®
Academic Research
Pubblication
&
Report
© Copyright 2010, BIOMA Agro Ecology CO AG Switzerland. All Rights Reserved
Sulphite Free
Organic Additives
to be use in
Wine Making Process
ELSEVIER
Journal of Food
Composition and
Analysis
SULPHREE
EU Research FP6
Consortium SULPHREE
GEOLIFE® PRÒTOS
Academic Research
Publications & Reports
Table of Contents
1. Introduction
2. Article published in the “Journal of Food Composition and Analysis:”
“A natural alternative to sulphur dioxide for red wine production: Influence on
colour, antioxidant activity and anthocyanin content.” 2008.
3. A Review of the European Union SULPHREE Research Project – Introduction
Calleja, G. “The content of sulfur dioxide in wine.” Undergraduate project in
partial fulfilment of the requirements of the Degree of Bachelor of Pharmacy
(Honours), University of Malta; 2011.
4. First SULPHREE report, presented at the CIGR Section VI International
Symposium on “Food and Agricultural Products: Processing and Innovations”, in
Naples, Italy, September, 2007
5. SULPHREE report: Toxicity Testing of Sulfite-Free Wines (2007-2008)
6. SULPHREE report: Toxicity Studies of the SULPHREE Additives (2008)
7. Toxicological Analyses of PRÒTOS by an accredited laboratory
8. Additional wine analyses done by the research partners of the SULPHREE
consortium on older wines (2007-2008)
9. Additional wine analyses done by the research partners of the SULPHREE
consortium on white wines with the new formulation, “PRÒTOS White” (20082009)
10. Excerpts from: Calleja, G. “The content of sulfur dioxide in wine.”
Undergraduate project in partial fulfilment of the requirements of the Degree of
Bachelor of Pharmacy (Honours), University of Malta; 2011.
APPENDIX:
•
Original abstracts, introduction, and bibliography to the first SULPHREE report,
presented at the CIGR Section VI International Symposium on “Food and
Agricultural Products: Processing and Innovations”, in Naples, Italy, September
2007
GEOLIFE® PRÒTOS – ACADEMIC RESEARCH -- INTRODUCTION
1
High-Quality Wines: Some Developments in Europe involving
Reduced Sulfites or No Sulfites Added
The search for auxiliary enological plant-based products which act as antioxidants and stabilizers
and can help to reduce or substitute sulfites, has been going on in Europe for many years.
In the year 2005 two academic-level research tests began in Europe on natural alternatives to
sulphur dioxide. Both of these research projects, described briefly below, gave very promising
results. Since these series of tests began in 2005, additional progress has been made, and this
®
year (2011) the GEOLIFE product named PRÒTOS – developed and manufactured by the
Swiss-based BIOMA Agro Ecology CO AG – is foreseen to come on the market in the EU.
An article published in the “Journal of Food Composition and Analysis” by scientists at the Wine
Institute / National Agricultural Research Foundation (Athens, Greece) and the Technological
Educational Institute (TEI) (Athens, Greece) describes their tests during the 2005 harvest on four
different red vinifications and their results. (Salaha, M.-I., et al., “A natural alternative to
sulphur dioxide for red wine production: Influence on colour, antioxidant activity and
anthocyanin content,” Journal of Food Composition and Analysis (2008).
The abstract states:
“In all cases, commercially acceptable red wines were produced, giving a possibility for
partial substitution of SO2 by a natural product.”
From the conclusion:
“The components of this alternative product have acknowledged beneficial properties in
contrast with potential health problems associated to sulphur dioxide. In this way, the
quantities of SO2 added during winemaking could be reduced following the need for the
production of a ‘healthier’ wine.”
The European Union itself has given substantial encouragement and support to the possibility of
replacing chemical additives with organic additives. Also beginning in 2005, the “SULPHREE”
Research Project (Sulphite-Free Organic Additives to be used in Wine-Making Process) was a
consortium project funded by the European Union. The SULPHREE Project was scheduled to
GEOLIFE® PRÒTOS – ACADEMIC RESEARCH -- INTRODUCTION
2
run for 2 years beginning in May 2005. Due to the encouraging results, the project was extended
for an additional six months until November 2007, and the research partners of the SULPHREE
consortium continued with additional post-project analyses. The SULPHREE reports have
undergone an additional academic review during 2010-11, which included translations and
editing for English language clarity. The SULPHREE reports include:
•
2007 – the first SULPHREE report with details confirming the technical validation of the
organic additives. From the conclusion:
“A natural additive able to mimic the SO2 effects in the wine-making process is being
pursued. Both aqueous extracts from plants and OPC additives were tested. Colour,
aromatic and anti-oxidant effects were analyzed in different microvinifications of several
white and red grapes. The chemical-physical characterization of the experimental wines
at different stages of the vinification process confirmed the possibility of using the new
formulation in wine avoiding oxidation and microbial spoilage. Also sensory analysis did
not show defects and/or alterations in the experimental wines.”
•
2007-2008: Toxicity Tests of the Sulphite-Free Wines
The conclusion states:
“All the above data – taken together – confirm the absence of toxicity of wines preserved
with the SULPHREE additives.”
•
2008: Toxicity Studies of the SULPHREE Additives
From the introductory explanation:
“Once the technical validation of the two organic additives had been confirmed, it was
then necessary to study the additives from the toxicological point of view – in order to
obtain experimental evidence that, in the doses incorporated in vinification, the additives
do not have any harmful health effects on the consumer.”
Conclusion:
®
“These experiments confirmed the hypothesis that Additive #1 – the GEOLIFE product
PRÒTOS – does not have any adverse toxicological effects on the cellular systems
tested.”
GEOLIFE® PRÒTOS – ACADEMIC RESEARCH -- INTRODUCTION
•
3
®
2007-2008: Additional Wine Analyses on Older Wines (preserved with GEOLIFE ’s
PRÒTOS, in 2003, 2004, and 2005)
The conclusion states:
“From all the experimental analysis carried out on the three different wines, a common
trend was observed: the additive was a suitable preservative during storage and imparted
better results when utilized to preserve red wines rather than white wines.”
•
2009: Additional white wine analyses with the new formulation PRÒTOS White
The conclusion states:
“The physicochemical, aromatic, and sensory analyses of the experimental wines as well as
the additives used in the vinification demonstrated that the additive enables obtaining white
wines of quality comparable to wines made with traditional vinification methods. Other
interventions (already planned) for improvements at the bottling stage and storage can also
lead to an improvement in quality.”
There is a growing community of consumers with a real interest in wines either with a minimum
of added sulfites or without added sulfites. Likewise, there are researchers and wine-producers
who continue to improve their efforts in producing high-quality wines – including high-quality
organic wines – without added sulfites, in order to satisfy this consumer request.
®
GEOLIFE PRÒTOS offers an alternative to sulfites – for those vintners who want to produce
wine without sulfites or with reduced sulfites and/or for those who want to produce truly organic
wine.
GEOLIFE® PRÒTOS – Preserving high quality wines . . . naturally
Journal of Food Composition and Analysis:
A natural alternative to sulphur dioxide for red wine production
Journal of Food Composition and Analysis
Volume 21 (2008) Pages 660 - 666
A natural alternative to sulphur dioxide for red wine production:
Influence on colour, antioxidant activity and anthocyanin content
Maria-Ioanna Salahaa, Stamatina Kallithrakaa, Ioannis Marmarasa,
Elisabeth Koussissib, Irini Tzouroua
a
Wine Institute, National Agricultural Research Foundation (NAGREF),
1, S. Venizelou Str. 141 23 Lykovryssi, Athens, Greece
b
Department of Oenology and Beverage Technology, Technological
Educational Institution (TEI) of Athens, Agiou Spiridona Str. 122 100 Eg
aleo, Athens, Greece
ARTICLE IN PRESS
Journal of Food Composition and Analysis 21 (2008) 660– 666
Contents lists available at ScienceDirect
Journal of Food Composition and Analysis
journal homepage: www.elsevier.com/locate/jfca
Original Article
A natural alternative to sulphur dioxide for red wine production: Influence on
colour, antioxidant activity and anthocyanin content
Maria-Ioanna Salaha a,!, Stamatina Kallithraka a, Ioannis Marmaras a,
Elisabeth Koussissi b, Irini Tzourou a
a
b
Wine Institute, National Agricultural Research Foundation (NAGREF), 1, S. Venizelou Str. 141 23 Lykovryssi, Athens, Greece
Department of Oenology and Beverage Technology, Technological Educational Institution (TEI) of Athens, Agiou Spiridona Str. 122 100 Egaleo, Athens, Greece
a r t i c l e in f o
a b s t r a c t
Article history:
Received 23 April 2007
Received in revised form
15 February 2008
Accepted 3 March 2008
An alternate to sulphur dioxide natural antioxidant was tested during 2005 harvest on four different red
vinifications, and was applied in each winemaking batch in combination with sulphur dioxide and on its
own. Responses measured and analysed with uni- and multivariate statistics were: anthocyanin
content, antioxidant capacity and classic oenological parameters. ANOVA revealed no significant effect
of treatment in the antioxidant values of RA or diphenylpicrylhydrazyl (DPPH) of wines, but a paired
t-test could differentiate wines treated only with SO2 from those treated only with the novel product
according to their RA values. Principal component analysis (PCA) of anthocyanin content of the treated
wines gave five significant components, explaining 100% of variance and differentiating products
treated only with SO2 from those treated only with new product. PCA of oenological data explained 72%
of variance in the first two components and wines were clearly differentiated on the basis of SO2 or
alternative treatment. In all cases, commercially acceptable red wines were produced, giving a
possibility for partial substitution of SO2 by a natural product.
& 2008 Elsevier Inc. All rights reserved.
Keywords:
Sulphur dioxide
Alternative wine preservative
Red wines
Antioxidant activity
Anthocyanins
HPLC
Food composition
1. Introduction
Sulphur dioxide is the main preservative, antiseptic and
antioxidant used in winemaking for the protection of wine from
alterations. It has long been used in winemaking to inhibit
oxidation (free SO2) and growth of undesirable micro-organisms
including wild yeast, and acetic and lactic bacteria (molecular
SO2) (Ough and Crowell, 1987; Fugelsang, 1989; KourakouDragona, 1998).
Nevertheless, and as a result of potential health problems that
may arise, the use of sulphur dioxide in wine has recently come
under review. Wines must now prominently display on the bottle,
next to restrictions required by law, the presence of total sulphites
in excess of 10 mg/l (European Union Regulation 1991/2004).
Winemakers are therefore faced with the problem of finding a
viable substitute for the antioxidant and antiseptic action of
sulphite in winemaking (Léglise, 1991; Eschenbruch, 1986).
There has been some research and work by commercial
companies on the reduction or replacement of sulphur dioxide
in winemaking; with regards to the production of biological wine,
alternatives have been sought that use substitute products
! Corresponding author. Tel.: +30 210 2828111; fax: +30 210 2844954.
E-mail addresses: [email protected], [email protected] (M.-I. Salaha).
0889-1575/$ - see front matter & 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2008.03.010
(Panagiotakopoulou and Morris, 1991; Main and Morris, 1991;
Marcillaud and Donèche, 1997) or radiation or electrochemical
treatment (Lustrato et al., 2003; Wilson et al., 2003). No effective
replacement has been suggested, however Legislation allows the
use of sorbic and ascorbic acid in order to increase the antimicrobial and antioxidant capacity of sulphur dioxide, respectively (Kourakou-Dragona, 1998). Ascorbic acid has long been used
in the wine industry as an antioxidant, the reason being its ability
to rapidly remove molecular oxygen from juice or wine. Therefore
ascorbic acid and its isomer erythorbic acid have been suggested
as alternatives to or adjuncts with sulphur dioxide (Fugelsang,
1989).
A novel, alternative product has recently been manufactured
and introduced as a stabilizer for the winemaking process
(GEOLIFE, 2004). Claims of the manufacturer include: acceleration
of different processing phases of winemaking in a non-toxic way,
substitution of the traditional chemical treatments, and better
sensory characteristics and preservation of the final products. This
product has been described as an energetic antioxidant of
biological origin, and contains mainly black radish (Raphanus
niger) extract and ascorbic acid (GEOLIFE, 2004). R. niger is a
variety of black radish that has been used since antiquity in folk
medicine as a natural remedy against many diseases (Bown, 1995;
Chevallier, 1996). First cultivated and used by the Egyptians in
2780 BC, the black radish root juice has always shown significant
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antioxidant and therapeutic properties, and this is also reported in
recent works (Lugasi et al., 1998, 2005). The plant, containing the
antibacterial and antifungal compound raphanin, inhibits the
growth of Staphylococcus aureus, Escherichia coli, Streptococci and
Pneumococci. It has also shown anti-tumour activity, contains high
amount of vitamin C and is known to contain mustard oils also
contributing to food preservation (Chevallier, 1996).
The primary objective of this study was to evaluate the use of
alternative antioxidant product (AAP) in red winemaking on its
own or in combination with different proportions of sulphur
dioxide, by measuring certain red wine quality parameters. These
were colour, phenolic content, antioxidant activity and classic
oenological parameters of the resulting wines. Furthermore, the
possibility of reducing the quantity of SO2 added to wines because
of the use of the novel product during winemaking without
deteriorating wine quality was explored. Greek regional wines are
produced from native varieties and/or from international cultivars, including Cabernet Sauvignon, Merlot and Cabernet Franc
(Koussissi et al., 2002, 2003). The present study included four
different red wine vinifications: two single varieties and two
co-vinifications.
2. Materials and methods
2.1. Grape samples and vinifications
All grapes used were of 2005 harvest. Four separate vinifications took place from the following grapes: (1) Cabernet
Sauvignon from the Peloponnese (CS), (2) Mourvèdre from
Peloponnese (Mo), (3) Cabernet Sauvignon, Cabernet Franc and
Merlot (co-vinification, mixture (1) all from the National vine
collection of the Vine Institute in Lykovryssi/Attica (Mix1)), (4)
Cabernet Sauvignon, Carignan and Syrah (co-vinification, mixture
(2) from northern Attica (Mix2)). Grapes were harvested at
technological maturity—based on indices of sugar content and
acidity established by the Wine Institute—and crushed, followed
by classic red fermentation (with wild yeasts) with must recycling
to keep skins wet, fermenting mass temperature: 28–32 1C, and
separation from skins after 7 days. The final reducing sugar
content was o2 g/l in all four cases. For each vinification the mass
of crushed grapes was divided into four batches, and four
conditions were subsequently created with addition of the
following: (a) 20 mg/l SO2 and 0.7 ml/l AAP, (b) 50 mg/l SO2 and
0.3 ml/l AAP, (c) 70 mg/l SO2 and (d) 1 ml/l AAP. A duplicate was
created for each treatment. Vinification and storage of the
resulting wines were carried out under identical conditions, to
ascertain minimal influence deriving from implementation of
techniques or conditions that could substantially alter wine
quality and composition and wines were analysed after 4 months
of storage.
661
acid, vitamin C, flavonoids, Raphanus niger extract and vegetables
extracts.
2.3. Classic oenological measurements
Classic parameters of wines were determined according to the
international methods of the OIV (International Organization of
Vine and Wine, 2005). All analyses were performed in triplicate.
Total polyphenol concentration was determined with the Folin–
Ciocalteu assay, with the microscale protocol previously reported
(Arnous et al., 2001). Total flavanol (F) concentration was
measured with the vanillin assay employing the optimized
protocol of Sun et al. (1998), adapted on a microscale.
2.4. Antioxidant activity
Reducing power of wines was estimated using the dipyridyl
method, according to Psarra et al. (2002), but with ascorbic acid as
the calibration standard instead of quercetin. The results were
expressed as millimoles of ascorbic acid equivalents (AAE). For the
determination of the antiradical activity, assays were performed
employing the DPPH stable radical. The methodology and the
analytical protocol used were as previously reported by Arnous
et al. (2001). All samples were tested after 1:10 dilution with 12%
EtOH. The results were expressed as millimoles of Trolox
equivalents (TE), using the calibration curve plotted with different
amounts of Trolox. In all cases, analyses were performed in
triplicate, the values were averaged and the standard deviation
was calculated.
2.5. HPLC determination of anthocyanins
Wine samples were filtered through 0.45 mm filters prior to
HPLC analysis. An equipment consisting of a HP chromatography
apparatus coupled to a diode array detector was employed.
Analyses were performed as demonstrated by Kallithraka et al.
(2005), on a Spherisorb ODS-2, 5 mm, 250 ! 4 mm, at a flow rate of
1 ml/min, using a 20 ml injection volume, detection at 520 nm and
the following elution programme: 95% eluent A for 1 min, then
from 95% to 50% in 25 min, from 50% to 5% in 3 min, which was
kept isocratic for further 3 min. Eluent A was 10% aqueous formic
acid and eluent B MeOH. Identification was based on comparing
retention times and UV spectra of the peaks detected with those
of commercial standards. Malvidin-3-O-glucose coumarate
(MvCoum) and malvidin-3-O-glucose acetate (MvAcet) were
tentatively identified based on previous observations (Arnous
et al., 2002). All peaks were quantified as malvidin-3-O-glucose
(Mv). Results were expressed as mg/l. Determinations were
carried out with external standard method (malvidin calibration
curve). All analyses were performed in triplicate.
2.6. Statistical analyses
2.2. Chemicals
Water for high-pressure liquid chromatographical (HPLC)
analyses was nanopure. Anthocyanins (ANs) standards were from
Polyphenols (Norway). Trolox and 2,20 -diphenylpicrylhydrazyl
(DPPH) radical were from Sigma Chemical Co. (St. Louis,
MO, USA). Folin–Ciocalteu reagent was from VWR International.
Iron (III) chloride, ascorbic acid, formic acid and EtOH were
from PRS Panreac (Spain) and 2,20 -dipyridyl was from Acros
Organics (New Jersey, USA). MeOH was HPLC grade from
Labscan (Ireland). The alternative preservative was purchased
from BIOMA Agro Ecology Co. (Switzerland) and contained: citric
One-way ANOVA was initially used to determine significant
differences amongst the samples due to their reducing power and
antiradical activity and subsequently to explore the effect of AAP
addition on the same. A paired t-test was then used to determine
differences on the reducing power and antiradical activity
between the SO2 only and AAP only treatments, respectively. All
univariate analyses were performed with MINITAB v13.1. Principal
component analysis (PCA; Unscrambler v 7.6, CAMO ASA, Norway)
was employed to study wine clustering and differentiation on the
basis of (a) AN content and (b) classic oenological data. Finally,
discriminant partial least squares (DPLS, Unscrambler v7.6)
regression (Koussissi et al., 2007) was used to investigate the
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M.-I. Salaha et al. / Journal of Food Composition and Analysis 21 (2008) 660–666
effect of different SO2 and AAP treatments on the AN content of
the wines.
3. Results and discussion
3.1. Classic oenological parameters
Values of certain important classic oenological variables of the
wines, namely: total and volatile acidity, free and total SO2,
tartaric and malic acid, colour intensity (CI), tonality, ionization
degree (ID) of ANs, total phenol (TP) and catechin content and
finally alcohol content (vol%) are given in Table 1. When the same
data were analysed with PCA two significant principal components could explain 72% of the total variance in the data. Plotting
PC1 (45% variance) vs. PC2 (27% variance) gave a space in which
products were clustered and differentiated as follows: All single
varietal Cabernet Sauvignon wines were closely clustered and
differentiated from the rest of the products, irrespective of SO2 or
AAP treatment with high scores on PC1 and negative scores on
PC2. Those wines had the highest loadings for CI, catechins and
TPs as well as volatile acidity and alcohol content. For the rest of
the wines, treatment did have an effect on their oenological
variables. Conditions A and D (higher proportion of AAP to SO2
and the one with no SO2 at all) had all negative scores on PC1 and
the highest loadings for tonality. On the contrary conditions B and
C (higher proportion of SO2 to AAP and SO2 only) had high scores
both on PC1 and PC2 and at the same time high loadings for: total
acidity, tartaric and malic acid and free and total SO2 (Fig. 1).
As expected (Fugelsang, 1989; Kourakou-Dragona, 1998;
GEOLIFE, 2004), since malolactic fermentation (MLF) is strongly
influenced by the presence of SO2 it occurs more easily in
treatments with lower SO2 doses and thus malic acid is
significantly lower in the correspondent wines consequently
affecting total acidity. As MLF usually causes an increase in
volatile acidity, wines produced with lower SO2 doses were
expected to contain relatively higher amounts of volatile acids,
although this was not always the case. Independent of treatment
A, B, C or D, volatile acidity did not exceed the amount expected
for a commercially acceptable red wine. The effect of treatment on
tonality reveals a higher tendency to oxidation for wines treated
with larger doses of AAP.
The grouping of single varietal Cabernet Sauvignon wines,
irrespective of treatment could be interpreted by their significantly higher phenolic content (CI, TPs and Fs) which offers
antioxidant protection to all wines, independently of treatment.
3.2. Antioxidant activity
The antiradical activity (AR) and the reducing power (PR) values
of the experimental wines are given in Table 2. Both values were
maximum in condition C samples (only SO2) (with the exception
of AR value of the wine produced by mixture (2)). Initially, oneway ANOVA was used to see whether there was a significant
difference in terms of measured wine antioxidant activity,
between wine samples from all treatments and vinifications and
indeed there were differences amongst the samples for both AR
and PR (P valueso0.000 in both cases); however, this should be
put on the account of the difference between the composition of
grapes of each vinification. One-way ANOVA was rerun to see the
effect of substitute treatment on AR and PR, values of the wines but
was not significant in either cases (P values were 0.411 and 0.625,
respectively).
Finally, in order to determine if there was a significant
difference between samples treated with only SO2 and those
treated with only AAP, a paired t-test was run for AR and PR,
respectively. There was no significant difference between the two
treatments in terms of the AR values of the wines (P ¼ 0.908). In
contrast, there was a significant difference between treatments C
and D in terms of their PR values, with those treated with SO2 only,
having higher PR values. The antioxidant potential of red wines is
believed to depend to a great extent on their F content (De Beer
et al., 2002), as well as on the chemical structure of the individual
F they contain. Total F concentration (Table 1), is highest for
treatment C (only sulphur dioxide) supporting this hypothesis. In
addition, the antioxidant activity of a wine is largely dependent on
Table 1
Composition of red wines produced with different treatments
Volatile acida
Tartaric acid (g/l)
Malic acid (g/l)
CI
Tonality
ID
Total phenols b(mg/l)
Flavanols c(mg/l)
Cabernet Sauvignon
A
14.4570.17
B
14.4270.16
C
14.7470.17
D
14.0070.16
0.6370.00
0.5270.01
0.5470.00
0.5670.02
1.970.01
2.170.00
2.270.01
1.870.02
0.570.01
0.870.02
0.770.03
0.970.00
16.0270.01
15.7570.28
18.0470.18
17.3070.39
0.6670.01
0.6170.02
0.6070.01
0.6070.00
22.870.3
23.470.21
25.270.24
23.670.21
1930738.6
1965734.3
2150741.8
2030729.4
34957139.9
29407117.5
36457144.2
2640798.3
Mourvèdre
A
B
C
D
10.8370.31
10.6370.28
12.0970.29
11.0070.20
0.6370.03
0.4570.01
0.4670.01
0.7570.01
2.270.02
2.270.03
2.370.00
2.470.01
0.370.00
1.470.00
1.470.01
0.270.01
8.3470.31
7.1670.21
10.9770.24
8.8770.18
0.7170.02
0.6770.03
0.6170.00
0.7670.02
24.970.29
27.870.3
26.870.28
23.970.21
1295726.1
1375725.4
1600730.8
1365727.2
1695750.8
2105748.1
3030739.2
2345747.3
Mixture 1
A
B
C
D
11.6170.13
11.9370.15
11.6370.13
11.4070.15
0.2470.02
0.2370.01
0.2570.00
0.4370.02
2.370.03
2.570.03
2.470.01
2.370.01
1.670.01
1.370.01
1.470.00
0.170.03
10.5270.14
11.1770.24
12.8270.17
9.9070.11
0.7570.03
0.6370.01
0.6270.01
0.6670.01
26.870.23
24.170.23
29.770.31
25.870.21
1180721.4
1140722.1
1320719.4
1070720.3
2435773.1
2710725.2
2805748.4
1825751.3
Mixture 2
A
B
C
D
12.2570.22
12.1370.23
12.4170.18
12.1570.27
0.3370.03
0.3770.00
0.4370.01
0.4470.01
2.070.02
2.370.00
2.270.01
1.870.03
0.570.01
0.470.01
1.770.01
0.270.00
6.5270.21
6.8270.23
7.4270.07
7.0570.18
0.7870.02
0.6970.00
0.6070.00
0.8470.01
13.770.18
16.770.31
19.570.22
14.870.23
1470713.4
1440724.8
1490727.1
1430726.9
2130761.4
2225758.6
2000759.5
1550746.0
Treatment
a
b
c
Alcohol (%vol)
Acetic acid (g/l).
Gallic acid.
Catechin.
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M.-I. Salaha et al. / Journal of Food Composition and Analysis 21 (2008) 660–666
3
PC2
Tartaric acid
Mix1 B
Mix 1 C
Malic acid
2
Mix2 C
Mix1 A
Free SO2
Total SO2
Total acidity
Mo C
Mo B
1
Mix2 B
0
Mix1 D
Mix2 A
Mo A
Catechins
Tonality
-1
CS B
Mo D
CI
CS C CS D
-2
Mix2 D
%vol
Total Phenols
Volatile acidity
CS A
-3
-4
-3
-2
-1
0
1
2
PC1
3
4
PC1: 45%, PC2: 27%
Fig. 1. PCA of classic oenological parameters of the wines: Bi-plot of scores and loadings for PC1 vs. PC2 product space.
3.3. Anthocyanin content
Table 2
Antioxidant capacity of red wines produced with different treatments
Antiradicala
Reducingb
Antiradicala
Reducingb
Cultivar
A
B
C
D
Cabernet Sauvignon
7.4770.10
7.0470.06
7.0770.23
6.7370.07
7.4770.18
7.3970.07
7.0370.31
7.0370.25
Mourvèdre
3.5270.07
2.1970.11
6.5870.56
5.6070.13
4.5270.13
5.1370.31
6.0770.05
4.9170.28
Cultivar
A
B
C
D
Mixture 1
5.7670.12
6.0670.12
6.3970.07
5.7970.55
Mixture 2
5.5070.18
6.4370.52
4.9170.40
5.0570.00
6.5770.21
6.7070.50
6.8170.39
5.0570.44
a
b
6.0570.26
6.1170.21
5.8970.11
5.7970.11
mM Trolox.
mM ascorbic acid.
its total phenolic content and composition, as different compounds and their combinations exhibit varying degrees of activity
(De Beer et al., 2002). Any variation in the vinification process
introducing a differentiation in the phenolic composition of the
wine influences its antioxidant activity. This hypothesis is also
supported by the observed maximum values (Table 1) of total
phenol concentrations of the wines produced with the sole
use of SO2.
In agreement with Alonso et al. (2002), AR was found to
be strongly correlated with TPs (r2 ¼ 0.5587), F (r2 ¼ 0.7245)
and ANs (r2 ¼ 0.6777), while the reducing power also exhibited correlation with these parameters: TP (r2 ¼ 0.6650); F
(r2 ¼ 0.6521) and AN (r2 ¼ 0.6892).
A detailed AN composition of all experimental wines is
presented in Table 3. The findings showed that malvidin-3-Oglucoside is the pre-dominant AN, as has been demonstrated for a
number of other V. vinifera species in the past (Lanaridis and BenaTzourou, 1997; Mazza et al., 2002; Kallithraka et al., 2005). The
profile of the other ANs was distinctive for each cultivar or
mixture of cultivars. PCA of AN concentrations of all wines gave
five highly significant components (all had Po0.000) explaining
100% of the variance in the data (PC1: 55%; PC2: 34%; PC3: 8%;
PC4: 2%; PC5: 1%). Plotting PC1 vs. PC2 (Fig. 2) showed initial
separation between products treated with SO2 only and AAP only,
on PC1. Wines treated with only SO2 had the highest scores on PC1
and subsequently the highest loadings on all measured ANs. On
the contrary the majority of products treated only with AAP were
negative on that component with very low loadings on all ANs.
The remaining two treatments (A and B) were not clearly
clustered and clustering of products was also based on varietal
content (Fig. 2).
In order to better understand the differentiation of wines on
the basis of their AN content related to the AAP treatment, a DPLS
(Koussissi et al., 2003) regression analysis was carried out. The
analysis gave a two-dimensional product space with both factors
highly significant from ANOVA (Po0.000). Total amount of
variance explained was 89% for X variables (ANs) and 16% for Y
variables (treatment) in the first two factors (Fig. 3). In that space
a clear separation between the conditions with little or no SO2 and
that of only SO2 treatment is observed with the latter giving wines
with the highest AN content (Fig. 3).
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Table 3
Anthocyanin content (in mg/l) of red winesa produced with different treatments
Treatment
Cyanidin
Petunidin
Peonidin
Malvidin
Malvidin acetate
Malvidin coumarate
Total anthocyanins
Cabernet Sauvignon
A
16.6370.04
B
20.7570.05
C
16.6070.11
D
13.6670.04
ND
2.0470.04
ND
ND
22.1970.50
22.4170.39
24.7770.28
20.2470.18
14.3570.28
23.7370.36
16.8470.42
12.7070.24
260.5170.44
266.7770.56
266.0670.35
225.5470.38
96.2970.43
73.1570.26
105.9070.32
83.3870.28
25.2370.23
28.0570.11
28.2970.28
22.2070.15
504
510
557
474
Mourvèdre
A
B
C
D
13.7970.23
8.4270.18
32.5670.30
19.0370.11
2.5070.06
1.4970.01
7.3970.28
4.8670.15
30.6670.25
20.7870.16
61.0771.00
35.4070.26
13.6470.37
10.5870.28
29.7170.24
15.5770.22
187.9570.53
148.1571.05
362.4570.41
200.8470.50
16.9470.43
10.5270.33
44.2270.17
18.2070.31
16.0970.14
9.3870.18
33.8570.45
16.2270.16
300
230
624
343
Mixture 1
A
B
C
D
6.6270.03
8.5270.02
7.8770.05
5.8870.03
ND
ND
ND
ND
12.5370.03
14.2070.01
12.4770.03
9.1570.02
12.5370.01
14.4270.01
14.3770.01
9.4470.02
204.5770.39
269.3070.29
281.8870.17
191.4970.04
69.1870.05
97.2670.07
111.0470.11
67.7170.33
19.9470.04
28.6670.04
32.6170.04
18.8970.06
360
490
512
337
Mixture 2
A
B
C
D
11.5970.35
8.2870.18
13.9170.16
9.7170.27
ND
ND
ND
ND
27.3770.03
22.8670.17
29.9270.02
24.0370.37
14.0270.31
12.1170.39
20.0470.18
13.3970.12
306.2470.24
298.9270.96
367.4970.53
285.4770.40
53.0370.11
49.6270.30
64.3770.25
50.2570.45
31.3370.38
34.4770.38
43.8770.16
27.7070.30
488
467
601
459
a
Delphinidin
HPLC determination of anthocyanins for each cultivar was performed in triplicate (n ¼ 3). Results are given as mean7SD.
3
Malvacet
Malvcoum
Mix1 C
2
Mix2 C
Total
Malvidin
Mix1 B
CS C
1
Mix2CS
BA
Mix2 A
CS D Mix2 D
Mix1 D Mix1 A
0
CS B
Peonidin
-1
Delphinidin
Petunidin
-2
Mo C
Mo A
Mo B
Cyanidin
-3
Mo D
-3
-2
-1
0
1
2
3
4
PC_01: 55%, PC_02: 34%
Fig. 2. PCA of anthocyanin content of the wines: Bi-plot of scores and loadings of PC1 vs. PC2.
5
6
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M.-I. Salaha et al. / Journal of Food Composition and Analysis 21 (2008) 660–666
0.4
PC2
X-loading Weights and Y-loadings
Malvcoum
Malvacet
Peonidin
0.2
Only SO2
0.3 AAP
Malvidin
0
0.7 AAP
Total
Only AAP
-0.2
Cyanidin
-0.4
Petunidin
Delphinidin
-0.6
PC1
-0.5
-0.4
-0.3
X-expl: 54%,35% Y-expl: 15%,1%
-0.2
-0.1
0
0.1
0.2
Fig. 3. DPLS product space (factor 1 vs. factor 2) for anthocyanin content of the wines coding for different degrees of AAP presence (0, 0.3, 0.7 and 1).
Although the ANs of grapes are genetically determined, the AN
composition of wines can be influenced by a wide range of factors.
Vineyard treatment, yeast strain, MLF, weather conditions at
harvest, harvest date, the extent of rot on the fruit, duration of
maceration, fining, the use of normal enzymes and mild oxidation,
all have no or small effect on wine AN composition. In contrast,
the use of pectolytic enzymes with acylase activities and
strong oxidation is known to considerably affect the wine ANs
(Eder, 2002). Since, as it was mentioned before, the experimental
wines were produced under similar oenological conditions, the
differences observed in their AN concentration could be attributed
to both the cultivar and treatment influences.
Clearly, the cultivar had a marked influence on the AN profile
of wines. However, it was difficult to clarify this effect, since in
two cases the wines were produced by co-vinification of grapes
coming from more than one cultivar. The differentiation of
Mourvèdre is significant, including the presence of cyanidin
(0.7–1.0%) which was not detected in the other three vinifications.
The presence of Cabernet Sauvignon as the single cultivar or part
of the mixture in the other three vinifications might be
responsible for this observed absence of cyanidin.
In each case, there was no significant differentiation among the
different SO2 treatments, concerning percentage of individual AN
distribution which was practically constant (percentage data not
shown). However, the concentration of major ANs, expressed as
mg/l, were always higher in the four different control samples
where only sulphur dioxide was used, as it can be seen in Table 2. It
is known that besides its protective role during vinification, SO2
also aids the extraction of pigments whose main sources are ANs.
Furthermore, SO2 reduces the rate of colour loss and phenolic
polymerization (Bakker et al., 1998). In addition, colour indexes
such as CI (sum of yellow, red and violet component: A420+A520+
A620) and ID of ANs (Table 1) were also higher in samples with
treatment C where only SO2 was used. Finally, TP and F concentrations were also higher in the C samples following the same
pattern with AN concentration. This could be attributed to the
reaction of ANs with phenols which is responsible for the formation
of pigmented polymers and stabilization of red wine colour
(Herderich and Smith, 2005). Recent research has confirmed that
a large portion of pigmented polymers are formed from ANs and
phenolic compounds during alcoholic fermentation, and that the
pigmented polymer concentration in red wine remains largely
unchanged after completion of MLF (Eglinton et al., 2004).
The results obtained regarding the concentration of total ANs,
TPs and total Fs were all within the range expected for
commercially acceptable red wines. CI and tonality (with the
exception of the wine produced by mixture 2, treatment D which
gave exceptionally high value) data fall also within the range
commonly found for red wines.
Thus, these wines might possibly have the potential for
commercial use, although their colour characteristics and antioxidant activity would be lower, comparatively to wines produced
with the sole use of SO2. In addition, since their total phenolic and F
content would be lower, they are expected to be less astringent and
bitter (Kallithraka et al., 1997) and thus an earlier introduction to
the market might be appropriate. However, a future sensory
evaluation of the wines is necessary, combined to further
investigation of the classic oenological parameters during the wine
ageing process, in order to have a clear picture regarding their taste
properties and consequently their acceptability by the consumers.
4. Conclusion
There were significant effects of SO2 level on the AN
composition in the wines of this experiment, with more ANs
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extracted in wines with higher SO2 level. The percentage distribution of AN composition was not affected by the treatments,
while extraction and ionization of total and individual ANs were
always higher when only sulphur dioxide was used. The same
pattern followed the concentration of TPs and Fs. Antioxidant
activity values also showed a similar trend, where the higher
values were observed in the wines containing only SO2. However,
all the above-mentioned parameters as well as volatile acidity
data exhibited values within the range expected for commercially
acceptable red wines.
Therefore, the use of SO2 remains dominant in winemaking,
although the results are not restrictive concerning the possibility
to use an alternative product which may lead, when combined
with SO2, to the production of commercially acceptable dry red
wines. The components of this alternative product have acknowledged beneficial properties in contrast with potential health
problems associated to sulphur dioxide. In this way, the quantities
of SO2 added during winemaking could be reduced following the
need for the production of a ‘‘healthier’’ wine.
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Eder, R., 2002. Successful application of the anthocyanin profile by HPLC for
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Mysteries. American Chemical Society, Washington, pp. 7–21.
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Kallithraka, S., Bakker, J., Clifford, M.N., 1997. Evaluation of bitterness and
astringency of (+)-catechin and (#)-epicatechin in red wine and in model
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Kallithraka, S., Mohdaly, A.A.A., Makris, D.P., Kefalas, P., 2005. Determination of
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A REVIEW OF THE SULPHREE PROJECT – INTRODUCTION
1
SULPHREE1
“Sulfite-free organic additives to be used in wine making process”
European Union consortium research project
A REVIEW OF THE SULPHREE PROJECT – INTRODUCTION2
1
EU Project reference number: 512721, Sixth Framework Programme (FP6)
Introduced, reviewed, and edited by: Calleja, G. The content of sulfur dioxide in wine.
Undergraduate project in partial fulfilment of the requirements of the Degree of Bachelor of Pharmacy
(Honours), University of Malta; 2011.
2
A REVIEW OF THE SULPHREE PROJECT – INTRODUCTION
2
An organic alternative to sulphite additives
in the manufacture of wine –
a review of the SULPHREE1 project2
Abstract
Sulfiting agents have been used for thousand of years as an integral component in the
vinification process. Recent reports of allergenic reactions to this compound have led to calls
for an organic alternative that would be as effective in preventing spoilage whilst preserving
the typical oraganoleptic characteristics of wine. The SULPHREE project, under the auspices
of the EU Research FP6 programme was entrusted with this exact objective.
Two different classes of natural additives were tested: the first were aqueous extracts from
plants and the second oligomeric proanthocyanidin complexes (OPCs). The formulations
were tested on both sample solutions and experimental wines; oenological analyses were
carried out to detect variations in physicochemical parameters including optical density,
odour, acidity, SO2 levels and toxicity.
The SULPHREE project demonstrates that it is feasible to replace sulfites with a natural
additive.
In the first report from the SULPHREE project, “Sulphite-Free Organic Additives To Be Used
in Wine Making Process” – presented at the 2007 CIGR Section VI International Symposium
on “FOOD AND AGRICULTURAL PRODUCTS: PROCESSING AND INNOVATIONS”,
24-26 September, 2007, in Naples, Italy – the physicochemical characteristics of the two
types of additives were analyzed and their technical efficacy was tested and confirmed. All
the tests showed a success in the use of these natural alternatives to preserve wine,
suppressing oxidation as well as microbial spoilage. Furthermore, no short comings were
observed when sensory analysis was carried out: the organoleptic qualities of the
experimental wines was unaltered.
1
SULPHREE- Sulfite-free organic additives to be used in wine-wine making process. European Union
onsortium research project between 11 organisations. Funded under the Sixth Framework Programme (FP6).
Project Reference number: 512721.
2
Introduced, reviewed, and edited by: Calleja, G. The content of sulfur dioxide in wine. Undergraduate project
in partial fulfilment of the requirements of the Degree of Bachelor of Pharmacy (Honours), University of Malta;
2011.
A REVIEW OF THE SULPHREE PROJECT – INTRODUCTION
3
Keywords:
Sulfur Dioxide, organic additive, wine and SULPHREE project
Introduction
Sulfur dioxide (SO2) has been the main preservative utilised in the winemaking process for
thousands of years (Vine et al., 1992; Goode, 2005; Jackson, 2008). It is produced
intrinsically as part of the vinification process itself (Romano and Suzzi, 1993; RibéreauGayon et al., 2006) and further quantities are added throughout different stages to prevent the
spoilage of the finished product (Clarke & Bakker, 2004; Jackson, 2008). Recently, the
potentially offending properties of SO2 have been highlighted in various studies (Dahl et al.,
1986, Taylor et al., 1986; Vally et al., 1999; Vally & Thompson, 2001, D’Mello, 2003), and
research has been directed at unearthing or formulating a safer and less irritant additive than
SO2, for use within the context of the winemaking industry.
The wine-making process and SO2
Microbial activity occurs at various stages of the wine manufacturing process (Ough, 1992;
Boulton et al., 1996, Bartowsky, 2008); if this is not adequately controlled it can lead to
deterioration in the quality of the final product. This deterioration can manifest itself in
various forms including a variation in taste, bouquet and appearance (Peynaud 1984; Romano
& Suzzi 1993; Smith, 2004). The safety aspect of the wines produced is also an issue as these
yeast moulds and bacteria produce biogenic amines and precursors or ethyl carbamate (Ough,
1983). It is to this aim of containing, reducing or preventing these effects that sulfur dioxide is
employed in the vinification process.
Sulfur dioxide is available in several commercial forms and is commonly incorporated
directly as a gas from cylinders, (available as pressurised liquid), as an aqueous solution
which may be prepared by bubbling gaseous SO2 into water, or as dissolved metabisulfite
(K2S2O5) salts (Beech et al.,1978; Ough et al.,1988).
Sulfur dioxide is a natural by-product of a few particular yeast strains during the fermentation
process and may accumulate to values ranging between 10 to 64 mg L-1. (Laure et al., 1985).
Major factors influencing the biosynthesis of sulfur dioxide are grape quality, yeast strain, and
fermentation temperature. However, these quantities do not provide adequate inhibition of
microbial activity and SO2 levels above 30 mg L-1 usually result from addition by the
oenologist at various stages of wine production.23 (Jackson, 2000).
A REVIEW OF THE SULPHREE PROJECT – INTRODUCTION
4
Although it is not easy to calculate the precise quantities required due to the complex
equilibrium of sulfites in wine (Taylor et al., 1986); accurate measurement, addition and
homogenous distribution of added sulfur dioxide to known volumes of must or wine are
satisfactory requisites of a wine producing facility.
SO2 is introduced immediately at the initial crushing phase, usually at a concentration of 5070 mg L-1 due to the pulp being highly susceptible to oxidation and microbial degradation
(Zoecklein et al., 1995). Further SO2 additions, executed prior to alcoholic or malolactic
fermentation have an effect on the wine’s final character. Accounting for pH, sulfite binding
(for instance residual sugar concentration of wine), sulfite oxidation (especially after racking)
and volatile depletion; sufficient additions are made periodically after sample analysis to
maintain the desired molecular SO2 concentration of 0.8 ppm (Rankine, 1989; Margalit 1990).
During bulk ageing, and before bottling, the wine is maintained at this same molecular SO2
level which may correspond and range to between 15 - 40 mg L-1 free SO2 (Jackson, 2008).
Sulfites are added to white and rosé wines in larger quantities than red wine since the latter
contains higher concentrations of anti-oxidative tannins (Henderson, 2009).
SO2 is also soluble in other aqueous solutions such as beer and juice; however the extent to
which this occurs (pKa) depends on the solutions’ composition. The dissociation constant, in
the presence of the alcohol and ions in wine, shifts to a value close to 1.8-2.0 (Boulton et al.,
1996).
H20 (l) + SO2 (aq)

HSO3 – (aq) + H+ (aq) 
pKa =1.86
SO32- (aq) + 2 H+ (aq)
pKa = 7.2
Following the equation above, the bisulfite anion, HSO3- is the dominant species between pH
levels of 1.8 and 7.2 and therefore in wine (pH 3.0 - 4.0) (King et al., 1981; Ough & Crowell,
1987).
Antimicrobial and antioxidant activity
Sulfur dioxide exerts an increased antimicrobial effect at low pH values (Rankine, 1977)
where it exists as undissociated molecular SO2 and as the bisulfite anion, HSO3-; it penetrates
the cells walls and membranes of the microbial population and its mutagenic effects result in
mRNA deactivation (Babich & Stotzky, 1980; Ough et al., 1988).
A REVIEW OF THE SULPHREE PROJECT – INTRODUCTION
5
SO2 also inhibits enzyme catalysed and non-enzymatic browning which have an obvious
advantage in maintaining the aesthetic quality of commercially produced wines (Taylor et al.,
1986; Bates et al., 2001). It also functions as an antioxidant preventing the development of
flavours as a by-product of oxidation (Jackson, 2008).
Safety issues
In recent times, questions regarding the safety of sulfites in food and drugs have arisen and
SO2 has been stigmatised for exacerbating respiratory, dermatological, cardiovascular and
gastrointestinal symptoms, manifested mainly as asthmatic-like reactions in sensitive
individuals (Yang & Purchase, 1985; Lester, 1995, Taylor et al., 2002; Simon, 2003).
Identified symptoms include asthma, nausea and vomiting, general malaise, headaches, hives
(anaphylactic, swelling, rash-like reaction) or a combination of these (Prenner & Stevens;
1976; Freedman, 1977; Jackson, 2008).
Thus, increasingly, consumers have been clamouring for natural, organic alternatives as
opposed to the chemical preservatives present in food products (Baker et al., 1981, Ough,
1983; Yang & Purchase 1985, ORWINE, 2009). In the context of the winemaking process,
the production of a natural preservative as a replacement for SO2 would result in a scenario
where the amount of added sulfites would be significantly reduced with a concomitant
reduction in the potential allergenic properties of this chemical additive.
Overview - SULPHREE Project
The SULPHREE project was scheduled to run for two years beginning May 2005. With the
encouraging results, the project was extended for an additional six months until the end of
October 2007, and also the research partners of the SULPHREE consortium continued with
additional testing after the project ended.
A number of documents generated from the scientific research of the SULPHREE project
were received. The first document includes the key scientific findings that resulted from the
FP6 funded project, collated from a paper that was presented at the CIGR Section VI
International Symposium on ‘Food and Agricultural Products: Processing and Innovations’ in
Naples, Italy, held between the dates of 24-26 September, 2007. The next documents consist
of additional toxicological and stability studies. All the documents were thoroughly reviewed
and collated as the Appendixes to this thesis.
SULPHREE PROJECT – FIRST REPORT (Sept, 2007)
1
SULPHREE1
“Sulfite-free organic additives to be used in wine making process”
European Union consortium research project
SULPHREE PROJECT – FIRST REPORT2
September, 2007
Tiziana Mariarita Granato3, Pasquale Ferranti*, Antonella Nasi, Liberata
Gualtieri, Catalina Valencia Peroni and Marco Mescia4
Presented at the
2007 CIGR Section VI International Symposium on
FOOD AND AGRICULTURAL PRODUCTS:
PROCESSING AND INNOVATIONS
Naples, Italy
24-26 September, 2007
1
EU Project reference number: 512721, Sixth Framework Programme (FP6)
Reviewed and edited by: Calleja, G. The content of sulfur dioxide in wine. Undergraduate project in
partial fulfilment of the requirements of the Degree of Bachelor of Pharmacy (Honours), University of
Malta; 2011.
3
Department of Food Science, University of Naples “Federico II” – Portici (NA) Italy
4
LABOR Srl – Via Giacomo Peroni 386 c/o Tecnopolo Tiburtino, 00131 Rome, Italy
2
SULPHREE PROJECT – FIRST REPORT (Sept, 2007)
2
THE SULPHREE ADDITIVES
The additives used for the experimental activities included two main classes of natural
compounds:
•
The first class included aqueous extracts from plants (ADD 1) composed of mixtures
of vegetal extracts, organic acids, tannins, pectins, etc. used as stabilisers in the
winemaking process because of their strong antioxidant properties. These products
can replace the traditional chemical treatments, thus enabling the improvement of
organoleptic characteristics and the preservation of wine.
•
The second class (ADD 2 and 3, and then later ADD 4), was composed of Oligomeric
Proanthocyanidin Complexes (OPCs), extracted and purified from grape skin extract.
Although primarily renowned for their anti-oxidant action, OPCs were chosen as they
have been reported to also exert antimicrobial activity.
SULPHREE EXPERIMENTAL WORK
•
Preliminary experimental analysis was executed on model, water/ethanol solutions (90/10
v/v) mimicking the wine composition and on two reference wines, one white and one red.
The tests included solubility, stability (precipitation reaction), physicochemical
parameters (total acidity, pH, turbidity, colour, polyphenolic composition and structural
characterization, aromatic compounds), and compatibility with SO2.
o The preliminary tests of ADD 2 and 3 were performed utilising varying
concentrations, ranging from 100 mg/l to 400 mg/l. This was done because the
dark colour of these additives (OPCs) could in turn influence the optical density
(colour) of white wines.
SULPHREE PROJECT – FIRST REPORT (Sept, 2007)
•
3
Polyphenols present in the polymeric tannins were extracted through a solid phase
extraction (SPE) process utilising a silica-based bonded phase cartridge (Sep-Pak C18)
and identified by different analytical techniques including Reverse Phase HPLC, HPLCMS and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.
(MALDI-TOF/MS)
•
Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS),
at different retention times (21.9, 25.19 and 27.68 minutes, respectively) was utilised to
detect trace amounts of anthocyanins.
•
Odoriferous compounds were assayed by the analytical approach of solid phase
microextraction (SPME) and GC-MS.
SULPHREE EXPERIMENTAL VINIFICATION
In order to confirm the capability of the additives for usage in the winemaking process, they
were introduced in the experimental microvinification of different red and white varietals:
•
Falanghina grape variety grown in Campania, Italy (fresh and lively white wines with
aromas and flavors of green apples and citrus fruits).
•
Tempranillo grapes, vinificated by Bodegas Roda (Spain) (red wine)
•
White grapes vinificated by Argiolas for the production of S’Elegas wine (Sardinia,
Italy).
The experimental vinification was carried out using ADD 1, 2, and 3 alone, as well as in
combination of reduced amounts of SO2 (20% of the concentrations normally used).
The possibility of making use of the novel formulations at different stages of the winemaking
process was assessed through the following experiments:
SULPHREE PROJECT – FIRST REPORT (Sept, 2007)
4
1. Correlation of each chemical property for each formulation to find the differences
between formulations in each variable.
2. Correlation in time for each formulation and similarity with traditional sulfite addition
to find the most similar additive.
3. Correlation between different formulations to identify any experimental difference
that exists between formulations.
SULPHREE RESULTS
•
The three additives (ADD 1, 2, and 3) were completely soluble both in ethanol solution
(pH 3.5) and in wine, but only ADD 1 was found to be stable over time.
•
The pH, total acidity, total phenolic content, colour and turbidity of the reference white
and red wines were not modified by the addition of ADD 1, neither in the absence nor in
the presence of reduced SO2 concentration.
•
ADD 2 and ADD 3 resulted in high optical densities and an increased content of total
polyphenols (316.27 and 556.55 mg mg/l). Low concentrations of ADD 2 modified the
turbidity of wine both in combination with SO2 and without SO2 addition. Increased
concentrations of ADD 2 had a negative influence on the colour of the wine; and the
influence on turbidity and colour became increasingly evident when the additive was used
in combination with SO2.
•
ADD 3 also darkened the colour of wine, but it resulted in a positive reduction in
turbidity. These effects were also observed when the additive was used in combination
with SO2, although they were less evident.
•
Due to the unfavourable effect of OPC additives on the optical density (colour) of the
reference wine, a newly formulated sample of proanthocyanidins with a much lighter
colour (ADD 4) was put through the same preliminary tests with the same analytical
approach as the other three formulations.
SULPHREE PROJECT – FIRST REPORT (Sept, 2007)
•
5
The addition of the modified OPC additive (ADD 4) generated positive results:
•
A lower optical density in the model solution, compared to the other OPC samples;
•
pH, total acidity, total phenolic content, and colour were found to be consistent and
unmodified in the absence as well as in the presence of a reduced amount of SO2;
•
Reduction in turbidity when used in combination with SO2.
Figure 1: the analysis of Additive 4, analysed using MALDI-TOF MS, shows peaks with m/z
values corresponding to flavan-3-ols (a class of flavonoids). These compounds were identified
to be an oligomeric series of catechins and epicatechins as well as their corresponding
catechin gallates (gallic acid ester derivatives), up to the dodecamer.
Fig. 1 Mass spectrum (MALDI-TOF) of ADD 4
SULPHREE PROJECT – FIRST REPORT (Sept, 2007)
6
Figure 2: Trace amounts of anthocyanins were identified as the 3-O-monoglucosides of
malvidin and peonidin on the basis of their UV/VIS and mass spectra (m/z 493, 463) and by
comparison with reference compounds. Additionally, 3-O-acetylglucosides and 3-O-pcoumaroylglucosides of malvidin were detected (m/z 535 and 639).
Fig. 2 – ESI mass spectra of peaks eluted at (a) 21.90 (b) 25.19 (c) 27.68 min of ADD 4. The
mass spectra correspond to (a) malvidin and peonidin-3-O-glucoside, (b) malvidin-3-O-(6-Oacetly)-glucoside and (c) malvidin-3-O-(6-O-p-coumaryl) glucoside.
SULPHREE PROJECT – FIRST REPORT (Sept, 2007)
7
Regarding the presence of odorous, aromatic compounds, the three additives, ADD 1, ADD 2
and ADD 3, were analysed by SPME and GC MS, and the results can be seen in Fig. 3, 4, and
5, The results included terpenes (such as limonene) and their ester derivatives (such as ethyl
octanoate and decanoate) and alcohols, differing for each additive and with their
corresponding aromatic descriptors displayed in Tables 1, 2 and 3.
Fig. 3 SPME- Total Ion Current Chromatogram ADD 1
Fig. 4 SPME- Total Ion Current Chromatogram ADD 2
8
SULPHREE PROJECT – FIRST REPORT (Sept, 2007)
Fig. 5 SPME- Total Ion Current Chromatogram ADD 3
The aromatic profile of the reference wine (S’Elegas), correlated well when compared to the
same wine fortified with ADD 3 (Fig. 6), further showing that the aromatic profile was not
altered by the ADD 3.
!"#$#%&!'('&))*'
!"#$#%&!'
Figure 6: Total Ion Current Chromatogram from SPME analysis of
S’ELEGAS alone and
Fig.
6 Total Ion
Current
Chromatogram
from SPME analysis of S’Elegas alone and S’Elegas
S’ELEGAS
added
with ADD
3
withbetween
ADD 3. wine and aromatic compounds from
However, interaction effects werefortified
observed
additives: some odorous compounds present in the additives, such as alpha-pinene and
limonene, were not observed in wine after addition of the additive, while they are observed in
model solution at the same concentration. This was due to the presence in wine of compounds
which interfere with the volatility of the compounds, probably by adsorption.
In order to test the capability of additive to be used in winemaking process, they were employed
in experimental microvinification of several white and red grapes:
! Falanghina grape variety grown in Campania. Today Falanghina is used to make
SULPHREE PROJECT – FIRST REPORT (Sept, 2007)
9
Nevertheless, interactions were observed between the wine and the analysed aromatic
compounds from the additives. For instance, α-pinene and limonene were not detected in the
wine after addition of the additive, even though they are present in the model solution at equal
concentrations. This results from interfering compounds present in the wine matrix that hinder
the volatility of the compounds, probably through adsorption reactions.
The three correlation approaches for the experimental vinification that outline the
physicochemical characteristics of experimental wines at different stages of the vinification
process, confirmed the possibility of using the new, selected formulations.
1. From the first correlation data set, the analysis confirmed that the chemical properties of
each different formulation at various stages of the vinification process do not depend on
the formulation itself. So the experimental data set is trust-worthy and all the chemical
analyses are accurate.
2. Comparison of additive properties to select the additives that closely mimic the
physicochemical properties of sulfur dioxide (serving as the control) when added to wine.
3. The relationship between the different formulations is identified for all four stages of the
winemaking process. The results, presented in the different matrices in Fig. 7 show that
the second additive (2nd variable of the matrix) has comparable behaviour to that of SO2
(1st variable present in each matrix), especially when analysing must characteristics. The
correlation factor, post-vinification process is higher than 0.8, confirming that there are no
significant alterations in the chemical characteristics of wine when preserved with the
formulations as compared to SO2.
SULPHREE PROJECT – FIRST REPORT (Sept, 2007)
Fig. 7. Correlation image with a scale. The 1st variable of each matrix portrayed is SO2 of the different
formulations analysed.
10
11
SULPHREE PROJECT – FIRST REPORT (Sept, 2007)
The results of the experimental data generated are summarized with the following
observations:
•
The OPC formulations used were incompatible with the concomitant use of SO2, on the
The results of the experimental work are summarized in the following sentences:
basis of different physico-chemical parameters (volatile acidity, polyphenols, optical
•
OPCs are not compatible with SO2, on the basis of different chemical-physical
density and
sensory(volatile
analysis).
parameters
acidity, polyphenols, optical density and sensory analysis)
!"#$%&'()*&&%+'
'+%!%&'),*-'&%-"
.!'&#$'*(&%$)$#-(
!"#$%&'()*+,$#-(
4,50
0,45
4,00
0,40
3,50
0,35
3,00
0,30
2,50
0,25
2,00
0,20
1,50
1,00
0,15
0,50
0,10
0,05
0,00
Mosto
Vino
Mosto
Vino ( I Travaso)
So2
Additivo 1
Additivo 1 + So2
Additivo 3 + So2
Additivo 2
Additivo 2 + So2
Additivo 3
Vino
Vino ( I Travaso)
So2
Additivo 1
Additivo 1 + So2
Additivo 3 + So2
Additivo 2
Additivo 2 + So2
Additivo 3
Fig 8:
8 Optical
density
and
Volatile
Acidity
of Falanghina
experimental
wines.
Figure
Optical density
and
volatile
acidity
of Falanghina
experimental
wines
•
•
OPCs darken white wines.
The OPC formulations used enhanced browning in white wines. This is explained through
This fact can be explained by the grapes maderization or by very high concentration of
additive,
unsuitable
for the
vinification
of white wines.
grape maderization
when
high
concentrations
of these additives were used, which were
Colour darkening during storage of experimental white wines resulted essentially from
deemed
unsuitable
for the
vinification
white wines.
The browning
phenomenon
during
chemical
reactions
involving
phenolicofcompounds,
in particular
the oxidation
of flavan-3-ol
derivatives. For this reason, these additives, that are oligomeric proanthocyanidin
complexes,
darkens the
white
wines
and shorten
their commercial
life. During
storage
storage
of experimental
white
wines
essentially
resulted
from chemical
reactions
of and
OPCs
aging of wine, polyphenolic compounds are gradually modified. Reactions among flavan-3ol, proanthocyanidins
and other
compounds,
as glyoxylic
acid, piruvic
acid and
involving
phenolic compounds,
in particular
the such
oxidation
of flavan-3-ol
derivatives.
This
acethaldehyde, and also between flavonols themselves are responsible for the appearance
of new pigments, and hence for the disappearance of oligomer proanthocyanidin from
ultimately gives rise to aesthetically unacceptable wines consequently shortening their
solution.
commercial lifespan. During storage and ageing of wine, polyphenols are gradually
modified. Reactions among flavan-3-ol, proanthocyanidins and other compounds, such as
glyoxylic acid, pyruvic acid and ethanal, and also between flavonols themselves are
responsible for the emergence of new pigments, and hence for the disappearance of
oligomer proanthocyanidin from solution.
10
SULPHREE PROJECT – FIRST REPORT (Sept, 2007)
•
12
The introduction of the OPC formulations used in red wines did not have a negative
influence on their colour and colloidal propertie, thus resulting in acceptable end
products. No significant differences were apparent for the chemical parameters and
organoleptic properties between the reference and experimental wines (Table 4). Sensory
analysis did not detect any alterations or flaws; ADD 2 used in the absence of sulfur
dioxide provided the best sensory characteristics.
SULPHREE PROJECT – FIRST REPORT (Sept, 2007)
•
13
ADD 1 shows synergetic effects when used in combination with SO2.
The vinification of the experimental S’Elegas using ADD 1 (listed as “BIOMA” in Table 5),
performed with about an 80% reduction in conventionally added SO2 concentration, imparts
the better results for white wines. Figure 10 displays experimental wines after 3 months of
storage; no alterations in chemical parameters (pH, volatile acidity, free SO2, total SO2,
optical density, etc.) nor in sensory perception were observed throughout the duration.
•
The antioxidant potency determination showed that the OPC formulations used had
optimal behaviour during the first stages of the vinification process, while ADD 1 was
found to have improved preservative and stabilising effects on wine during storage.
These results pave the way for the possibility of combining both classes of additives:
incorporation of OPCs could be optimally included during the early stages of vinification
(grape pressing or prior to alcoholic fermentation), whereas ADD 1 can be used in the
final phases (refining, prior to bottling and ageing purposes), taking advantage of its
stabilising effects.
SULPHREE PROJECT – FIRST REPORT (Sept, 2007)
14
DISCUSSION
In this first report from the SULPHREE Project, natural organic additives have been identified
and pursued to an extent that favourable results on physicochemical and stability parameters
have emerged from their meticulous analysis. Both aqueous extracts from plants and OPC
additives were tested. Colour, aromatic profile and antioxidant strength were evaluated for the
different microvinifications of a variety of white and red wines. The physicochemical analysis
of experimental wines at different stages of the vinification process confirmed the possibility
of implementing the newly selected formulation in wine making. Consolidating the
information and facts from the experimental data, all the tests showed a success in the use of
these natural alternatives to preserve wine, suppressing oxidation as well as microbial
spoilage. Furthermore, no short comings were observed when sensory analysis was carried
out: the organoleptic quality of the experimental wines was unaltered. Moreover, one
additive shows a synergetic effect with SO2, advocating the possibility of including both
classes of additives at the appropriate winemaking stages.
CONCLUSION
The studies undertaken have provided a firm basis for further work: the additives show
promise as alternatives to the currently accepted standard of the utilisation of sulfur dioxide as
a preservative within the wine-making process.
The next step in the SULPHREE Project was the execution of toxicological analyses – on
wines preserved with the additives, as well as on the additives themselves – in order to
understand the safety of these products and their possible permissibility as food additives.
Further tests were then also done on older wines and white wines.
TOXICITY TESTING OF THE SULPHREE WINES
1
SULPHREE1
“Sulfite-free organic additives to be used in wine making process”
European Union consortium research project
TOXICITY TESTING OF THE SULPHREE WINES
Overseen by: Professor Pasquale Ferranti and Dr. Tiziana Granato
Department of Food Science, University of Naples “Federico II”, Italy2
2007 – 2008
1
2
EU Project reference number: 512721, Sixth Framework Programme (FP6)
Reviewed and edited by: Calleja, G. The content of sulfur dioxide in wine. Undergraduate project in partial
fulfilment of the requirements of the Degree of Bachelor of Pharmacy (Honours), University of Malta; 2011.
TOXICITY TESTING OF THE SULPHREE WINES
2
The potential toxicity of the wines produced within the framework of the SULPHREE project was
examined through biochemical assays that tested the action of wines on selected cell systems.
These studies were aimed to evaluate the cytotoxic effects of the sulfite-free, experimental wines
and compare them to the reference wines containing sulfites.
EXPERIMENTAL
Cell lines and wines
The cell systems HL-60 (Human leukemia) and HT-29 (intestinal tumor cells) cultured in the
laboratory were used for toxicity tests. The wines tested were a white varietal, Nuragus of Cagliari
(S’Elegas), produced in 2007 in the Argiolas winery – one using a preliminary OPC formulation,
and the other using Bioma Protos.
These wines were selected since they yielded the most promising and favourable results when
processed using the alternative additives.
Cell viability assay
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) is a dye commonly used to
measure cell viability. The test is based on the conversion of yellow, water-soluble MTT to the
purple, water insoluble end product, formazan, by the enzymatic action of mitochondrial
dehydrogenase. The amount of formazan formed is proportional to the number of metabolically
active cells (Mossman, 1983).
The key steps in the assay are:
• incubation of cells with MTT
• extraction of the formazan crystals from the cells
• dissolution of formazan
• measurement of the absorbance at 570 nm.
The cell line HT-29 was welled in Multi-well 96® and treated with wine at varying concentrations
(1%, 5%, and 10%). After 24h hours elapsed, a solution of MTT (10%) was added to all wells and
the cells were incubated for 4 hours at 37°C. The formazan crystals formed were then dissolved in a
solution of isopropanol/HCL 1N (10%).
The absorbance of each well was determined by using an automatic ELISA reader set at 570 nm test
wavelength and a reference wavelength of 655 nm. The percentage of cell proliferation was
calculated as follows:
(Absorbance of treated cells) / (Absorbance of CTR ) x 100%
where CTR: Control, non-treated cells (absence of wine).
Oxidative stress analysis by flow cytometric technique
HT-29 cells were incubated in culture medium (without serum) in both the presence and absence of
wine (control sample) and were treated in a solution containing the radical sensitive dye
hydroethidine (2,5 mg/ml stock solution). The increase of the intracellular fluorescence of the
oxidative product of hydroethidine, ethidium, was taken as a measure for intracellular free radicals
that were generated. Hydroethidine is a neutral, blue fluorescent compound produced by the
TOXICITY TESTING OF THE SULPHREE WINES
3
reduction of ethidium bromide. It is incorporated into viable cells where it undergoes enzymatic
dehydrogenation or oxidation through the action of free radicals. Due to its cationic nature the
product, ethidium, becomes trapped within the cell. The cells were incubated in hydroethidine (20
ng/ml) for an hour and then washed twice in PBS. Fluorimetric analysis was carried out using
FACScan® flow cytometer (FACScan Becton Dickinson) which makes use of CellQuest® software.
For each sample measurement, about 10,000 events were acquired.
Thiobarbituric acid (TBA) assay
The thiobarbituric acid (TBA) method is the most widely used test for measuring the extent of
oxidative deterioration of lipids. In this test the TBA reacts at 95°C with malondialdehyde (MDA),
a degradation product of lipid hydroperoxides, to give a red fluorescent 1:2 MA/TBA adduct
(TMT), which is quantified spectrophotometrically at 525-532 nm.
The wine samples (100 µL) were dissolved in an aqueous solution of an antioxidant agent such as
Butylated Hydroxy Toluene (BHT) 7.2% and centrifuged at 4° C, set at 13,000 rpm for 10 minutes.
The samples were then incubated with TBA 0.2% in HCl at 100°C for 30'. The reaction was
stopped in cold water and the adduct was quantified by measuring its adsorption at a wavelength of
532 nm.
RESULTS
Figure 1. % of HT-29 cell proliferation after 24 h of incubation with varying concentrations (1-10%
v/v) of different wines with: OPC, PROTOS, and reference (with traditional sulfite concentration)
As can be seen in Figure 1, after 24 hours the percentage of cell proliferation increased
proportionally with increasing wine concentrations. Slight variations were observed, although no
marked differences of cell vitality were identified for wines prepared with the SULPHREE additives
compared to the reference wine.
TOXICITY TESTING OF THE SULPHREE WINES
4
Figure 2. Mithocondrial oxidative stress of HT-29 cells calculated by flow cytometric technique with
hydroethidine
Figure 2 displays histograms relative to the cell mitochondrial oxidative stress measured in the
presence of the experimental wines. The data clearly show that wine prepared using the
SULPHREE additives have comparable (OPC) or even higher (Protos) activity against oxidative
stress, corresponding to comparable or slightly lower toxic effect compared to the reference wine.
This behaviour was also confirmed by direct analysis of the additives in their pure state at the same
concentrations used for winemaking.
Figure 3. Reactive aldheydes (malondialdehyde) (MDA) of different wines by TBA test
The above conclusion were further supported by the biochemical assay of the reactive aldehydes
carried out by means of the TBA test. This analysis (Figure 3) shows that the two SULPHREE
wines both present a lower level of reactive aldehydes than the control wine; and that the wine
preserved with Protos generated the best results.
CONCLUSION
Holistically, all the above data, confirms the absence of toxicity of wines preserved with the
SULPHREE additives.
TOXICITY STUDIES OF THE SULPHREE ADDITIVES
1 SULPHREE1
“Sulfite-free organic additives to be used in wine making process”
European Union consortium research project
TOXICITY STUDIES OF THE SULPHREE ADDITIVES
(“Studio della tossicità degli additivi – attività anno 2008”)
Overseen by: Professor Pasquale Ferranti and Dr. Tiziana Granato
Department of Food Science – University of Naples “Federico II” –
Italy2
2008
1
2
EU Project reference number: 512721, Sixth Framework Programme (FP6)
Translated and reviewed by: Calleja, G. The content of sulfur dioxide in wine. Undergraduate
project in partial fulfilment of the requirements of the Degree of Bachelor of Pharmacy (Honours),
University of Malta; 2011.
2 TOXICITY STUDIES OF THE SULPHREE ADDITIVES
SUMMARY OF TECHNICAL EFFICACY OF THE TWO ADDITIVES
The technical efficacy of the additives – preliminary OPC (Oligomeric Proanthocyanidin
Complex) formulations and Protos by Bioma – was tested within the scope of the
SULPHREE
Project.
These
additives
were
incorporated
in
the
experimental
microvinification of two white wines typical of the Italian region of Campania
(Catalenesca and Falanghina), one white wine from Sardinia (Nuragus di Cagliari), and
one red wine from Spain (Tempranillo).
The physicochemical and sensory analysis of the experimental musts and wines did not
reveal any serious defects or alterations – with the exception of the OPC formulations in
the white wines, as they had an unsuitable impact on the colour, due to the concentrations
utilised. Whereas, the plant extract additive, Protos by Bioma, yielded the better results in
the white wines. Additionally, Protos resulted in a synergistic effect when used in
combination with a substantially reduced quantity of sulfites, specifically only 20% of
the normal sulfite concentration.
More specifically, the analyses of the antioxidant strength of the experimental wines
demonstrated a difference in behaviour between the two additives, depending on the
phase in the vinification process. The proanthocyanidin additive (OPC) showed a greater
antioxidant strength in the initial phases of vinification, whereas the additive Protos
seemed to carry out the antioxidant function better in the conservation phase of wine.
This would suggest the possibility of using the two additives at different stages of wine
production.
TOXICITY STUDIES OF THE ADDITIVES
Once the technical validation of the two organic additives had been confirmed, it was then
necessary to study the additives from the toxicological point of view – in order to obtain
experimental evidence that, in the doses incorporated in vinification, the additives do not
have any harmful health effects on the consumer.
3 TOXICITY STUDIES OF THE SULPHREE ADDITIVES
For this purpose, toxicity testing was carried out in vitro. The medium selected for this
first experimental model were Caco-2 cells, derived from human colon carcinoma. These
cells serve as a good benchmark to test the effects which the additives, once ingested, can
impart on human intestinal cells. The first experiment was to verify whether the addition
of the two additives, together with the incubation of the cells in the culture medium, could
in any way hinder the growth of the cells.
For this reason, the Caco-2 cells were
!"#$"%&'(%)*+,,)-)'"#)'."(/)'!+'.(%0.+-)'12#+-2()#!2'3%('45'6+2(#+'*%'.%**"*%'+#."7)-%'.2#'
incubated for a duration of 10 days together with the additives, in the same concentrations
6*+' )!!+-+/+' )**%' .2#.%#-(),+2#+' "-+*+,,)-%' +#' 8)0%' !+' /+#+8+.),+2#%' 941*:;' 3%(' <(2-20=' >55'
utilised in the vinification process (1 ml/liter of PROTOS and 40 mg/liter of OPC).
16:;'3%('6*+'?<@AB';)'."(/)'!+'.(%0.+-)&'120-()-)'+#'8+6"()'4)'120-()'.C%'6*+')!!+-+/+'!%**)'
*+#%)'
!"#$#%'
#2#'
*)' #2(1)*%'
.%**"*)(%&'
"#)'
Figure
1 shows
theC)##2'
growth)*-%()-2'
curve Caco-2
(“Curva .(%0.+-)'
di Crescita
Caco-2”).2#0%#-%#!2'
for this period,
and
12*-+3*+.),+2#%'
.%**"*)(%'
3)()62#)7+*%'
2' 0"3%(+2(%'
)*' .2#-(2**2'
)**%' .%**"*%'
.2#'
clearly demonstrates
that
the two Protos
additives
(“N” and%'“S”)
did not-()--)-%'
alter normal
1%-)7+02*8+-2'!+'32-)00+2B''
cellular proliferation, thus allowing equal or superior cell growth as opposed to the
' control and also to cells combined with potassium metabisulfite (“bisolfito”).
CURVA DI CRESCITA CACO2
2500
3
n° celllule x 10
2000
controllo
4914 1X
1500
4914 0,1X
4914 0,01X
protos N
1000
protos S
bisolfito
500
0
tempi
'
Fig 1 Time of analysis (“tempi”) on the horizontal axis, vs the numerical count of Caco-2 cells on the
vertical
axis. delle ascisse sono riportati i tempi di analisi, sull’asse delle ordinate sono riportati i valori numerici
Fig.
1. A Sull’asse
corrispondenti al numero di cellule CaCo2 contate. '
'
In order to verify whether these results were confined to the type of cell culture selected,
<%(' /%(+8+.)(%' 0%' *D%88%--2' 120-()-2' 8200%' !+3%#!%#-%' 2' 1%#2' !)*' 12!%**2' .%**"*)(%' 0.%*-2&'
the same experiment was carried out using murine fibroblast cells: NIH3T3. The results,
-)*%'0)66+2'E'0-)-2'%0%6"+-2')#.C%'0"'"#)'*+#%)'.%**"*)(%'!+'8+7(27*)0-+'1"(+#+&'FGHIJIB'
shown in Figure 1b, confirm the same observations demonstrated in the culture medium
G'(+0"*-)-+'2--%#"-+&'120-()-+'+#'K+6"()'47&'.2#8%(1)#2'3%('$"%0-2'0+0-%1)'.%**"*)(%'$")#-2'
in the previous experiment.
200%(/)-2'3%('*%'.%**"*%'@).2LMB'
4 TOXICITY STUDIES OF THE SULPHREE ADDITIVES
CURVA DI CRESCITA NIH3T3
3.500
n° cellule x 10
3.000
3
controllo
2.500
4914 1x
4914 0,1 X
2.000
4914 0,01 X
1.500
protos n
protos s
1.000
bisolfito
500
0
tempi
Time of delle
analysis
(“tempi”)
on the horizontal
the numerical
count ofsono
Caco-2
cellsi on
the
Fig.Fig1. 1b
B Sull’asse
ascisse
sono riportati
i tempi di axis,
analisi,vs sull’asse
delle ordinate
riportati
valori
vertical axis.
numericicorrispondenti al numero di cellule NIH3T3 contate.
!
Additionally,
a test regarding
proliferation
(“cell titer”)
was ()(2*.&$!
performed*#!
on both
of the
"#$%&'(!
)*! (#&'+,-(!
%(! %.#((!cell
/(%%*%+'.!
)(%(0.$+#&(!
1! )&+&$!
)+22.$!
3.!
cellular lines. A cell titer is a colorimetric test which enables testing to determine if the
4'$%.5('+0.$#(!/(%%*%+'(!67(%%!8.&('9:!!
cells are metabolically active after incubation with a substance in different concentrations
"%! 7(%%! 8.&('! 1! *#! )+22.$! /$%$'.,(&'./$! /;(! 4(',(&&(! 3.! &()&+'(! )(! %(! /(%%*%(! )$#$!
or time periods. The cells, which have been subjected to toxicity to a lesser manner, were
,(&+-$%./+,(#&(!
+&&.<(! 3$4$!
*#+! of
)$)&+#0+!
+! /$#/(#&'+0.$#.!
deemed metabolically
active%=.#/*-+0.$#(!
and therefore /$#!
capable
oxidising
NADH to NAD$!+.&(,4.!
The
extent>(!
of/(%%*%(!
oxidation
was measured
to the,.#$'(!
bio-reduction
a coloured
3.<(').:!
/;(! ;+##$!
)*-.&$! .#!due
,+#.('+!
%=(55(&&$!of
&$))./$!
3.! *#+!substance
)$)&+#0+!
capable,(&+-$%./+,(#&(!
of emitting florescence
at 490
nm. .#! 2'+3$! 3.! $)).3+'(! .%! ABCD! +! ABCE:! 8+%(!
)+'+##$!
+&&.<(! (?!
@*.#3.?!
$)).3+0.$#(! <.(#(! ,.)*'+&+! 2'+0.(! +%%+! -.$'.3*0.$#(! 3.! *#! 4'$3$&&$! /$%$'+&$! .#! 2'+3$! 3.!
Both cell types – Caco-2 and NIH3T3 – were incubated with the additives for varying
(,(&&('(!5%*$'()/(#0+!+!FGH!#,:!!
time parameters: 24, 48 and 72 hours. Figure 2 demonstrates that no variation was found
>(!/(%%*%(!7+/$IJ!(!A"DK8K!)$#$!)&+&(!.#/*-+&(!/$#!2%.!+33.&.<.!4('!&(,4.!3.55('(#&.L!JF?!FM!
between the cells incubated with Protos and those with potassium metabisulfite and the
untreated control cells.
(!NJ!$'(:!>+!5.2*'+!J!,$)&'+!/;(!%+!4('/(#&*+%(!<.&+%(!3(%%(!/(%%*%(!.#/*-+&(!/$#!%=+33.&.<$!
!"#$#%! (3! .%! ,(&+-.)$%5.&$! 3.! 4$&+)).$! 1! *2*+%(! +! @*(%%+! 3(%%(! /(%%*%(! 3.! /$#&'$%%$?! #$#!
&'+&&+&(:!
8+%(!'.)*%&+&$!/$#5(',+!%=.4$&().!/;(!%=+33.&.<$!O!6&'()*+!"#$#%+,'+-'#.*9?!+!3.55('(#0+!3(2%.!
+33.&.<.!!#$#!+--.+!+%/*#!(55(&&$!&$))./$!)*.!).)&(,.!/(%%*%+'.!.#!()+,(:!
5 TOXICITY STUDIES OF THE SULPHREE ADDITIVES
CONCLUSION
In conclusion, these experiments confirmed the hypothesis that the additive #1, Protos
line by Bioma – as opposed to the other additives tested – does not have any adverse
toxicological effects on the cellular systems
tested.
!"##$%&'&"(%!)!*+
0/,
!"##$%&'&"(%!)!*+
0.,
0-,
0/,
>%?;7@<;7A
0+,
0.,
B+%C
0,,
0-,
-/%C
>%?;7@<;7A
0+,/,
B+%C
0,,.,
+-C
-/%C
/,-,
+-C
.,+,
-, ,
!
+,
-10-
-10-%,203 -10-%,2,03 456768%9
456768%$
:;86<=;76
,
!
-10-
-10-%,203 -10-%,2,03 456768%9
456768%$
:;86<=;76
!"##%&'&"(%9'DE&E
0.,
!"##%&'&"(%9'DE&E
0-,
0+,
0.,
B+%C
0+,/,
-/%C
> ?;7@<;7A
> ?;7@<;7A
0,,
0-,
+-C
0,,.,
B+%C
/,-,
-/%C
.,+,
+-C
-, ,
+,
!
-10-
-10-%,203
-10-%,2,03
456768%9
456768%$
:;86<=;76
,
Fig. 2 Clustered!bar graph-10displaying-10-%,203
percentage-10-%,2,03
vitality of Caco-2
cells (panel
A) and:;86<=;76
NIH3T3 (panel B)
456768%9
456768%$
on the
vertical axis
forordinate
the different
additives
(listed on the
horizontal
axis), CACO2
analyzed(pannello
after 24, 48
72
Fig.
7 Sull’asse
delle
è indicata
la percentuale
vitale
delle cellule
A) and
e NIH3T3
(pannello B
hours.
per tempi diversi di trattamento, sulle asse delle ascisse sono indicati gli additivi analizzati.
Fig. 7 Sull’asse delle ordinate è indicata la percentuale vitale delle cellule CACO2 (pannello A) e NIH3T3 (pannello B)
per tempi diversi di trattamento, sulle asse delle ascisse sono indicati gli additivi analizzati.
Dipartimento di Scienze degli Alimenti - University of Naples “Federico II” – Parco Gussone, 80055 Portici (Italy)
TOXICOLOGICAL ANALYSES OF PROTOS
1
TOXICOLOGICAL ANALYSES OF PROTOS1
Research Centre Biotecnologie BT srl
Perugia, Italy
December, 2006
1
Reviewed and edited by: Calleja, G. The content of sulfur dioxide in wine. Undergraduate project in
partial fulfilment of the requirements of the Degree of Bachelor of Pharmacy (Honours), University of
Malta; 2011.
TOXICOLOGICAL ANALYSES OF PROTOS
2
Toxicological analyses of PROTOS 1 (N) and PROTOS 2 (S)
As a required part of the SULPHREE Research Project, toxicological analyses were
performed. These were done by:
Research Centre Biotecnologie BT srl,
in Perugia, Italy,
December, 2006
The parameters analysed included:
1) Determination of Nitrates/Nitrites utilising Ion Chromatography
Results: PROTOS 1 (N)
NO2: ND (< 0.1mg/l)
NO3: 0.261 mg/l
Results: PROTOS 2 (S)
NO2: ND (< 0.1mg/l)
NO3: 1.217 mg/l
2) Residual phytosanitary for PROTOS 1 (N) and 2 (S)
Organochlorurate insecticides
-dicofol <0.01 mg/Kg
-dieldrin <0.01 mg/Kg
-endrin <0.01 mg/Kg
-methoxychlor <0.01 mg/Kg
-o,p’-DDD <0.01 mg/Kg
-o,p’-DDE <0.01 mg/Kg
-o,p’-DDT <0.01 mg/Kg
-p,p’-DDD <0.01 mg/Kg
-p,p’-DDE <0.01 mg/Kg
-p,p’-DDT <0.01 mg/Kg
3) Heavy metals: PROTOS 1 (N) and 2 (S)
Parameters
Values (µg/l)
Analytical technique
Cadmium
<0.031
GFAAS
Lead
<0.84
GFAAS
Mercury
<0.1
HVG
Arsenic
<1.49
GFAAS
4) Nitrosamine concentration
PROTOS 1 (N) and 2 (S): < 0.1mg/litre
TOXICOLOGICAL ANALYSES OF PROTOS
3
5) Ocratoxin A:
PROTOS 1 (N): 0.94 ppb
PROTOS 2 (S): 0.62 ppb
6) Dioxin (PCB):
Parameters Values Technique
Formulation
Parameters
Values (µg/l)
PROTOS 1(N)
<0.067
PCB
PROTOS 2(S)
0.16
Gas Chromatography-Electrochemical Detection
Analytical technique
GC-ECD
GC-ECD
7) Fumosine for PROTOS 1 (N) and 2 (S)
Fumosine B1 <50 µg/l (ppb)
Fumosine B2 <50 µg/l (ppb)
Fumosine B3 <50 µg/l (ppb)
The results generated from the toxicological analyses show that the formulations
PROTOS 1 (N) and PROTOS 2 (S) conform to acceptable standards.
ppb
ADDITIONAL WINE ANALYSES ON OLDER WINES
SULPHREE1
“Sulfite-free organic additives to be used in wine making process”
European Union consortium research project
ADDITIONAL WINE ANALYSES ON OLDER WINES
Overseen by: Professor Pasquale Ferranti and Dr. Tiziana Granato
Department of Food Science, University of Naples “Federico II”, Italy2
Completed: January, 2008
1
2
EU Project reference number: 512721, Sixth Framework Programme (FP6)
Reviewed and edited by: Calleja, G. The content of sulfur dioxide in wine. Undergraduate
project in partial fulfilment of the requirements of the Degree of Bachelor of Pharmacy
(Honours), University of Malta; 2011.
1
ADDITIONAL WINE ANALYSES ON OLDER WINES
2
The “reference wines” were preserved with conventional sulfite concentrations and
the “experimental wines” were preserved with the plant-based additive, Protos by
Bioma. These analyses were done on older wines, in order to determine the
possibility of using Protos in preserving wines over a number of years.
A white wine, FENDANT, produced in 2003 and stored for about 4 years
Table 1 shows the physicochemical properties of the Fendant. The evaluation of
these analytical parameters did not show significant differences between the samples
– except for total acidity, volatile acidity, and optical density. The total acidity was
higher in the experimental wine: this fact could be very important for the
preservation / suppression of microbial spoilage in the experimental NSA (No Sulfites
Added) wine. A residual value of free SO2 was observed in the wine treated with the
additive, probably caused by the interference of phenolic compounds with the
procedure for SO2 determination. This interference further effected the values for
unstable and stable SO2. Additionally, the absorbance at 420 nm shows two different
degrees of browning in the samples: optical density of the experimental wine was
higher than that of the reference wine, due to the grape maderization.
FENDANT 2003
Reference wine
Experimental wine
Total Acidity (g L-1)
3,72
5,02
Volatile Acidity (g L-1)
0,44
0,72
pH
3,83
3,99
Free SO2 (mg L-1)
9,6
6,4
Bound unstable SO2 (mg L-1)
52,48
5,76
Bound stable SO2 (mg L )
23,04
7,04
Total SO2 (mg L )
85,12
19,2
Optical density
0,1563
0,3340
% V/V
12
12,2
Total Polyphenols (mg L-1)
177,42
151
-1
-1
Table 1 Comparative physicochemical parameters of Fendant wines (2003)
The red wines (Nebbiolo d’Alba and Dolcetto)
Unlike the white wine variant, the two red wines showed little variation in the
comparative analysis between the reference and experimental wines.
ADDITIONAL WINE ANALYSES ON OLDER WINES
3
Red wine, NEBBIOLO D’ALBA, produced in 2004 and stored for about 3 years
As can be observed in Table 2, the Nebbiolo preserved with Protos showed very
slight discrepancies – if any at all – when compared with the reference wine.
NEBBIOLO D’ALBA 2004
Reference wine
Experimental wine
Total Acidity (g L-1)
5,625
5,175
Volatile Acidity (g L-1)
0,79
0,816
pH
3,0
3,03
Free SO2 (mg L-1)
23,7
27,2
Bound unstable SO2 (mg L-1)
62,78
8,37
Bound stable SO2 (mg L )
31,4
13,95
Total SO2 (mg L-1)
117,88
49,52
Abs 420 nm
1,901
1,877
Abs 520 nm
1,680
1,810
Colour Density
3,581
3,687
Tint
1,13
1,037
% V/V
14
13,8
Total Polyphenols (mg L-1)
484,14
314,57
-1
Table 2 Comparative physicochemical parameters of Nebbiolo d’Alba wines (2004)
Red wine, DOLCETTE D’ALBA, produced in 2005 and stored for about 2 years
As can be seen in Table 3, the Dolcetto demonstrated a significant difference in
optical density. The absorbance at 420 nm shows a higher extent of browning (grape
maderization) in the experimental sample. The colour of red wines is mainly due to
the anthocyanins and polymeric compounds they contain and depends on several
factors, including the variety and age of the wines (McCloskey & Yengoyan, 1981).
During storage, the typical purple-red colour of young red wines is lost as it changes
to orange-red. These changes, more evident in the experimental wine, are mainly due
to the displacement of the monomeric pigments of anthocyanins to more stable
oligomeric forms. Additionally, the volatile acidity was higher in the experimental
wine than in the reference wine, and this finding was also confirmed by sensorial
analysis, as the acetic acid affected the taste of the experimental wine.
ADDITIONAL WINE ANALYSES ON OLDER WINES
DOLCETTO D’ALBA 2005
Reference wine
Experimental wine
Total Acidity (g L-1)
6,037
6,45
Volatile Acidity (g L-1)
1,128
1,33
pH
3,40
3,27
Free SO2 (mg L-1)
30,69
15,34
Bound unstable SO2 (mg L-1)
42,55
22,32
Bound stable SO2 (mg L-1)
64,17
45,34
Total SO2 (mg L-1)
137,41
83
Abs 420 nm
5,153
2,667
Abs 520 nm
6,360
3,381
Colour Density
11,51
6,048
Tint
0,810
0,78
% V/V
13,5
13,2
Total Polyphenols (mg L-1)
413,71
459,28
4
Table 3 Comparative physicochemical parameters of Dolcetto d’Alba wines
Figures 1, 2, 3, and 4
Figures 1, 2, 3 display the total ion current (TIC) chromatograms, obtained by means
of Headspace Solid Phase Micro Extraction (HS-SPME) analysis.
Figure 4 shows the quantitative determination of aromatic compounds in the analysed
samples.
The Fendant white wine samples (2003) are shown in Figure 1: for reference wine
(added sulfites: REF, 1a) and experimental (NSA: EXP, 1b).
Some differences in the volatile composition can be observed in Figure 1, as well as
in Figure 4.
Higher quantities of phenylethylalcohol, 3-methyl-1-butanol were observed for the
experimental wine when compared to reference sample: this can derive from the
oxidative deamination of free amino acids precursors (leucine and phenylalanine
respectively (Câmara et al, 2006).
Higher concentrations of 1-hexanol, diethyl succinate, ethyl acetate and lower content
of a few ethyl esters including hexyl acetate and isoamyl acetate were detected. It is
highly likely that such alterations stem from a more advanced hydrolysis on these
ester compounds.
Regarding the Dolcetto D’Alba red wine samples (2005), the differences in the aroma
composition decreased, as can be seen in Figure 3 and Figure 4.
Only in the Nebbiolo d’Alba experimental red wine (2004) produced without sulfites
(Figure 2 and Figure 4), were the concentrations of some ethyl esters (ethyl
hexanoate and ethyl butanoate) higher in comparison with the reference samples,
while the content of phenylethylalcohol was found to be slightly lower.
Fig 1 - 1a Fendant (2003) Reference wine
1b Fendant (2003) Experimental wine
ADDITIONAL WINE ANALYSES ON OLDER WINES
5
Fig 2 - 2a Nebbiolo (2004) Reference wine
2b Nebbiolo (2004) Experimental wine
ADDITIONAL WINE ANALYSES ON OLDER WINES
6
Fig 3 - 3a Dolcetto d’Alba (2005) Reference wine
3b Dolcetto d’Alba (2005) Experimental wine
ADDITIONAL WINE ANALYSES ON OLDER WINES
7
ADDITIONAL WINE ANALYSES ON OLDER WINES
Fig. 4 Quantitative determination of aromatic compounds in the analysed
samples
A Fendant wine (2003)
B Dolcetto wine (2005)
C Nebbiolo wine (2004)
8
ADDITIONAL WINE ANALYSES ON OLDER WINES
9
Conclusion
With the white Fendant wine, significant differences – especially for properties
affecting colour and aromatic composition – were observed between the reference and
experimental samples. It was found that the browning reactions and vivid colour were
more prominent in the experimental wine, even though it could be considered as an
acceptable colour of a white wine, since the wine was stored for a duration of 4 years.
As expected, a higher amount of oxidation products and enzymatic reactions were
detected in the experimental Fendant wine.
On the other hand, for the red wines, the experimental Nebbiolo was almost
analogous to the reference wine; while the Dolcetto d’Alba experimental wine
showed very minor modifications in volatile acidity and colour density. However the
volatile composition was only subject to slight variations, unlike the white wine.
From all the experimental analysis carried out on the three different wines, it can be
concluded that a common trend was observed: the additive Protos was a suitable
preservative during storage and imparted better results when utilised to preserve red
wines rather than white wines.
ADDITIONAL WHITE WINE ANALYSES WITH NEW FORMULATION “PROTOS WHITE”
1 SULPHREE1
“Sulfite-free organic additives to be used in wine making process”
European Union consortium research project
ADDITIONAL WHITE WINE ANALYSES
WITH THE NEW FORMULATION, “PROTOS WHITE”
(“Vinificazione con Impiego di Additivi PROTOS”, 2008 - 2009)
Overseen by: Professor Pasquale Ferranti and Dr. Tiziana Granato
Department of Food Science, University of Naples “Federico II”, Italy2
2008 - 2009
1
EU Project reference number: 512721, Sixth Framework Programme (FP6)
2
Translated and reviewed by: Calleja, G. The content of sulfur dioxide in wine. Undergraduate
project in partial fulfilment of the requirements of the Degree of Bachelor of Pharmacy (Honours),
University of Malta; 2011.
1 ADDITIONAL WHITE WINE ANALYSES WITH NEW FORMULATION “PROTOS WHITE”
2 The
data generatedCON
and the
sensoryDI
analyses
proved
to be very
satisfactory,
thus further
VINIFICAZIONE
IMPIEGO
ADDITIVI
PROTOS,
Università
di Napoli
confirming the positive results.
A) Analisi chimica e aromatica degli additivi
Physicochemical
and-(./)!
aromatic
analysis
of the
additives
#$! %$&$''(&)**$*)+,(!
$--)')0)!
-(//$! /),($!
123435!
67849! :$! ;&(0)<'+! /=$,$/)<)! -(//$!
%+>;+,(,'(! ;+/)?(,+/)%$@! -(./)! $%)-)! +&.$,)%)! (! -(//(! >+/(%+/(! +-+&+<(! >(-)$,'(! /=)>;)(.+! -)!
The characteristics of the PROTOS WHITE line of additives – specifically the polyphenolic
-)! <;(''&+>('&)$!
B$//+! <;(''&+!
CD#B8E43F!
-(//=$--)')0+! 123435! "!
and'(%,)%:(!
odoriferous
compounds-)!–>$<<$A!
were analysed
via mass
spectrometry.
67849@! &);+&'$'+! ),! ?).G&$! "$@! <)! +<<(&0$! /$! ;&(<(,*$! -)! ;+/)>(&)! -(//$! %$'(%:),$! (! -(//$!
MALDI-TOF
mass spectrum of the PROTOS 1 WHITE, displayed in Figure 1a, verifies the
%$'(%:),$!.$//$'+!%+,!G,!H$<<+!.&$-+!-)!;+/)>(&)**$*)+,(@!),!;$&')%+/$&(!;+/)>(&)!-(//$!%$'(%:),$!
presence of catechin polymers. These compounds were identified to be an oligomeric series of
?),+!$/!'('&$>(&+!(!-(//$!%$'(%:),$!.$//$'+!?),+!$/!;(,'$>(&+@!%+,!,G>(&+!0$&)$H)/(!-)!&(<)-G)!-)!
catechins,
specifically up to the tetramer and catechin gallates up to the pentamer (gallic acid
.$//+)/$*)+,(A!
ester derivatives) with a variable number of gallic acid residues.
!
!
!
!
!
!
$I!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!
!"#$%&'
()*$(+",)
-%"#$%&'
()*$(+",)
-$*%)#$%&'
()*$(+",)
-$*%)#$%&'
."/)00)*&
579.21
-$*%)#$%&'
."/)00)*&
1/0"(&,$
("),".",)
HI!
1/0"(&,$
8$*6,".",)
1/0"(&,$
#)09".",)
2"),".",) 345'/06(&7".$
:$&,".",) 345'/06(&7".$
:$*6,".",) 345'/06(&7".$
;)09".",) 345'/06(&7".$
Figure 1. Mass spectrum MALDI-TOF of the additives
F).G&$!"A!5;(''&+!-)!>$<<$!CD#B8!43F!-(//=$--)')0+!$I1&+'+<!"!6:)'(!(!HI1&+'+<!J!6:)'(!
a)Protos 1 White, and b)Protos 3 White
!
"
2 ADDITIONAL WHITE WINE ANALYSES WITH NEW FORMULATION “PROTOS WHITE”
3 Analysis of the other two additives tested (PROTOS 2 WHITE and PROTOS 3 WHITE)
demonstrated that they contained polyphenolic compounds typical of red grapes. In Figure 1b
one can see the MALDI mass spectrum of the additive PROTOS 3 WHITE. The spectrum
illustrates the presence of cyanidin-3-O-glucoside, peonidin-3-O-glucoside, petundin-3-Oglucoside, malvidin-3-O-glucoside and their respective aglycones (non-sugars). An analysis
of the organic acids showed that in each of the three additives, citric and malic acids were
found to be present, and in PROTOS 1 WHITE traces of oxalic acid were also detected.
In order to confirm the presence of molecules in the PROTOS WHITE additives that could
have an influence on the natural aromatic characteristics of the wines, the following analysis
was done. Each of the 3 PROTOS WHITE additives were added to a model solution made up
of 85% water and 15% ethanol – in the same concentrations as recommended for use in must
and wine – and analyzed using HS-SPME-GC/MS. In the formulations of all three additives,
a variety of odoriferous compounds were found: in particular some terpenes were identified
including α-pinene and β-pinene, other terpene derivatives such as isobromile acetate (absent
in PROTOS 1 WHITE), and others such as eucalyptus and a synthetic antioxidant (BHT)
present in trace amounts. These odorous substances can be naturally inherent in a wine: in
particular the terpene composition of a wine is usually associated with the specific varietal
sensory characteristics of the aromatic bouquet.
Experimental vinification
To verify the efficacy in the vinification process, the additives being tested were involved in
an experimental microvinification using a white grape variety – Catalanesca (2008).
The must obtained was subdivided into four parts and the vinification was done with the
following additives:
• one part with 3 g / hl SO2 (“Reference wine: SO2”)
• one part with 1 ml / liter PROTOS 1 WHITE (“Experimental wine: PROTOS
WHITE”)
• the other two parts with the same concentration of PROTOS 1 WHITE, but combined
with a reduced quantity of normal SO2 concentrations: 20% and 50% of the dosage
used in the reference, respectively.
The additive was added to the must and again to the wine at the end of the fermentation
process, at the same time when the SO2 was added to the reference.
The analytical data obtained for the reference and experimental wines are recorded in Table 1.
The must showed a reduction in total acidity (6.39 g / liter of tartaric acid), which is a typical
characteristic of the must of this grape variety, with a pH value of 3.4 and 0.09 g / liter
volatile acidity. So, the process began with the raw material in good condition, and care was
taken during handling and transport to avoid the initiation of fermentation or other altering
processes. Regarding sugar content, the Brix value was found to be 20.3, corresponding to
19.48 kg / hL sugar content and therefore a potential alcohol percentage of 11.70 v/v
obtainable after fermentation. Polpyphenol and catechin content was found to be 293.71
mg/L and 110.34 mg/L, respectively. The analysis of the color revealed an optical density of
0.91. In the non-sulfited must it was also possible to measure out a content of total SO2 equal
to 1.6 mg/liter.
3 ADDITIONAL WHITE WINE ANALYSES WITH NEW FORMULATION “PROTOS WHITE”
4 Reference wine: SO2
Parameters
Must
2/10/08
Must in
fermentation
6/10/08
Experimental wine: PROTOS WHITE
Wine after Wine after
Wine after Wine after
Must in
Wine
the 1st
the 3rd
the 1st
the 3rd
fermentation
racking
racking
racking
racking
17/11/08
17/11/08
6/10/08
16/02/09 30/04/09
16/02/09 30/04/09
Wine
3.4
3.55
3,55
3.51
3.47
3.48
3,45
3.46
3.45
Total Acidity (g
tartaric aid)
Volatile Acidity (g L-1
acetic aid)
6.39
5.8
5,89
4.9
4.85
5.89
5,42
5.36
5.28
0.09
0,37
0,41
0.42
0.39
0,31
0,31
0.42
0.39
Total SO2 (mg L-1)
9.8
28,8
34,56
34.56
29.76
22,08
9,28
9.6
12.16
Free SO2 (mg L-1)
1.6
2,56
2,56
2.56
3.52
2,24
1,6
1.92
1.28
Brix
20.3
14
10,2
11.3
11.5
pH
L-1
15.8
% alcohol v/v
10.1
11
11.2
Total polyphenol
content ( mg L-1
gallic acid)
293.05
139,45
132,95
146.95
142.25
147,15
123,95
123.55
126.65
Catechin (mg L-1)
110.34
116,25
60,80
41.67
42.11
98,53
47,88
46.78
40.08
Optical density
0.9135
/
0,2113
0.2180
0.1658
2,2141
0,1442
0.2156
0.1704
Parameters
Must
2/10/08
Experimental wine:
Experimental wine:
PROTOS WHITE + 20% SO2
PROTOS WHITE + 50% SO2
Must in
fermentation
6/10/08
Wine
17/11/08
Wine after
the 1st
racking
16/02/09
Wine after
Wine after Wine after
Must in
Wine
the 3rd
the 1st
the 3rd
fermentation
racking
racking
racking
17/11/08
6/10/08
30/04/09
16/02/09 30/04/09
pH
3.4
3.47
3,43
3.42
3.41
3.49
3,45
3.43
3.46
Total Acidity (g L-1
tartaric aid)
Volatile Acidity (g L-1
acetic aid)
6.39
6.24
5,89
5.03
4.94
5.78
5,65
5.45
5.33
0.09
0,27
0,34
0.36
0.36
0,37
0,44
0.42
0.42
Total SO2 (mg L-1)
9.8
22,4
10,56
10.88
12.16
22,08
16,32
11.84
28.16
Free SO2 (mg L-1)
1.6
2,88
1,6
1.92
1.6
2,56
1,6
1.6
2.56
Brix
20.3
14.5
10,2
11.1
11.2
% alcohol v/v
14
10,1
11.2
11.1
Total polyphenol
content ( mg L-1
gallic acid)
293.05
297,15
235,15
112.95
123.35
253,85
272,15
196.15
174.95
Catechin (mg L-1)
110.34
139,16
48,42
44.51
40.24
92,66
62,77
48.34
41.68
0.9135
/
0,1676
0.1576
0.2268
2,301
0,2716
0.1394
0.2158
Optical density
Table 1 Physicochemical properties of reference wine (preserved using SO2) and experimental
wines, preserved with PROTOS WHITE additives either used alone or in combination with
varying, reduced concentration of SO2 (20% and 50%)
4 ADDITIONAL WHITE WINE ANALYSES WITH NEW FORMULATION “PROTOS WHITE”
5 After the addition of SO2 and the additives, the four musts were allowed to ferment at a
controlled temperature of 18°C. After fermentation terminated, the wines were racked several
times in order to aerate and their additive content was adjusted. Samples were then withdrawn
for analyzing the characteristics.
A comparison of all the chemical parameters at the end of fermentation, including total and
volatile acidity, pH, and alcohol content, did not result in any significant differences between
the reference wine and the three experimental wines. The results confirm the possibility of
utilizing the PROTOS additives in wine production. Some incompatibility was seen in the use of the additives combined with SO2, especially in
the final stabilization process. As expected, in the case of the reference wine and in that
produced with the exclusive help of the additive, the optical density continued to decrease
from the end of the fermentation process up to the last racking. In the first case this is due to
the clarifying ability of sulfite, while in the additive-containing wine, this was probably due to
successive racking through which the tannic, proteinaceous and polysaccharide components
were eliminated. During the final phase of conservation of the wines obtained through the
combined use of SO2 and the additives, an increase in optical density was observed. This was
probably a result of partial oxidation due to the proanthocyanidin component of the additive
and/or of the wine which led to the formation of polymers and brown pigments.
Aromatic and sensory profiles of the experimental wines
In order to comprehend the possible influences on the aromatic profile caused by the additive
during the fermentation process, and to understand how the odoriferous molecules present in
the additives could possibly affect the aromatic bouquet of the finished product, analysis was
carried out on the aromatic profile of the experimental wines. The following histograms
compare the odoriferous molecules in the wine with sulfites with similar molecules in the
wine with the additives.
The first clear observation is that the potentially odorous volatile compounds present in the
headspace of the model solution containing the additive are not noticeable in the wine’s
headspace, but have resulted instead from interactive effects between the wine’s “texture” and
the odorous compounds contained in the wine. Such effects tend to retain the odoriferous
molecules within the wine matrix; for this reason the olfactory impact of the additives in wine
is presumably negligible, compared to the olfactory impact of the 85% water / 15% ethanol
solution.
The same compositions of odoriferous molecules were found in both the experimental and the
control wines, resulting in no qualitative changes in their aromatic profile. It was, however,
possible to perceive quantitative differences related to the fermentation odours, particularly
with the terpene compounds. Fermentation esters were found in higher quantities in the
experimental wines with the additives, compared to the reference wine. This experimental
evidence is closely connected with the development of the fermentation, which is clearly
influenced by the presence of the additive.
5 ADDITIONAL WHITE WINE ANALYSES WITH NEW FORMULATION “PROTOS WHITE”
6 Ethyloctanoat
e Protos White Protos White 20% SO2 Protos White 50% SO2 SO
2 In the wines with the combined use of the additive and SO2 concentration, the characteristic
varietal aromas of this wine, including limonene and benzaldehye, are present in extremely
minute quantities. The reason for this noticeable loss in these two samples could be attributed
to being wines with overall less stability. In support of this theory is also the more advanced
state of oxidation of these wines, observed through the increased optical density mentioned
above as well as the more conspicuous quantity of molecules derived from oxidation reactions
of amino acids, specifically phenylethylalcohol and 3-methyl-butan-1-ol.
6 ADDITIONAL WHITE WINE ANALYSES WITH NEW FORMULATION “PROTOS WHITE”
7 Phenylethylalcohl
Protos White Protos White 20% SO2 Protos White 50% SO2 SO2 3-methyl-1-butanol
Protos White Protos White 20% SO2 Protos White 50% SO2 SO2 The sensory analyses did not show any substantial differences for the wines with the additive,
compared to the reference wine. However, it should be emphasized that the wines produced
with the additives had a positive increment in fruity and floral descriptions.
Conclusion
The physicochemical, aromatic and sensory analyses of the experimental wines as well as the
additives used in the vinification demonstrated that the PROTOS additives allowed obtaining
white wines of quality comparable to wines made with traditional vinification methods. Other
interventions (already planned) for improvements at the bottling stage and storage can also
lead to an improvement in quality.
7 THE CONTENT OF SULFUR DIOXIDE IN WINE
Excerpts from
THE CONTENT OF SULFUR DIOXIDE IN WINE
Ghislaine Calleja
Undergraduate project
in partial fulfilment of the requirements of the
Degree of Bachelor of Pharmacy (Honours)
University of Malta
2011
1 THE CONTENT OF SULFUR DIOXIDE IN WINE
2 Chapter 1.13, “The SULPHREE Project,” pg 34:
“The European SULPHREE consortium research project approved under the auspices of the
Sixth Framework Programme (FP6) initiated in 2005 and concluded in 2007. The
organisations and institutions involved merged their erudition and expertise with the initiative
to replace or significantly reduce sulfites with natural occurring plant metabolites….
“The ingredients comprising the SULPHREE organic additives have undergone rigorous and
exhaustive physicochemical and toxicological analyses to ensure that they are safe and do
indeed impinge beneficial effects when introduced in wine for its consumption. Their
scientific endeavours are summarised through a review of their work in the following
chapters.”
Chapter 2.5, “Collaborative study with organisations involved in the SULPHREE
project,” pg 70:
“Although the food industry is faced with the issue of microbial deterioration and oxidation,
which would lead to a subsequent degradation in the taste, colour, odour and texture of wine,
increasing legislative pressure is restricting the use of synthetic, potentially allergenic
chemical additives such as sulfur dioxide. Part of the literature review, outlined in Chapter
One, Section 1.7, was also directed towards possible alternative additives or measures to
reduce the concentration of sulfur dioxide in wine. This research was indicative of the fact
that food formulators are seeking natural alternatives to preserve the quality of their end
products.”
Chapter 2.5, “Collaborative study with organisations involved in the SULPHREE
project,” pg 71:
“ …. members of the SULPHREE consortium: Professor Pasquale Ferranti and Dr. Tiziana
Granato at the Department of Food Science, Dipartimento di Scienza degli Alimenti (DSA),
University of Naples “Federico II”, Italy. Widely recognized throughout the scientific
community, the DSA contributed to the SULPHREE project with its expertise in many areas
of food research, its state-of-the-art laboratories, and its accredited staff of researchers under
the guidance of Professor Ferranti.”
“BIOMA is involved in numerous research and development projects and offers organic
solutions in the areas of agriculture, environmental remediation, and natural food and
beverage preservation, amongst others.”
THE CONTENT OF SULFUR DIOXIDE IN WINE
3 Chapter 4.5, “Future endeavours regarding the sulfite additive controversy,” pg 182-­3: “Owing to the increased rate reporting of adverse effects attributed to sulfite additives, the
industrial trend is to minimise as much as possible the use of sulfite additives. This
pharmacovigilant mounting reinforces the need for more detailed information to accompany
commercially available products. This is of particular importance to an allergic consumer
cohort that are hypersensitive to specific causative agents. The proportion of the population
suffering from food intolerance or allergies is continuously increasing and new allergen
sources are relentlessly emerging. The lack of detailed information means patrons remain
uninformed as to the product’s safety. A complete and precise labelling system would rectify
this and there is no reason why one should not be introduced. Modern-day consumers yearn to
be better informed about the foodstuffs they purchase, specifically about their composition,
even if full ingredient labelling will inevitably make ingredient lists longer.59
“European legislation is consequently becoming more stringent with permitted concentrations
of total SO2 being constantly reviewed and reduced. A decline in the maximum permitted
levels was duly noted after the millennium reported in OJ EU L 127, 15/05/2008 and more
recently OJ EU L 193, 24/07/2009….”
Chapter 4.5, “Future endeavours regarding the sulfite additive controversy,” pg 187:
“Viable alternative measures or products to sulfites should be investigated and given due
consideration. Such projects are definitely worthy of investment under the umbrella of
European and other international networks.
“Some commercial companies and research-based consortiums have thus taken on the mantle,
investing time and money, together with incentive aid, in research in order to reduce or
replace sulfur dioxide in wine production through two different approaches:
•
The technological impact on processing techniques involved in winemaking.
•
The development of viable alternative products, preferably of organic origin, bar the
induction of noxious effects.”
59
Commission Press Release (IP/03/1310). Labelling- Food without fear: new food labelling rules will improve consumer
information on food ingredients, in particular allergens. 2003 Sep 23; [cited 2011 Mar 28]. Available from: URL:
http://www.reading.ac.uk/foodlaw/news/eu-03076.htm
4 THE CONTENT OF SULFUR DIOXIDE IN WINE
Chapter 4.5, “Future endeavours regarding the sulfite additive controversy,” pg 189-91:
“European research has been carried out and remains ongoing with the anticipation of
developing non-sulfite-containing, naturally derived additives to preserve wine without
affecting its final nutritional value, taste and aroma.
“The SULPHREE consortium research project made important steps toward identifying an
alternative organic additive for wine processing. The product Protos – one of the GEOLIFE
®
line products developed by BIOMA Agro Ecology CO AG in Switzerland – demonstrated
promising results. The SULPHREE formulations, which included Protos, were tested on both
sample solutions (water and ethanol matrix) and experimental wines; oenological analyses
were carried out to detect variation in physicochemical parameters including optical density,
odour, acidity, SO2 levels and toxicity. The SULPHREE additives demonstrated the feasibility
of either replacing sulfites completely, or reducing the sulfite concentrations to a mere 20% of
the traditionally used amounts.
“Once the technical validation of the SULPHREE additives had been confirmed, the additives
were studied from the toxicological point of view. The experiments confirmed that both the
additives themselves, as well as the wines preserved with the additives, were absent of
toxicity. Toxicological analyses were also performed by an accredited laboratory, further
ensuring the safety and permissibility of the additives. Along with the toxicological analyses
results, the data clearly showed that wine prepared with Protos had “higher activity against
oxidative stress, corresponding to comparable or slightly lower toxic effect compared to the
reference wine” (with sulfites). Stability tests, done after four years of storage, showed that
red experimental wines exhibited negligible variation from the reference samples.
“After termination of the SULPHREE project, BIOMA continued to develop its product, and
the range of Protos additives has recently been expanded. Post-project analyses done by the
Department of Food Science at the University of Naples, and overseen by Professor Pasquale
Ferranti, demonstrated that the newly formulated “Protos White” enables obtaining white
wines of quality comparable to conventionally produced, sulfite-containing wines. Wines
vinified with Protos may be inherently more wholesome and vivacious as a result of the
natural substances utilised in the formulation of this additive.61 This promising product is
scheduled to come on the market in the EU during the year 2011.”
61
BIOMA products. Available from: URL: http://www.bioma.com/europe/english/menu_principale.htm
THE CONTENT OF SULFUR DIOXIDE IN WINE
5 Chapter 4.6, “Conclusion,” pg 191-2:
“Moderate wine consumption is actually recommended for a healthy lifestyle due to its
multiplicity of antioxidants namely proanthocyanidins (present in higher concentrations in red
wines which have been allowed to ferment with grape skins), such as resveratrol. Studies have
demonstrated a spectrum of potentially beneficial clinical effects noted to decrease oxidative
stress in circulation (Micallef et al., 2007).”
Chapter 4.7, “Recommendations for further studies,” pg 193-6:
“In an effort to improve on the work undertaken in this study the following recommendations
have been put forward:
•
....
•
To obtain specimens of the Protos formulations developed by BIOMA Agro Ecology
CO AG, utilise in experimental vinification of local endemic and international grape
varieties and carry out subsequent analyses on these experimental wines.
To obtain the GEOLIFE® organic input for vineyards, developed by the same Swiss
•
company as Protos. According to the manufacturer, effects of using GEOLIFE® in
vineyards include: better health of the soil leading to better health of the vines; highquality organic grapes, better organoleptic characteristics; long-term regeneration of the
soil. Ideally, the grapes produced with GEOLIFE® could then be preserved with Protos
- which should result in a high quality, organic wine, preserved safely without added
sulfites.
•
….
“Incorporating the aforementioned changes in local approaches to and cognisance of sulfite
sensitivity can make for a more contemporary and health-oriented conception of wine
production. It is inevitable that health implications spur the need for further research and
development of rapid, simple, reliable and cost-effective analytical procedure(s) that cater for
the requirements of the local scenario to critically reduce or do without sulfite addition to
wine.”
GEOLIFE® PRÒTOS
Academic Research
Publications & Reports
APPENDIX
1. SULPHREE Abstract
2. SULPHREE Abstract, Introduction, and Bibliography – presented at the CIGR Section
VI International Symposium on “Food and Agricultural Products: Processing and
Innovations”, in Naples, Italy, September, 2007
APPENDIX
Sulphite-Free Organic Additives To Be Used In Wine
Making Process
Pasquale Ferranti*, Tiziana Mariarita Granato1, Catalina Valencia Peroni and
Marco Mescia2.
1.
Dipartimento di Scienze degli Alimenti - University of Naples “Federico II” – Parco Gussone,
80055 Portici (Italy)
2.
LABOR Srl – Via Giacomo Peroni 386 c/o Tecnopolo Tiburtino, 00131 Roma (Italy)
June, 2007
Keywords. White and red wines, sulphites, additives, vinification process
Abstract. Sulphites are extensively used as additives in many foodstuffs including wines,
beer, cider, fruit juices, dried fruits, biscuits and vegetables. Currently all the wine
produced in the world involves the use of sulphur dioxide and/or sodium, potassium salts
of hydrogen-sulphite in various stage of winemaking process for their technological
efficacy (antioxidant power, antimicrobial agents, enzyme inhibitors, control of enzymatic
and non-enzymatic browning reactions, pro-fermentative and colour-stabilising effect).
Since 1959 sulphating agents have been listed in the US Code of Federal Regulation
(CFR) as GRAS (generally recognized as safe): the GRAS status came under question
when in the early 1980s, sulphite additives were implicated as the major cause of wine
induced severe asthma attacks and anaphylactic reactions. Sulphites are also known to
present some cytotossic, mutagenic and antinutritional effects (Stammati et al.,1992).
For these reasons winemakers and researches are trying to obtain an added-sulphites free
wines by using modern wine making techniques (hyperoxigenation, flotation and
tangential microfiltration). Nowadays neither validated methodology has been developed.
The final object of this research work was to develop a natural additive able to mimic the
SO2 effects and to preserve the typical organoleptic characteristics of the white and red
wines. Qualitative and quantitative composition of the new formulation (organic acids,
flavonoids, catechins, and oligomeric proanthocyanidins) was defined on the basis of
preliminary microbiological, chemical, biochemical, sensorial and toxicological analysis by
using an innovative analytical approach based on the “Experimental Design”, a
methodology allowing to minimize the number of experiments and maximise the
resources. The effectiveness of the designed mixture was tested “on field” in real
vinification process of several variety of white and red grapes (Catalanesca, Falanghina,
Tempranillo). Polyphenols, tannins, procyanidins and other components and their
influence on wine stability was assayed by conventional tests as well as instrumental
techniques (HPLC e HPLC-MS). The odorous compounds were analyzed by the analytical
approach of solid phase micro-extraction (SPME) and GC MS. All field tests results
showed a success in the use of this formulation to preserve wine avoiding oxidation and
microbial spoilage. Also sensory analysis did not show defects and/or alterations in
experimental wines.
Sulphite-Free Organic Additives To Be Used In Wine Making
Process
Tiziana Mariarita Granato1, Pasquale Ferranti*, Antonella Nasi, Liberata
Gualtieri,Catalina Valencia Peroni and Marco Mescia2.
1.
University of Naples “Federico II” – Portici (NA) Italy
2.
LABOR Srl – Via Giacomo Peroni 386 c/o Tecnopolo Tiburtino, 00131 Roma – Italy
Written for presentation at the
2007 CIGR Section VI International Symposium on
FOOD AND AGRICULTURAL PRODUCTS: PROCESSING AND INNOVATIONS
Naples, Italy
24-26 September 2007
Abstract.
Sulphites are extensively used as additives in many foodstuffs including wines, beer, cider, fruit
juices, dried fruits, biscuits and vegetables. Currently all the wine produced in the world involves the
use of sulphur dioxide and/or sodium, potassium salts of hydrogen-sulphite in various stage of
winemaking process for their technological efficacy (antioxidant power, antimicrobial agents, enzyme
inhibitors, control of enzymatic and non-enzymatic browning reactions, pro-fermentative and colourstabilising effect). Sulphite additives were also implicated as the major cause of wine induced severe
asthma attacks and anaphylactic reactions and presented some cytotossic, mutagenic and
antinutritional effects: for these reasons winemakers and researches are trying to obtain an addedsulphites free wines by using modern wine making techniques (hyperoxigenation, flotation and
tangential microfiltration). Nowadays neither validated methodology has been developed.
The final object of this research work was to develop a natural additive able to mimic the SO2 effects
and to preserve the typical organoleptic characteristics of white and red wines. Qualitative and
quantitative composition of the new formulation (organic acids, flavonoids, catechins, and oligomeric
proanthocyanidins) was defined on the basis of microbiological, chemical, biochemical, sensory and
toxicological data by using an innovative analytical approach based on the “Experimental Design”, a
methodology allowing to minimize the number of experiments and maximise the resources. The
effectiveness of the designed mixture was tested “on field” in real vinification process of several
variety of white and red grapes. Polyphenols, tannins, procyanidins and other components and their
influence on wine stability was assayed by conventional tests as well as advanced instrumental
techniques (HPLC e HPLC-MS). The odorous compounds were analyzed by the analytical approach
of solid phase micro-extraction (SPME) and headspace GC MS. All field tests showed a success in
the use of this formulation to preserve wine avoiding oxidation and microbial spoilage. Also sensory
analysis did not show defects and/or alterations in experimental wines.
Keywords. White and red wines, sulphites, additives, vinification process.
Proceedings of the 3rd CIGR Section VI International Symposium on
FOOD AND AGRICULTURAL PRODUCTS: PROCESSING AND INNOVATIONS
Naples, Italy, 24-26 September 2007
1. Introduction
The winemaking process includes multiple stages at which microbial spoilage can occur,
altering the quality and hygienic status of the wine and rendering it unacceptable (1). The faults
caused include bitterness and off-flavours (mousiness, ester taint, phenolic vinegary, buttery,
geranium tone), and cosmetic problems such as turbidity, viscosity, sediment and film formation.
These spoilage organisms can also affect the wholesomeness of wine by producing biogenic
amines and precursors of ethyl carbamate. The judicious use of chemical preservatives, mainly
sulphur dioxide during the winemaking process decreases the risk of microbial spoilage.
Currently, the forms employed include sulfur dioxide gas, and the sodium and potassium salts of
sulfide, bisulfite or metabisulfite. In aqueous solution sulfur dioxide and sulfite salts form
sulfurous acid, and ions of bisulfite and sulfite (2).
SO2+ H2O = H2SO3
2H2SO3 = H+, HSO3- + 2H+,SO32The relative proportion of each form depends on the pH of the solution, and at pH 4,5 or lower
the HSO3- ion and undissociated sulfurous acid predominate. It has been shown that sulfur
dioxide is most effective as an antimicrobial agent in acid media, and this effect is believed to
result from undissociated sulfurous acid, which is the dominant from below pH 3,0.
The enhanced antimicrobial effect of sulfur dioxide at low pH values may result because
undissociated sulfurous acid can more easily penetrate the cell wall. Sulfurous acid inhibits
yeasts, molds, and bacteria, but not always to the same degree. This is particularly true at high
pH values, where it has been suggested that the HSO3- ion is effective against bacteria but not
against yeasts (2). Postulated mechanisms by which sulfurous acid inhibits microorganisms
include the reaction of bisulfite with acetaldehyde in the cell, the reduction of essential disulfide
linkages in enzymes, and the formation of bisulfite addition compounds that interfere with
respiratory reactions involving nicotinamide dinucleotide.
Of the known inhibitors of non-enzymatic browning in wine, sulfur dioxide is probably the most
effective. The chemical mechanism by which sulfur dioxide inhibits nonenzymatic browning is
not fully understood, but it probably involves bisulfite interactions with active carbonyl groups.
Bisulfite combines reversibly with reducing sugars and aldehydic intermediates, and more
strongly with -dicarbonyls and  -unsaturated aldheydes. These bisulfite addition products
appear to retard the browning process, which when coupled with the bleaching action of sulfur
dioxide on pigments, results in effective inhibition of non-enzymatic browning.
Sulfur dioxide also inhibits certain enzyme-catalyzed reactions, notably enzymic browning. The
production of brown pigments by enzyme-catalyzed oxidation of phenolic compounds can lead
to a serious quality problem in winemaking.
Sulfur dioxide also functions as an antioxidant in wine and beer, avoiding the developments of
oxidized flavours during storage. Also, sulfur dioxide in combination with buffering agents is
applied to prevent browning and to induce oxidative bleaching of nathocyanin pigments: the
resulting properties are desired in products, such as those used to make white wines and
maraschino cherries (3).
Sulfur dioxide and sulfites are metabolized to sulphate and are excreted in the urine without any
obvious pathological results. However, the safety-related aspects of sulfur dioxide and its
derivatives are undergoing extensive reviews because of reports of severe reactions in some
asthmatics upon consumption of wine, and also because of potential mutagenicity.
2
For these reasons, there is mounting consumer bias against chemical preservatives and the
subsequent request of use of natural preservatives in complying with the consumers’ demand
for “clean and green” products (1-5-7).
All food additives must have a demonstrated useful purpose and undergo a rigorous scientific
safety evaluation before they can be approved for use: the aim of this work was the study the
molecular and functional characteristics of natural additives and the definition of the strategies
for employing these compounds in the technological process of winemaking. The natural
vegetable products for wine processing should be able to mimic the effect of the SO2 in white
wine making process, thus guaranteeing the antioxidant and antibacterial action. The use of
such products could decrease the amount of added sulphites during vinification and to obtain,
as final result, a concentration of volatile sulphites lower than 10 ppm in white wines.
Both critical chemical and biochemical parameters of new formulation and experimental wines
were assayed by conventional tests as well as analytical methods based on high resolution
chromatographic techniques in combination with structural analysis by mass spectrometry (GCMS, HPLC e HPLC-MS).
3


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ELSEVIER
Journal of Food
Composition and
Analysis
Sulphite Free
Organic Additives
to be use in
Wine Making Process
SULPHREE
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