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October 2006 Brett Project Update Poster (4Nov06) .pdf


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THE UNIVERSITY OF BRITISH COLUMBIA

Current Research Efforts Regarding
4-Ethylphenol and 4-Ethylguaiacol in Wines from
the Okanagan Valley Region of
British Columbia, Canada
(October 2006 Update)
Sierra Rayne and Nigel Eggers
Chemistry, Earth & Environmental Sciences
The University of British Columbia at Okanagan
1

THE UNIVERSITY OF BRITISH COLUMBIA

Introduction to 4-Ethylphenol, 4-Ethylguaiacol, and
the Brettanomyces/Dekkera Connection











Brettanomyces (“Brett”) is a wild yeast implicated in the spoilage of red wine and has long been associated almost
exclusively with European wines, but in recent years is considered to occur in wines from the New World
Two compounds that are widely considered to be primarily responsible for the ‘Brett’ odour are
4-ethylphenol and 4-ethylguaiacol
OCH3
Brettanomyces (the asexual, nonsporulating form) and Dekkera (its sexual, sporulating form) are
OH
OH
ubiquitous in the vineyard and winery, and are likely to be present in the water, the soil, the grapes 4-Ethylphenol
4-Ethylguaiacol
and must, and throughout the winery – and their presence can be monitored, but not controlled (Arvik and Henick-Kling,
2002)
Once this yeast is established in a winery, it is difficult to eliminate  spoilage of wine by Brettanomyces can be
devastating and wineries have had to shut down to remove this contaminant
Maintaining appropriate sulfur dioxide (SO2) levels, lower temperatures (generally <20°C), filtration, excluding oxygen
ingress into the wine during topping up of barrels (and keeping the barrels topped up), and general winery hygiene have
all been noted as methods to keep “Brett” problems minimized (Arvik and Henick-Kling, 2002)
However, infected barrels cannot effectively be sterilized due to their large internal surface areas and porosity, whether it
be by washing with sulfited water, shaving and firing, or ozone treatment (Arvik and Henick-Kling, 2002 and their
citation of Kunkee, 2001; Pollnitz et al., 2000a), with Brettanomyces found as deep as 8 mm into the oak wood (MalfeitoFerreira et al., 2004)
For these reasons, we recently (in May 2006) began a comprehensive two-year study based in the Okanagan Valley wine
region of British Columbia, Canada, that is targeted at better understanding the levels, distributions, fate, and factors
governing the production of the two most well-known Brettanomyces metabolites: 4-ethylphenol and 4-ethylguaiacol
2

THE UNIVERSITY OF BRITISH COLUMBIA

Overview of the Okanagan Valley Wine Industry




The Okanagan Valley is located in south-central British Columbia, Canada,
approximately 300 kilometers from the Pacific Ocean, is long and narrow, and runs
northward for 160 kilometers from the US border at 49 to 50°N latitude
Within the valley, there are significant climatic differences from north to south, with
the following five designated agricultural sub-regions (BCWI, 2006a):
1.
2.

3.
4.
5.





Kelowna: 1200 degree days; heavier soils with sandy loam, clay and limestone; common
varietals: Pinot Noir, Pinot Gris, Pinot Blanc, Riesling, and Chardonnay.
Penticton/Naramata: 1320 degree days; long frost-free autumn due to lake proximity and
sloping aspect; common varietals: Pinot Noir, Pinot Gris, Pinot Blanc, Chardonnay, and
Merlot.
Okanagan Falls: 1410 degree days; diverse soils and aspects, with some vineyards on
terraced slopes; common varietals: Riesling, Gewurztraminer, and Pinot Noir.
Oliver/Golden Mile: 1480 degree days; well-drained gravel, clay and sandy soils; common
varietals: Merlot, Chardonnay, and Gewurztraminer.
Black Sage/Osoyoos: 1490 degree days; soils are very deep sand; common varietals:
Bordeaux varieties, Chardonnay, and Syrah

Grape and wine production has increased steadily from 4850 tons in 1992 to a high
of 16 900 tons in 2003, with the value of British Columbia VQA (Vintners Quality
Alliance) wines sold in the province also increasing from CDN$6.9 million in
1991/1992 (BCWI, 2005) to CDN$131 million in 2005 (BCWI, 2006b)
The top five red varieties produced in British Columbia during 2005 were as follows:
Merlot, 2784 tons; Cabernet Sauvignon, 1054 tons; Pinot Noir, 963 tons; Cabernet
Franc, 594 tons; and Syrah, 408 tons (BCWI, 2006c).

N
Okanagan Lake

Vernon
Okanagan
Centre
Peachland

Kelowna

Skaha Lake
Okanagan Falls
Summerland

Vaseaux Lake

Penticton
Osoyoos

Oliver
Canada
United States of
America

3

THE UNIVERSITY OF BRITISH COLUMBIA

Wine Sampling and Analysis








190 red wine samples from five major varieties (Merlot, Cabernet Sauvignon, Syrah, Pinot Noir, and Cabernet Franc) were
collected using sterile glassware from ~220 L oak barrels at 10 small through large-scale wineries in the Okanagan Valley
10 commercially available un-oaked bottled Riesling wines from Canada and Europe (vintage years 2002 through 2004) were
also analyzed
Sample volumes ranged from 50 mL to 250 mL, were collected using glass pipettes, stored at 4°C without headspace in precleaned (Alconox detergent, following by sequential rinses with tap water, deionized water, dichloromethane, toluene, and
hexanes) and pre-baked (300°C for 12 hours) amber glass jars until analysis
At the time of sampling, dissolved oxygen and temperature were measured at half-depth in the barrel using a commercial meter.
Other variables (e.g., barrel type and details, cellar temperature and humidity, barrel hygiene and history, etc.) were obtained via
direct observation and/or personal communications with individual winemakers.
The corresponding stable isotope derivatives (4-ethylphenol-d3 and 4-ethylguaiacol-d3) of the target analytes were synthesized
using base-assisted deuterium exchange on the terminal methyl group from 4-hydroxyacetophenone and acetovanillone,
respectively, followed by palladium-catalyzed hydrogenation of the carbonyl group:
O

CH3

O

NaH/D2O/

OH
4-Hydroxyacetophenone





CD3

CD3

O

CH3

H2/Pd on C

OH
4-Hydroxyacetophenone-d3

O
NaH/D2O/

OH
4-Ethylphenol-d3

CD3

CD3
H2/Pd on C

OCH3

OCH3

OCH3

OH

OH

OH

Acetovanillone

Acetovanillone-d3

4-Ethylguaiacol-d3

Wines were analyzed by spiking a known quantity (5 mL) of sample with the isotopically labeled internal standards, saturating the
solution with salt (NaCl) to obtain a consistent ionic strength between samples, extracting with diethyl ether (2 mL), and
centrifugation to break the partial emulsion and maximize analyte recoveries
The resulting isolated organic extracts were dried under ambient air for 12 h to evaporate the solvent, reconstituted in 50 μL of
diethyl ether or THF, and 5 μL was injected into a gas-chromatograph/mass-spectrometer (GC-MS; Saturn 3800/2000 or Thermo
4
Trace DSQ) running in selected-ion-monitoring (SIM) mode

THE UNIVERSITY OF BRITISH COLUMBIA

Results to Date and Comparison with Levels from
Other Worldwide Winemaking Regions





~30% of the samples contained 4-EG concentrations below the 1 μg/L MDL




80% of samples had a 4-EG level below the lower bound of the odor threshold range

Median 4-EP and 4-EG concentrations are 29 μg/L and 15 μg/L, respectively

5

4000

3950

3900

600

550

500

450

400

350

300

250

200

150



10

100



4-Ethylphenol Concentration (µg/L)
Odor Threshold Range
50
40
30
20
10

1300

1250

400

350

300

250

200

150

100

50

0

0

Chatonnet et al. (1992) analyzed 137 red and white wines from France, and found that
red wines contained an average of 440 μg/L 4-ethylphenol (range: 1-6047 μg/L),
compared to white wines having an average of 3 μg/L 4-ethylphenol (range: 0-28 μg/L)
 some French wineries contained up to half of their vintages with 4-ethylphenol
sourced “Brett” organoleptic defects
In a survey of barrelled (1998 vintage) and bottled (1986-1996 vintages) red wines from
Australia, Pollnitz and co-workers reported barrelled 4-ethylphenol ranges of 385-680
μg/L and bottled ranges of 2-2660 μg/L (mean=795 μg/L) (Pollnitz et al., 2000a, 2000b)
 corresponding ranges of 4-ethylguaiacol were 28-45 μg/L in barrelled reds and 1-437
g/L (mean=99 μg/L) in bottled reds.
In a subsequent survey of Cabernet Sauvignon and Cabernet Sauvignon-Merlot wines
from the vintages 1996-2002 in Australia, Henschke et al. (2004) found that mean 4ethylphenol concentrations in the years between 1996-2000 were not different (range
from 864 to 1164 μg/L), but that concentrations decreased to an average of 490 μg/L for
the following 2001 and 2002 vintages  Australia is observing a decreasing incidence
of red wines with 4-ethylphenol concentrations above 800 μg/L after 2001 (Coulter et
al., 2003; Hayasaka et al., 2005).

15

0

In comparison with concentrations reported from other worldwide winemaking
regions, the levels of 4-ethylphenol and 4-ethylguaiacol we found in the
Okanagan barrelled reds are quite low:


20

50

Averages are 77 μg/L (4-EP) and 35 μg/L (4-EG)

25

0





98% of samples had a 4-EP level below the lower bound of the odor threshold range

Odor Threshold Range
30

% Frequency of Occurrence



Concentrations of both 4-ethylphenol (4-EP) and 4-ethylguaiacol (4-EG) were
determined in barrelled red wines analyzed in 190 samples from 10
commercial wineries in the Okanagan Valley
~50% of the samples contained 4-EP concentrations below the 10 μg/L method
detection limit (MDL)

% Frequency of Occurrence



4-Ethylguaiacol Concentration (µg/L)

5

THE UNIVERSITY OF BRITISH COLUMBIA

Relationships Between 4-EP/4-EG Levels and
Dissolved Oxygen, Temperature, and Humidity

10000
1000
100
10
1
0.1
0.1

0.2

0.3

0.4

0.5

0.6

1000
100
10
1
0.1
12

14

10000
1000
100
10
1
0.1
0

0.1

0.2

0.3

0.4

Dissolved Oxygen (mg/L)

18

20

22

24

10000

1000

100

10

1
45

50

0.5

0.6

10000
1000
100
10
1
0.1
12

14

16

18

20

Sample Temperature (°C)

55

60

65

70

75

80

75

80

Cellar Relative Humidity (%)

Sample Temperature (°C)

4-Ethylguaiacol Concentration (µg/L)

4-Ethylguaiacol Concentration (µg/L)

Dissolved Oxygen (mg/L)

16

22

24

4-Ethylguaiacol Concentration (µg/L)

0

10000

4-Ethylphenol Concentration (µg/L)

To date, we have found no significant relationships (p<0.05) between 4-ethylphenol and 4-ethylguaiacol concentrations and
levels of dissolved oxygen in the barrel, temperature of the barrelled wine, or the cellar humidity

4-Ethylphenol Concentration (µg/L)

4-Ethylphenol Concentration (µg/L)



10000
1000
100
10
1
0.1
45

50

55

60

65

70

Cellar Relative Humidity (%)

6

THE UNIVERSITY OF BRITISH COLUMBIA

Relationships Between 4-EP/4-EG Levels and
Length of Time in Barrel, Age of Barrel, and
Number of Rackings

10000
1000
100
10
1
0.1
200

400

600

800

1000

1000
100
10
1
0.1
2000

2001

10000
1000
100
10
1
0.1
0

200

400

600

800

Sample Temperature (°C)

2003

2004

2005

2006

10000

1000

100

10

1
0

Year of Barrel Production

1000

4-Ethylguaiacol Concentration (µg/L)

4-Ethylguaiacol Concentration (µg/L)

Length of Time in Barrel (days)

2002

10000
1000
100
10
1
0.1
2000

2001

2002

2003

2004

Year of Barrel Production

1

2

3

Number of Rackings

2005

2006

4-Ethylguaiacol Concentration (µg/L)

0

10000

4-Ethylphenol Concentration (µg/L)

To date, we have found no significant relationships (p<0.05) between 4-ethylphenol and 4-ethylguaiacol concentrations and
length of time in the barrel, year of barrel production, or the number of rackings prior to sampling

4-Ethylphenol Concentration (µg/L)

4-Ethylphenol Concentration (µg/L)



10000
1000
100
10
1
0.1
0

1

2

3

Number of Rackings

7

THE UNIVERSITY OF BRITISH COLUMBIA

Relationships Between 4-EP/4-EG Levels and
Variety and Winery ID


To date, we have found no significant relationships (p<0.05) between 4-ethylphenol
and 4-ethylguaiacol concentrations and variety or the winery ID
4-Ethylphenol Concentrations (μg/L)
Cabernet
Franc

Cabernet
Sauvignon

Merlot

Pinot
Noir

Syrah

Median

15

27

25

42

32

Mean

28

65

48

148

Minimum

<10

<10

<10

Maximum

69

413

212

4-Ethylphenol Concentrations (μg/L)
W1

W2

W3

W4

W5

W6

W7

W8

W9

W10

Median

<10

21

<10

40

15

40

37

47

155

53

66

Mean

<10

33

<10

81

33

194

64

55

147

71

<10

<10

Minimum

<10

<10

<10

11

<10

<10

<10

14

90

<10

3930

238

Maximum

<10

128

29

310

203

3930

413

137

213

238

4-Ethylguaiacol Concentrations (μg/L)
Cabernet
Franc

Cabernet
Sauvignon

Merlot

Pinot
Noir

Syrah

Median

15

27

25

42

32

Mean

28

65

48

148

66

Minimum

<1

<1

<1

<1

<1

Maximum

69

413

212

3930

238

4-Ethylguaiacol Concentrations (μg/L)
W1

W2

W3

W4

W5

W6

W7

W8

W9

W10

Median

<1

<1

<1

33

18

41

14

24

40

17

Mean

<1

5

3

42

24

83

23

49

49

34

Minimum

<1

<1

<1

<1

<1

<1

<1

<1

30

<1

Maximum

<1

16

20

122

109

1273

140

296

80

96

8

THE UNIVERSITY OF BRITISH COLUMBIA

Relationships Between 4-EP/4-EG Levels and
Oak Type and Toasting Level


As well, we have found no significant relationships (p<0.05) between 4-ethylphenol
and 4-ethylguaiacol concentrations and oak type or the toasting level
4-Ethylphenol Concentrations (μg/L)
American
Oak

French
Oak

Median

25

37

Mean

48

Minimum
Maximum

4-Ethylphenol Concentrations (μg/L)
Medium

Medium
Plus

Median

40

35

103

Mean

114

61

<10

<10

Minimum

<10

<10

233

3930

Maximum

3930

213

4-Ethylguaiacol Concentrations (μg/L)
American
Oak

French
Oak

Median

12

29

Mean

28

Minimum
Maximum

4-Ethylguaiacol Concentrations (μg/L)
Medium

Medium
Plus

Median

29

29

45

Mean

53

33

<1

<1

Minimum

<1

<1

296

1273

Maximum

1273

109

9


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