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Thesis defence for MSc
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Sensory, chemical and consumer analysis of Brettanomyces
spoilage in South African wines
Janita BothaThesis defence
17 February 2009
Research Team:
M Muller, W J du Toit, A J de Villiers & A G J Tredoux
Project background
FermentationMalo-lactic Fermentation
Barrel Aging
Bottle
Saccharomyces cerevisiae
Oenococcus oeni
Other LAB
Brettanomyces/
Dekkera
Ethanol
CO2
Lactic acid
Diacetyl
Furfural
Guaiacol
Eugenol
Etc
Brett-related spoilage compounds
+
Sensory studies about Brett
Chatonnet et al. (1992): Linked 4-ethylphenol and 4-ethylguaiacol to
BrettanomycesDetection thresholds of 4-EP and 4-EGCommonly quoted study
Licker et al. (1999): Linked isovaleric acid to Brettanomyces
Hesford & Schneider (2004): Linked 4-ethylcatechol to Brettanomyces
Recent sensory studies
Curtin et al. (2008):Detection thresholds of 4-EP, 4-EG and 4-ECProfiling of certain combinationsConsumer analysisRecommend further studies
Romano et al. (2009):Found further strong links between 4-EP and
isovaleric acid Investigated effect of isovaleric acid on
detection thresholds of 4-EP and 4-EG
Isovaleric acid?
4-ethylcatechol?
Research Aims
Determine the sensory
detection thresholds of 8 Brett-related compounds
To determine the sensory profiles of 4 Brett-related
compounds
To determine the sensory interactions of 4 Brett-
related compounds
To determine the consumer preference of 4 Brett-related
compounds
Chapter 1
Chapter 2
Chapter 3
To investigate realationships between 8
Brett-related compounds in selected South African
wines
Chapter 4
Research Chapter 1:Detection Thresholds
Materials and Methods
Method based on ASTM E 679 – 04
3-Alternative Forced Choice (3-AFC)
3 glasses of wine presented, 1 contains the Brett compound
8 levels of 8 compounds tested
Difference known, judges therefore
trained
Materials and Methods
Concentration increases with a constant MF factor on log scale
Two calculation methods investigated: ASTM and median
0
400
800
1200
1600
2000
1 2 3 4 5 6 7 8 9 10
Level
Co
nc
en
tra
tio
n (
μg
/L)
Results: 4-ethylphenol
LLLL
ASTM
Median
UL
UL
0
50
100
150
200
250
300
350
400
Median ASTMCalculation method
Con
cent
ratio
n (μ
g/L)
Level 5
Level 2
Level 4
Level 3
Level 1
Outlier
Larger range
Results: 4-ethylcatechol
LLLL
ASTMMedian
UL
UL
0
200
400
600
800
1000
1200
Median ASTMCalculation method
Con
cent
ratio
n (μ
g/L)
Level 3
Level 4
Level 5
Level 6
Level 7
Level 8
True rangeSimplified
range
Conclusions
Concept of “detection threshold” is limited and obscures information
The use of a detection threshold range more accurate
Range provided by median more informative about population
Research Chapter 2: Singular Profiling
Materials and Methods:Central composite design
Com
poun
d 1
Compound 2
Compound 3Singular profiling exploration step
before compounds can be tested in
combination
Materials and Methods: Singular profiling
4-EP
4-EG
4-EC
Isovaleric acid
0 2 431 5
DTValues
determined used as guide
Levels pre-screened to
confirm suitability for use in new
medium
Example of linear results: 4-EP
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12 14Level of 4-ethylphenol
Inte
nsi
ty o
f d
escr
ipto
r
Elastoplast
Leather
Berry-like
Sick-sweet
Mirroring
Following
Example of linear results: 4-EP
0
10
20
30
40
50
60
70
0 2 4 6 8 10 12 14Level of 4-ethylphenol
Inte
nsi
ty o
f d
escr
ipto
r
Elastoplast
Leather
Berry-like
Sick-sweet
Example of Discriminant Analysis: 4-EP
0000
0
000
0
0
1
1
1
1
11 1
1
11
2 2
2
22
2
2
2
2
2
33
3
L3
3
3
3
3 3
3
444
4
444
4
44
5
5
55
5
5
5
5
5
5
5
4
3
21
0
-6
-4
-2
0
2
4
6
-6 -4 -2 0 2 4 6
F1 (78.64 %)
F2
(19.
88 %
)
012345Centroids
Level
Example of PCA: 4-EP
5
5
55
5
5
5
5
5
5
4
4
4
4
44
4
4
4
4
33
3
3
3
3
3
33
3
2
2
2
22
2
2
2
2
2
1
1
1
1
1 11
1
1
1
00000
0000
0
Elastoplast
Leather
Sick-sweet
Berry-like
-1.5
-0.5
0.5
1.5
-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8
F1 (86.81 %)
F2
(8.9
0 %
)Similar pattern in univariate results
Overall results
4-EP 4-EG 4-EC Isovaleric acid
Elastoplast Leather Medicinal Smoky Savoury Pungent
Sick-sweetBerry-like
Not in literature
Clove-like??
More detail obtained about the contributions of the respective compounds
Research Chapter 3:Combination Profiling
Materials and Methods:Design
Com
poun
d 1
Compound 2
Compound 3
Samples
Materials and Methods: Samples
24444444
424422444424242442242224444224424242224244222422422222225333133335333133
33533313
333533313333
Isovaleric acid4-EC4-EG4-EP 1 Centre Sample
8 Star Samples
16 Cube Samples
Results: Interaction
Elastoplast: 4-EP*4-EG*4-EC4-EP*4-EG*ISOV
Medicinal:4-EG cause increase4-EC and isovaleric acid cause decrease
Pungent:ISOV*4-EG4-EP cause increase
All four compounds affect intensity of Elastoplast descriptor
Compounds have different effects at different levels
Intensity affected by other compounds present
“Sweaty leather” aroma attributed to Brett?
PCA vs PARAFAC: sensory data
Judges
Sam
ples
Attributes
Sam
ples
Attributes
Judge 1 Judge 2 Judge n
PCA: Analysis of (unfolded) 2-way data
PARAFAC: Analysis of data cube
Effects of judges not ignored Often simplified by finding mean over judges
PCA vs PARAFAC
1st factor calculated
Subsequent orthogonal factors
calculated
1st factor calculatedSubsequent factors
calculated in sucessive directions
of variance
Factors recalculated untill they converge
PCA
PARAFAC
Less noise modelled
Biplot (axes F1 and F2: 67.45 %)
5333
4444
4442
4424
4422
42444242
4224
4222
3533
33533335
3333
33313313
3133
24442442
2424
2422
2244
2242
22242222
1333
Medicinal
Savoury
Pungent
Elastoplast
Sick-sweet
Berry-like
-4
-3
-2
-1
0
1
2
3
4
-5 -4 -3 -2 -1 0 1 2 3 4 5 6
F1 (37.92 %)
F2
(2
9.5
3 %
)
PCA of profiles
Biplot (axes F1 and F2: 67.45 %)
5333
4444
4442
4424
4422
42444242
4224
4222
3533
33533335
3333
33313313
3133
24442442
2424
2422
2244
2242
22242222
1333
Medicinal
Savoury
Pungent
Elastoplast
Sick-sweet
Berry-like
-4
-3
-2
-1
0
1
2
3
4
-5 -4 -3 -2 -1 0 1 2 3 4 5 6
F1 (37.92 %)
F2
(2
9.5
3 %
)
Detection threshold change
PARAFAC – factor 1 vs 2
Berry-like
ElastoplastMedicinal
Pungent
Sick-sweetSavoury
Factor 1: ElastoplastFactor 2: Berry-like
PARAFAC – factor 2 vs 3
Pungent
Berry-like
Sick-sweet
SavouryMedicinal
Elastoplast
Factor 3: Pungent, Medicinal, SavouryPungent related to group with highest levels of 4-EP
Conclusions: Combination profiling
Elastoplast
Berry-like
PungentMedicinalSavoury
Sick-sweet
Hierachy of descriptors:
Results of PARAFAC complementary to PCA and univariate results
4-EP related to pungency
Consumer analysis
Some “Brett” wine liked better than the control
Difference found in terms of wine knowledge
Results not conclusive
Research methodology: Test “fuzzy” concepts
Research Chapter 4:Chemical Analyses
Materials and methods
Set of 32 South African winesWines known to be spoiltWines on the marketQuestionable wines
GC-MS4-ethylphenol, 4-ethylguaiacol, isovaleric acid, isobutyric
acid, acetic acid, 4-vinylphenol and 4-vinylguaiacol
HPLC-MS-MS4-ethylcatechol
Cultivar and mildly spoilt wines
Biplot (axes F1 and F2: 85.20 %)
B6
B4
C6
M3
B3
S6S4
C4
M2
P6P5
P2
B2
4-EC
4-VG
4-EP4-EG
Isovaleric acidIsobutyric acid
Acetic acid
4-VP
-5
0
5
-7 -2 3 8 13
F1 (62.71 %)
F2
(22.
50 %
)
Pinotage?
Same enzymatic pathways
Conclusions: Chemical analyses
First South African study to analyse 4-ethylcatechol
Strong correlations between ethylphenols and isovaleric/isobutyric acid
Sample set too small to draw valid conclusions regarding cultivar
Recommended that larger study be undertaken to investigate this aspect further
Conclusions
Conclusions
Threshold value limiting concept, range more appropriate
Focus should shift to ranges
More research should be done for detection threshold methodology
All four compounds tested interact, especially in terms of the most NB descriptors
All four compounds should therefore be included in future sensorystudies
Conclusions cont.
Consumer analysis not conclusive
Use better testing methodologies
Use of central composite design limiting but necessary for investigating interactions
Further studies with a more “complete” design
Future research
4-ethylcatechol should be tested in future studies concerning Brettanomyces
Help clear up discrepancies
Study indicates direction of future research
Acknowledgements
Adriaan Oelofse & Jan Bester
Thomas Skov – Parafac
Prof van Aarde – Statistics help
Marietjie Stander – Chemical analyses
Distell
Friends & Family
Heavenly Father
Conclusions
Discriminant analyses and Linear LSD’s have similar results
Same underlying function
PCA complement linear statistics by means of providing insight into relationships
Overall – techniques complement one another
More detail about the profile of “Brett character” (Smoky/Savoury)
Materials and Methods: Samples
24444444
424422444424242442242224444224424242224244222422422222225333133335333133
33533313
333533313333
Isovaleric acid4-EC4-EG4-EP