thesis defence
DESCRIPTION
Thesis defence for MScTRANSCRIPT
![Page 1: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/1.jpg)
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
![Page 2: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/2.jpg)
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
+
![Page 3: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/3.jpg)
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
![Page 4: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/4.jpg)
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?
![Page 5: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/5.jpg)
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
![Page 6: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/6.jpg)
Research Chapter 1:Detection Thresholds
![Page 7: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/7.jpg)
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
![Page 8: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/8.jpg)
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)
![Page 9: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/9.jpg)
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
![Page 10: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/10.jpg)
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
![Page 11: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/11.jpg)
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
![Page 12: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/12.jpg)
Research Chapter 2: Singular Profiling
![Page 13: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/13.jpg)
Materials and Methods:Central composite design
Com
poun
d 1
Compound 2
Compound 3Singular profiling exploration step
before compounds can be tested in
combination
![Page 14: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/14.jpg)
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
![Page 15: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/15.jpg)
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
![Page 16: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/16.jpg)
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
![Page 17: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/17.jpg)
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
![Page 18: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/18.jpg)
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
![Page 19: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/19.jpg)
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
![Page 20: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/20.jpg)
Research Chapter 3:Combination Profiling
![Page 21: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/21.jpg)
Materials and Methods:Design
Com
poun
d 1
Compound 2
Compound 3
Samples
![Page 22: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/22.jpg)
Materials and Methods: Samples
24444444
424422444424242442242224444224424242224244222422422222225333133335333133
33533313
333533313333
Isovaleric acid4-EC4-EG4-EP 1 Centre Sample
8 Star Samples
16 Cube Samples
![Page 23: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/23.jpg)
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?
![Page 24: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/24.jpg)
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
![Page 25: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/25.jpg)
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
![Page 26: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/26.jpg)
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
![Page 27: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/27.jpg)
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
![Page 28: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/28.jpg)
PARAFAC – factor 1 vs 2
Berry-like
ElastoplastMedicinal
Pungent
Sick-sweetSavoury
Factor 1: ElastoplastFactor 2: Berry-like
![Page 29: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/29.jpg)
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
![Page 30: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/30.jpg)
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
![Page 31: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/31.jpg)
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
![Page 32: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/32.jpg)
Research Chapter 4:Chemical Analyses
![Page 33: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/33.jpg)
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
![Page 34: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/34.jpg)
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
![Page 35: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/35.jpg)
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
![Page 36: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/36.jpg)
Conclusions
![Page 37: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/37.jpg)
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
![Page 38: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/38.jpg)
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
![Page 39: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/39.jpg)
Future research
4-ethylcatechol should be tested in future studies concerning Brettanomyces
Help clear up discrepancies
Study indicates direction of future research
![Page 40: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/40.jpg)
Acknowledgements
Adriaan Oelofse & Jan Bester
Thomas Skov – Parafac
Prof van Aarde – Statistics help
Marietjie Stander – Chemical analyses
Distell
Friends & Family
Heavenly Father
![Page 41: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/41.jpg)
![Page 42: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/42.jpg)
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)
![Page 43: Thesis Defence](https://reader035.vdocuments.us/reader035/viewer/2022062405/556956c7d8b42ad3278b4cec/html5/thumbnails/43.jpg)
Materials and Methods: Samples
24444444
424422444424242442242224444224424242224244222422422222225333133335333133
33533313
333533313333
Isovaleric acid4-EC4-EG4-EP