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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

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Submit to: Louise Kotzé

Suite 275, Postnet X5061 Stellenbosch, 7599

Tel: +27 (0)21 882 8470/1

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DFTS Dried Fruit Technical Services

Submit to: Dappie Smit


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Winetech

Submit to: Jan Booysen


Tel: +27 (0)21 807 3324

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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


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.


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


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


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


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


“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


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


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.


3.4. 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.


3.7. Submission of the final report. 3.7. Objective completed.

3.8. Establishment of an authorative legal-technical isotopic database of


Progress report 10


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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 7 2008/09 273304 273304

TOTAL 1931763 2670105 4602229

Progress report 33


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

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