Thesis Defence

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Thesis Defence

  1. 1. Sensory, chemical and consumer analysis of Brettanomyces spoilage in South African wines Janita Botha Thesis defence 17 February 2009 Research Team : M Muller, W J du Toit, A J de Villiers & A G J Tredoux
  2. 2. Project background Bottle Saccharomyces cerevisiae Oenococcus oeni Other LAB Ethanol CO 2 Lactic acid Diacetyl Furfural Guaiacol Eugenol Etc Brett-related spoilage compounds + Fermentation Malo-lactic Fermentation Barrel Aging Brettanomyces/ Dekkera
  3. 3. Sensory studies about Brett <ul><li>Chatonnet et al . (1992): </li></ul><ul><ul><li>Linked 4-ethylphenol and 4-ethylguaiacol to Brettanomyces </li></ul></ul><ul><ul><li>Detection thresholds of 4-EP and 4-EG </li></ul></ul><ul><ul><li>Commonly quoted study </li></ul></ul><ul><li>Licker et al . (1999): </li></ul><ul><ul><li>Linked isovaleric acid to Brettanomyces </li></ul></ul><ul><li>Hesford & Schneider (2004): </li></ul><ul><ul><li>Linked 4-ethylcatechol to Brettanomyces </li></ul></ul>
  4. 4. Recent sensory studies <ul><li>Curtin et al . (2008): </li></ul><ul><ul><li>Detection thresholds of 4-EP, 4-EG and 4-EC </li></ul></ul><ul><ul><li>Profiling of certain combinations </li></ul></ul><ul><ul><li>Consumer analysis </li></ul></ul><ul><ul><li>Recommend further studies </li></ul></ul><ul><li>Romano et al . (2009): </li></ul><ul><ul><li>Found further strong links between 4-EP and isovaleric acid </li></ul></ul><ul><ul><li>Investigated effect of isovaleric acid on detection thresholds of 4-EP and 4-EG </li></ul></ul>Isovaleric acid? 4-ethylcatechol?
  5. 5. 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
  6. 6. Research Chapter 1: Detection Thresholds
  7. 7. Materials and Methods <ul><li>Method based on ASTM E 679 – 04 </li></ul><ul><li>3-Alternative Forced Choice (3-AFC) </li></ul><ul><li>3 glasses of wine presented, 1 contains the Brett compound </li></ul><ul><li>8 levels of 8 compounds tested </li></ul>Difference known, judges therefore trained
  8. 8. Materials and Methods <ul><li>Concentration increases with a constant MF factor on log scale </li></ul><ul><li>Two calculation methods investigated: ASTM and median </li></ul>
  9. 9. Results: 4-ethylphenol LL LL ASTM Median UL UL 0 50 100 150 200 250 300 350 400 Median ASTM Calculation method Concentration (μg/L) Level 5 Level 2 Level 4 Level 3 Level 1 Outlier Larger range
  10. 10. Results: 4-ethylcatechol LL LL ASTM Median UL UL 0 200 400 600 800 1000 1200 Median ASTM Calculation method Concentration (μg/L) Level 3 Level 4 Level 5 Level 6 Level 7 Level 8 True range Simplified range
  11. 11. Conclusions <ul><li>Concept of “detection threshold” is limited and obscures information </li></ul><ul><li>The use of a detection threshold range more accurate </li></ul><ul><li>Range provided by median more informative about population </li></ul>
  12. 12. Research Chapter 2: Singular Profiling
  13. 13. Materials and Methods: Central composite design Compound 1 Compound 2 Compound 3 Singular profiling exploration step before compounds can be tested in combination
  14. 14. Materials and Methods: Singular profiling 4-EP 4-EG 4-EC Isovaleric acid 0 2 4 3 1 5 DT Values determined used as guide Levels pre-screened to confirm suitability for use in new medium
  15. 15. Example of linear results: 4-EP 0 10 20 30 40 50 60 70 0 2 4 6 8 10 12 14 Level of 4-ethylphenol Intensity of descriptor Elastoplast Leather Berry-like Sick-sweet Mirroring Following
  16. 16. Example of linear results: 4-EP 0 10 20 30 40 50 60 70 0 2 4 6 8 10 12 14 Level of 4-ethylphenol Intensity of descriptor Elastoplast Leather Berry-like Sick-sweet
  17. 17. Example of Discriminant Analysis: 4-EP 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 3 3 L3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 4 3 2 1 0 -6 -4 -2 0 2 4 6 -6 -4 -2 0 2 4 6 F1 (78.64 %) F2 (19.88 %) 0 1 2 3 4 5 Centroids Level
  18. 18. Example of PCA: 4-EP Similar pattern in univariate results 5 5 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 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 (8.90 %)
  19. 19. Overall results 4-EP 4-EG 4-EC Isovaleric acid Elastoplast Leather Medicinal Smoky Savoury Pungent Sick-sweet Berry-like Not in literature Clove-like?? More detail obtained about the contributions of the respective compounds
  20. 20. Research Chapter 3: Combination Profiling
  21. 21. Materials and Methods: Design Compound 1 Compound 2 Compound 3 Samples
  22. 22. Materials and Methods: Samples 2 4 4 4 4 4 4 4 4 2 4 4 2 2 4 4 4 4 2 4 2 4 2 4 4 2 2 4 2 2 2 4 4 4 4 2 2 4 4 2 4 2 4 2 2 2 4 2 4 4 2 2 2 4 2 2 4 2 2 2 2 2 2 2 5 3 3 3 1 3 3 3 3 5 3 3 3 1 3 3 3 3 5 3 3 3 1 3 3 3 3 5 3 3 3 1 3 3 3 3 Isovaleric acid 4-EC 4-EG 4-EP 1 Centre Sample 8 Star Samples 16 Cube Samples
  23. 23. Results: Interaction <ul><li>Elastoplast: </li></ul><ul><li>4-EP*4-EG*4-EC </li></ul><ul><li>4-EP*4-EG*ISOV </li></ul><ul><li>Medicinal: </li></ul><ul><li>4-EG cause increase </li></ul><ul><li>4-EC and isovaleric acid cause decrease </li></ul><ul><li>Pungent: </li></ul><ul><li>ISOV*4-EG </li></ul><ul><li>4-EP cause increase </li></ul>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?
  24. 24. PCA vs PARAFAC: sensory data Judges Samples Attributes Samples 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
  25. 25. PCA vs PARAFAC 1 st factor calculated Subsequent orthogonal factors calculated 1 st factor calculated Subsequent factors calculated in sucessive directions of variance Factors recalculated untill they converge PCA PARAFAC Less noise modelled
  26. 26. PCA of profiles Biplot (axes F1 and F2: 67.45 %) 5333 4444 4442 4424 4422 4244 4242 4224 4222 3533 3353 3335 3333 3331 3313 3133 2444 2442 2424 2422 2244 2242 2224 2222 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 (29.53 %)
  27. 27. Detection threshold change Biplot (axes F1 and F2: 67.45 %) 5333 4444 4442 4424 4422 4244 4242 4224 4222 3533 3353 3335 3333 3331 3313 3133 2444 2442 2424 2422 2244 2242 2224 2222 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 (29.53 %)
  28. 28. PARAFAC – factor 1 vs 2 Berry-like Elastoplast Medicinal Pungent Sick-sweet Savoury <ul><li>Factor 1: Elastoplast </li></ul><ul><li>Factor 2: Berry-like </li></ul>
  29. 29. PARAFAC – factor 2 vs 3 Pungent Berry-like Sick-sweet Savoury Medicinal Elastoplast <ul><li>Factor 3: Pungent, Medicinal, Savoury </li></ul><ul><li>Pungent related to group with highest levels of 4-EP </li></ul>
  30. 30. Conclusions: Combination profiling <ul><li>Hierachy of descriptors: </li></ul><ul><li>Results of PARAFAC complementary to PCA and univariate results </li></ul><ul><li>4-EP related to pungency </li></ul>Elastoplast Berry-like Pungent Medicinal Savoury Sick-sweet
  31. 31. Consumer analysis <ul><li>Some “Brett” wine liked better than the control </li></ul><ul><li>Difference found in terms of wine knowledge </li></ul><ul><li>Results not conclusive </li></ul><ul><li>Research methodology: Test “fuzzy” concepts </li></ul>
  32. 32. Research Chapter 4: Chemical Analyses
  33. 33. Materials and methods <ul><li>Set of 32 South African wines </li></ul><ul><ul><li>Wines known to be spoilt </li></ul></ul><ul><ul><li>Wines on the market </li></ul></ul><ul><ul><li>Questionable wines </li></ul></ul><ul><li>GC-MS </li></ul><ul><ul><li>4-ethylphenol, 4-ethylguaiacol, isovaleric acid, isobutyric acid, acetic acid, 4-vinylphenol and 4-vinylguaiacol </li></ul></ul><ul><li>HPLC-MS-MS </li></ul><ul><ul><li>4-ethylcatechol </li></ul></ul>
  34. 34. Cultivar and mildly spoilt wines Biplot (axes F1 and F2: 85.20 %) B6 B4 C6 M3 B3 S6 S4 C4 M2 P6 P5 P2 B2 4-EC 4-VG 4-EP 4-EG Isovaleric acid Isobutyric acid Acetic acid 4-VP -5 0 5 -7 -2 3 8 13 F1 (62.71 %) F2 (22.50 %) Pinotage? Same enzymatic pathways
  35. 35. Conclusions: Chemical analyses <ul><li>First South African study to analyse 4-ethylcatechol </li></ul><ul><li>Strong correlations between ethylphenols and isovaleric/isobutyric acid </li></ul><ul><li>Sample set too small to draw valid conclusions regarding cultivar </li></ul><ul><li>Recommended that larger study be undertaken to investigate this aspect further </li></ul>
  36. 36. Conclusions
  37. 37. Conclusions <ul><li>Threshold value limiting concept, range more appropriate </li></ul><ul><li>Focus should shift to ranges </li></ul><ul><li>More research should be done for detection threshold methodology </li></ul><ul><li>All four compounds tested interact, especially in terms of the most NB descriptors </li></ul><ul><li>All four compounds should therefore be included in future sensorystudies </li></ul>
  38. 38. Conclusions cont. <ul><li>Consumer analysis not conclusive </li></ul><ul><li>Use better testing methodologies </li></ul><ul><li>Use of central composite design limiting but necessary for investigating interactions </li></ul><ul><li>Further studies with a more “complete” design </li></ul>
  39. 39. Future research <ul><li>4-ethylcatechol should be tested in future studies concerning Brettanomyces </li></ul><ul><li>Help clear up discrepancies </li></ul><ul><li>Study indicates direction of future research </li></ul>
  40. 40. Acknowledgements <ul><li>Adriaan Oelofse & Jan Bester </li></ul><ul><li>Thomas Skov – Parafac </li></ul><ul><li>Prof van Aarde – Statistics help </li></ul><ul><li>Marietjie Stander – Chemical analyses </li></ul><ul><li>Distell </li></ul><ul><li>Friends & Family </li></ul><ul><li>Heavenly Father </li></ul>
  41. 42. Conclusions <ul><li>Discriminant analyses and Linear LSD’s have similar results </li></ul><ul><li>Same underlying function </li></ul><ul><li>PCA complement linear statistics by means of providing insight into relationships </li></ul><ul><li>Overall – techniques complement one another </li></ul><ul><li>More detail about the profile of “Brett character” (Smoky/Savoury) </li></ul>
  42. 43. Materials and Methods: Samples 2 4 4 4 4 4 4 4 4 2 4 4 2 2 4 4 4 4 2 4 2 4 2 4 4 2 2 4 2 2 2 4 4 4 4 2 2 4 4 2 4 2 4 2 2 2 4 2 4 4 2 2 2 4 2 2 4 2 2 2 2 2 2 2 5 3 3 3 1 3 3 3 3 5 3 3 3 1 3 3 3 3 5 3 3 3 1 3 3 3 3 5 3 3 3 1 3 3 3 3 Isovaleric acid 4-EC 4-EG 4-EP

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