other Italian red varieties (Provenzano, El
Bilali et al. 2010), the final copper in the wines was not at levels harmful to human health. In
other research carried out by Pessanha et al. (2010) almost no changes in the copper
concentrations were found in the grapes, must or the wine from plants with a higher
concentration in their leaves due to the sprays applied during the vegetation season
(Pessanha, Carvalho et al. 2010).
Although chemical products can lead to an increase in the copper content of the grape
juice, it must not be forgotten that there is a significant decrease of the final concentrations
found in the wine because of the quantities used by the yeast cells during the alcoholic
fermentation (Scollary 1997). Taking this into account, we can suppose that the key source of
copper in the final product is often related to the wine-making techniques and products. The
removal of hydrogen sulfide (H2S) by adding copper sulfate (CuSO4) to the wine is the main
source of high residual copper. Such a misbalance in the wine content can contribute to a
stronger oxidative spoilage, which in white wines is the cause for a particular “browning”
effect. This phenomenon can be activated by certain metallic ions, such as copper, iron or
manganese. When it occurs, wines have been found to not only appear brown in colour but
also lose some of their aromatic compounds, thus changing the organoleptic characteristics of
the wine. After a certain amount of time condensed phenolic materials will eventually start to
precipitate in the bottle as a result of long oxidation process. In order to avoid such faults the
copper(II) content in the bottled wine should remain below 0.3 mg/L as higher levels has
been found to cause excessive oxidative colouration in model white wine. In the model white
wine where 0.3 mg/L copper(II) was added, there was a significant shift in the colour towards
15
brown after 29 days, but no precipitation was observed and the model wine didn’t become
cloudy. There was almost no difference in the “browning” which developed in the wines
without any addition and those with 0.1 mg/L copper(II) added (Clark and Scollary 2002),
which suggests that a low copper(II) concentration doesn’t accelerate the oxidative
colouration process.
What is more, it isn’t only the final copper concentration in the wine, which is important,
but it is also the levels found in the grape juice before the fermentation that influence the
development and outcome of the fermentation. Copper is an important ingredient for the
growth of Saccharomyces cerevisiae yeast (Brandolini, Tedeschi et al. 2002). If certain levels
of copper are reached in the juice, it has been observed that the element can slow down the
activity of the yeast and thus become a limiting factor strongly related to the specific strain of
Saccharomyces cerevisiae used (Serrano, Bernal et al. 2004). Copper resistance is recognised
as a characteristic of the yeast and is believed to be related to the environmental conditions to
which they are exposed. In a study carried out by Milanović in 2009/2011 it was discovered
that applying copper and sulfur (S) fungicides in the vineyard had a stronger inhibitory effect
on the diversity and abundance of all the yeast found on the berry including Saccharomyces
cerevisiae (Milanović 2012).
In another study Brandolini et al. (2002) studied two strains of Saccharomyces cerevisiae
– one which was described as copper sensitive and the other one as resistant. Their results
clearly showed how the sensitive strain slowed down its fermentative activity after an
addition of 32 ppm of copper(II) to the must and was completely inhibited at a higher copper
concentration - 320 ppm. On the other hand, the resistant strain showed almost no difference
in its performance when comparing the control (without any addition) to the juice with 32
ppm copper, and a slower but still complete fermentation when the concentrations of the
metal was highest in the must. This difference in the fermentation between the two yeast
strains was also related to the amount of copper which was accumulated during the process.
Although the sensitive strain accumulated the copper naturally present in the must (the
control), it wasn’t able to do so in the presence of both higher copper levels. The resistant
strain showed very good accumulative capabilities reducing the copper from 35 to 3.4 ppm
and from 323 to 193 ppm of copper respectively in the first and second additions taking into
account the concentrations at the beginning and the end of the alcoholic fermentation
(Brandolini, Tedeschi et al. 2002).
16
The final concentration of copper in the wine may be reduced if it can react with the
hydrogen sulfide (H2S) in wine and form a black insoluble precipitate. This is more likely to
happen when copper(II) sulfate (CuSO4) is actually added to the wine in order to eliminate
some off-aromas caused by hydrogen sulfide (H2S). This addition of copper(II) to the wine
can cause excess levels of the element in final product, which can lead to turbidity (RibéreauGayon, Glories et al. 2006).
In wines which are in contact with the air, copper is mostly found in an oxidized form –
Cu2+. Over a longer period of storage when there is very little and sometimes almost no air in
the tanks and bottles a copper casse may form. At a certain point when the cation’s oxidationreduction potential drops and in the presence of sulfur dioxide (SO2), the divalent cation is
reduced to monovalent – Cu+. These conditions initiate the turbidity associated with the
copper casse fault. The development of the phenomenon is accelerated by high temperature
and bright light (Ribéreau-Gayon, Glories et al. 2006).
Iron in wine
The iron content in wines originates from either from the grapes or through a
contamination during the production process, transportation and/ or storage when the wine
comes in contact with iron containing alloys. Iron can accelerate oxidation and at low
concentrations this may be a desired effect which can help the wine develop new sensory
characteristics. However, iron can also form complexes with tannins and phosphates, which
leads to haze formation, known as “iron casse”.
As early as the beginning of the 20th century scientists have been interested in the levels
of iron in grapes, must and wine and have been investigating the effect they could have in the
wine. This is because there are certain amounts of iron in the grapes – which can significantly
vary – that will eventually make their way into the must (Byrne, Saywell et al. 1937). Both
ion forms of iron – ferrous (Fe2+) and ferric (Fe3+) – are found in wine and the ratio between
them is influenced by the amount of free sulfur dioxide (SO2) and the storage conditions
(Ribéreau-Gayon, Glories et al. 2006)
In grapes, iron is normally a part of certain enzymes, but some climatic and
environmental conditions can alter its concentration. Some of these include the stage of
maturity of the grape, the soil type, soil pH and chemical residues in the soil, climate changes
17
and additional sources of contamination in the vicinity of the vineyard (Fernández 1988). An
increase in the levels of iron in the must can be a result of some of the technical processes
during the winemaking. One which is considered to be the most significant is the pressing of
the grapes, but some iron can be accumulated in the wine during its storage in certain metal
containers or could even come from the metal tapes which cover the cork if it doesn’t seal the
bottle completely. Iron salts are also present in the coloured bottles which are often used to
protect the wine from ultraviolet rays and can also contribute to high final concentrations of
the metal (Fernández 1988; Etiévant, Schlich et al. 2006).
As with many other metals, iron (Fe) is also an essential and necessary element for the
growth and activity of Saccharomyces cerevisiae during the alcoholic fermentation. The yeast
is known to be able to survive in environments diverse in their content of bioavailable iron.
Research carried out in 2012 revealed surprising information about the ability of the wine
yeast to grow in conditions of excess iron. During their work they observed that the
microorganisms actually grew faster when in an environment considered as an “iron-surplus
medium”. Their trials included FeCl3 additions of 0, 28, 56, 112, 280 mg/L and the higher the
iron concentration, the more active the yeast. It was concluded that excess iron concentration
enhanced the cell growth and didn’t cause the otherwise expected metal stress. The growth
rate of the yeast increased in these conditions as they were able to perform aerobic
respiration(Du, Cheng et al. 2012).
A ferric casse can occur after aeration of the wine and can be explained with the reaction
between Fe3+ and phosphates (PO43−) which forms an unstable colloid. Afterwards, it
interacts with some proteins in the wine, flocculates and precipitates. The precipitate has an
intense white colour, which is why this phenomenon is also known as “white casse”
(Ribéreau-Gayon, Glories et al. 2006). This reaction is possible only in high acid media and
in the presence of higher levels of phosphoric acid, which makes it white-wine-specific. In
the “Handbook of Enology, Volume 2” the authors suggest that knowing the total
concentration of the metal is not the only factor for determining whether or not there is a risk
of casse. They explain that turbidity has been observed in wine with 6-8 mg/L iron content,
and at the same time none has occurred in other wines with concentrations of 25 mg/L
(Ribéreau-Gayon, Glories et al. 2006).
18
In order to protect the wine from a ferric casse it is possible to increase the ferric (Fe3+)
ions in soluble complexes by the addition of citric acid (C6H8O7), or to decrease the Fe3+ total
levels by adding ascorbic acid (C6H8O6). Another option is to add gum arabic, which will act
as a protection and stop the precipitation of the ferric colloid. The other type of haze which is
induced by Fe3+ is known as “blue casse”. It involves Fe3+ complexing and precipitating with
phenolic tannin material (Ribéreau-Gayon, Glories et al. 2006)
Iron (Fe) is also involved in wine oxidation and has been found to have a contribution to
diverse stages throughout the process. Some researchers propose that it is the relation
between iron (Fe) and tartaric and malic acids which determines the redox capabilities of the
ion by altering the reduction potential of the couple ferric (Fe3+) to ferrous (Fe2+) ions in
connection to that of the couple between oxygen (O2) and catechol (C6H4(OH)2) (Danilewicz
2013). The catalytic action of metal ions during wine oxidation reactions can lead to a
“browning” of the wine during ageing which is one of the major problems experienced by
winemakers. It is important to predict and take precautions of protecting the wine from this
spoilage in order to avoid changes in both colour and sensory characteristics (Li, Guo et al.
2008).
Metal-induced spoilage
Turbidity
Diverse chemical reactions occur in the wine during all stages of its production from the
crushing of the grapes throughout the alcoholic fermentation and then during the storage
whether in tanks or bottles.
Not all reactions are, however, favourable to the wine
characteristics and thus may have an undesirable influence on its quality. They may lead to
spoilage in the wine and certain steps and actions should be taken in order to prevent, reduce
or eliminate their effects.
Metal ions, organic acids and phenolic compounds are found in abundance in all types
and styles of wine and put under specific conditions, which include exposure to light, contact
with oxygen (O2), temperature and other variable parameters, can be responsible for a
negative alteration in the colour and flavour of wines.
One of the essential characteristics of wine, which is of significant importance to wine
consumers, is its clarity. It is even more important for white wines which are traditionally
19
stored in lightly coloured or transparent bottles and any alterations during storage are easily
visible. For example, a haze can result due to particles in suspension in the wine. Since
naturally there is a high amount of particles found in young wines originating from the lees or
some other small grape debris there is a variety of winemaking techniques which are applied
to clarify the wines such as racking, filtration, and centrifugation (Ribéreau-Gayon, Glories et
al. 2006).
Metals have been found to generate irreversible turbidities and sedimentation in some
alcoholic beverages, including wine (Tariba 2011). There is a group of factors which
influence this process which are not limited to the metal content of the wine, but include
some other naturally present wine compounds, the pH, the temperature during the
winemaking process and storage and the oxygen availability (Cacho, Castells et al. 1995;
Scollary 1997; Viviers, Smith et al. 2013). Even though for the moment overall limits in
regards to the metal concentrations in the wine and other alcoholic beverages are not
available, most often it has been observed that turbidity increases with metal concentrations,
especially above a certain threshold (Ibanez, Carreon-Alvarez et al. 2008). There already are
certain metallic ions such as copper, aluminium, iron, zinc, nickel, and tin, which have been
related to haze formation during the wine fermentation and storage (Galani-Nikolakaki,
Kallithrakas-Kontos et al. 2002; Ibanez, Carreon-Alvarez et al. 2008).
Turbidity can be explained as a result of the conditions under which some particles in the
wine increase in size and either flocculate or sediment. In the “Handbook of Enology Volume
2: The Chemistry of Wine Stabilisation and Treatments” the authors have presented the main
aspects in practical winemaking which can lead to the appearance of this colloidal
phenomenon:
-
Clarification and limpidity; Metallic precipitation (ferric casse and copper casse);
Protein turbidity of white wines and bentonite treatment; Precipitation of colloidal
colouring matter in red wines; Fining wine; Involvement of protective colloids in
clarification problems and the tartrate precipitation mechanism; Treating wines with
gum arabic(Ribéreau-Gayon, Glories et al. 2006).
It was outlined in the two previous sections of this literature review that both copper and
iron contribute to the formation of a haze in wine under specific conditions. In order to
prevent either of these spoilages from affecting the wine it is important that the winemaker
20
takes prevention and limitation actions. The most effective approach to protecting the wine
from a metallic casse is to limit the metal concentrations left in the wine after the alcoholic
fermentation. Both copper and iron have been found to have negative effects on the wine
when present in higher concentration (Zoecklein, Gump et al. 1999). Some of these include
cupric and ferric cloudiness and unpleasant and astringent taste. Ibanez et al. (2008) found
that as little as 3 mg/L of copper can cause significant turbidity in the wine after five months
of storage in several alcoholic beverages (Ibanez, Carreon-Alvarez et al. 2008). These
reactions were attributed to the reaction between the copper cations and the hydrogen-sulfide
(HS) groups from denaturated wine proteins. Some other sources recommend keeping the
copper concentration in the wine even lower to ensure a reasonable wine shelf life. A
concentration below 0.3–0.5 mg/L are referred to as “safe” with respect to the contribution of
the metal to wine oxidation(Rankine 1991).
In regards to the iron content of wines, the frequency of the so-called “white casse” has
been reported to increase significantly at concentration around and above 15-20 mg/L.
However, it is still hard to outline any reference levels for the appearance of the other
possible iron-induced fault in wine – “blue casse” (Jackson 2008).
The recent development of the viticultural and oenological techniques implemented by
wine producers have had a major influence on lowering the total copper and iron content in
wines. Thanks to the use of mainly stainless steel equipment and to the optimisation and thus
decrease in the number and concentrations of the chemical products applied in the vineyard
or added to the wine, winemakers have been able to produce wines which are much more
stable with respect to metal haze formation and require little or no further metal-reduction
treatment. This is most certainly the best and preferred way to protect the wines from
experiencing any metal-induced spoilage during their storage.
Oxidation
Winemaking is a long process which involves a series of techniques and manipulations
during which the wine can come in contact with oxygen which reacts with certain compounds
in the wine. During the ageing of the wine the level of oxidation can be monitored and
controlled by the winemaker. Ageing in barrels, racking and even controlled introduction of
oxygen can all promote the aeration of wine (Ribéreau-Gayon, Glories et al. 2006). This
process is responsible for changes in the colour and flavour which can be both beneficial and
21
undesirable in regards to the quality of the final product. The positive effects are mostly
related to red wines, where some undesirable aromas can be removed, the colour could
become more stable and the overall mouthfeel can be improved. On the contrary, oxygen
additions generally have a negative influence on white wines and can lead to loss in fruity
aromas, freshness, colour changes (browning) and phenolic precipitation(Reynolds 2010).
Transition metals, and copper, and iron more specifically are thought to be the main
catalysts for nonenzymatic wine oxidation even in trace quantities (Danilewicz 2003).
Oxygen in its ground state cannot react with other organic molecules in wine and needs to be
excited to a singlet state in order for such reactions to take place. Phytochemical excitation is
one possibility for this to happen, but it has been stated that it is the reduction by transitional
metal catalysts which most likely promotes the process (Danilewicz 2003).
Hydrogen peroxide (H2O2) which is formed after the reduction of oxygen in the presence
of ferric ions (Fe3+) and its reaction with wine phenolics, is believed to be reduced by either
Fe2+ or Cu+ ions through a considerably rapid reaction in the absence of bisulphite. This
reaction is known as a Fenton reaction and its final products are the very active hydroxyl
radicals (HO•), the main target of which is the ethanol being the most abundant organic
component in the wine (Danilewicz 2003; Kreitman, Cantu et al. 2013). The hydroxyl radical
can also target tartaric acid and form a precursor to yellow/brown wine colour pigments
(Clark 2008).
Another possible outcome from nonenzymatic oxidation is the formation of quinones – a
result of the oxidation of catechol or pyrogallol bearing phenolics. These quinones are
responsible for certain wine defects, which include browning and the loss of important
aroma-active thiols (Li, Guo et al. 2008; Laurie, Zúñiga et al. 2012).
Both wine oxidation products, H2O2 and quinones, can react with the main preservative
used in wine, sulfur dioxide (Danilewicz and Wallbridge 2010). This results in a loss of
sulfur dioxide during wine oxidation and once it gets below a certain concentration microbial
spoilage and/or aromas and colour defects will occur in the wine (Barril, Clark et al. 2012;
Grant-Preece, Fang et al. 2013).
Up to 19 new odorants have been found to occur in bottled wine which has been oxidised
and other odorants have been found to disappear. Smells of rancid oil, cider, rotten food,
22
meat, cooked vegetables, chemicals, sherry-like, vinegary, and metallic smells are some of
those aromas associated to oxidative spoilage. What is more, oxidised wines are commonly
described as becoming “flat”, lacking in flavour and are usual subject to changes in colour
(Escudero, Cacho et al. 2000).
All of these undesirable effects due to the oxidation of a wine are frequently catalysed by
transition metals. The best way to prevent any metal-induced oxidation reaction is to limit
oxygen exposure and then to achieve low copper and iron concentrations in the wine.
Danilewicz et al. have shown that the progressive removal of these two metals decreases and
eventually stops wine oxidation, but their complete removal can have some negative effects
on the wine quality as well and is thus not recommended (Danilewicz and Wallbridge 2010).
Reductive process
There is a certain group of aromas referred to as “reductive” which appear when the wine
has essentially no oxygen during the winemaking process or when there is once again little
oxygen available after bottling. These unpleasant odours are most commonly due to the
presence of hydrogen sulfide (H2S), methanethiol (MeSH) and/or dimethyl sulfide (DMS)
which account for rotten eggs, rubber or natural gas, and canned corn or vegetal aromas,
respectively. Mainly formed as a by-product of yeast activity during alcohol fermentation,
these aromas are suspected to be formed from amino acids or even pesticides, which contain
sulfur, and lack of oxygen plays an important role in their formation (Viviers, Smith et al.
2014). It has been shown on several occasions that the lower the level of oxygen exposure
after bottling, the more “reductive” aromas can accumulate in a wine (Ugliano, Kwiatkowski
et al. 2011; Ugliano, Dieval et al. 2012). The effects of low oxygen concentrations in the
bottle are even more pronounced and easily perceptible in wines bottled with efficient oxygen
removal and under screw cap, which has been found to isolate almost completely the wine
thus not allowing any oxygen to come in contact with the wine after bottling (He, Zhou et al.
2013).
Copper and iron are among the metals which can contribute to not only an oxidative, but
also a reductive process in the wine when present in sufficient concentrations. In an
experiment by Viviers et al. in 2013 the formation of the three main volatile sulfur
compounds (VSC) was measured in 31 treatments on white and red wines with different
amounts and combinations of 5 of the metals, which are thought to have the most significant
23
influence on wine reduction and oxidation – copper, iron, manganese, zinc, and aluminium.
After 12 months of bottle maturation the concentration of volatile compounds was measured
and an increase in hydrogen sulfide and dimethyl sulfide compounds in white wine and
hydrogen sulfide and methanethiol in red wines was observed. Some of the treatments which
showed the most significant increase in the concentration of volatile sulfur compounds were
most associated with the presence of copper and iron. Thus, an interesting conclusion could
be made, that in samples containing only zinc, manganese and aluminium there were also
high levels of hydrogen sulfide in the white and methanethiol in red wines (Viviers, Smith et
al. 2013). It is important, however, to note that in this study the metal additions which were
made resulted in total metal concentrations considered as higher than the average commonly
reported for commercial wine in literature. What is more, even though the sample preparation
for all the treatments was performed in the presence of air, the bottles were afterwards
flushed with 98% nitrogen (N2) gas for 30 s. This step led to very low concentrations of
dissolved oxygen available in the headspace which were on average 1.11 mg/L for the
Chardonnay and 1.43 mg/L for the Shiraz control samples. The samples were then stored in
anaerobic conditions and it was reported that the entirety of the initially introduced oxygen
was consumed by the fourth month of the experiment.
Other studies on reductive aromas have also proven the increase of mostly hydrogen
sulfide in wines during bottle storage with an increase in the copper concentration (Ugliano,
Kwiatkowski et al. 2011; Ugliano, Dieval et al. 2012; Viviers, Smith et al. 2014).
Furthermore, there was a difference when comparing the wine that had undergone protein
stabilisation with bentonite prior to bottling and the one that had been left untreated. Levels
of hydrogen sulfide were found to be significantly higher in the untreated samples. An
explanation was found with further analysis on the wines which revealed that the copper/iron
ratio was another factor which contributed to the formation of hydrogen sulfide. The addition
of bentonite had led to an increase in the iron concentration on those wines and thus to a
lower ratio between the two metals (Viviers, Smith et al. 2014).
Bentonite
Addition of bentonite to wines is one of the most common methods used by winemakers
protect the wine from a very specific turbidity which can form in the bottle, known as
“protein haze”. This phenomenon is due to the aggregation of wine proteins during storage,
24
which form a deposit and has a negative effect on the clarity of the wine. The proteins slowly
unfold during the maturation period of the wines and precipitate in the bottle. This fault is
mostly seen in white wines as the proteins in red wines flocculate with tannins during
alcoholic fermentation. The protein precipitation has been shown to be significantly affected
by changes in temperature during storage (Sauvage, Bach et al. 2010).
Bentonite is a volcanic aluminium/magnesium silicate clay and is used as a fining agent
due to its capability to reduce the protein content in wines, thanks to its exchangeable cationic
components (Zoecklein, Gump et al. 1999). There are three types of bentonite which can be
utilised for this treatment: natural sodium bentonite, natural calcium bentonite, and activated
calcium bentonite. The latter is the most commonly used as it has a high percentage of
exchangeable sodium and calcium which makes it more efficient (Moreno and Rafael 2012).
It is interesting to note, that depending on the type of bentonite used it can have a more or
less significant contribution to the metal content of wine with possible increase in the
concentration of aluminium, calcium, iron and others (Catarino, Madeira et al. 2008).
Except for its main role as a fining agent bentonite has also been reported to accelerate the
natural clarification and stabilisation of the wine. Recommended doses should not be greater
than 60 to 1800 mg/L because when found in excess in the wine it can react with certain
aromatic compounds and decrease its fruity character (Butzke July 2010).
One study investigated the probable beneficial effect of bentonite in reducing the
“browning” in white wines. Even though the colour intensity seemed to be lower in
comparison to the control where no addition was made, it was concluded that bentonite didn’t
have a sufficient effect on its own. However it could be considered as an addition to enforce
the reduction of brown colour when using sulfur dioxide (SO2) which is the general practice
when treating such a fault in white wines (Main and Morris 1991).
What is more, the influence of bentonite on the prevention of ferric casse has been
recorded to be very small to none, due to the fact that the reaction between iron(III) and the
phosphate anion responsible for the ferric casse is generally negatively charged and thus
cannot react with the bentonite (Moreno and Rafael 2012). It can, however, limit the copper
casse in certain white wines as it binds with the proteins which favour the flocculation of the
copper complex responsible for this spoilage. The treatment is applicable in wine with copper
25
concentrations below 1 mg/L, but above this concentration additional actions should be taken
to protect the wine (Moreno and Rafael 2012).
Metal removal
In their review on metal in alcoholic beverages, Ibanez et al. (2008) have presented some
of the most popular and efficient actions which may be applied to reduce the metal
concentrations in wine. One interesting method combines an increase in the pH of the wine
with sodium bicarbonate (NaHCO3) or calcium carbonate (CaCO3) to 4.5-5 in combination
with the addition of tannins or tannic acids. After the wine is left to settle for several days,
gelatine and bentonite are added. At this stage it is important to stir the wines, so that the
gelatine and bentonite react with the metal tannates, followed by decanting and filtering of
the wine. This technique has been reported to significantly decrease the copper, iron and zinc
concentrations in wine (Bakalov, Angelov et al. 1989)
Another option is the use of ion exchange resins for the removal of copper, iron and
manganese. A chelating resin and an acidic cation exchange resin both proved to be highly
effective in lowering the amounts of these three metals from white wine. However, Benítez et
al. (2002) discovered that there were some changes in the organoleptic characteristics of the
treated wines – a reduction in the polyphenolic and aromatic profiles. At the same time, this
reduction in the polyphenol levels was suggested as the possible reason for the increased
stability of the wines to browning (Benítez, Castro et al. 2002).
Detering et al. (1999), utilising chelating agents, observed no organoleptic alterations
after the clarification of the wines and the wine retained its typical aroma (Detering, Sanner et
al. 1999).
Potassium ferrocyanide (K4[Fe(CN)6].3H2O) is an efficient removal agent of copper and
iron cations from wine, but can also remove some heavy metals like mercury, lead and
arsenic. It has been officially approved by the Office Internationale de la Vigne et du Vin
(OIV) as a legal additive against metal-induced faults (COEI-1-POTFER: 2000), (OIV
2010). It forms a cupric, ferro, or ferriferrocyanide salt that precipitates and can be removed
by settling and filtration (Ribéreau-Gayon, Glories et al. 2006).
A danger with this technique is the release of cyanide into the wine if it is performed in
highly oxidising conditions. For this reason over-fining with potassium ferrocyanide should
26
be avoided and cyanide levels measured. It has been suggested to performed additional
techniques, such as the addition of ascorbic acid, for example, which reduces the
concentration of Fe3+ in the wine and favours the formation of ferrous ferrocyanide
complexes thus making them more easily precipitable. This approach will result in the use of
smaller quantity of potassium ferrocyanide to stabilise the wine and decrease the possible
amount of residual hydrogen cyanide left in the wine (Moreno and Rafael 2012).
Methods for metal determination (Cu and Fe more specifically) in grape
juice and wine
It is of major importance to keep track of the levels of these elements both in the juice and
wine. As already discussed, metals can cause undesirable changes in the colour and taste of
wine (Tariba 2011) as well as be potentially toxic for humans.
In order to optimise the determination of the metal content in wine and grape juice,
scientists have performed a series of experiments applying different analytical methods. For
the determination of copper, for example, one of the most frequently used methods is atomic
absorption spectrometry (AAS) (Riganakos and Veltsistas 2003; Espinoza, Olea Azar et al.
2008). One variation of this method, which is flame atomic absorption spectrometry (FAAS)
is also the official method employed by the Office International de la Vigne et du Vin (OIV)
(Recueil OIV ed. 1990, revised by 377/2009) and the European Union (Commission
Regulation (EEC) No 2676/90). FAAS has also been applied for the determination of iron
species in wine and several modifications have been developed for the technique over the
years to optimise the measurement process (de Campos and Araújo 2001).
Other methods for metal determination include inductively coupled plasma mass
spectrometry (ICP-MS) (Hague, Petroczi et al. 2008; Viviers, Smith et al. 2013), inductively
coupled plasma optical emission spectroscopy (ICP-OES) (Provenzano, El Bilali et al. 2010)
and stripping potentiometry (SP) (Green, Clark et al. 1997; Clark and Scollary 2000).
For almost all of the methods cited here-above there can be a matrix effect that can
interfere with the signal intensity during the analysis (Pyrzynska 2007). It is why every
method requires a specific sample preparation before the use of the instrument. Decomposing
the samples is one of the most frequent techniques especially when the concentrations of
analyte are close to the detection limits of the specific instrument. Taking into account the
27
consistency of wine, a wet digestion can be performed on the samples after several reagents
are added (nitric acid (HNO3), hydrogen peroxide (H2O2), etc.) (Castiñeira, Brandt et al.
2001; Galani-Nikolakaki, Kallithrakas-Kontos et al. 2002). Recent studies on metal
determination have focused on lowering the detection limits when using inductively coupled
plasma mass spectrometry (ICP-MS), thus making it more precise and easily applicable in a
wider range of experiments. Others have worked on optimising the technique by separating
metals from sample matrices with capillary electrophoresis prior to detection. Increasing the
sensitivity of the electrospray in mass spectrometry for the detection of trace elements in
complex matrices is another recent achievement in the field. All of these have led to
researchers being able to obtain more precise and reliable data (Pyrzynska 2007).
The necessary time for sample preparation has been decreased with the use of sample
decomposition with microwave energy or UV photolysis (Almeida, Vasconcelos et al. 2002;
Riganakos and Veltsistas 2003). The treatment of samples with lower concentrations prior to
their analysis may include preconcentration, separation and dilution of each sample (Bhatt
and Agrawal 2004; Catarino, Pimentel et al. 2005). However, it is important to point out that
the more pretreatment steps utilised the greater the risk of contamination and/or reduction in
analytical precision.
In their review published in 2011 regarding the utilisation of atomic spectrometry
techniques for wine research, Grindlay et al. (2011) state that atomic spectrometry techniques
are preferred when studying trace and ultratrace elements in wine. According to their research
around 10% of the elemental wine analysis from 2000 onwards have used or referred such a
method. They were also able to put them in order according to their popularity, which is
presented here-below (Grindlay, Mora et al. 2011):
1. Inductively coupled plasma mass spectrometry (ICP-MS)
2. Inductively coupled plasma atomic optical spectrometry (ICP-OES)
3. Electrothermal atomic absorption spectrometry (ETAAS)
4. Flame atomic absorption spectrometry (FAAS)
Some other less utilised techniques include total reflection X-ray fluorescence (TXRF)
spectrometry, volatile compound generation-based techniques: hydride generation atomic
absorption spectrometry (HGAAS), hydride generation atomic fluorescence spectrometry
28
(HGAFS) and cold vapour atomic absorption spectrometry (CVAAS) (Grindlay, Mora et al.
2011).
In order to develop a reliable analytical method for metal detection, it is important to
understand and take into account the complex matrix of wine especially if sample
pretreatment is to be minimised. From a more general point of view we can say that wine is a
complex water-ethanol (H2O-C2H5OH) mixture, which includes two main groups of chemical
species – volatile and non-volatile; and salts which can be divided into organic and nonorganic. Negative matrix effects have a wide variation and are specific to the matrix
component, the analytical technique and the sample introduction device utilised (Grindlay,
Mora et al. 2011). Another important aspect which should be accounted for is the addition of
chemical compounds to the wine samples during their preparation (Todolí, Gras et al. 2002).
Even though atomic absorption methods or atomic fluorescence based technique are
commonly referenced by the Office International de la Vigne et du Vin (OIV) and the
European Union (EU) for the detection and quantification of a range of elements, these
instruments allow the determination of only one element at a time. This is one of the main
reasons why an increasing number of researchers prefer to implement multi-element
techniques such as inductively coupled plasma mass spectrometry, inductively coupled
plasma optical emission spectrometry and total reflection X-ray fluorescence spectrometry in
their work. For instance, Anjos et al. (2003) analysed both red and white Brazilian wines in
2003 and obtained information about phosphor (P), sulfur (S), chlorine (Cl), calcium (Ca),
titanium (Ti), chromium (Cr), manganese, iron, nickel (Ni), copper, zinc, rubidium (Rb) and
strontium (Sr) after a simple sample preparation and subsequent analysis by total reflection
X-ray fluorescence using synchrotron radiation.
Plasma based methods have been chosen by other scientist to analyse a number of
different elements at the same time. Gonzálvez et al. (2009) performed analysis on 38
elements at the same time in Spanish wines from the region of Valencia using inductively
coupled plasma optical emission spectrometry. What is more, as many as 63 elements were
simultaneously analysed with an inductively coupled plasma mass spectrometry in German
wines by a group of scientist led by Castiñeira (2004). The last method mentioned also
presents the possibility to obtain isotopic information about the wine samples.
29
Matrix effects, which are one of the main issues encountered during elemental
determination studies of wines samples, have been successfully overcome by the use of
standard additions when using inductively coupled plasma instruments. For example, a
matrix effect originating from the ethanol concentration of the samples was accounted for by
the use of standard additions by Grindlay et al. (2009) in the work on lead, selenium (Se) and
arsenic (As). Also, for ICP analysis the ethanol content causes instability in the plasma.
Consequently, specialised sample introduction chambers are required or reduction of the
ethanol content in the sample.
Another methodology which has been successfully implemented with the use of
inductively coupled plasma techniques is the use of internal standards. In this case the
references (meaning the prepared standards) and the analysed samples are measured
simultaneously, thus providing the necessary information for any necessary corrections to the
results. It has primarily been used when working with plasma based methods, although it is
difficult to identify internal standards which can account for all the elements of interest
analysed with the instrument (Grindlay, Mora et al. 2011). Even though in their study on
Spanish red wines Iglesias et al. (2007) used two internal standards with both ICP-MS and
ICP-OES for the analysis of major, minor and trace elements, most authors have found that
having only one internal standard is enough to obtain good reliability and repeatability with
Inductively coupled plasma instruments (Castiñeira, Brandt et al. 2001). It is very important,
however, to make sure that all the samples, with the standards as well as the analytes, behave
in the same or very similar way when coming in contact with the plasma.
Fractionation and Speciation
In wine, metals can be found in the form of free ions, in complexes with organic acids or
complexes with peptides and/or macro molecules including pectic polysaccharides, proteins
and polyphenols. The bioavailability of the metal is not a constant parameter, and is not
always linked to the total concentration of metal but can be dependent upon the different
forms of the metal. This is why it is important to point out that it is not only the total
concentration which should be taken into account when examining the wine, but also the way
it is distributed between its different species. This information is expected to be crucial for
determining the possible effect and potential damage the metal content can have on the wine.
Another interesting aspect of metal speciation is that it has been linked to the geographical
30
origin of the wine and in this way be used to distinguish and identify wines from different
regions mainly regarding the differences in the soil composition (Riganakos and Veltsistas
2003).
In reference to metal-induced spoilage, Fe2+ has been found to be the more abundant of
the oxidation states of free iron in wine through the use of speciation procedures (Tašev,
Karadjova et al. 2006). In another study, no oxidation took place in samples where the
concentrations of Fe2+ and Fe3+ were in equilibrium (Fe2+/Fe3+≈1), but once this state was
disrupted by sulphites, nucleophiles or other compounds the oxidation was once again
initiated (Kreitman, Cantu et al. 2013).
As mentioned previously metals are present in various forms in the wine where numerous
free elements as well as complexes can be found. This makes the determination of a specific
form of a certain metal a difficult and time-consuming challenge.
Karadjova et al. (2002) presents a detailed resin-based method for fractionation and
speciation of wine samples for the determination of copper, iron and zinc and their analysis
with a flame atomic absorption spectrometry and electrothermal atomic absorption
spectrometry. For their wine component fractionation they used three types of commercial
resins – Amberlite® XAD-8, Dowex 50-x8 and Dowex 1-x8. The samples were filtered and
then passed through each of the three columns in the order given above.
The results allowed them to conclude that labile iron, copper and zinc species can be
determined through the use of a Dowex 50-x8 resin with analysis by flame atomic absorption
spectrometry or an electrothermal atomic absorption spectrometry. Some modifications were
applied with success in their methodology. For example, for the negatively charged portion of
the metals they found that adding tartaric acid to the sample was a reliable and adequate
sorption method. An acetone precipitable fraction was chosen by the team of scientists led by
Karadjova to separate the organically bonded species of the metals (Karadjova, Izgi et al.
2002).
The separation of wine polyphenols proved to be a bigger challenge as the method
normally used involves methanol (CH3OH) extraction. It is not advised, however, to use this
technique due to the fact that most of the other fractions of copper, iron and zinc will also be
extracted by methanol (Karadjova, Izgi et al. 2002). The preferred method was to pre-treat a
31
hydrophobic resin with bismuth(III) nitrate (Bi(NO3)3) to eliminate cation exchange sites on
the resin before passing the wine through and to target the extraction of phenolic metal
complexes.
As a summary, the data obtained at the end of the research showed that almost 30% of the
iron was in a complex with polyphenols and proteins in wine samples which were only
filtered and not treated with any stabilising agents. The portion was lower (around 20%) in
finished wines which were filtered and stabilised prior to bottling. The polysaccharide
fraction was almost inexistent, as only 5% of the iron was detected in it. For copper it was
observed that the majority (approximately 50%) was in the labile fraction, which was
suspected to be the most reactive fraction. The polyphenol/protein fraction was where the
organically-bound copper was located with less than 5% detected in the polysaccharide
fraction.
The utilisation of a physical method to separate the metal fractions has been utilised in
wine elemental studies due to the possibility of obtaining additional information about the
different forms of trace elements. Some of these techniques include fractionation through
ultrafiltration, which allows the determination of the distribution of different particle size
fractions (McKinnon and Scollary 1997). In the case of non-volatile dissolved organic
compounds and metal species similar results have been obtained through the use of a multistage ultrafiltration coupled to ICP-MS (Castiñeira, Burba et al. 2003). Murányi et al. (1998)
demonstrated that it was possible to separate the colloidal and the suspended forms of a
specific metal element and that their results were strongly dependant on the previous
treatments of the wine and its storage (Murányi and Papp 1998).
Throughout the speciation procedure it is important to note that the species of the element
of interest have different biological properties and toxicity (Pyrzynska 2007). Thus in order to
obtain accurate results it is important to limit impact of the actual speciation measurement
technique on the distribution of metal forms in the samples (Grindlay, Mora et al. 2011).
Given that some perturbation of the metal species will occur during the measurement, the
speciation measures are very dependent on the methodology used and hence are often termed
operationally defined speciation measures.
Inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma
optical emission spectrometry (ICP-OES) have been successfully coupled with high
32
performance liquid chromatography (HPLC) as the former techniques were not always
sufficiently sensitive to detect metals in the samples. This is one of the techniques referred to
as “hyphenated” and other possibilities include the separation of sample components with gas
chromatography (GC) and capillary electrophoresis (CE) prior to analysis by ICP (Caruso,
Wuilloud et al. 2005).
Iron (Fe) speciation
A clear and structured example for a speciation procedure for iron analysis was previously
presented in the report by Karadjova et al. (2002) where two methods were used – flame
atomic absorption spectrometry and electrothermal atomic absorption spectrometry.
Furthermore, a solid phase extraction (SPE) approach was developed and combined with
FAAS to separate the labile ferrous (Fe2+) and ferric (Fe3+) species in wine (Tawali and
Schwedt 1997).
Being a reference method for metal determination in general, flame atomic absorption
spectrometry has also been used for iron speciation after fractionation of the wine sample.
One pre-treatment is the use of micelles of a non-ionic surfactant mixture to precipitate the
tannins and other phenolic and insoluble compounds. In this type of study a significant
difference was found between the amount of iron which was in a complex with tannins and
other similar complexes in commercial white wines and domestic red wine – 5% and 30%,
respectively (Paleologos, Giokas et al. 2002).
Liquid/ liquid extraction techniques and solid phase extraction techniques have also been
coupled with the flame atomic absorption spectrometry in other research. One of them
determines the species of iron found in Bulgarian and Macedonian wines and revealed that
the smallest amount of iron was in the form of ferrous ions (Fe2+) – about 5%, followed by
the ferric ions (Fe3+) with 30-50% and the largest concentration was the organically stable
bonded iron – 30-50% (Tašev, Karadjova et al. 2006). The same methodology was utilised by
Pohl et al. in their study in 2009 where they used two separate columns - Amberlite® XAD7HP followed by Dowex 50W-x8-200. This allowed them to distinguish three iron species –
a phenolic fraction which included the hydrophobic species of iron bound to phenolic
substances and related species; a cationic fraction comprising simple iron ions and labile iron
forms; and a residual fraction with the anionic and/or neutral Fe complexes with organic
acids (Pohl and Prusisz 2009).
33
Although these are some of the most common techniques for iron speciation, further
approaches can been found in the literature, such as: molecular absorption spectrometry,
fluorescence spectrometry and electrochemical detection also combined with the flame
atomic absorption spectrometry (Grindlay, Mora et al. 2011).
Copper (Cu) speciation
For copper speciation research, the same methodology has been predominantly used as for
iron. For example, in a study of Pohl et al. (2009), they were able to obtain three groups of
copper species as discussed for iron on lager beer and red wine. In the red wine samples,
copper seemed to be most strongly bound to various flavonoids and other polyphenols,
located in the polar and non-cationic fractions (Pohl and Sergiel 2009).
A speciation procedure was also performed on a wide range of Port wines (white, singleyear and blended aged red, and young red wines) with the aim to evaluate the bioavailability
of copper and lead. Azenha et al. (2000) were able to gather detailed information through the
use of electrochemical analysis and flame atomic absorption spectrometry about how the
metals were distributed among the separate groups of compounds of different molecular
weights and/or polarities in the different bands separated by reverse phase high-performance
liquid chromatography (RP-HPLC) as well as the total metal concentration in the wines. They
were able to conclude that copper was predominantly complexed with higher molecular
weight compounds with apolar character in comparison to a similar study carried out by the
same research team on table wines (Azenha and Vasconcelos 2000).
Another study looked into the complexometric properties of four varietal red Spanish
wines toward copper. The samples were titrated through an ion selective electrode
potentiometric titrator and the final results showed that the copper present in the studied
wines is strongly bound with organic ligands as in the previous study (Vasconcelos, Azenha
et al. 1999).
Weise et al. in 1997 separated the ionic, labile and tightly bound species of copper
combining potentiometry, anodic stripping voltammetry (ASV) and kinetic photometry. It
was revealed that the free copper ion fraction varied between 3.3 and 31% of the total
concentration, while the labile fraction was significantly higher for all the samples with
values between 33 and 86%. An interesting conclusion from this study also revealed that in
34
the wines, where the tightly bound species was dominant, the total copper concentration was
lower, but the amount of proteins was higher (Wiese and Schwedt 1997).
When using stripping potentiometry for the determination of labile lead and copper in
wine Green et al. (1997) found that this method doesn’t require any pre-treatment of the
samples, but that it is necessary to use a different chemical oxidant, required for the
measurement phase of the technique, in the place of oxygen (O2), and in this case it was
mercury(II) (Hg).
This methodology was further developed by Clark et al. (2006), when a medium exchange
stripping potentiometry was utilised for the measurement of electrochemically defined labile
and non-labile fractions of copper in 16 white wines. This technique enabled direct analysis
of the wines without any pre-treatment. It overcame the common interference of organic
compounds on the electrode surface by utilising an exchange step between enriching and
stripping copper from the electrode. The exchange step involved movement of the electrode
from the wine to an ammonium acetate medium via rising steps (Clark and Scollary 2006).
35
Materials and methods
Winemaking stage
At the beginning of our study we received 180 L of Chardonnay juice from the
Commercial Winery at the NWGIC in Wagga Wagga. The grapes originated from the region
of Orange, NSW which is a well-known cool climate grape growing region situated about
300 km North-East from Wagga Wagga. The juice was left to settle for 1 day in a
temperature control room at the Experimental Winery of the NWGIC, where all the following
stages of the winemaking took place.
The experiment involved 6 different treatments with 3 repetitions per treatment. 12 L
demi-johns were used for the alcoholic fermentation which were washed and then rinsed with
Milli-Q water before the addition of the juice. Samples were taken from each repetition
before the inoculation to determine their initial metal concentrations. Afterwards different
metal additions were made to each treatment as follows:
-
Treatment 1 – no addition (control)
-
Treatment 2 – 2.9 mg/ L (low copper)
-
Treatment 3 – 5.8 mg/L (high copper)
-
Treatment 4 – 6.3 mg/L (low iron)
-
Treatment 5 – 12.5 mg/L (high iron)
-
Treatment 6 – 5.8 mg/L Cu and 12.5 mg/L Fe (high copper and high iron)
Copper was added as copper(II) sulfate pentahydrate (249.69 g/mol) and iron was added
as iron(II) sulfate heptahydrate (278.01 g/mol).
Sub-samples of juice (˜200 mL) were taken from each treatment after the metal additions
which was followed by the addition of SO2 (200 mg/L SO2 which is 347.1 mg/L K2O5S2
(222.4 g/mol) to sub-sample and storage at 4C. These sub-samples were utilised to confirm
the metal concentrations in the juice after the metal additions. The bulk juice treatments were
then inoculated with Lalvin DV10™ commercial yeast. It is approved for the production of
dry white wines as well as base wines for sparkling. It is produced by the company
Lallemand S.A.S. which describes it as strain with strong fermentation kinetics over a wide
temperature range and relatively low nitrogen demands. It is a clean fermenter that respects
36
varietal character and avoids bitter sensory contributions (Product specifications, Lallemand
S.A.S. official website).
The inoculation solution was prepared by dissolving 65 g of GO-FERM® (yeast nutrient
from Lallmand S.A.S.) in 650 mL of water at a temperature of 43oC. After 10 mins when the
temperature had dropped down to 30oC 65 g of Lalvin DV10™ yeast (Saccharomyces
cereviseae ex. bayanus) was added. The solution was stirred and then left for 20 mins to
activate the yeast. It was then equally divided and added to each demi-john. The inoculated
treatments were then put in a temperature control room at 18oC for the first 24 h to start the
alcohol fermentation. The temperature was then dropped to 16oC to insure a good and stable
yeast activity throughout the process.
After a drop of 3o Baume (5.4o Brix) the appropriate amounts of Fermaid A (yeast nutrient
by Lallemand S.A.S., 13% N concentration) was added to each trial to adjust the YAN to 200
ppm. To determine the end of the alcoholic fermentation samples were analysed on a Digital
Density Meter which provided more precise information about the concentration of both
fructose and glucose in the samples. After a concentration of below 0.5 g/L of sugars
(fructose and glucose) was reached, the demi-johns were moved to a temperature control
room set at 4oC to induce settling.
The wines were racked twice before bottling to release them from the lees. Between the
two rackings cold and heat stability tests were performed. Half of the volume of each wine
was bottled before the addition of bentonite and left for storage in the cellar. The second half
of the wines was transferred to 5 L demi-johns which were washed and rinsed with Milli-Q
water. The necessary amounts of calcium bentonite were calculated according to the results
obtained from the Bentonite stability and turbidity tests and the equal amounts (1.6 g/L) of
SIHA ActivBentonit G (calcium bentonite by EnolTech) were added to each sample. These
wines were left to settle at a temperature of 4oC before bottling.
The bottling of all wines was executed manually, transferring the wines from the demijohns to the bottles through a simple gravitation method. The bottles were cleaned with a
bottle-rinsing product and hung on a bottle rack upside down prior to bottling. In order to
remove the oxygen in the bottles and thus protect the wines from any spoilage during storage,
the bottles were also flushed with nitrogen gas just before the wine was poured into them.
The wine was poured almost to the top of the bottle to limit the headspace left. Just before
37
installing the screw cap the bottles were flushed once again with nitrogen gas. The utilisation
of this simple, but time consuming technique ensured maximum protection for the wines
against microbial and oxidative spoilage.
All treatments were bottled in 375 mL bottles to minimise wastage of wine during the
analysis phase of the experiment.
Analytical stage
Solid Phase Extraction (SPE) usage
An accurate and sensitive determination of the speciation of heavy metals is a crucial part
of the activities included in this research project. Atomic absorption spectrometry is
considered to be the most suitable instrument quantification of total metal concentration in
wine because of its fairly simple usage and cheap cost (Tuzen, Saygi et al. 2008). For this
reason flame atomic absorption spectrometry (FAAS) was initially utilised in the project for
determination of total concentrations of metals in juice. Nonetheless, this instrumentation
cannot provide determination of metal species via direct analysis of wine. The wine first
requires treatment to enable the different forms of metal ions to be separated prior to analysis
by such instruments as FAAS. For the purposes of this project, a solid phase extraction (SPE)
with different adsorbing/ion exchanging resins has been chosen to enable separation of
different metal fractions. SPE is a fairly simple, but fast extraction system, as well as being
adaptability to pre-concentration and can involve the use of a flow injection analysis
technique for elution through the SPE cartridge. After fractionation of the metals by different
SPE cartridges, the FAAS determination of the different metal species, often at very low
levels, such as μg/L, is not possible without pre-concentration of the speciation samples, due
to insufficient sensitivity of FAAS and the effect of 1A and 2A group elements on the
determination process (Hosseini, Raissi et al. 2001; Ivanov and Kochelayeva 2006; Rezaei,
Meghdadi et al. 2007; Tuzen, Saygi et al. 2008). Consequently, inductively coupled plasma –
optical emission spectroscopy (ICP-OES) was adopted over FAAS for the quantification of
metal species in the wine samples due to its superior sensitivity and lack of interference from
1A and 2A group elements.
38
SPE column preparation
A polymeric resin (Amberlite® XAD16, Sigma Life Science) and a cation-exchange resin
(Dowex 50WX8 hydrogen form, Dow Chemical, Sigma-Aldrich®) were used in the first
stages of the experiment. The polymeric resin has a non-ionic, hydrophobic, cross linked and
polyaromatic surface (dipole moment 0.3) and is suitable for adsorption of hydrophobic
compounds with small to medium molecular weight (up to 40,000 MW) with a mesh of 2060. It is reported to be more efficient than XAD-2 thanks to its higher surface area (SigmaAldrich® Product Information Sheet). The strong acid cation-exchange resin has 100-200
mesh in bead size and is suitable for fine chemical and pharmaceutical separations (SigmaAldrich® product information sheet).
The Amberlite® XAD16 resin was firstly dried in a controlled oven at 100 oC for 4 hours.
1 g of each resin was weighted out after being kept in a desiccator with a small quantity of
desiccant on the bottom to prevent the absorption of moisture from the surrounding air. The
regeneration of both resins was executed as advised by the producer. This included a
sequence of washing steps for the polymeric resin: 20 mL methanol and twice with 20 mL
ultrapure Milli-Q water with stirring and leaving the resin for 20 minutes. The resin was then
poured into a 6 mL glass column with a porous glass frit as a slurry and placed under vacuum
(Varian Vac Elute™ 20 HPLC Lab Vacuum Manifold with 20 ports). The polymeric resin
was rinsed further with acid (10 mL 1M HCl), and 20 mL of 12% aqueous ethanol (pH 3.2,
0.011 M potassium hydrogen tartrate, 0.007 M tartaric acid). The resin was then dried under
vacuum.
The following steps were required for preparation of the cationic resin: wetting twice with
20 mL ultrapure Milli-Q water, with stirring and leaving the resin for 20 minutes between
washing. The resin was then poured into a 6 mL glass column with a porous glass frit as a
slurry and placed under vacuum (Varian Vac Elute™ 20 HPLC Lab Vacuum Manifold with
20 ports). The cationic resin was rinsed further with acid (10 mL 1M HCl), and 20 mL of
water, then with 10 mL 1 M NaOH and finally with 20 mL water. The resin was then dried
under vacuum.
The resins mentioned above were utilised in various trials with ultrapure Milli-Q water
and model wine during the development and testing of the method. After numerous tests
which included the application of several preparation and usage methodologies, it was
39
decided that in order to ensure a better homogeneity extraction conditions, the Amberlite ®
XAD16 and Dowex® 50WX8 resin would be replaced by commercially prepared pre-packed
SPE cartridges.
For the purposes of our project the following two SPE cartridges were chosen, tested and
then utilized for the experiment. The first SPE was the Strata® C18-E. It is a hydrophobic
resin with an end-capped silica-based sorbent that offers strong hydrophobic retention with
negligible secondary polar interactions from active silanol groups. It was used in the place of
Amberlite® XAD16 in the analysis of all the wine samples. Its preparation is identical to the
one of Amberlite® XAD16 with the only difference that all the wetting stages were prepared
via passing solutions through the SPE cartridge via vacuum, and also no transferring or
packing of columns with wet resin was required.
The second cartridge – the Strata® SCX, has a benzene sulfonic acid group which is
bonded to the surface of the silica particle, giving it strong cation-exchange selectivity. It
replaced the Dowex® 50WX8 resin, with the only difference that all the wetting stages were
prepared via passing solutions through the SPE cartridge via vacuum, and also no transferring
or packing of columns with wet resin was required.
The samples were extracted as per the scheme shown in Figure 1.The ready cartridges
were then mounted on a peristaltic pump and 30 mL (using a 50 mL burette) of each filtered
wine sample (0.45 μm PES) were passed through the Strata® C18-E cartridge at a rate of 1
mL/min. 20 mL of this post-C18-E sample were then passed through the second column – the
Strata® SCX again at a rate of 1 mL/min, whilst the remaining 10 mL eluting through the
Strata® C18-E cartridge would be utilised for metal quantification and calculation of the
‘hydrophobic’ fraction of the metal ions (i.e., hydrophobic metal = total metal – (metal
eluting from Strata® C18-E)). The 20 mL eluting from the cationic resin was quantified for
metal to be termed ‘residual’ metal. In order to obtain the third and final fraction of Cu and
Fe found in the sample, 10 mL of 2M HCl was passed through the Strata ® SCX column at a
rate of 1 mL/min. This 10 mL fraction was collected and quantified for metal to be termed
‘cationic’, after being corrected for a 2-fold concentration in the SPE cartridge. Following
this procedure allowed us to obtain three separate speciation solutions for each sample which
divided provided quantification of three metal fractions – hydrophobic, residual and cationic,
respectively.
40
Metal Speciation Procedure
Step 1
30 mL Filtered (0.22µm) wine
Strata C18 SPE cartridge
10 mL Post-C18 wine
Non-hydrophobic
metal fraction
Step 2
20 mL Post-C18 wine
Step 3
10 mL 2M HCl
Strata SCX SPE cartridge
20 mL Post-SCX wine
Residual
metal fraction
10 mL Post-SCX 2M HCl
Cationic metal
fraction (2-fold
concentrated)
Hydrophobic fraction =
Total – Non-hydrophobic
metal fraction
Figure 1: Scheme of the metal speciation procedure for both copper and iron samples
Instruments
DMA 35N Anton Paar GmbH portable density meter
During the alcoholic fermentation the development of the wines was followed twice daily
with the use of a DMA 35N Anton Paar GmbH portable density meter. The use of this
instrument allowed the measurement of the density of the wine in g/cm3 or kg/m3 according
to the oscillating U-tube principle. Temperature influence is compensated by the set
temperature coefficient α and the sugar concentration is automatically calculated and
displayed on the screen in °Baumé. Apart from the density, the instrument also showed
41
information about the current temperature. The measuring range of the instrument is 0 to
1.999 g/cm3 for the density and 0 to 40 °C for the temperature. Its accuracy is ± 0.001 g/cm3
for density and ± 0.2°C for the temperature, thus assuring the precise and accurate tracking of
these parameters (Source: official “Anton Paar GmbH Instruction Manual for DMA 35N
Portable Density, Specific Gravity and Concentration Meter”).
The sugar concentration and the temperature were measured two times a day (8 AM and 4
PM) during fermentation and the data was recorded in order to assess the development of the
alcoholic fermentation for each sample separately.
Robotic Sample Processor XL titrator and Flow Injection Analysis Sulfur
Dioxide Analyser
Once the wine had been left to settle post fermentation, samples were taken once again
from all treatments for chemical analyses. A 10 mL aliquot of each wine were analysed with
a 789 Robotic Sample Processor XL titrator (Metrohm AG, Switzerland) for pH and titrable
acidity. The concentrations of total and free sulfur dioxide (SO2) was measured with a flow
injection analyser (FIAstar™ 5000, FOSS, Denmark), connected to an auto-sampler (5027,
FOSS, Denmark).
Flame Atomic Absorption Spectroscopy (FAAS)
For the analysis of total metal concentrations in juice and during method development of
speciation measures, a Varian, Inc. SpectrAA 50B FAAS was used. It is a stand-alone system
with a single beam which allowed determining the Cu and Fe in the samples. The instrument
uses hollow cathode lamps which were adjusted to provide maximum sensitivity, and left to
warm up. The Cu and Fe concentrations were measured at an absorbance of 324.8 nm and
248.3 nm, respectively, as an average of 3 repetitions. An external calibration graph was
utilised for the quantification of the concentration of Cu and Fe in samples. The external
calibration standards were prepared in a synthetic juice matrix (200 g/L glucose, pH 3.2
(tartaric acid) and diluted 4 mL in 10 mL with Milli-Q water prior to analysis. Samples were
diluted similarly prior to analysis by FAAS.
Inductively coupled plasma optical emission spectrometry (ICP-OES)
ICP-OES was conducted on a Varian 710 ICP-OES with utilisation of a High Salts
Nebuliser/Spray Chamber.
42
Cu was monitored at 324.754 nm and Fe at 259.940 nm. The instrumental conditions were
plasma power 1.30 kW, plasma flow 15.0 L/min, auxiliary flow 1.5 L/min, nebuliser pressure
200 kPa, stabilisation time 15 s, sample uptake 60 s, rinse time 60 s, pump rate 15 rpm and
fast pump off. Quantification was conducted via the use of Lu (261.541 nm) as an internal
standard.
During the preparation and method-development stage for the purpose of this analysis 4
mL of each sample were diluted with 12 mL of 5% HNO3 to be analysed with the ICP-OES.
The nitric acid solution was freshly prepared by combining 18 mL of concentrated (70%)
nitric acid with ultrapure Milli-Q water (made up to 250 mL). Another method of sample
preparation was also tested and finally adopted for subsequent research. The hydrophobic and
residual samples, which contain ethanol, were 4-fold diluted: 2.5 mL of solution was added to
a 10 mL calibrated volumetric flask and topped with 5% HNO3 to 10 mL. The cationic
solutions were analysed by an 8-fold dilution to account for the two-fold concentrating
resulting from the SPE extraction and elution. In this case 1.25 mL of sample was added to
calibrated 10 mL volumetric flasks, along with 1.00 mL of 1.0 M NaOH and 0.25 mL of
Milli-Q water. The flasks were then made up to 10 mL with 5% HNO3.
Table 3: Chemical elements and their wavelength [nm] determined by the 710-ES ICP-OES
Element Wavelength (nm) Element Wavelength (nm)
Ag
328.07
Mg
279.55
Al
396.15
Mn
257.61
As
188.98
Mo
202.03
Au
242.79
Na
589.59
B
249.77
Ni
231.60
Ba
455.40
P
177.43
Be
313.11
Pb
220.35
Bi
223.06
S
181.97
Ca
396.85
Sb
206.83
Cd
214.44
Se
196.03
Ce
418.66
Si
251.61
Co
238.89
Sn
189.93
Cr
267.72
Sr
407.77
Cu
324.75
Ti
334.94
Fe
238.20
Tl
190.79
Hg
184.89
V
292.40
K
766.49
Zn
213.86
Li
670.78
Zr
343.82
43
High Performance Liquid Chromatography (HPLC)
The wines used in our study were also analysed with a High Performance Liquid
Chromatography (HPLC) for quantification of organic acids, sugars, ethanol and glycerol.
Ion exclusion chromatography (IEC) with photodiode array (PDA) detection was
conducted using a Waters 2690 Separation Module, run by Millennium32 software, that was
connected to a Waters 2996 photodiode array detector and Waters 410 refractive index
detector. The chromatography was performed on two 300 x 7.8 mm Aminex HPX-87H
organic acid analysis cation exchange columns (Bio-Rad Laboratories), connected in series,
with a guard column of the same stationary phase. The IEC analyses were carried out as
described by Frayne (1986) with a sample injection of 10 microL and flow rate of 0.7 ml/min,
column temperature of 65oC and with an isocratic elution using 0.085% phosphoric acid in
water (0.75 mL of concentrated phosphoric acid in 1.0 L of Milli-Q water). Quantification of
organic acids was performed using peak areas of the 210 nm chromatogram (PDA detector),
whilst the sugars, glycerol and ethanol were quantified using peak areas obtained from the
refractive index detector. An external calibration graph of each commercially purchased
organic acid, sugar and ethanol standard was utilised for quantification.
Konelab™ 20 Clinical Chemistry Analyzer
After alcoholic fermentation, samples from all treatments were analysed on a Thermo
Scientific™ Konelab™ 20 Clinical Chemistry Analyzer for sugar concentrations. The
instrument features automated operation, as well as continuous sample and reagent loading.
Through the use of specifically designed Konelab™ Arena System Reagents the colorimetric
and enzymatic measurement of fructose, glucose, and saccharose were obtained for each
treatment.
Oxygen decay analysis by luminescence analysis (Fibox 3 LCD trace)
A Fibox 3 LCD trace, manufactured by Precision Sensing GmbH, was utilised for
measurement of dissolved oxygen concentrations. It is a stand-alone fibre optic oxygen
transmitter with temperature compensation and used with oxygen sensors based on a 2 mm
optical fibre. It can be controlled via an integrated, programmable LCD control panel. For the
purposes of the current study the Fibox 3 LCD was used with sensors of the type PSt3
44
(detection limit 15 ppb, 0 - 100% oxygen). The sensors come pre-calibrated from the
manufacturer.
For oxygen decay experiments, the wines were placed in an incubator at 25.5  0.1oC
(uncertainty = standard deviation) at least 24 h before the start of the experiment. Afterwards
they were transferred into 500 mL Schott bottles and were shaken for 5 mins to increase the
dissolved oxygen concentration of the wines. Once aerated the wines were then left to settle
for 20 mins in the incubator at 25oC before sample preparation to allow dissipation of bubbles
in the wine. The wines were then transferred to 195 mL bottles. The oxygen sensors,
necessary for the measurements, were pre-installed inside the neck of the bottles utilising
food grade adhesive glue. The bottles were entire covered with aluminium foil to prevent any
effect of light on the oxidation process. The wines were poured into the bottles and 0.4 mL of
106 g/L ascorbic acid and 0.63 mL of 109 g/L sodium metabisulfite were added to each one.
This provided concentrations of ascorbic acid of 216 mg/L and a sulfur dioxide addition of
232 mg/L. These solutions were prepared by dissolving 1.000 g of ascorbic acid and 1.000 g
of sodium metabisulfite, respectively, in 9.000 mL of ultrapure Milli-Q water to give final
volumes of 9.4 and 9.2 mL, respectively. The solutions of ascorbic acid and sodium
metabisulfite were then shaken and covered with aluminium foil to limit any outside light
influence on the solutions. Fresh solutions were made on the day required for the experiment.
The bottles were filled to the top, making sure there is not oxygen left in the headspace.
They were then transferred in the incubator and kept at a constant temperature of 25 oC for 2
days. The oxygen concentrations were carefully followed with regular measurements 24 h per
day. Before each measurement the reading temperature on the Fibox 3 LCD was adjusted to
an accuracy of 0.1oC according to an electronic thermometer installed inside the incubator.
Utilising the same methodology additional samples were prepared at the beginning and
the end of this part of the study for each wine which were then analysed by HPLC.
Reagents and solutions
Deionized water (Milli-Q Millipore 18.2 MΩ/cm conductivity, Milli-Q® Direct Water
Purification System) was used for all dilutions. All the plastic and glassware were cleaned by
soaking overnight in dilute 10% HNO3 bath and were rinsed six times with ultrapure Milli-Q
water prior to use.
45
All solutions were prepared with analytical reagent grade and fresh ultrapure Milli-Q
water. A stock solution of Fe(II) with concentration of 10 mg/L and Cu(II) with concentration
of 20 mg/L were prepared by dissolving the appropriate amount of FeSO4.7H2O and
CuSO4.5H2O respectively with model wine (12.5% alc.) in a 100 mL volumetric flask. Then
20 mL of each were then diluted in a separate 100 mL volumetric flask with model wine. The
final standard was obtained by taking 10 mL of the last solution and combining it with
ultrapure Milli-Q water to the mark (100 mL). The same procedure was followed for the
preparation of Fe(II) and Cu(II) standards with a final dilution in wine base instead of water
which was used in the testing and preparation of the method. The wine base was prepared by
mixing 4.1 g of potassium hydrogen tartrate, 2.4 g of tartaric acid and 250 mL of ethanol in a
2 L Schott bottle which was topped with ultrapure Milli-Q water to make a 2 L solution of
12.5% wine base (Clark 2007). Once prepared it was left stirring overnight covered with
aluminium foil to avoid any influence from direct light.
All the prepared solutions were kept in closed storage rooms without direct sunlight and
at room temperature to ensure their proper preservation. Fresh standard solutions were made
before each experimental session.
46
Results and discussions
Wine production
Grape juice – treatment and measurements
The wine for this experiment was produced in the experimental winery at the NWGIC on
the campus of CSU in Wagga Wagga, Australia. The initial grape juice from the variety
Chardonnay was obtained from NWGIC’s commercial winery and originally came from the
region of Orange, situated around 300 km North-East from Wagga Wagga. Grape juice (220
L) was separated into 12 L demi-johns – 6 different treatments with 3 repetitions per
treatment – and the appropriate metal additions were made to each sample as follows:
-
Treatment 1 (T1) – Control, no additions, 3 replicates;
-
Treatment 2 (T2) – Low copper, 2.9 mg/L Cu, 3 replicates;
-
Treatment 3 (T3) – High copper, 5.8 mg/L Cu, 3 replicates;
-
Treatment 4 (T4) – Low iron, 6.3 mg/L Fe, 3 replicates;
-
Treatment 5 (T5) – High iron, 12.5 mg/L Fe, 3 replicates;
-
Treatment 6 (T6) – High copper and high iron, 5.8 mg/L Cu and 12.5 mg/L Fe.
The copper and iron additions to the grape juice were based on a literature review of
various studies that assessed total copper and iron concentrations in grape juice.
The
concentrations of iron quoted for juice ranged from 1.02 to 21.0 mg/L in juice, and the
concentrations of copper in juice ranged from 1.5 to 7.3 mg/L (Byrne, Saywell et al. 1937;
Feng, Mei et al. 1997; Olalla, González et al. 2000; Cavazza 2013).
The juice was analysed for its metal (copper and iron) content before and after the metal
additions. Samples were taken from each treatment and were analysed by FAAS. The results
are presented in Table 4 for copper and Table 5 for iron, showing the actual concentrations of
copper added to every sample, as well as the final total concentrations.
47
Table 4: Copper concentrations in grape juice on average for each treatment before and after metal
additions [mg/L]. The uncertainty indicated is the standard deviation.
Copper [mg/L]
Sample
Initial
concentration
Actual
Addition
Total/Final
Concentration
T1
T2
T3
T4
T5
T6
0.80 ± 0.01
0.80 ± 0.01
0.80 ± 0.01
0.80 ± 0.01
0.80 ± 0.01
0.80 ± 0.01
0
2.90 ± 0.01
5.80 ± 0.01
0.00 ± 0.01
0
5.83 ± 0.06
0.80 ± 0.01
3.70 ± 0.01
6.60 ± 0.01
0.80 ± 0.01
0.80 ± 0.01
6.63 ± 0.06
Table 5: Iron concentrations in grape juice on average for each treatment before and after metal
additions [mg/L]. The uncertainty indicated is the standard deviation.
Iron [mg/L]
Sample
Initial
concentration
Actual
Addition
Total/Final
Concentration
T1
T2
T3
T4
T5
T6
1.08 ± 0.01
1.08 ± 0.01
1.08 ± 0.01
1.08 ± 0.01
1.08 ± 0.01
1.08 ± 0.01
0
0
0
6.24 ± 0.01
12.5 ± 0.01
12.51 ± 0.02
1.08 ± 0.01
1.08 ± 0.01
1.08 ± 0.01
7.32 ± 0.01
13.58 ± 0.01
13.59 ± 0.02
Alcoholic fermentation
The fermentation process was closely followed with regular sugar concentration and
temperature measurements using a hand held density meter (see experimental section for
details). The measurements are displayed in Figure 2 (and in Appendix A) and follow the
change in the parameters from the first until the last day of the fermentation process. The
sugar concentrations are presented in ⁰Baumé, which is the official measuring unit for wine
in Australia. In order to obtain the concentration in the official sugar concentration unit for
European countries (oBrix) the following formula should be applied:
48
1⁰Baumé = 1.80oBrix
Figure 2: Density measurements taken during alcoholic fermentation in ⁰Baumé.
Toward the end of the fermentation, 10 mL samples were taken from all wines and their
sugar levels were measured enzymatically which enabled more accurate and sensitive reading
of this parameter than the hand held density meter. The results revealed that for treatments 2,
3 and 6 the sugars had been consumed almost entirely by March 17 th (11 days since
commencement of the fermentation) and this was not the case for the remaining three
treatments (Table 6). The wines corresponding to treatment 2, 3 and 6 were then transferred
to a temperature control room allowed to settle at low temperature (3-4oC). 1.92 g of
potassium metabisulfite (PMS) was added to each repetition to protect it from oxidation and
the development of any undesired microbial species.
For the other three treatments – 1, 4 and 5 – however, the alcoholic fermentation was not
finished by March 17th as the sugar levels had not dropped sufficiently (i.e. below 1.5 mg/L
fructose). For this reason these three treatments were kept in a separate temperature control
room at 16oC which allowed sufficient residual sugar to be consumed and for the alcoholic
fermentation to finish. The sugar levels of the ferments were monitored over the subsequent
week to assess completion of the fermentation (Table 6). Once the alcoholic fermentation was
deemed finished (March 24th, day 21 after commencement of fermentation), the same amount
of potassium metabisulfite (PMS) was added to the last three treatments and the wines were
left to settle at low temperature (3-4oC).
49
Table 6: Sugar measurements for fructose and glucose in wine samples [mg/L]. The uncertainty
indicated is the standard deviation.
Date
17/03/2014
19/03/2014
21/03/2014
24/03/2014
Sugars D-Fructose D-Glucose D-Fructose D-Glucose D-Fructose D-Glucose D-Fructose D-Glucose
T1
T2
[g/L]
[g/L]
[g/L]
[g/L]
[g/L]
[g/L]
[g/L]
[g/L]
1.8 ± 0.5
0.06 ± 0.03
1.2 ± 0.3
0.04 ± 0.01
1.5 ± 0.4
0
1.1 ± 0.2
0.05 ± 0.1
0
0
0
0
0
0
0.47 ± 0.03 0.07 ± 0.04
T3
0.2 ± 0.1
0.06 ± 0.05
0
0
0
0
0
0
T4
3.2 ± 0.3
0.08 ± 0.01
2.2 ± 0.2
0.06 ± 0.01
1.8 ± 0.3
0
1.6 ± 0.1
0.05 ± 0.1
T5
1.9 ± 0.1
0.04 ± 0.01
1.5 ± 0.1
0.04 ± 0.01
1.3 ± 0.2
0
1.3 ± 0.1
0.05 ± 0.1
0
0
0
0
0
0
T6
0.12 ± 0.05 0.04 ± 0.04
These results show that juices with high copper concentration (i.e. 3-6 mg/L) actually led
to quicker ferments than for the juices without copper additions.
Iron, on the other hand, had negligible effect on the process.
Literature has shown that much higher copper concentrations in juice (i.e., > 10 mg/L
copper) can result in stuck ferments (Soares 2003). However, our results are the first to show
that more than 1 mg/L and below 6 mg/L can aid the efficiency of yeast towards the end of
the fermentation (i.e., specifically the commercial yeast strain: Lalvin DV10™). This could
potentially be due to the copper concentrations of 1-6 mg/L selecting the most viable and
efficient yeast – more able to tolerate (12-13 %v/v) ethanol concentration, hence aiding the
fermentation at the higher alcohol conditions. This is consistent with the fact that the copper
concentrations of 3-6 mg/L only hastened the end date of fermentation (Table 6), whilst the
overall kinetics for the early stages of the fermentation were similar (Figure 2).
These results show that wines were able to be produced utilising different concentrations
of metal ions in the juice, with the only variation in the wines being slightly higher fructose
concentrations in the treatments, with less than 2.5 mg/L copper added to the juice.
Wine composition
After settling samples were analysed for free and total sulfur dioxide concentrations as
well as pH and titratable acidity (g/L tartaric acid equivalents (TA) (Table 7).
50
Table 7: Titratable acidity (TA), pH, free SO2 (FSO2) and total SO2 (TSO2) measurements for all wine
samples after the end of the alcoholic fermentation. The uncertainty indicated is the standard deviation.
TA
FSO2
TSO2
[g/L]
[mg/L]
[mg/L]
3.13 ± 0.01 6.3 ± 0.2 41.3 ± 0.6
165 ± 2
9/04/2014 T1
3.13 ± 0.01 6.5 ± 0.01 40.3 ± 2.9
173.7 ± 0.6
9/04/2014 T2
3.13
±
0.01
6.4
±
0.1
37.3
±
1.2
177 ± 2
9/04/2014 T3
3.12 ± 0.01 6.1 ± 0.2 35.7 ± 9.3
160 ± 13
9/04/2014 T4
3.12 ± 0.01 6.4 ± 0.1 41.7 ± 1.5
168.3 ± 0.6
9/04/2014 T5
3.12 ± 0.01 6.5 ± 0.01 39.3 ± 1.2
171.7 ± 0.6
9/04/2014 T6
All wines had similar chemical composition which can be regarded as typical for white
Date
Sample
pH
Chardonnay wine from this region in Australia Table 7. The pH was almost identical for all
treatments and remained around 3.13 ± 0.1. The titratable acidity (TA) had a slight variation
across different trials but all wines had a total TA between 6.0 and 6.5 g/L. The free and total
sulfur dioxide were sufficient to ensure oxidative and microbial stability of the wine. The SO2
concentration provided an average ratio of free/total SO2 = 0.23 ± 0.02.
Other wine parameters were also analysed by HPLC (ion exclusion chromatography),
including organic acids, ethanol, and glycerol, however the samples exhibited no significant
differences over treatments (Appendix B).
Copper and Iron speciation
Optimisation of methodology and approach
The first part of the project focused on analysing the concentrations of copper and iron in
wine not only as total concentrations, but also the different forms in which the metals can be
found as well as the ratio between these forms. For this purpose, a specific analytical method
was developed in order to ensure the accurate and precise collection of data. Specific resins
were utilized to separate the metal species in the wine so as to allow quantification of
different forms.
The initial approach was to prepare the wine samples with the use of a polymeric resin
and a cation-exchange resin. These are some of the most commonly used resins in metal
speciation research (Pohl and Sergiel 2009), as they efficiently separate metal species into
different groups – hydrophobic, residual and cationic forms. Although the results obtained
showed a separation of the metal ions, it is important to note that the preparation technique
51
was quite labour intensive. It also had the potential for variations in the amount and
consistency of the resin due to the number and diversity of steps required before the resin
usage. This inevitably led to some variability in the results which were obtained on a test
wine and a wine base. For example, the average relative standard deviation for the copper
speciation was 41.3%, 17.1% and 43.7% for the hydrophobic, residual and cationic fractions
respectively. Consequently, it was decided that a set of commercially prepared resins, in preprepared solid phase extraction (SPE) cartridges, with more consistent and repeatable
chemical and physical characteristics would be utilised instead. The conditioning technique
for the SPE cartridges remained the same as for the resins with the exception that no
weighing, pre-wetting or transferring of the SPE material was required. This abbreviated pretreatment allowed for a much better repeatability. For example, an average relative standard
deviation of 3.2%, 1.8% and 11.7% was obtained for the hydrophobic, residual and cationic
copper fractions, respectively, on a test wine sample. This approach significantly limited the
possibility of differences and variations of the resins. All subsequent wine samples were
examined with the use of commercially prepared SPE cartridges.
Metal concentration in grape juice
After the additions of copper and iron to the grape juice, the latter was analysed to
measure the exact concentrations of the two metals. All samples and associated replicates
were analysed on a FAAS after a two-fold dilution with water and quantification was
performed by calibration graph. The calibration standards were 0.4, 1.2 and 2.4 mg/L for both
copper and iron and they were prepared in a model juice system that was also diluted two
fold before addition of copper or iron (i.e., 200 g/L glucose, pH 3.2). The calibrations graphs
Absorbance (248.3 nm)
Calibration graph, Iron
0,35
0,3
0,25
0,2
0,15
y = 0,0875x + 0,0021
0,1
0,05
0
0
0,5
1
1,5
2
2,5
3
3,5
Iron concentration [mg/L]
Calibration Fe
Linear (Calibration Fe)
Figure 3 : Calibration graph for iron in grape juice.
52
for both metals can be observed in Figure 4 (for copper) and Figure 3 (for iron). Preliminary
studies showed no significant difference (p=0.05) between quantification by calibration graph
versus calibration graph via internal standards.
Calibration graph, Copper
Absorbance (324.8nm)
0.3
0.25
0.2
0.15
y = 0.0863x + 0.0011
0.1
0.05
0
-0.05 0
0.5
1
1.5
2
2.5
3
3.5
Copper concentration [mg/L]
Calibration Cu
Linear (Calibration Cu)
Figure 4 : Calibration graph for copper in grape juice.
The results from the FAAS analysis on the grape juice can be seen in Figure 6 and they
show low uncertainty (i.e., see standard deviation error bars) in the metal concentrations
within treatments. The control juice (treatment 1) as well as the treatments where no copper
additions were made (treatment 4 and 5) all have a copper concentration around 0.76 ± 0.05
mg/L (i.e., all values quoted are mean ± standard deviation (n=3)). The low copper treatment
(treatment 2) averages 3.61 ± 0.09 mg/L and the high copper treatments (treatment 3 and 6)
average 6.35 ± 0.03 mg/L and 6.52 ± 0.06 mg/L of copper respectively, which is consistent
with the actual additions. When looking into the iron concentrations we can once again see
that the control and the treatments without iron additions (treatment 1, 2 and 3) show very
close results with 0.35 ± 0.05 mg/L. Treatment 4 was the low iron trial where 6.03 ± 0.02
mg/L of iron was measured. The higher iron additions were made to treatments 5 and 6 which
were found to have 11.11 ± 0.03 mg/L and 11.31 ± 0.06 mg/L of iron, respectively. The
results confirm that addition of the metal ions to the juice provided total juice concentrations
as expected and no precipitation of metals occurred in the juice before measurement of the
total concentrations. In the following Figure 5 the metal concentrations detected in all wine
samples after the alcoholic fermentation.
53
Figure 6 : Copper and iron concentrations [mg/L] in grape juice after metal additions. Error bars
indicate the standard deviation.
Figure 5 : Copper and iron concentrations [mg/L] in wine after alcoholic fermentation. Error bars
Copper and iron speciation in wine
As mentioned earlier (see Material and methods) all further metal analysis of the wine
after the alcoholic fermentation were carried out with the use of an ICP-OES due to the low
concentrations of the metals, especially in the treatments where no copper or iron additions
were made. This method proved to be much more sensitive, accurate and reliable, compared
to FAAS, when analysing trace elements in the wine at low concentrations.
For each treatment and repetition, five samples were prepared. These preparations
included the hydrophobic, residual and cationic speciation fractions as well as a sample of the
54
initial wine before and after filtration. This approach enabled the measurement of the
different species of metal ions in the wine. Filtration had no significant impact (p=0.05) on
the total metal concentration (data not shown) throughout the study and will not be
commented on further. It does indicate that the metal in the wines was in a form that was able
to pass through 0.45 µm filters of polyethylsulfone (PES) membrane composition.
Copper speciation
Table 8: Total and speciation copper concentrations [mg/L] in wine all samples (T1-T6). The
uncertainty indicated is the standard deviation. Means with the same letters are not significantly different
(SD) at p=0.05.
Sample
T1
T2
T3
T4
T5
T6
The results
Hydrophobic
Total Cu
Cu
Residual Cu
a
a
0.083 ± 0.008
0.019 ± 0.006
0.039 ± 0.007a
0.233 ± 0.005c
0.068 ± 0.024b
0.061 ± 0.006b
0.293 ± 0.006d
0.060 ± 0.024b
0.073 ± 0.006b
0.068 ± 0.014ab
0.008 ± 0.004a
0.031 ± 0.007a
0.052 ± 0.011b
0c
0.015 ± 0.007c
0.288 ± 0.007d
0.032 ± 0.028abc 0.068 ± 0.012b
from the metal speciation presented several interesting
Cationic Cu
0.041 ± 0.005a
0.109 ± 0.022b
0.155 ± 0.013c
0.027 ± 0.002d
0.028 v 0.004d
0.164 ± 0.038bc
tendencies for copper
(Table 8, Figure 5 and Figure 7). In terms of total copper, the concentration had vastly
decreased after fermentation (compare Figure 6, Figure 5 and Figure 7), particularly for
treatments 3, 4 and 6. It has been found in past research that the copper content in wine is
significantly lower than the corresponding grape juice. The reason given for this decrease is
most often the yeast activity during the alcoholic fermentation and the formation of insoluble
sulphides, removed through sedimentation with the yeast and lees (Ribéreau-Gayon, Glories
et al. 2006).The result is a drop in concentrations from 5.04 ± 0.03 mg/kg (García-Esparza
2006) and 4.95 ± 0.08 mg/kg (Provenzano, El Bilali et al. 2010) in the grapes to 1.01 ± 0.95
mg/L (García-Esparza 2006) and 0.18 ± 0. 69 mg/L (Bukovčan 2009) in the wines,
respectively.
55
Copper concentation [mg/L]
Copper speciation concentrations in wine
0,35
0,30
0,25
0,20
Cationic
0,15
Residual
0,10
Hydrophobic
0,05
0,00
T1
T2
T3
T4
T5
T6
Sample
Figure 7: Copper speciation concentrations [mg/L] for all samples (T1-T6). Error bars indicate
the standard deviation.
Even though after the alcoholic fermentation the copper concentrations had significantly
decreased due to the yeast activity, the low detection limits of the instrument (ICP-OES
Minimal Detection Limit for Cu – 0.002 mg/L) made it still possible to quantify the different
fractions. Apart from treatment 5, none of the samples had a cumulative total of copper
species (hydrophobic + residual + cationic) that were significantly different (p=0.05) than the
measured total concentration (Table 8, measured total corresponded to 100%). For treatment
5, the low concentrations of copper may have contributed to it having a lower cumulative
total of copper species than expected, especially given that the concentration of residual
copper was below the limit of detection. This general agreement in total copper concentration
with the cumulative total of copper species supported the validity of the speciation approach
taken.
For copper, in the samples without copper addition to the juice (T1, T4 and T5), there was
a trend towards higher cationic fractions, compared to the other speciation fractions,
regardless of the initial metal additions made to the grape juice (Figure 7). Furthermore, there
was not a large difference in the copper speciation, in terms of percentage, over the different
treatments. For example, in treatments 4 and 5 the cationic fraction was 40 ± 9% and 55 ±
5%, respectively, out of the total copper concentration. In T4 the concentration of cationic
and residual copper fractions were similar, representing more than 85% of the total copper
concentration. The major difference detected was in treatment 5 where the two fractions of
56
Figure 8 : Copper fractions in wine as percentage of the total concentration. Error bars indicate the
standard deviation.
residual and cationic accounted for all the copper as no hydrophobic fraction was detected.
The cationic fraction was also dominant in the control treatment with 50 ± 7% of the total
concentration.
It should be noted that due to the yeast activity after the alcoholic fermentation the
amount of copper ions in the treatments with copper additions was almost the same for the
wines with both low and high metal additions – T2, T3 and T6. It is once again evident that
the cationic copper species are the most abundant form and similar for all 6 treatments and
represent between 40% and 60% of the total copper. This is not the case for the residual
fraction where treatments 1 (control) and 4 (low iron) seem to have a more significant
percentage of their copper concentration in this fraction 47 ± 9% and 45 ± 3%, respectively,
compared to other samples which are generally less than 40% of this fraction. Overall, these
results (Figure 8), show that generally the most significant portion of the copper which is
found in the wine after alcoholic fermentation is in the cationic fraction and that this species
doesn’t seem to be overly affected by the initial copper concentration in the juice – varying
from 0.70 mg/L to 6.52 mg/L.
57
Iron speciation
Table 9: Total and speciation iron concentrations [mg/L] in wine all samples (T1-T6). The uncertainty
indicated is the standard deviation. Means with the same letters are not significantly different (SD) at
p=0.05.
Sample
T1
T2
T3
T4
T5
T6
The data
Total Fe
Hydrophobic Fe
Residual Fe
Cationic Fe
a
a
a
0.0423 ± 0.006
0.097 ± 0.027
0.279 ± 0.016
0.079 ± 0.014a
0.0425 ± 0.009a
0.016 ± 0.022b
0.200 ± 0.016b
0.216 ± 0.020b
0.0421 ± 0.006a
0.008 ± 0.004b
0.203 ± 0.040b
0.224 ± 0.16c
6.200 ± 0.046b
0.293 ± 0.046c
1.345 ± 0.084c
4.428 ± 0.061c
11.693 ± 0.140c
0ab
0.788 ± 0.246d
10.095 ± 0.480d
11.773 ± 0.083c
0abc
0.643 ± 0.203d
10.202 ± 0.295d
gathered from the ICP-OES revealed some interesting results regarding the iron
total concentrations and speciation (Table 9, Figure 9 and Figure 10). The same three metal
species were examined as for copper – hydrophobic, residual and cationic – as well as the
total iron concentration. The division between fractions for the iron ions in the wines seemed
to be similar to the one already presented for copper. In the treatments without iron additions
(T1, T2 and T3) the total concentrations were almost identical and significantly lower than
Figure 9: Iron speciation concentrations [mg/L] for all samples (T1-T6). Error bars indicate the
standard deviation.
the other three trials where defined metal additions were made (T4, T5 and T6). The first
three treatments were found to have an average total iron concentration of 0.423 ± 0.002
mg/L. Despite these low concentrations, a difference is of note in the speciation results when
comparing the control (T1) to T2 and T3. The most significant species of iron in the T1 wine
sample was the residual (66 ± 3%), followed by the hydrophobic (23 ± 5%) and the cationic
58
accounting for the smallest portion (19 ± 3%) (Table 6 and Figure 9). However, the
remaining treatments (T2-T6) have differing speciation profiles to T1 but profiles that are
similar to each other. For T2 and T3, where the total iron was very low, the most dominant
fraction showed to be the cationic with more than 50% of the total (51 ± 6% and 53 ± 4%,
respectively). The residual iron was slightly lower in concentration – 47 ± 3% in T2 and 48 ±
9% in T3 than for the cationic fraction, whilst the hydrophobic form was less than 5% for
both. This suggested that copper addition to the juice tended to decrease the amount of iron
found in the hydrophobic form in the wine. Comparing T2 and T3 samples with the high iron
samples (T4, T5 and T6) a similar proportional division between the iron species can be
highlighted. The difference in this case is that the cationic fraction accounts for a more
significant part of the total concentration (72 ± 2% for T4, 86 ± 5% for T5 and 87 ± 2% for
T6). The dominance of cationic iron species is obvious, and leads to only 22 ± 2%, 7 ± 2%
and 6 ± 1% of the total for the residual fraction in T4, T5 and T6, respectively. Once again,
the proportion of iron in the hydrophobic form is less than 5% in all samples apart from the
control (T1) and the sample with low iron addition (T4).
Analysing the results for the iron speciation obtained in the case of the current study it
became clear that the cationic species of iron are the most predominant form for iron in all
but one of the wine samples. The proportion of cationic iron, compared to the total
concentration, varies from 51% to 87% with the exception of the control wine where it
represent 19 ± 3% of the total. It would appear that copper addition to the juice or iron
addition to the juice favours a decrease in the proportion of iron in the hydrophobic form and
Figure 10: Iron fractions in wine as percentage of the total concentration. Error bars indicate
the standard deviation.
59
increase in the proportion of iron in the cationic form. In the sample emanating from juice
without copper or iron addition, most of the iron is found in the residual form (46 ± 3%).
Copper and iron speciation in wine before and after bentonite fining
The second aspect of iron and copper speciation in white wine which was investigated
was to compare the impact of protein stabilisation on metal speciation measures. The wine
samples are the same as in the previous section with the only difference being the addition of
calcium bentonite. This is a common technique amongst wine makers, also known as
bentonite fining or protein stabilisation, which significantly reduces the amount of proteins in
the wine and thus makes it more stable during storage. The entire process is presented in
detail in the “Material and method” section of this report.
The wine samples with and without bentonite additions were analysed for metal
speciation profiles utilising the same technique as for the samples in the first part of the
study. This approach allowed the bentonite fining process to be the only difference between
the two groups of samples. A subset of the samples analysed in the first part of the study were
selected to allow insight into the effects of bentonite treatment on copper and iron speciation.
By following this approach it was possible to represent each analysed wine sample as an
average of three replicates and thus significantly increasing the reliability and
representativeness of the results for the wines with and without bentonite. The three wine
samples which were chosen and analysed in this part of the study were treatment 1 (control),
treatment 3 (high copper) and treatment 6 (high copper and high iron). For each of these
treatments, one experimental replicate was randomly chosen and they were as follows: T1R3,
T3R2 and T6R1 respectively.
Bentonite has already been reported to influence the metal content of wine having
different effects on the total content of various elements present in the wine, including iron
and copper (Catarino, Madeira et al. 2008). The aim of this study was to investigate how
bentonite impacts not only the total content, but more specifically the three fractions of
copper and iron ions.
60
Copper speciation and influence of bentonite fining
Table 10: Copper speciation concentrations [mg/L] before and after bentonite addition. The
uncertainty indicated is the standard deviation.
Sample
T1R3 No
Bentonite
T1R3 With
Bentonite
T3R2 No
Bentonite
T3R2 With
Bentonite
T6R1 No
Bentonite
T6R1 With
Bentonite
Total Cu
Hydrophobic Cu
Residual Cu
Cationic Cu
0.076 ± 0.001a
0a
0.032 ± 0.003a
0.048 ± 0.002a
0.034 ± 0.002b
0.005 ± 0.001b
0.017 ± 0.003b
0.014 ± 0.003b
0.290 ± 0.002c
0.021 0.019a,b,c
0.061 ± 0.018c
0.236 ± 0.026c
0.046 ± 0.002d
0.009 ± 0.005b,d
0.024 ± 0.002b
0.024 ± 0.004d
0.354 ± 0.023e
0.065 ± 0.035c,d
0.074 ± 0.029a,c
0.251 ± 0.030c
0.092 ± 0.003f
0.050 ± 0.004c
0.029 ± 0.009a,b,c
0.014 ± 0.012b,d
A comparison between the ICP-OES results, before and after bentonite fining, revealed
some changes in both the total and metal speciation copper. It must firstly be noted that there
was a significant decrease in the total copper for all three different treatments (Table 10). For
the control wine (treatment 1) the difference was smaller (a change of 0.041 ± 0.001 mg/L)
which is due to the fact that the copper content in this wine was initially very small (only
0.076 ± 0.002 mg/L) after the alcoholic fermentation due to the yeast activity. It is more
interesting to note that for the other two analysed treatments, the total copper content in the
wine with bentonite addition had significantly decreased such that the concentration of
copper became very close to the control. The decrease was 0.243 ± 0.001 mg/L and 0.262 ±
0.02 mg/L for treatments 3 and 6, respectively. To sum up, this led to similar final total
copper concentration for all three wine samples of around 0.06 ± 0.03 mg/L.
Looking more closely into the copper speciation results, it was evident that the decrease
of the copper content had affected all three copper species (Figure 11 and Table 10). What is
more important to note is that the relative amounts of the metal species had been influenced
by the addition of bentonite. In the wines without bentonite treatment, the hydrophobic
fraction was the lowest concentrated copper species: none was detected in treatment 1 and
less than 20% was evident in treatment 3 and 6 (Figure 11). After the bentonite addition,
however, the hydrophobic fraction increased in proportion to total copper. In treatment 1 and
3, where there was almost no hydrophobic copper before the bentonite addition, this fraction
represented 15-20% of the overall copper in the two wines respectively. The increase was
even bigger in treatment 6 (where the hydrophobic fraction was 55 ± 4%) after bentonite
61
fining which also had the highest copper concentration. In absolute terms, the actual
concentration (mg/L) of the hydrophobic fraction of copper only increased significantly in T3
(p=0.05) due to bentonite treatment, whilst in the other samples it was not significantly
different (Table 10) due to bentonite treatment. In fact, in T6 the concentration of the residual
copper was less (albeit not significantly) after bentonite treatment, but because the total
Figure 11: Copper speciation as percentage of total [%] before and after bentonite addition. Error
bars indicate the standard deviation.
concentration of copper in the wine was also reduced, the proportion (% of total) of
hydrophobic copper increased.
The bentonite fining was found to have a similar impact on the residual copper fraction in
all samples. For the control treatment (T1) it had a trend towards higher concentrations from
42 ± 4% of the total concentration to 50 ± 10%, albeit not a significant increase (p=0.05). In
the high copper treatment (T3) this fraction also represented around half of the copper in the
wine which was fined with bentonite (50 ± 10%), which was, however, a considerable
increase in comparison to the same wine that had no bentonite addition made, where it
represented 21 ± 9% of the total. This was the biggest difference detected for the residual
species of copper in the wines, since in the third treatment analysed (T6), the residual fraction
increased to be around a third of the total copper (21 ± 9% versus 30 ± 10%) in the treated
wine (Figure 11). Therefore, the treatment of wines with bentonite showed a trend towards a
higher proportion of copper being in the residual copper form. Although, the difference in
residual copper due to bentonite treatment was not significant (p=0.05) in any sample, in
62
proportional terms, T1 and T3 had significant differences in residual copper due to bentonite
treatment in absolute terms (Table 10). The impact of bentonite showed a significant (p=0.05)
reduction in the concentration of residual copper. Given that bentonite is efficient in
removing protein and does have the ability to undergo ion exchange with cations, the
decrease in residual copper could be as a consequence of removal of either of these forms. .
The further analysis of the speciation data showed a significant decrease in the cationic
portion of copper in all samples due to bentonite treatment (Table 10 and Figure 11). The
cationic form of copper was the predominant copper species in the wines without any
bentonite additions – 63 ± 2% in treatment 1, 81 ± 8% in treatment 3 and 71 ± 5% in
treatment 6. The drop was quite similar for the first two treatments where the bentonite
addition had led to 41 ± 7% and 50 ± 10% of the copper being in this fraction in treatments 1
and 3, respectively. Once again the most significant difference was detected in the high
copper and high iron treatment (T6) which ended up having only 15 ± 13% cationic copper
species due to the addition of calcium bentonite to the wine before bottling.
Iron speciation and influence of bentonite fining
The distribution of all three iron fractions is presented in Table 11 as concentrations in
mg/L and in Figure 12 as a percentage of the total concentration on average from the three
replicates which were analysed for each treatment.
Table 11: Iron speciation concentrations [mg/L] before and after bentonite addition. The uncertainty
indicated is the standard deviation
Sample
Total Fe
Hydrophobic Fe
Residual Fe
Cationic Fe
0.530 ± 0.001a
0a
0.37 ± 0.01a
0.22 ± 0.01a
T1R3 With Bentonite
1.65 ± 0.02b
1.60 ± 0.03b
0.40 ± 0.07a
1.40 ± 0.05d
T3R2 No Bentonite
0.54 ± 0.01a
0a
0.20 ± 0.05b
0.41 ± 0.04b
T3R2 With Bentonite
1.81 ± 0.03c
0.12 ± 0.10a,b
0.36 ± 0.08a
1.57 ± 0.12d
T6R1 No Bentonite
13.4 ± 0.2d
0.62 ± 0.86a,b
0.64 ± 0.40a,b
13.50 ± 0.35c
T6R1 With Bentonite
14.3 ± 0.3e
0.46 ± 0.28b
0.83 ± 0.50a,b
14.55 ± 0.42e
T1R3 No Bentonite
63
Adding bentonite to the wines , in order to achieve protein stability, has been found to
influence the total content of iron ions in wine (Catarino, Madeira et al. 2008). The iron
speciation analysis which was performed in this study revealed similar results for the total
iron concentration content, but at the same time providing further information about the
distribution of iron between the hydrophobic, residual and cationic fractions.
In the case of total iron concentration, bentonite treatment on average increased iron by
1.08 ± 0.20 mg/L over all three treatments and the increase was significant (p=0.05) in all
cases. The hydrophobic fraction significantly increased in treatment 1 and had trends to
higher concentrations for treatments 3 and 6 although the differences were not significant.
There appeared to be some high variability in the measurement of the hydrophobic fraction in
treatments 3 and 6. Treatment 3 was also found to have a concentration of total iron below 1
mg/L, which increased after the fining to 1.81 ± 0.03 mg/L (Table 11). In the last sample,
which initially had a very high iron concentration (13.4 ± 0.2 mg/L), the total concentration
in the wine after the bentonite addition had also increased and was measured to be 14.3 ± 0.3
mg/L (Table 11).
In terms of proportions (as a % of total), the hydrophobic form of iron was only a minor
species, contributing less than 7% of iron in all cases. In absolute concentration (mg/L) there
was a trend to higher concentrations of residual iron after addition of bentonite, although this
was only significant for treatment 1. In terms of proportions (% of total iron) the residual iron
species actually decreased after bentonite addition for treatments 1 and 3 and was steady for
treatment 6 (Figure 12). Treatment 6 most likely didn’t change as the iron concentration was
already very high before the bentonite addition, whilst for the other samples the total iron
concentration increased far more than the increase observed in the residual iron
concentration.
64
Iron fractions [%] of total
Figure 12: Iron speciation as percentage of total [%] before and after bentonite addition. Error bars
It was revealed that for treatment 6, which initially had the highest copper and iron
additions to the grape juice, the proportion of cationic fraction remained almost unchanged
after the addition of bentonite before bottling. This is where practically all of the iron was
identified to be present – 102.1 ± 2.6% before and 100.1 ± 2.9% after the treatment. For
treatments 1 and 3 this winemaking procedure increased the proportion of the cationic iron
species in the total concentration. For the first wine samples (T1) the increase was double
from 41 ± 4% to 85 ± 3%, but much less important for treatment 3 where from 75 ± 11% it
changed to 86 ± 7%.
When talking about iron speciation in the case of the current study it could be said that
there is a definite and easily distinguishable effect of the addition of calcium bentonite to the
white wine samples. In terms of total copper concentration a significant decreased is observed
and for iron a significant increase is observed. For copper this resulted in a decrease in the
mg/L concentration of most species apart from the hydrophobic form, whilst for iron
increases in species were generally observed.
Impact of copper and iron speciation in wine on the oxygen decay rate
The third and final part of the study was designed to measure and compare oxygen decay
rate for each treatment. The aim was to investigate whether there was a difference in the
oxygen consumption rates in samples and if so, whether it could be related to the copper and
iron speciation of these wines.
65
The rate of the oxygen decay is presented for separate treatment as an average of the data
gathered from all three replicates for each treatment. The results provide the average decay
rate, calculated as first order rate constants (1/min) as such rate order reaction kinetics fitted
the data best. The oxygen concentration measurements were taken regularly, after saturating
the wines with oxygen, and adding ascorbic acid (216 mg/L) and sulfur dioxide (228 mg/L)
to the wines to be in excess over the dissolved oxygen present. It should be noted, however,
that the preparation method of the samples included them being intensively aerated (by
shaking) for 5 mins which led to an increase in their oxygen concentration and the presence
of micro-bubbles. After gathering and carefully analysing all the data for the samples, it was
clearly visible that there was a certain period of time during which the concentrations of
oxygen were unstable and had a tendency to fluctuate by ± 0.50 mg/L. This was attributed to
the wine degassing and achieving stable dissolved oxygen concentrations. Taking this into
consideration, the calculation of oxygen consumption rates was performed from 500 mins
after addition of ascorbic acid sulfur dioxide, until around 2300-2500 minutes.
Table 12: The first order oxygen decay rates presented in [1/min] for all samples (T1-T6). Means with
the same letters are not significantly different (SD) at p=0.05.
Sample
Oxygen decay rate
[min-1]
T1
T2
T3
T4
T5
0.00020 ± 0.00003a
0.00045 ± 0.00002b,c
0.00054 ± 0.00007b
0.00038 ± 0.00006c
0.00038 ± 0.00005c
0.00072 ± 0.00003e
T6
* different superscripts indicate a
significant difference (p=0.05)
Studying the results for the oxidation process of the wine samples it can be pointed out
that the control wine (T1) has the slowest decrease in oxygen (0.00020 ± 0.00003 min-1).
Looking into the results obtained in the first two parts of the study, it could be noted that this
is the wine with the smallest concentrations of both copper and iron. The next two treatments
(T2 and T3) which had copper added to the juice before the fermentation (the additions were
2.90 and 5.90 mg/L, respectively) show a very similar oxidation rate, which is significantly
(p=0.05) higher in comparison to the control. Consistent with this is the fact that T2 and T3
also had similar copper speciation profiles.
66
Treatment 4 (low iron) and Treatment 5 (high iron) gave similar oxygen decay rates to
each other and had faster oxygen decay than the control but slower decay than the samples
with only added copper (T2 and T3), although only T3 was significantly different. This
suggested that for a given [mg/L] concentration, copper was impacting the rate of oxygen
decay more critically than iron. Treatment 6 (high copper and high iron) showed significantly
faster oxygen decay than any other sample consistent with its elevated copper and iron
concentrations (Table 9).
The aim of the experiment was, however, not to only compare the oxidation to total
concentrations, but to relate the oxidation to the iron and copper speciation data, gathered in
the previous parts of the study. A comparison has been made between the total metal
concentration and the separate metal fractions.
Influence of copper speciation on oxidation
In Figure 13 the oxygen decay rate (first order) are compared to the total copper
concentrations for the treatment 1-3 samples. The data reveals a very good correlation
between the total concentration for treatments 1-3 and the oxidation rates. It is evident that
the higher the concentration of copper the faster the oxygen consumption, which is consistent
with the results obtained in previous studies (Cacho, Castells et al. 1995; Clark and Scollary
2002; Danilewicz 2013). The results reveal that the more residual copper left in the wine at
Figure 13: Correlation between the oxygen decay rate [1/min] and the total copper concentrations
[mg/L].
67
bottling, the faster the oxidation of the wine will occur, provided it has excess of oxygen, and
thus alter the organoleptic characteristics of the wine in the bottle.
The data gathered throughout this study gives also the possibility to compare for the first
time not only the total available copper, but also the three different species in which it can be
found in the wine – hydrophobic, residual and cationic. The latter copper species have been
related to the oxygen decay rates in treatments 1-3 and a graphical presentation is given in
Figure 14. This representation of the results shows that the hydrophobic fraction, accounting
for the smallest part of the copper in the finished wine, seems to have the least correlation
with the speed of the oxidation process. The cationic fraction has improved correlation, and it
is the most dominant in almost all treatments. These two fractions do have positive
correlations to the oxygen consumption rates but do not correlate as highly as the residual
copper concentration.
The strongest correlation was found between the residual copper fraction and the oxygen
decay rates in the studied wines. Again a positive correlation can be seen between the
residual copper and oxygen consumption rate, which shows an increase in the speed of the
oxygen decay with the rise of the concentration of residual copper. It can be assumed that this
fraction contributes significantly to the linear relationship between the total copper and the
oxidation rate in Figure 13.
Figure 14: Correlation between the oxygen decay rate [first order] and the hydrophobic, residual
and cationic copper fractions [mg/L] in the wine.
Influence of iron speciation on oxidation
Examining the relationship between the total iron concentration in treatments 1, 4 and 5,
and the oxygen decay rate, which is presented in Figure 16, it can be said that there is some
linear correlation between the two – the oxidation process seems to be faster as the iron
68
concentrations in the wine increase. However, it should be noted that there is a variation in
the results and the correlation is not as strong as was observed for copper (Figure 13). It is
possible that for iron, its impact on oxygen consumption is not as strong as for copper,
especially when iron reaches a certain concentration.
The iron speciation was related to the oxidation rates utilising the same approach as for
the copper speciation. The hydrophobic fraction had poor positive correlation with oxygen
decay rate (Figure 15). However, it should be noted that it accounted for the smallest portion
of the iron in the wine and its concentrations were less than 1mg/L for all treatments.
Figure 16: Correlation between the oxygen decay rate [first order] and the total iron concentration
[mg/L].
Figure 15: Correlation between the oxygen decay rate [first order] and the hydrophobic, residual and
cationic iron fractions [mg/L] in the wine.
69
In Figure 15 it can be seen that there is a much better correlation between the residual iron
species and the oxidation rate. The treatments where a higher concentration of the latter was
detected were also found to have undergone faster oxidation.
The cationic fraction of iron was the highest in concentration for treatments 1, 4 and 5.
However, the correlation of the cationic fraction of iron was not as strong as for the residual
fraction (Figure 15). The results show that as for copper, the residual iron content correlates
best with the oxygen consumption rate followed by the cationic fraction.
Influence of bentonite addition on oxidation
In this third part of the study a further investigation on the oxidation process was made
looking into a possible influence of the addition of calcium bentonite to the wine on the
oxygen decay rates. This part of the experiment was carried on three representative
treatments – treatment 1 replicate 3 (T1R3), treatment 3 replicate 2 (T3R2) and treatment 6
replicate 1 (T6R1). Each one was performed in three replicates before and after the bentonite
fining.
As shown in Table 13 a difference in the oxidation rates was observed in the wine
samples with and without bentonite. For T1R3 (control) it can be concluded that the bentonite
treatment has significantly (p=0.05) increased the rate almost double – from 0.00023 ±
0.00003 min-1 to 0.00043 ± 0.00005 min-1. The reason for this could possibly be found in the
fact that in addition to the removal of proteins from the wine the bentonite treatment was also
responsible for an increase in the total concentration of iron. A good correlation between the
latter and the oxygen decay rate was already discussed in the previous section.
Table 13 : Comparison between oxygen decay rates [first order] of wines before and after bentonite
addition. Means with the same letters are not significantly different (SD) at p=0.05.
Sample
Oxygen decay rate [min-1]
T1R3 Bentonite
T3R2 Bentonite
T6R1 Bentonite
T1R3
T3R2
0.00043 ± 0.00005a
0.00054 ± 0.00005a,b
0.00061 ± 0.00002b,c
0.00023 ± 0.00003d
0.00046 ± 0.00001a
0.00064 ± 0.00002c
T6R1
* different superscripts indicate a significant difference (p=0.05)
70
In T3R2 (high copper) an entirely different impact was discovered. In the wine sample
with addition of bentonite, the oxygen decay rate was no longer significantly different. In this
case it can be suggested that the significant removal of copper from the wine due to the
bentonite additions was offset by the contribution of iron that bentonite provides the wine.
Consequently, little change in the oxygen consumption rate in the sample occurred after
treatment with bentonite. This decrease in the copper concentration has been reported
previously (Catarino, Madeira et al. 2008).
T6R1 had the biggest additions of both copper and iron made to the grape juice, and thus
had the highest iron and copper concentrations in the wine in comparison to all other
treatments. When comparing the oxygen decay rates there was no significant change in the
rate of oxidation based on bentonite treatment.
It can be concluded, that using calcium bentonite to stabilise the wines before bottling, by
removing most of the proteins from the wine, can influence the rate of oxygen decay in a
wine. However, the effect was only significant for a wine that had low iron and low copper
concentrations prior to bentonite addition. Alternatively, for wines already high in iron (> 7
mg/L) bentonite fining had little impact on the rate of oxygen decay in the wines.
The speciation data for the bentonite fined wines was not related to the oxygen decay
rates as they were complicated by the significant changing concentrations of both Cu and Fe
between bentonite treated samples.
71
Limitations
The project design for the research project was carefully considered to ensure a good
overall outcome. The initial juice-derived experiment was carried out on 6 separate
treatments and each treatment was prepared in 3 replicates (18 wine samples in total) which
significantly increased the pertinence of the obtained results. In order to best control the
development of the wines, the winemaking process was monitored frequently and consistent
production protocols were maintained for all treatments/replicates. Nonetheless, working
with such a large number of samples, in combination with growth of a microbial organism
(i.e., fermentation yeast), resulted in slight differences between replicates, which was
accounted for in the calculated uncertainty ranges for treatments, and enable statistical
considerations of significant differences between treatments.
The Chardonnay grapes used for the preparation of the wine samples, were from a single
vintage (2014). Although the vintage was reported as a “typical” for the region of origin, a
difference between vintages is always present and thus the results revealed in the current
work cannot be treated as generally applicable, but should be regarded as relevant to the
appropriate vintage (2014).
Regarding the analytical part of the experiment, it must be pointed out that the use of SPE
columns for the separation of the wine samples into three fractions requires a multitude of
measurements (i.e., weighing) and volume delivery/preparation critical to the final result.
What is more, further treatment of the samples was necessary in order for them to be
analysed by FAAS and ICP-OES. These are both reference instruments for such type of
research since they provide good precision and accuracy. However, it is probable the multistage and labour intensive sample preparation required could be responsible for some
variation between samples.
Conclusion and future scope of work
Conclusion
First of all, it was observed that regardless of the quantity of copper added to the grape
juice, after the alcoholic fermentation its concentrations had significantly decreased due to
72
the yeast activity. The opposite tendency was found for iron, as almost all of the iron which
was detected in the juice reached the finished wine.
Furthermore, the methodology applied for the speciation of both copper and iron was
significantly optimised in comparison to previous research. This led to a precise fractionation
and thus measurement of hydrophobic, residual and cationic species of the metals.
The copper speciation revealed that it was the cationic species which were predominant
for all treatments, and that the differences in the copper additions made to the grape juice had
no significant influence on the distribution of the copper species for all samples.
The cationic fraction also accounted for the majority of iron in all, but one wine sample. It
was more than half of the total iron concentration for these samples, varying from 51% to
87%. It could be assumed that either the copper or iron additions to the grape juice result in a
decrease the proportion of hydrophobic iron species and increase the proportion of cationic
species. In the samples without any initial copper and iron addition, it was the residual form
of iron which was predominant (46 ± 3%).
Additionally, the oxygen decay rates in the wines showed a good correlation with the total
concentration for copper in the wine samples. However, a stronger correlation was discovered
between the oxygen decay rates and the residual species of copper. A similar match was
found for iron, for both total and residual concentrations versus oxygen decay rates, although
not as strong as for copper.
Furthermore, the influence of calcium bentonite, which is one of the most frequently used
agents for protein stabilisation in white wine, on the oxygen decay rate was investigated. It
was determined that the addition of bentonite prior to bottling was variable but often led to an
increase in the speed at which the oxygen concentration decreased in wine samples. This was
particularly the case for the sample which had the lowest copper and iron concentrations
before bentonite addition.
In summary, it can be highlighted that the results gathered from this research are the first
to show the copper and iron speciation in white wine as a function of juice metal
concentrations. In addition, it is also the first time that the impact of bentonite on the copper
and iron speciation profile of white wines has been studied. Last but not least, never before
had the link between the speciation of copper and iron in white wine and the rate of oxidation
73
been studied and thus this work is the first to reveal interdependence between the three metal
species and the oxygen decay rate.
Future scope of work
The data from this study can be used as a good base for future research in the field. One
suggestion for such a continuation could be the investigation of the actual chemical
composition of different metal species (i.e. copper and iron). Such a study could focus on the
link between the specific metals and their complexation by macromolecules (including
proteins, polysaccharides, phenolic compounds). Another interesting aspect of such a
research could be the relationship of the different metal species to metal sulfides (i.e. copper
sulfide (CuS)) in wine.
Further studies investigating oxygen decay rates and its correlation to the different copper
and iron species in a larger range of white wine is advisable. This will establish a clearer link
between speciation measures and the ability of the metal ions to catalyse oxidation reactions.
Other possible future work stemming from this research could be the investigation of how
metal (i.e. copper and iron) speciation in red wines could be influenced by the initial juice
metal concentrations and also how this speciation could impact on the oxidation process.
Finally, the impact of copper and iron additions to grape juice before alcoholic
fermentation on other cations present in white wine is an important aspect in winemaking
which should be investigated. The work which was carried out in the current study enabled us
to gather a large data set relevant to other metal cations in the wine, due to the simultaneous
analysis capabilities of the ICP-OES. A thorough analysis of this data is to be initiated in the
near future.
74
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88
Appendix A: Temperature and sugar concentrations during alcoholic fermentation
Table 1(A): Temperature (in oC) and sugar concentration (in ⁰Baumé) for treatments 1, 2 and 3 taken twice a day – 8AM and 4PM
T&B Table
Day 1
04/03
Day 2
05/03
Day 3
06/03
Day 4
07/03
Day 5
08/03
Day 6
09/03
Day 7
10/03
Day 8
11/03
Day 9
12/03
Day 10
13/03
Day 11
14/03
T ⁰C
⁰Baumé
T ⁰C
⁰Baumé
T ⁰C
⁰Baumé
T ⁰C
⁰Baumé
T ⁰C
⁰Baumé
T ⁰C
⁰Baumé
T ⁰C
⁰Baumé
T ⁰C
⁰Baumé
T ⁰C
⁰Baumé
T ⁰C
⁰Baumé
T ⁰C
⁰Baumé
Rep 1
AM PM
18.5 16.6
11.6 11.8
16 16.4
11.5 11.1
16.6 16.5
10.5 10
16.9 15.5
8.3
7.8
15.3 15.9
6.4
5.8
15.1 14.9
4.8
4.5
15
15
3.6
2.5
15 15.4
2.5
2.2
16.5 16.1
1.4
1.1
16.1 15.9
0.4
0.2
15.9 15.6
-0.1 -0.3
Treatment 1
Rep 2
Rep 3
AM PM AM PM
18.2 17 17.9 17
11.5 11.7 11.8 11.9
16.3 16.4 16.2 16.3
11.4 11.2 11.4 11.2
16.8 16.9 16.8 16.8
10.4 9.7 10.4 9.9
17.3 16.3 17.4 16.2
7.6
7.2
7.7
7.4
15.6 15.2 15.5 15.4
5.8
5.2
5.9
5.4
15.3 15 15.3 15
4.2
3.8
4.3
4
15.3 15.1 15.1 15.1
3
2.4
3.2
2.6
15.1 15.6 15.1 15.5
1.9
1.3
2
1.7
16.4 16.3 16.3 16.2
0.8
0.5
1
0.7
16 15.9 16 15.8
0
0
0.1
0
16.2 15.6 16.1 15.3
-0.3 -0.5 -0.2 -0.3
Rep 1
AM PM
17.9 16.5
11.7 11.7
16 16.1
11.5 11.3
16.3 16.1
10.7 10.3
16.6 15.1
9
8.5
14.9 14.5
7.2
6.6
15.2 14.6
5.2
5.2
14.7 14.6
4.3
3.9
14.9 15.2
3.1
2.7
16.1 16
1.9
1.5
16.1 15.7
0.7
0.5
16.2 15.6
-0.1 -0.2
Treatment 2
Rep 2
Rep 3
AM PM AM PM
17.8 16.9 17.7 16.9
11.7 11.8 11.6 11.7
16.1 16.2 16.1 16.2
11.4 11.3 11.4 11.3
16.5 16.5 16.5 16.4
10.6 10.1 10.6 10.2
17.6 16
17
16
8.1
8.1
8.9
8.2
15.5 15.2 15.7 15.1
6.6
6
6.7
6.1
15.2 14.8 15.2 14.9
4.9
4.6
5
4.6
15.1 15
15 14.8
3.7
3.2
3.7
3.3
15 15.5 15 15.2
2.5
2.1
2.5
2.1
16.1 16.2 16.1 16
1.3
1
1.4
1
16.1 15.9 16.2 15.7
0.3
0
0.3
0
16.2 15.6 16 15.5
-0.2 -0.6 -0.2 -0.4
Rep 1
AM PM
17.7 16.7
11.7 11.8
15.9 16.1
11.5 11.3
16.3 16.1
10.7 10.3
16.4 15.1
9.3
8.8
15 14.4
7.6
7
14.8 14.5
5.9
5.5
14.7 14.6
4.6
4.1
14.6 15.4
3.3
3
16 15.9
2.1
1.7
16 15.6
0.9
0.5
16.1 15.5
0
-0.2
Treatment 3
Rep 2
Rep 3
AM PM AM PM
17.7 16.9 17.6 16.9
11.5 11.8 11.6 11.8
16.2 16.2 16
16
11.5 11.2 11.5 11.3
16.5 16.4 16.4 16.3
10.6 10.2 10.6 10.2
16.8 15.7 16.6 15.5
9.1
8.5
9.2 8.7
15.5 15.5 15.6 15
7.1
6.5
7.3 6.7
15.2 14.9 15.1 14.9
5.3
4.9
5.5
1
15 14.8 14.8 14.7
4
3.6
4.8 3.6
15.2 15.5 15.3 15.3
2.8
2.4
2.9 2.5
16.1 16.2 16.3 15.9
1.6
1
1.6 1.2
16.2 15.9 16.2 15.7
0.5
0.2
0.5 0.2
16.2 15.5 16.1 15.4
-0.2 -0.4 -0.2 -0.4
89
Table 2(A): Temperature (in oC) and sugar concentration (in ⁰Baumé) for treatments 4, 5 and 6 taken twice a day – 8AM and 4PM
T&B Table
Day 1
04/03
Day 2
05/03
Day 3
06/03
Day 4
07/03
Day 5
08/03
Day 6
09/03
Day 7
10/03
Day 8
11/03
Day 9
12/03
Day 10
13/03
Day 11
14/03
Rep 1
AM PM
T ⁰C 17.7 17
⁰Baumé 11.6 11.8
T ⁰C 16.2 16.3
⁰Baumé 11.4 11.2
T ⁰C 16.8 16.6
⁰Baumé 10.3 9.7
T ⁰C 17 15.9
⁰Baumé 8.1 7.3
T ⁰C 15.4 14.9
⁰Baumé 5.8 5.3
T ⁰C 15.1 14.8
⁰Baumé 4.2 3.9
T ⁰C 15 14.6
⁰Baumé 3.1 2.7
T ⁰C 14.9 15.2
1.7
⁰Baumé 2
T ⁰C 16.3 15.9
0.7
⁰Baumé 1
T ⁰C 15.8 15.6
0
⁰Baumé 0.2
T ⁰C 15.9 15.4
⁰Baumé -0.2 -0.3
Treatment 4
Rep 2
Rep 3
AM PM AM PM
17.6
17
17.6 16.7
11.3 11.8 11.6 11.8
16.1 16.2 15.9 15.9
11.5 11.2 11.5 11.3
16.7 16.7 16.3 16.3
10.4 9.8 10.6
10
17.4 16.1
17
15.7
8
7.3
8.3
7.7
15.6 14.8 15.2 14.8
5.8
5.2
6.2
5.6
15 14.7 14.9 14.7
4.2
3.9
4.5
4.2
15.1 14.5 14.7 14.5
3.1
2.7
3.3
3
14.7 15.1 14.8 15.2
2
1.7
2.2
1.9
16.2 15.8 16.1 15.7
1
0.7
1.2
0.9
15.9 15.5 15.7 15.4
0.2
0.1
0.3
0.2
15.9 15.3 15.6 15.1
-0.2 -0.3 -0.1 -0.2
Rep 1
AM PM
17.7
17
11.7 11.8
16.2 16.3
11.5 11.2
16.8 16.6
10.4 9.6
17.1 16.1
8.1
7.3
15.8 15.1
5.7
5.1
15.1 14.9
4
3.6
15
14.7
2.8
2.4
14.9 15.3
1.7
1.3
16.1 15.9
0.7
0.4
15.7 15.6
0
-0.1
15.8 15.3
-0.4 -0.5
Treatment 5
Rep 2
Rep 3
AM PM AM PM
17.7
17
17.5 16.7
11.7 11.8 11.5 11.8
16.1 16.2 15.8 15.8
11.5 11.2 11.5 11.3
16.7 16.8 16.3 16.3
10.4 9.8 10.5 9.9
17.4 16.2 16.9 15.6
8
7.3
7.5
7.7
15.6 15.1 15.2 14.7
5.7
5.1
6.1
5.5
15
14.8 14.7 14.6
4
3.6
4.4
4
15.2 14.5 14.9 14.3
2.7
2.4
3.1
2.7
14.6 15.2 14.7
15
1.7
1.3
2
1.6
16.1 15.8
16
15.6
0.7
0.4
0.9
0.6
15.7 15.5 15.5 15.3
0
-0.1 0.1
0
15.6 15.2 15.5
15
-0.4 -0.5 -0.3 -0.7
Rep 1
AM PM
17.8
17
11.5 11.8
16.1 16.1
11.4 11.2
16.6 16.4
10.4
10
16.9 15.8
8.6
8
15.4
15
6.5
5.8
15.6 14.8
4.2
4.2
15.1 14.5
3.3
2.9
14.9 15.2
2
1.7
16.2 15.9
0.9
0.5
15.8 15.6
0
-0.2
15.8 15.3
-0.5 -0.7
Treatment 6
Rep 2
Rep 3
AM PM AM PM
17.6 16.9 17.5 16.9
11.6 11.8 11.6 11.8
16.1 16.1 15.9 15.9
11.5 11.2 11.5 11.3
16.5 16.4 16.4 16.2
10.5 10.1 10.6 10.1
16.8 15.7 16.5 15.4
8.7
8.1
8.9
8.3
15.3
15
15.5 14.7
6.6
6
6.9
6.2
15
14.8 14.9 14.6
4.8
4.4
5.1
4.7
14.7 14.6 14.6 14.5
3.5
3.1
3.7
3.3
14.6 15.1
15 15.1
2.3
1.9
2.5
2.1
16.1 15.8 15.9 15.6
1.1
0.7
1.3
0.9
15.8 15.5 15.6 15.3
0
-0.1 0.2
0
15.7 15.2 15.6 15.2
-0.4 -0.6 -0.3 -0.2
90
Appendix B: Measurements for all wine samples (T1-T6) by
High performance liquid chromatography (HPLC)
Table 3(A): Lactic, tartaric, citric and malic acid [g/L] measurements for all samples (T1-T6)
by HPLC
Sample
T1
T2
T3
T4
T5
T6
T1R1
T1R2
T1R3
T2R1
T2R2
T2R3
T3R1
T3R2
T3R3
T4R1
T4R2
T4R3
T5R1
T5R2
T5R3
T6R1
T6R2
T6R3
Lactic
Tartaric Citric
Malic
0,295
0,199
0,224
0,222
0,203
0,213
0,212
0,246
0,218
0,218
0,193
0,214
0,283
0,257
0,246
0,285
0,282
0,277
2,977
2,959
2,872
2,857
2,838
2,949
2,978
2,804
2,993
2,932
2,908
2,912
2,757
2,849
2,848
2,816
2,783
2,914
2,303
2,535
2,037
2,275
2,298
2,446
2,392
2,268
2,372
2,449
2,298
2,229
2,018
2,024
2,033
2,252
2,242
2,234
0,361
0,547
0,323
0,358
0,362
0,546
0,538
0,361
0,536
0,545
0,66
0,474
0,405
0,402
0,401
0,353
0,351
0,345
91
Appendix C: Speciation Procedure Protocol
1. SPE Preparation
Samples passed through SPE columns utilising a peristaltic pump.
-
Strata C18 SPE cartridge utilised (Phenomenex, C18-E (55microm, 70A) 1000mg/6mL
(8B-S001-JCH).
Cartridge washed with methanol (20 mL at 1-2 mL/min).
Cartridge washed with water (40 mL at 1-2 mL/min).
Excess water was drained and resin bed washed with 10 mL of 1 M HCl.
Finally resin rinsed with 20 mL Model Wine.
Cartridge dried thoroughly by leaving peristaltic pump to run (1 min).
-
Strata SCX cartridge utilised (Strata SCX (55um, 70A) 1000mg/6mL)
Wash with 20mL water (40 mL at 1-2 mL/min).
Wash with 10 mL 1 M HCl, and then 20 mL water.
Wash with 10 mL 1M NaOH solution and then 20 mL water.
Cartridge dried thoroughly by leaving peristaltic pump to run (1 min).
2. Fractionation procedure
Solutions run through peristaltic pump at a rate around 1 mL/min.
-
Filter samples. Syringe filter prior to analysis (0.22 microm, regenerated cellulose).
Discard first 5 mL or so of filtered wine and then collect remaining.
Allow 30mL of filtered wine to pass through the Strata C18 SPE cartridge. (Delivery via
burette).
Take 10 mL of eluent and place it aside for metal analysis (i.e., Post-C18/ Hydrophoic).
Take the remaining 20 mL and pass it through SCX SPE cartridge. The 20 mL eluent
from the SCX SPE will be analysed for metal analysis (i.e., Residual Metal).
Elute the cationic species retained on the SCX SPE cartridge by passing 10.0 mL of 2.0 M
HCl through the SPE cartridge (i.e., Cationic eluent).
3. Solution Preparation prior to ICP-OES analysis
-
All samples containing ethanol (post-C18/ hydrophobic, residual metal) will be analysed
by 4-fold dilution with 5% HNO3. That is, 4.0 mL sample + 12.0 mL 5 % HNO3.
The acidic chloride samples will be made up to 20 mL with addition of 8.0 mL of 1.0 M
NaOH and 2.0 mL water.
92
-
-
For the acidic chloride samples, initial experiments will be conducted to compare the
quantification by standard additions with quantification via external calibration graph
(internal standard). Control (~10mL), standard 1 (5mL) and standard 2 (5mL).
Analyse original wine in triplicate. That is, 3 x (4.0 mL sample + 12.0 mL 5 % HNO3).
4. Samples
-
Calculation of Limit of Detection (LOD) and Limit of Quantification (LOQ) on ICP-OES
via the injection of 5 x (4.0 mL water + 12.0 mL 5 % HNO3).
Initially, 3x model wine. This will allow assessment of concentrations of contamination
from resins.
18 x prepared wines.
93
Gantt Chart, Winemaking
17/02
09/03
29/03
18/04
08/05
28/05
17/06
Introduction to winery facilities and staff
Winery training
Preparation work
Harvest of the grapes
Collection of grape juice
Preparation of experiment samples
Yeast nutrient addition
SO2 addition 1
SO2 addition 2
Wine sample storage
Racking 1
Cold stability test
Heat stability test
D.1: Winemaking
Appendix D: Work programme
Alcoholic fermentation of samples
Bottling 1
Bentonite addition
Settling after bentonite
Bottling 2
Start date
Duration
94
Gantt Chart, Part 1
07/12
27/12
16/01
05/02
25/02
17/03
06/04
26/04
16/05
05/06
Introduction
Laboratory analysis of initial juice
Laboratory analysis of juice after metal additions
Laboratory analysis of samples during AF2
Glassware and reagent preparation
Procedure development and tests
Metal speciation analysis T1, T2, T3
D.2: Part 1
Metal speciation - additional
Start date
Duration
95
Gantt chart, Part 2
06/06
11/06
16/06
21/06
26/06
Start date
Duration
01/07
06/07
11/07
16/07
21/07
ICP-OES sample preparation
ICP-OES sample preparation
ICP-OES analysis
Data gathering
D.3: Part 2
ICP-OES analysis
Data analysis
96
Gantt chart, Part 3
27/07
29/07
31/07
02/08
04/08
06/08
08/08
10/08
12/08
14/08
16/08
Wine sample preparation 1
Oxygen decay measurements 1
Data gathering
D.4: Part 3
Data analysis
Start date
Duration
97

COPPER AND IRON SPECIATION IN WHITE WINE: IMPACT ON

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