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WW 08/26 – Dr. F.P. van Jaarsveld
CFPA Canning Fruit Producers’ Assoc.
Submit to: Wiehahn Victor
PO Box 426 Paarl, 7620
Tel: +27 (0)21 872 1501
DFPT Deciduous Fruit Producers’ Trust
Submit to: Louise Kotzé
Suite 275, Postnet X5061 Stellenbosch, 7599
Tel: +27 (0)21 882 8470/1
DFTS Dried Fruit Technical Services
Submit to: Dappie Smit
PO Box 426 Paarl, 7620
Tel: +27 (0)21 872 1501
Winetech
Submit to: Jan Booysen
PO Box 825 Paarl, 7624
Tel: +27 (0)21 807 3324
x
Indicate (�) client(s) to whom this final report is submitted. Replace any of these with other relevant clients if required.
FINAL REPORT
FOR 2007/8
PROGRAMME & PROJECT LEADER INFORMATION
Programme leader Project leader Title, initials, surname Dr. O. P. H. Augustyn Dr. F. P. van Jaarsveld Present position Research leader Senior Researcher Address Private Bag X5026
Stellenbosch 7599
Private Bag X5026 Stellenbosch 7599
Tel. / Cell no. (021) 809 3010 (021) 809 3052 Fax (021) 809 1400 (021) 809 3002 E-mail [email protected] [email protected]
PROJECT INFORMATION
Project number WW 08/26
Project title Authenticity of South African wines
CFPA DFPT DFTS Winetech Production Technology
Industry programme
Other Fruit kind(s) Grapes (wine)
Start date (dd/mm/yyyy) 01/04/2002 End date (dd/mm/yyyy) 31/03/2008
WW 08/26 – Dr. F.P. van Jaarsveld
FINAL SUMMARY OF RESEARCH PROJECT
PROGRAMME & PROJECT LEADER INFORMATION
Programme leader Project leader Title, initials, surname Dr. O. P. H. Augustyn Dr. F. P. van Jaarsveld Institution ARC Infruitec-Nietvoorbij ARC Infruitec-Nietvoorbij Tel. / Cell no. (021) 809 3010 (021) 809 3052 E-mail [email protected] [email protected]
PROJECT INFORMATION
Project number WW 08/26
Project title Authenticity of South African wines
Fruit kind(s) Wine grapes
Start date (dd/mm/yyyy) 01/04/2002 End date (dd/mm/yyyy) 31/03/2008
The aim of this project is to create a database with which the possible adulteration of
local wine with foreign ethanol/sugar, wine (2H/1H en 13C/12C ratios) or water (18O/16O
ratio) can be detected. The possibility to use isotopes for origin of appellation purposes
is also being investigated (2H/1H, 13C/12C, 18O/16O ratios).
Chaptalisation of grape musts with crystallised cane sugar led to significant (p < 0.05)
increases in the (D/H)I and δ13C values of wine ethanol. Decreases in the R value,
except for (D/H)II, were observed. δ13C without doubt proved to be the best and most
sensitive parameter for the detection of adulteration by small additions of cane sugar.
Non-grape sugar ethanol sources could quite clearly be differentiated from wines with a
multi-isotopic approach using site-specific nuclear magnetic resonance (SNIF-NMR)
and isotope-ratio mass spectometry (IRMS).
The detection of adulteration in the form of dilution with water was possible using
oxygen isotope ratios. Serial dilutions of red and white wines showed linear responses
upon increasing addition of water. Dilution with water had very little to no effect (p >
0.05) on the (D/H)I, (D/H)II, R and δ13C values of wine ethanol, but caused a rapid and
significant (p < 0.05) lowering of the obtained δ18O value of wine. Using δ
18O-IRMS,
addition of water to wine can be detected effectively.
WW 08/26 – Dr. F.P. van Jaarsveld
Identification of geographical origin of wines was possible with respect to the larger and
some smaller geographical areas. South African wines can clearly be differentiated
along larger and some smaller geographical lines using a multi-isotopic approach,
and/or multivariate statistical and discriminant analysis. Wines from the two main
geographical units, i.e. Western- and Northern Cape, could clearly be differentiated.
Other analytical parameters, i.e. biogenic amines, inorganic parameters (ash, alkalinity,
calcium, sodium, potasium, magnesium), rare earth elements and classical paremeters
(sugar, acid, organic acids, volatile components, phosphates, sulphates, etc.), in
addition to natural isotopes, clearly showed promise in wine authentication and in origin
(botanical and geographical) determination.
Although the focus of this study revolves mainly around the investigation of detection of
foreign sugar, wines and water to South African wines, the other main aim, i.e. upkeep
of a legal-technical database is ongoing. An authoratative database of deuterium/
hydrogen (D/H) and carbon-13/12 for the ethanol in authentic South African brandies,
and oxygen-18/16 ratios for wine water, including all variations caused by grape cultivar,
geographic location and vintage, has been compiled over five years. Checking the
correctness and accuracy of five years’ worth of data and information, entered manually
into the system by various data capturers, however, proved to be a major task and is
nearing complesion.
All main objectives of project WW 08/26 have been attained.
The isotopic wine database is in Microsoft Access format and can be uploaded onto a
network for use by one or multiple authorised users with the necessary access and
passwords. Typically, unknown or suspicious commercial wines will be analysed
isotopically by means of SNIF-NMR and IRMS by accredited laboratories, and the
results compared to the authentic and/or unadulterated commercial databases.
Samples clearly adulterated with one or more sources of non-grape alcohol, and more
than two standard deviations out of spec as compared to the authentic and
unadulterated commercial samples, will be reported to be adulterated with one or more
adulterants. A report, with average, minimum, maximum, upper & lower limits, and
WW 08/26 – Dr. F.P. van Jaarsveld
number of samples, is generated by the database. This report can then be attached to
a report on the unknown sample. The report on the unknown sample states various
details about the sample and allows for calculation of the percentage adulteration and
comments regarding its authenticity. Graphical representations allow the sample(s) to
be screened visually for any obvious adulteration(s).
Several publications are in preparation and will follow the final report.
Progress report 5
WW 08/26 – Dr. F.P. van Jaarsveld
FINAL REPORT
1. Problem identification and objectives State the problem being addressed and the ultimate aim of the project.
The global wine market has expanded rapidly over the last 10 years or so with the
increased accession of the New/Third World and Eastern European countries (old
east-block countries) into the free and traditionally more West European markets.
This has also brought about increased awareness of the authenticity of wine. Wine
forgery is indeed a very tempting criminal activity. Forgery has been very difficult to
detect, let alone prove beyond reasonable doubt in a court of law, but science is
coming to the rescue. Today, many European countries, as well as the USA, have
mechanisms in place to test for the authenticity of this beverage. In the future
European Union regulations may require full authentication of imported wines and
spirits before sale. Certification of authenticity, like certification of origin, will thus be
highly likely in the future. It would be beneficial to the industry if South Africa can
supply information about the authenticity of its wines if and when this type of
information is requested.
Although wines can be adulterated in various ways, the main adulterations
internationally seem to be the addition of water, cheaper foreign ethanol, sugar
(chaptalisation), colourants and synthetic flavourants (all alcoholic beverages, except
where allowed and correctly labelled), organic acids, as well as the wrong indication
of geographical and varietal origin, and vintage. The dilution of wine with water is
one form of adulteration that was addressed by this project and could, until recently,
not be pinpointed locally. Also, illegal blending with lesser quality foreign wines
could, until recently, not be detected. To protect the Wine Industry, consumer and
States’ income from excise, it has become very important to establish a database for
South Africa against which the authenticity of South African wines can be tested
analytically and adulterations prevented. Proving authenticity is becoming more and
more important with regard to the country’s niche or export markets, and to ensure
that wine exports are not disadvantaged by aspects regarding authenticity, a wine
database was established.
The main purpose of this project is to create a database with which it can be proved
whether water has been added to wine (18O/16O ratios) and whether chaptalisation or
Progress report 6
WW 08/26 – Dr. F.P. van Jaarsveld
addition of foreign wine to the local product has occurred (2H/1H, 13C/12C and 18O/16O
ratios). The possibility to distinguish between different regions of appellation will also
be investigated (2H/1H, 13C/12C, 18O/16O ratios). Such a database will reflect all
possible variations found in South African wines.
Objectives for the current year (2008/09):
• Enter last isotopic data into the database.
• Check correctness of all data and information entered into the database.
• Publication of research results.
2. Workplan (materials & methods)
List trial sites, treatments, experimental layout and statistical detail, sampling detail, cold storage and examination stages and parameters.
Documentation
Documentation accompanied the sampling bottles delivered to producers. The
documentation included a sampling procedure, drivers log, description sheet and
written report or spreadsheets. During the EU study (2003-2005) producers were
asked to provide the necessary information electronically using a simpler
spreadsheet format. Producers had to complete the documentation/forms/
spreadsheets and return it with the samples or as soon as possible to the ARC
Infruitec-Nietvoorbij.
Sampling
In order for the database to reflect all possible geoclimatological variations found in
South Africa, grape, wine and water (irrigation- and cellar) samples representative of
the various wine growing regions in South Africa, were collected. Various cellars and
farms/estates, representative of all wine-growing regions and districts in South Africa,
were approached for collaboration in supplying grape, wine and water samples.
Grape/wine samples representative of the more common and most popular cultivars
found in most wine-growing regions were collected. These cultivars include the white
varieties Chardonnay, Chenin blanc and Sauvignon blanc, and the red varieties
Cabernet Sauvignon, Merlot, Pinotage and Shiraz. Adulterated wines were received
from a cellar (following a complaint) and from an official regulatory body.
Progress report 7
WW 08/26 – Dr. F.P. van Jaarsveld
“Authentic” wines
Authentic data in the reference isotopic database must be representative of
unchaptalised certified wines containing grape alcohol only, with origin, vintage and
cultivar known. Unblended wines, representing single vineyard blocks, were initially
collected from tanks or vats. The method of sampling, however, changed. As a
direct outflow of the EU participation, a shift from the collection of tank wines,
representative of specific vineyard blocks or defined areas, towards the collection of
grapes and microvinification of the corresponding grape musts, took place. Based on
the motivation in the “Request for Extension of Project” submitted in April 2006,
grape samples were microvinified to wine in order to bring the methodology and
project more in line with the officially recognised EEC Regulatory method of sample
preparation in Europe. Microvinified wines were made according to the same
standard method under the same conditions, thus eliminating possible anthropogenic
effects. The cost of collecting grape samples and microvinification, however, did
make this method of sampling and preparation more expensive. Results were taken
up as representative of the authentic dataset in the isotopic databank at the institute.
Commercial wines
Commercial wines were obtained from various sources, including wine sales shops
at farms/estates and from retailers. All samples were analysed for their stable
isotope ratios.
Water
At each cellar, water was collected at taps or points generally used during
operational wine-making activities. Irrigation water was collected from various
sources, including dams, rivers and boreholes. Generally, water was sampled from
irrigation sources used for the irrigation of vineyard blocks from which grapes were
collected. Water samples are used as reference or control in the calculation of
adulteration of wines by dilution with water. Water samples were analysed for their
stable oxygen isotope ratios.
Chaptalisation of wines
Healthy Cabernet Sauvignon grapes (640 kg) from block number J1 on the
Nietvoorbij experimental farm were harvested at 16.25°B, pressed and the grape
Progress report 8
WW 08/26 – Dr. F.P. van Jaarsveld
must adulterated by chaptalisation with table (cane) sugar with 1°B increments to
24°B in 20 L cannisters. Unchaptalised and chaptalised musts were fermented into
wine during the harvest of 2006. Wines were bottled and analysed for their isotopic
ratios, i.e. (D/H)I, (D/H)II, δ13C and δ18O.
Intentional dilution of wines with water
Wines were diluted with tap, distilled and deionised water in dilution experiments.
Possibly adulterated wines
Wines possibly adulterated by dilution with water were received from a cellar
(following a complaint) (case study A) and from an official regulatory body (case
study B).
Chemical analysis
In a multi-isotopic approach authentic, commercial and adulterated (including
chaptalised and diluted) wine samples were analysed for their (D/H)I and (D/H)II
ratios using site-specific nuclear magnetic resonance (SNIF-NMR) and for their δ13C
and δ18O isotope ratios using isotope-ratio mass spectometry (IRMS). All water
samples were analysed for their δ18O isotope ratios using isotope-ratio mass
spectometry (IRMS). The site-specific quantitative analysis of deuterium at the
methyl (D/HI) and methylenic (D/HII) positions of ethanol was carried out at the
Istituto Agrario di San Michele all’Adige, Italy with an AMX 400 Bruker NMR
instrument, in accordance with the EC method (EC Regulation n° 2676/90) and with
a line broadening of 0.5 Hz (Monetti et al., 1994). Results were expressed as parts
per million (ppm). δ13C of ethanol and δ18O of irrigation/cellar and wine water were
measured with IRMS (SIRA II VG mass spectrometer) according to the Italian official
method incorporating the EC Reg. N° 822/97 (Versini et al., 1997), literature
references (Epstein and Mayeda, 1953; Office International de la Vigne et du Vin, FV
N°919, 1955/220792) and EC Reg. N° 822/97. The results were expressed as ‰
scale against international standards PDB for carbon and V-SMOW for oxygen
isotopes. Analytical errors are within the range fixed by the quoted methods.
Progress report 9
WW 08/26 – Dr. F.P. van Jaarsveld
Statistical procedures
The variables measured were subjected to Analysis of Variance (ANOVA), using
GLM (General Linear Models) procedure of SAS statistical software version 8.2 (SAS
Institute Inc., Cary, NC, USA) (SAS, 2000). The Shapiro-Wilk test was performed to
test for normality (Shapiro, 1965). Fisher’s t-least significant difference (LSD) was
calculated at the 5% level to compare treatment means. A probability level of 5%
was considered significant for all significance tests. This univariate approach was
introductory, in order to understand the behaviour of the variables. Once statistically
significant differences were found, multivariate analysis of variance (MANOVA) with
linear (LDA)/stepwise/parametric/canonical discriminant analysis were performed on
the parameters (D/H)I, (D/H)II, δ13C‰ and δ
13O‰ in order to maximise the
discrimination and to verify the possibility of regional characterisation. The ratio R,
derived from the (D/H)I and (D/H)II values, was not considered. A cross-validation
summary using the linear discriminant function was performed for discrimination at
the geographical unit level because only two variables (only two geographical units)
were considered, whereas a cross-validation summary using the quadratic function
was considered for discrimination at the regional and district level as more variables
were considered.
3. Results and discussion State results obtained and list any benefits to the industry. Include a short discussion if applicable to your results. This final discussion must cover ALL accumulated results from the start of the project, but please limit it to essential
information.
Milestone Achievement
3.1. Collection of wine, grape and water samples.
3.1. Objective completed.
3.2. Chaptalisation of wines. 3.2. Objective completed.
3.3. Intentional dilution of wines with foreign water.
3.3. Objective completed.
3.4. Possibly adulterated wines 3.4. Objective completed.
3.5. Appellation 3.5. Objective completed.
3.6. Isotopic and routine analysis of wine and water samples.
3.6. Objective completed.
3.7. Submission of the final report. 3.7. Objective completed.
3.8. Establishment of an authorative legal-technical isotopic database of
3.8. Objective completed.
Progress report 10
WW 08/26 – Dr. F.P. van Jaarsveld
deuterium/hydrogen (D/H) and carbon-13 ratios for the ethanol, and oxygen-18 ratios for the water in authentic South African wines, inclusive of all variations caused by grape cultivar, geographic location and vintage.
3.9. Objectives not realised. 3.9. All main objectives realised.
3.10. Objectives completed. 3.10. All objectives completed.
3.11. Future objectives. 3.11.
• Implementation and upkeep of the wine isotopic database.
• Routine application of the database to investigate and prove possible adulteration of commercial wines.
3.12. Publication of research results. 3.12. Several publications are in preparation.
RESULTS
Milestone 3.1. Collection of wine, grape and water samples
Six hundred and seventy two authentic (530 x grape and 142 x certified tank) and
338 commercial wine samples from vintages 2002-2007 and representative of all
South African wine-growing regions/ districts, were collected/ received or
microvinified and analysed. Grape samples received were microvinified into wines.
One hundred and thirteen water samples were collected during the same period.
Milestone 3.2. Chaptalisation of wines
After chaptalisation of grape must with cane sugar, the original (D/H)I ratio of the
must increased linearly (initially) and significantly (p < 0.05) in relation to the amount
of chaptalisation, levelling off at high sugar concentrations (Fig. 1A). The increase in
the (D/H)I ratio upon addition of cane or corn sugar is well documented (AOAC
Official Method 995.17). The (D/H)II ratio seems unaffected and is not changed
significantly by chaptalisation, whereas the R-value decreases significantly (Fig. 1C).
The effect of chaptalisation on the original δ13C-IRMS values of a white wine is
clearly illustrated in Figure 1D and F, with linear and significant (p < 0.05) increases
observed upon increased additions of crystallised cane sugar to grape must before
fermentation. The slightly decreased δ13C values and leveling off at 7-8% w/v sugar
additions can be attributed to the fact that at these concentrations not all the sugar
Progress report 11
WW 08/26 – Dr. F.P. van Jaarsveld
could be utilised by the yeast cells, reflected by increased levels of unutilised sugar
and a leveling off in the alcohol production and ultimately in the ethanol-δ13C values.
The increased levels of wine extract observed at these high(er) concentrations,
reflect the high levels of cane sugar addition and unfermented sugar remaining in the
wine. Noticeably, changes in the δ13C ratios generally are paralleled by changes in
the alcohol concentration (Fig. 1D).
Although added cane or corn of >0% (v/v) can be detected with significance using the
δ13C-IRMS method(Fig. 1D), it must be remembered that in the example of this study,
the unchaptalised wine was taken as reference to which all chaptalised wines were
compared. In practice this detection limit might vary due to various factors, such as
whether or not a database is in place with representative and sufficient number of
wines from the same vintage and production area. The reported detection limit is
approximately 10%.
Chaptalisation or addition of crystallised cane sugar to the grape must did not bring
about significant changes in the δ18O values of wines (Fig. 1E).
Progress report 12
WW 08/26 – Dr. F.P. van Jaarsveld
aa
b
c
bb
d
e
e
R2 = 0.9465
105.00
105.50
106.00
106.50
107.00
107.50
108.00
108.50
0 2 4 6 8 10
Added sugar (%w/v)
(D/H
) I - p
pm
0
5
10
15
20
25
30
Ro
utin
e a
na
lysis
me
asu
rem
en
t
(D_H)I
Reducing sugars
Invert sugar
Alcohol
Extract
A
a
a
a
a
a
a
a
aa
133.00
133.20
133.40
133.60
133.80
134.00
134.20
134.40
134.60
0 2 4 6 8 10
Added sugar (%w/v)
(D/H
) II -
pp
m
0
5
10
15
20
25
30
Ro
utin
e a
na
lysis
me
asu
rem
en
t
(D_H)II
Reducing sugars
Invert sugar
Alcohol
Extract
B
Progress report 13
WW 08/26 – Dr. F.P. van Jaarsveld
decde
e e
bcdbc bc
ab
aR2 = 0.8497
2.460
2.470
2.480
2.490
2.500
2.510
2.520
2.530
2.540
2.550
0 2 4 6 8 10
Added sugar (%w/v)
R
0
5
10
15
20
25
30
Ro
utin
e a
na
lysis
me
asu
rem
en
t
R
Reducing sugars
Invert sugar
Alcohol
Extract
C
hg f
e dc
bb a
dd d cdcd cbb
a
aih g
f ed c b a
b b b bb b
b
a
a
R2 = 0.9993
-30.00
-25.00
-20.00
-15.00
-10.00
-5.00
0.00
0 2 4 6 8 10
Added sugar (%w/v)
δ13C
(‰
)
0
5
10
15
20
25
30
Ro
utin
e a
na
lysis
me
asu
rem
en
t13C
Reducing sugars
Invert sugar
Alcohol
Extract
D
Progress report 14
WW 08/26 – Dr. F.P. van Jaarsveld
b
b
bab
ababab
a
ab
R2 = 0.5733
8.60
8.80
9.00
9.20
9.40
9.60
9.80
0 2 4 6 8 10
Added sugar (%w/v)
δ18O
(‰
)
0
5
10
15
20
25
30
Ro
utin
e a
na
lysis
me
asu
rem
en
t
18O
Reducing sugars
Invert sugar
Alcohol
Extract
E
-27.00
-26.00
-25.00
-24.00
-23.00
-22.00
-21.00
-20.00
0 0.5 1 1.5 2 2.5 3 3.5
Adulteration (%)
δ13C
(‰
)
F
FIGURE 1 Effect of chaptalisation of grape must with cane sugar on the A, (D/H)I, B, (D/H)II, C,
R and D, δ13C ratios of ethanol and E, wine water δ
18O isotope ratios, and routine (reducing sugars, invert sugar, alcohol and extract) analysis measurements of wines prepared from Cabernet sauvignon grape must (16.3°B) by chaptalisation with crystallised table (cane) sugar at 1°B increments up to the equivalent of 24.3°B, with 10g/L = 1°B. Analyses shown are relevant to the wines. Treatments with the same letters do not differ significantly (p > 0.05). The significance of differences between different sugar additions for routine analysis measurements are only presented in graph D. All values represent the average of replicate data ± the standard error of the mean (error bars). Graph F shows the calculated percent adulteration using the
referenced cane sugar δ13C values for the standard and not the actual crystallised
cane sugar used in the chaptalisation experiment.
Progress report 15
WW 08/26 – Dr. F.P. van Jaarsveld
Non-grape sugar ethanol sources could quite clearly be differentiated from wines with
a multi-isotopic approach using site-specific nuclear magnetic resonance (SNIF-
NMR) and isotope-ratio mass spectometry (IRMS) (Fig. 2).
-35
-30
-25
-20
-15
-10
-5
90 95 100 105 110 115 120 125 130 135 140
(D/H)I - ppm
13C
‰
Argentina Bolivia Central EuropeChile France GermanyItaly New Zealand RomaniaSouth Africa Southern Europe SpainUnknown Uruguay L_Chapt. wine_50%BeetCaneL_Chaptalized wine L_Citrus honey L_Cotton honeyL_Eucalyptus honey L_Field flower honey L_Onion honeyL_Thorns honey L_Zaatar honey S-Cane sugar_ethanolSL_ milk_ray grass SL_50%maize_50%C3plants SL_grapefruitSL_Lactose_alpha SL_Lactose_Lactoseserum SL_Maple syrupSL_maple syrup_authentic SL_maple syrup_commercial SL_milk_ sugar beetSL_milk_C3 (100%) SL_milk_C3 MeadowBreeding SL_milk_C3-C4 (50/50)SL_milk_maize (silage) SL_Orange SL_Orange_A.I.JuiceSL_orange_authentic_conc SL_Orange_conc SL_Orange_Lab squeezedSL_wheat-barley (50/50) SL-C3_Apple SL-C3_GrapeSL-C3_Sugarbeet(root) SL-C4_Maize SL-C4SugarcaneSL-Synthetic W-Chaptalised0% W-CommercialW-Microvinified W-Tank
FIGURE 2
Ethanol δ13C plotted as a function against ethanol (D/H)I for various botanical and
synthetic sources of ethanol. Abbreviations: W, wine; SL or L, literature. Country names indicate the origin of the wine. W-commercial, W-microvinified and W-tank are commercial, microvinified and tank wines, respectively, from South Africa, representative of local wine-growing regions.
Milestone 3.3. Intentional dilution of wines with foreign water.
δ18O‰ or the H2O
18/H2O16 ratio is influenced by various processes, i.e. dilution,
concentration by distillation or evaporation, freezing, etc. The experiment was first
performed at higher levels of dilution (0-100% water addition) and repeated with
lower levels of dilution with water (0-30% added water). In the first experiment, red
and white wines were serially diluted with distilled water (Fig. 3 A-E). In the second
Progress report 16
WW 08/26 – Dr. F.P. van Jaarsveld
experiment, intentional dilution of wine with tap, distilled and deionised water was
performed at a lower dilution range (Fig. 4 A-F). All diluted wines were shown to be
out of spec as compared to their undiluted counterparts. Dilution by tap, distilled and
deionised water addition was apparent and caused a rapid and significant (p < 0.05)
lowering of the obtained δ18O value of wine (Figs 3E & 4E). Decreases in the δ18O
value upon dilution accompanied observed linear decreases in alcohol contents (Fig.
3E). The method is sensitive to water additions of 4% (v/v) and more, with significant
changes observed at these and higher levels of water additions to wine (Fig. 4E).
The 18O/16O ratios of ground/irrigation/cellar water are lower and sometimes negative
compared to that of wine water (Fig. 5), thus explaining the observed decrease with
addition of water.
Dilution with water had very little to no effect (p > 0.05) on the (D/H)I, (D/H)II, R and
δ13C values of wine ethanol (Figs 3A-D and 4A-D), even at higher levels of dilution
(between 30 and 100 % water). (D/H)I-NMR, (D/H)II-NMR and δ13C-IRMS and not
accepted EU Regulation methods for the detection of addition of water to wine.
Progress report 17
WW 08/26 – Dr. F.P. van Jaarsveld
a
ab abcabc bc
bcc
100
101
102
103
104
105
106
107
108
0 20 40 60 80 100 120
Wine (%)
(D/H
) I - p
pm
White wine
Red wine
A
a
ab
ab
ababbb
b
133
133.5
134
134.5
135
135.5
136
136.5
0 20 40 60 80 100 120
Wine (%)
(D/H
) II - ppm
White wine
Red wine
B
bab
ab abab
ab
a
a
2.5
2.51
2.52
2.53
2.54
2.55
2.56
2.57
2.58
0 20 40 60 80 100 120
Wine (%)
R
White wine
Red wine
C
a
cc
b abab a ab
-28
-27.5
-27
-26.5
-26
-25.5
-25
0 20 40 60 80 100 120
Wine (%)
Rela
tive c
arb
on-1
3 c
onte
nt
(per
mil)
White wine
Red wine
D
Progress report 18
WW 08/26 – Dr. F.P. van Jaarsveld
aa
ab
bb
c
d
e
e
R2 = 0.7894
R2 = 0.9662
R2 = 0.9967
R2 = 0.996
-6.00
-4.00
-2.00
0.00
2.00
4.00
6.00
8.00
10.00
12.00
0 20 40 60 80 100 120
Wine (%)
Rela
tive o
xygen-1
8 c
onte
nt (p
er
mil)
-2
0
2
4
6
8
10
12
14
16
Alc
ohol (
Vol%
) 18O - w hite
18O - red
Alcohol - w hite
Alcohol - red
E
FIGURE 3
Effect of dilution with distilled water on the A, (D/H)I, B, (D/H)II, C, R and D, δ13C
ratios of wine ethanol, and (E) wine water δ18O isotope ratios, and routine analysis measurements of wine. Treatments with the same superscript do not differ significantly (p > 0.05) and was calculated on the average values for red and white wines. The R parameter (graph 2C) is represented by the following equation: [(D/H)II/(D/H)I x 2]. Red wine, 2002 Shiraz; white wine, 2002 Chardonnay. The "Wine %" indicates the volume percentage actual wine after dilution, the remainder being added water.
Progress report 19
WW 08/26 – Dr. F.P. van Jaarsveld
aa
aa a
a
a
a
a
a a
105.00
105.20
105.40
105.60
105.80
106.00
106.20
106.40
106.60
106.80
107.00
100 99.8 99.5 99 98 96 92 90 85 80 70
% Wine
(D/H
) I -
pp
m Deionsed water
Tap water
Distilled water
Average
A
aa
a a
aa
aa
aaa
134.00
134.50
135.00
135.50
136.00
136.50
137.00
100 99.8 99.5 99 98 96 92 90 85 80 70
% Wine
(D/H
) II -
pp
m Deionsed water
Tap water
Distilled water
Average
B
Progress report 20
WW 08/26 – Dr. F.P. van Jaarsveld
a a
a
aaaaa
aaa
2.525
2.530
2.535
2.540
2.545
2.550
2.555
2.560
2.565
2.570
100 99.8 99.5 99 98 96 92 90 85 80 70
% Wine
R
Deionsed water
Tap water
Distilled water
Average
C
ababc
a
bcbc bccabc abcbcabc
-26.60
-26.40
-26.20
-26.00
-25.80
-25.60
-25.40
100 99.8 99.5 99 98 96 92 90 85 80 70
% Wine
δ13C
(‰
)
Deionsed water
Tap water
Distilled water
Average
D
Progress report 21
WW 08/26 – Dr. F.P. van Jaarsveld
g
fed
cc
h
aba
bab
-6.00
-4.00
-2.00
0.00
2.00
4.00
6.00
8.00
10.00
100 99.8 99.5 99 98 96 92 90 85 80 70 0
% Wine
δ18O
(‰
) Deionsed water
Tap water
Distilled water
E
g
fed c
c
h
ab ab
ab
0
20
40
60
80
100
120
-5.00 0.00 5.00 10.00
δ18O‰
% W
ine
-10
0
10
20
30
40
50
60
70
80
90
100
110
Ad
ulte
ratio
n (
%)
Deionsed water
Tap water
Distilled water
Adulteration (%)
F
FIGURE 4 Effect of dilution with various types of water on the A, (D/H)I, B (D/H)II, C, R and D,
δ13C ratios of wine ethanol, E, wine water δ18O isotope ratios, and routine analysis
measurements of Chenin Blanc wine. Treatments with the same letters do not differ significantly (p > 0.05). The R parameter (graph C) is represented by the following equation: [(D/H)II/(D/H)I x 2]. The percentage wine represents the amount of wine after dilution, the remainder being water used for the dilution. Graph F shows the percentage calculated adulteration.
Progress report 22
WW 08/26 – Dr. F.P. van Jaarsveld
Although added water of >4% (v/v) can be detected with significance (Fig. 2E), it
must be remembered that in the example of this study, the undiluted wine was taken
as reference to which all diluted wines were compared. In practice this detection limit
might increase due to various factors. The reported detection limit is approximately
15% (Versini, personal communication), depending on various factors such as
whether or not a database is in place with representative and sufficient number of
wines from the same vintage and production area, and whether or not it is
compulsory to declare the vintage and, therefore, if the vintage is known.
-4
-2
0
2
4
6
8
10
South Africa
Type of sample
δδ δδ1
8O
‰
Authentic water - H2O-Cellar water
Authentic water - H2O-Irrigation water
Authentic Wine - W-Microvinified
Authentic Wine - W-Tank
Commercial Wine Unadulterated - W-Commercial
Wine Literature - W-Literature
FIGURE 5
δ18O isotope ratios of wine and water representative of some of the SA wine-growing
regions for vintages 2002-2007.
Milestone 3.4. Possibly adulterated wines – case studies
Case study A
A clearly adulterated wine obtained from a cellar that received a complaint from the country
of export, as well as a retention wine, was analysed isotopically for (D/H)I, (D/H)II, R, δ13C
and δ18O. The pinkish colour of the wine and very low alcohol content already served as the
first proof of adulteration. The retention wine served as a control. The questions were, (i) is
the wine diluted, and (ii) if so, with water from where? The claim was that the dilution had to
take place at or near the vicinity of the cellar. Upon analysis the alcohol was reconfirmed to
Progress report 23
WW 08/26 – Dr. F.P. van Jaarsveld
be very low and as a result the (D/H)I, (D/H)II and δ13C‰ values for wine ethanol could not be
determined, but only the δ18O‰ values of wine water. Comparison of the δ18O‰ values of
the retention wine (9.76‰) with that of the “suspicious” wine (-6.12‰), clearly and
significantly (p < 0.05) showed the “suspicious” wine to be diluted. The values for wines
obtained from the larger, but still the same geographical area, and not just that of the cellar
itself, was used to draw comparisons in order to increase the sample size for statistical
evaluation. All wines compared significantly for all isotopic parameters (p > 0.05). Knowing
now that the “suspicious” wine was definitely adulterated, the question was if the dilution
could possibly have occurred at or near the vicinity of the cellar in question. Comparison of
the δ18O‰ values of the wine water from the diluted/adulterated “wine” (-6.12‰) with that of
the local irrigation water (-2.4‰) from the same vintage, clearly indicated the two values to
be significantly different, leaving two possibilities, (i) that the wine was diluted with water from
another region, or possibly during winter time, but not during the harvesting season. Water
with such low values clearly indicates an origin relating to colder regions.
Case study B
Tank wines suspected to be adulterated by dilution with water, and delivered by a regulatory
body to ARC Infruitec-Nietvoorbij, were analysed for their isotopic contents. Using values
from the authentic isotopic South African database for wine and water from the same
vintage, the possible adulterations (percent added water) were calculated. The δ18O
contents of these wines, and their respective alcohol contents, decreased linearly with
increased adulteration or added water (Fig. 6A-C). Both the alcohol and δ18O ratios are thus
influenced linearly with increased addition of water.
Progress report 24
WW 08/26 – Dr. F.P. van Jaarsveld
R2 = 0.9012
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 14
Alcohol (vol%)
Rela
tive o
xygen-1
8 c
onte
nt
(per
mil)
JVS12
JVS15
JVS14
JVS13
JVS16
JVS10
±0%
±1%
±28%
28%
±34%
±25%
±10%
JVS11
A
R2 = 1.0000
0
5
10
15
20
25
30
35
0 1 2 3 4 5 6 7 8
Relative oxygen-18 content (per mil)
% A
dultera
tion
JVS12
JVS15
JVS14
JVS13JVS16
JVS10
JVS11
B
Progress report 25
WW 08/26 – Dr. F.P. van Jaarsveld
R2 = 0.9012
0
5
10
15
20
25
30
35
0 2 4 6 8 10 12 14
Alcohol (vol%)
% A
dultera
tion
JVS12
JVS15
JVS14
JVS13
JVS16
JVS10
JVS11
C
FIGURE 6 (A) Relationship between alcohol- and oxygen-18 contents for wines collected at a cellar/ bottling site. Percentages indicated represent the amounts of water possibly added. (B) Percentage adulteration or possible added water plotted as a function against oxygen-18 values of wine water. (C) Percentage adulteration or added water plotted as a function against alcohol content of wine. Samples JVS10-16 represent the wines analysed. All wines were from the 2003 season.
Milestone 3.5. Appellation
South African wines can clearly be differentiated from wines from other countries.
The discrimination of South Africa from its Eastern European counterparts, i.e. the
Czech Republic, Hungary and Romania, was straightforward and, considering all the
variables, could sufficiently be achieved with one isotopic ratio, i.e. (D/H)I or (D/H)II.
Using a multi-elemental, multivariate statistical and chemometric approach,
distinction of wine-producing areas within South Africa, with it’s variety of climatic
and/or soil features, was possible, with some production areas showing very
satisfactory discrimination and unambiguously distinction (Fig. 7). Based on
statistical evaluations using analytical data, discrimination is not always 100%
quantitative based on presently defined Wine of Origin appellations, and in some
cases geographical lines might have to be redrawn/shifted somewhat in order to get
as close as possible to a hundred percent classification.
Progress report 26
WW 08/26 – Dr. F.P. van Jaarsveld
Argentina
Austria
Brazil
Chile
Cyprus
Italy
Spain
USA (Washington State)
Italy
SA - commercial
Australia
Bulgaria
Canada
Czech Republic
France
Germany
Hungary
IsraelM exico
M oldova
M orocco
New ZealandSlovenia
SA - literature
Uruguay
USA (Califo rnia)
SA - microvinified
SA - tank
120
122
124
126
128
130
132
134
136
96 98 100 102 104 106 108
(D/H)I - ppm
(D/H
) II -
ppm
Argentina Australia AustriaBrazil Bulgaria CanadaChile Cyprus Czech RepublicFrance Germany HungaryIsrael Italy MexicoMoldova Morocco New ZealandSlovenia SA - literature SpainUruguay USA (California) USA (Washington State)Italy SA - microvinified SA - tankSA - commercial
A
All - Region - 2001-2005 - Authentic & Commercial - 77
variables
-6
-4
-2
0
2
4
6
-100 0 100 200 300
Can 1
Can 2
Overberg/Walker
Bay/Elim
Orange River
Olifant River
Breede River Valley
Coastal
B
♦, 71%; ■, 100%; ▲, 67%; х, 88%; ж, 90%
Progress report 27
WW 08/26 – Dr. F.P. van Jaarsveld
Districts - Authentic & Commercial - White
11
111
1
1
2
2
2
2 2
22
222
2
2
22
2
22
22
22
22
2
2
2
2
2
2
22
222
2
2
2
2
3
3
3
3
3
33
3
3
3
3
3
33
3
33
3
33
3
33
33
333
3
33
3
33
3
3
33
44
4
444
4
4
4
4
4
4444
44
4
444 444
4
44
44444
4444
4
4
4
4
4
5555
55
55
5
555
5
5
55
5
5
555
55
5
5
5
5
5
5
5
5
55
5
66
6
6
66
7
777
777
7
77
7
7
7
77
7
7
7
7
7
7
7
7
7
77
7
7
7
7
7
777
7
77
7
77
7
7
7
88
8
8
8
8
88
88 8
8
99
99
9
9
9
99 999
10
10
10
1010
10
10
-5
-4
-3
-2
-1
0
1
2
3
4
5
-10 0 10 20 30 40 50
Can 1
Can 2
1
2
3
4
5
6
7
8
9
10
C
1 - 86%, 2 - 79%, 3 - 44%, 4 - 95%, 5 - 67%, 6 - 17%, 7 - 71%, 8 - 100%, 9 - 83%, 10 -
86%
FIGURE 7 Graph A, (D/H)II plotted as a function against (D/H)I for wines from various countries. Graphs B & C, canonical variate analysis of wines from different appellations and vintages for different combinations and corresponding Stepdisc selected variables. 1, Overberg/Walker Bay; 2, Swartland; 3, Paarl; 4, Robertson; 5, Stellenbosch; 6, Tulbagh; 7, Worcester; 8, Orange River; 9a, Lutzville Valley; 9b, Vredendal; 10, Tygerberg(Durbanville). Graph B, 2001 to 2004 vintages for red and white wines using 77 different variables; Graph C, authentic and commercial white wines of all vintages analysed together. Discriminant analysis based on Stepdisc selected variables: LRb, Wine_d18O, LB, Et_13C, LCs, LZn, LS, LMn, LSi, LGlu_Acid, LShi_Acid, LAl, LP, LSr, LBa, Vol_Mass, LInvert_S, LMal_Acid, LCl, LZn, LBr. Percentages indicate the error rates of LDA or correct classification rates (%) by region (graph B) and district (graph C).
Milestone 3.6. Isotopic and routine analysis of wine and water samples.
All authentic and commercial wines, collected during the 2002-2007 vintages, were
analysed for their (D/H)I, (D/H)II, δ13C and δ
18O isotope ratios. Chaptalised and
diluted wines were also analysed isotopically and for routine parameters, i.e. alcohol,
reducing and invert sugars, and extract. Water was analysed isotopically for its δ18O
ratios. All results were received and incorporated into the database and this report.
Milestones 3.7-3.12.
See table of milestones and achievements under point 3 above.
CONCLUSIONS AND RECOMMENDATIONS
Progress report 28
WW 08/26 – Dr. F.P. van Jaarsveld
All the main aims as set out at the beginning of project WW 08/26 have been met.
Using a multi-isotopic approach and the authentic database constructed since the
onset of the project as foundation, several aspects can be investigated, i.e. to
− Screen commercial wines to ensure legality with regard to composition, i.e.
100% grape ethanol.
− Determine whether or not wines were chaptalised by addition of non-grape
sugar before or during fermentation.
− Determine whether or not wines were diluted with water.
− Screen wines to aid confirmation that wines are from the country or region of
origin and vintage as specified.
Differences between plant metabolisms in conjunction with SNIF-NMR and IRMS
enable characterisation and discrimination of alcoholic beverages. It is clear that
metabolism and plant physiology have a drastic influence on the deuterium
distribution since C3, CAM and C4 plants are characterised with well defined ranges
of values: C3 tubers (91.5 to 93), aerial plants (97-102), CAM (107±0.1) and C4 (109-
111). Non-grape sources of ethanol could clearly be differentiated from wine ethanol.
Adulteration with non-grape sources of ethanol, bring about shifts in values not
typical of grape ethanol or wine, revealing not only adulteration of the product, but
also the possible botanical origin of the adulterant.
Both the SNIF-NMR method for the determination of ethanolic (D/H)I, and the IRMS
method for the determination of ethanolic δ13C, are effective for the determination of
chaptalisation with cane sugar, δ13C-IRMS being the best and most sensitive with
regard to addition of C4 sources like cane and maize. The δ13C-IRMS method, also
an officially recognised EU method, should be applied to detect addition of cane and
grain (maize) sugars to South African wine.
δ18O-IRMS clearly is the best and accepted method for accurately measuring and
detecting adulteration by dilution of wine with water. The δ18O-IRMS method, also an
officially recognised EU method, should be applied for the detection of addition of
water to or the dilution of South African wines.
Progress report 29
WW 08/26 – Dr. F.P. van Jaarsveld
A nice to have would be to have the database being able to graphically show the
95% confidence limits. This way a sample can visually and immediately be seen as
falling outside of the confidence borders/lines presented graphically. This add-on
ability will entail some designing/programming/statistical knowledge and is certainly
recommended as an additional user-friendly function making the work of the officials
at regulatory bodies or that of the authenticity expert easier.
This study revolved mainly around the investigation of detection of foreign sugar,
wines and water to South African wines, and the construction of a legal-technical
database. Decisions as to whether or not the vast amount of data generated by the
EU participation will have to be incorporated into the existing isotopic database
(project WW 08/26) (will involve some structural changes), or if a separate database
must be constructed, will have to be made. In addition to isotopic data, classical and
inorganic parameters, and biogenic amines were also generated by the three year
EU collaboration. Structural changes to the existing database, or construction of a
new database, with accompanying programming/database/visual basic knowledge,
and manual input of data since no systems for the automatic upload of data into
databases exist, will involve time and expense. Since the application and importance
of such a multi-elemental database, combining isotopic data and other parameters, to
the authentication of South African wines, is obvious, it is recommended to that these
upgrades get the necessary attention.
Due to year-by-year climatological fluctuations, global warming and climatic change,
different and changing suppliers of wines, new brands coming onto the market,
operating conditions, etc., the database should be kept-up/maintained yearly in order
to maintain representivity and legal-technical trustworthiness in the long run.
Upkeep/maintenance and in-house facilitation of the database will have financial
implications.
Several publications are in progress and will follow this final report, the time-
consuming part being the literature review and references.
Progress report 30
WW 08/26 – Dr. F.P. van Jaarsveld
4. Accumulated outputs List ALL the outputs from the start of the project. The year of each output must also be indicated.
Technology developed
An authorative database of deuterium/hydrogen (D/H) and δ13C ratios of ethanol, and
δ18O ratios in wine water in authentic South African wines, inclusive of all variations
caused by grape cultivar, geographic location and vintage.
Human resources developed/trained
Three seasonal workers.
Patents
Publications (popular, press releases, semi-scientific, scientific)
Presentations/papers delivered
Winetech Terroir Program, 18 September 2003. Slideshow 4. Title: Authenticity of
S.A. wines. Authors: F.P. van Jaarsveld. Venue: Olive Grove, Infruitec, Stellenbosch
7600.
The Beverage Industry Symposium 2002, 14 - 15 October 2002. Title: Improving
competitiveness and global market share by ensuring the authenticity of SA alcoholic
beverages. Presenter: F.P. van Jaarsveld. Venue: Stellenbosch Country Lodge
Hotel.
End of the First Year Meeting, 22-23 May 2003. Project/Program: European Union
Wine Database Project, Competitive and Sustainable Growth Program. Title:
Presentation of First Round of Samples – South Africa. Presenter: F.P. van
Jaarsveld. Venue: Federal Institute for Risk Assessment (BfR), Berlin, Germany.
Mid-term Meeting, 13-14 November 2003. Project/Program: European Union Wine
Database Project, Competitive and Sustainable Growth Program. Title: Presentation
of First & Second Round of Sampling – South Africa. Presenter: F.P. van Jaarsveld.
Venue: Vrije Universiteit Brussels, Brussels.
Progress report 31
WW 08/26 – Dr. F.P. van Jaarsveld
End of the Second Year Meeting, 13-14 May 2004. Project/Program: European
Union Wine Database Project, Competitive and Sustainable Growth Program. Title:
Presentation of Second Round of Sampling – South Africa. Presenter: F.P. van
Jaarsveld. Venue: Laboratorul de Oenolgoie U.S.A.M.V., Iasi, Romania.
Final Meeting, 3-4 November 2005. Project/Program: European Union Wine
Database Project, Competitive and Sustainable Growth Program. Title: Presentation
of Final Round of Sampling and Technical. Review of Results – South Africa.
Presenter: F.P. van Jaarsveld. Venue: Laboratorul de Oenolgoie U.S.A.M.V., Iasi,
Romania.
Winetech Terroir Program Meeting, 22 May 2006. Title: Authenticity of S.A. wines.
Authors: F.P. van Jaarsveld. Venue: Olive Grove, Infruitec, Stellenbosch 7600.
Radio Talk Shows: Duration: 3 minutes. Title: Authenticity aspects. Recorded: 9
April 2001 by Chris Viljoen from Radio Elsenburg. Broadcasted: 27 April 2001, 12h30
on "Landbou Oorsig" Radio Sonder Grense.
© Agricultural Research Council, 2007 The content of this document may constitute valuable Intellectual Property and is confidential. It may not be read, copied, disclosed or used in any other manner by any person other than the addressee(s) and specifically not disclosed to another party submitting a proposal herein. Unauthorised use, disclosure or copying is strictly prohibited and unlawful.
Progress report 32
WW 08/26 – Dr. F.P. van Jaarsveld
4. Total cost summary of project (ww0826)
Year CFPA DFPT DFTS Winetech THRIP Other TOTAL
Total cost in real terms for year 1 2001/02 163374 245602 409336
Total cost in real terms for year 2 2003/04 304637 372338 676975
Total cost in real terms for year 3 2004/05 265468 328960 594428
Total cost in real terms for year 4 2005/06 328903 401992 730895
Total cost in real terms for year 5 2006/07 340902 401992 742894
Total cost in real terms for year 6 2007/08 528479 645917 1174397
Total cost in real terms for year 7 2008/09 273304 273304
TOTAL 1931763 2670105 4602229
Progress report 33
WW 08/26 – Dr. F.P. van Jaarsveld
5. Budget for the following year: 2008/09
CFPA DFPT DFTS Winetech THRIP Other TOTAL
FUNDING REQUIRED FOR FOLLOWING YEAR: TOTAL
Overheads (only if part of project cost)
Personnel costs
Running costs
Local travel and accommodation
Local conferences (only specify separately for THIRP purposes)
Equipment (capital items*): pH meter, bench-top centrifuge
Other
* Industries will only fund capital items under exceptional circumstances