1. ©Copyright 2013 All Rights Reserved 2012-2013 Wine Flavor 101C The Impact of Phenolic Management on Wine Style Options Making the Most of a Partial Extraction: Phenolics Profiling in Real-Time UC Davis, February 15, 2013

2. OVERVIEW Red Wine Phenolic Extractions What We Measure and How Repeatability & Reproducibility is Key Multivariate Analysis & Chemometrics VESUVVIO © - Our UV-Vis Based Red Wine Phenolics Profiling Platform Tasting with Phenolic Profiles Assessment ©Copyright 2013 All Rights Reserved

3. DOES THIS LOOK FAMILIAR? ©Copyright 2013 All Rights Reserved

4. RED WINE MACERATIONS ©Copyright 2013 All Rights Reserved Grape supply and management is the single most important step of winemaking, both in terms of quality and cost. Are you getting the most and the best out of your fruit?

5. THE REALITY ©Copyright 2013 All Rights Reserved The single most important fact to remember is that red wine macerations are only PARTIAL extractions. Anthocyanins: 20% -50% Tannins: 5% - 40% Total Phenolics: 20% - 50%

6. IMPORTANT FACTORS Incoming Fruit Characteristics & Quality Movement – Punchdowns / Pumpovers, Delestage Fermenter Temperature Profiles pH, Ethanol Concentration,… Yeast Strains,… Maceration Time Post-Maceration Conditions ©Copyright 2013 All Rights Reserved

7. Non-flavonoids benzoic acids (C 6 -C 1 ) hydroxycinnamic acids (C 6 -C 3 ) oak (poly-)phenolics Flavonoids ( C 6 -C 3 -C 6 ) anthocyanins flavonols flavanols proanthocyanidins, Condensed Tannins p-coumaric acid WHAT WE DO NOT MEASURE flavonoid backbone anthocyanins aglycone flavonols aglycone flavan-3-ol monomer flavan-3-ols oligoner/polymer

8. WHAT’S THE REASON? Needs technologically advanced instrumentation; costly. Requires highly skilled laboratory personnel. Time consuming. Not timely for production environment. Rather removed from sensory assessments. Cannot preferentially extract individual compounds during maceration. Works best for fundamental research & development of applied technologies. ©Copyright 2013 All Rights Reserved

9. WHAT WE DO MEASURE Pigments – Free and Total Anthocyanins: basis of color perception and key structural ingredients. Bound Anthocyanins: Anthocyanins linked to tannins that form stable color and add textural qualities. Tannins: structural building blocks, major sensory effects – very important to wine style and ageability. Total (Iron-Reactive) Phenolics: amount of all phenolic compounds in the wine; useful when comparing maceration protocols or blends. ©Copyright 2013 All Rights Reserved

10. REPRESENTATIVE LEVELS Total Anthos Free Anthos Bound Anthos Tannins Total IRPs 12CS 95970023110262386 12ME 6344791388061968 12PS 1338101829017163524 12ZI 316254535281529 12PN 406334502661366 Current Vintage, Post-Fermentation ©Copyright 2013 All Rights Reserved

11. HOW WE MEASURE THEM Wet Chemistry - We use an extended version of the Harbertson & Adams Phenolics Assay New parameters reported – Total Anthocyanins and Bound Anthocyanins New extended H&A Assay SOP has resulted in significant reduction of execution time, and increased repeatability resulting in error standard deviations within 5% Execution time - 24 samples in 4 to 5 hours ©Copyright 2013 All Rights Reserved Harbertson, Adams et al, American Journal of Enology and Viticulture, 2002 & 2003 Six Sigma Quality Systems Methodology, Gage Repeatability & Reproducibility

12. Gage R&R - REPEATABILITY Op/Tech Free Anthos SPPLPPTannins Total IRPs GC 3.22.28.13.51.5 O/T 2 7.76.714.88.512.8 O/T 3 4.52.66.73.62.4 O/T 4 2.11.94.74.01.0 O/T 5 2.45.012.84.92.8 SIX WINES, 4 REPS, AVERAGE PERCENT ERROR ©Copyright 2013 All Rights Reserved

13. UV-Vis SPECTROSCOPY Primary Fermentation - One Maceration over Six Days ©Copyright 2013 All Rights Reserved

14. UV-Vis SPECTROSCOPY Later During Primary Fermentation-One Maceration Days 8-12 ©Copyright 2013 All Rights Reserved

15. MODEL-BASED H&A ASSAY 24 samples in 1 hour Total Anthocyanins Free Anthocyanins Bound Anthocyanins © (p-p) Tannins Total (i-r) Phenolics ©Copyright 2013 All Rights Reserved

16. MV ANALYSIS & MODELING Specific Focus – CHEMOMETRICS Technology Openly Available and Widely Used in the Process Industries Applications Include: Laboratory Analyses, On-line Virtual Sensors, Process Optimization Steps: measurements, calibration, internal validation, deployment, field validation. ©Copyright 2013 All Rights Reserved

17. CALIBRATION & VALIDATION PC / Notebook process analytical instrumentation (at-line or in-line; UV/Vis, IR, …) standardized measurements SAMPLE RESULTS SAMPLES SPECTRA MEASURED VALUES laboratory analytical instrumentation (Wet Chemistry [H&A Assay] or HPLC, GC/MS, …) model building & deployment (multivariate; PCR, PLS, ANN,… ) “RMSEC” & “RMSEV” ©Copyright 2013 All Rights Reserved

18. DEPLOYMENT PC / Notebook process analytical instrumentation (at-line or in-line; UV/Vis, IR, …) SAMPLE RESULTS SAMPLES SPECTRA model building & deployment (multivariate; PCR, PLS, ANN,… ) “RMSEP” ©Copyright 2013 All Rights Reserved

19. PREDICTIVE STATISTICS Cabernet Sauvignon - During Elevage (Napa) ©Copyright 2013 All Rights Reserved

20. VESUVVIO © The UV-Vis Based Red Wine Phenolics Assay, … no wine sample alterations, fast, precise, and timely. ©Copyright 2013 All Rights Reserved Harbertson, Adams et al, American Journal of Enology and Viticulture, 2002 & 2003 Skogerson, Boulton et al, American Journal of Enology and Viticulture, 2007

21. PROTOCOL Obtain Wine Samples To Be Profiled Centrifuge Wine Samples for 5 Minutes Prime Flow-Through Cell & Run UV-Vis Spectrophotometer Extract (Electronically) Samples Spectra Run Multivariate Calibration Report Measured Wine Phenolic Profiles ©Copyright 2013 All Rights Reserved

22. REQUIRED EQUIPMENT PC / Notebook / Laptop UV-Vis Spectrophotometer Micro-Centrifuge Sipper Automatic Sample Changer (OPTIONAL) ©Copyright 2013 All Rights Reserved

23. KEY ADVANTAGES Reduced Assay Execution Time compared to original wet chemistry method - 24 samples per hour per spectrophotometer. Very Timely Measurements for Operational Decision-Making. Increased Repeatability & Reproducibility – over 95% precision. No sample transfer: upload spectra electronically, receive results within minutes. ©Copyright 2013 All Rights Reserved

24. APPLICATION AREAS Vineyard Performance and Grower Feedback: Possible Contractual Element Maceration and Elevage Wine Evaluation & Classification Blending Fining: Measurable Impact Bottled Wine and Vintage Comparisons Vintage Quality Assessment ©Copyright 2013 All Rights Reserved

25. MACERATION ©Copyright 2013 All Rights Reserved

26. MACERATION ©Copyright 2013 All Rights Reserved

27. MACERATION ©Copyright 2013 All Rights Reserved

28. MACERATION ©Copyright 2013 All Rights Reserved

29. ANTHOCYANINS IN TIME ©Copyright 2013 All Rights Reserved

30. COMPARISON OF PEER GROUP 10 CS NAPA Total Anthos Free Anthos Bound Anthos Tannins Total IRPs Jones102074422811352448 Bear65543212810892354 Charlie Block 4 8975681559082155 Charlie Block 7 7454291338442033 Seven Oaks 8286081559332178 Paris10098331889922311 Johnson64439811811892449 ©Copyright 2013 All Rights Reserved * Assayed on 02/2011

31. Total Antho’s Free Antho’s Bound Antho’s Tannins Total IRPs 12 PN A Sonoma 283219443391703 12 PN B Carneros 405307695372012 12 CS A Napa 7355891368181932 12 CS B Napa 48234912914922832 11 PS A California 7055901095551621 12 PS B Mendocino 69651015713743061 2008 CS “CULT” 5262362759562137 ©Copyright 2013 All Rights Reserved * Assayed on 02/09/2013 TASTING SESSION WINES

32. Total Antho’s Free Antho’s Bound Antho’s Tannins Total IRPs 12 PN A Sonoma 283219443391703 12 PN B Carneros 405307695372012 TASTING SESSION: 2012 PN * Assayed on 02/09/2013 ©Copyright 2013 All Rights Reserved

33. Total Antho’s Free Antho’s Bound Antho’s Tannins Total IRPs 12 PN A Sonoma 283219443391703 12 PN B Carneros 405307695372012 - ‘Total Anthocyanins’: ‘B’ 43% higher than ‘A’ - ‘Shift’ from ‘Total to Free’ is 23% and 24%, respectively - ‘Tannins’ / ‘Total IRP’s’: 20% and 27%, respectively - ‘Tannins’ very similar to ‘Total Anthocyanins’ for both - Verdict: different grape sources, both wines are balanced TASTING SESSION: 2012 PN * Assayed on 02/09/2013 ©Copyright 2013 All Rights Reserved

34. Total Antho’s Free Antho’s Bound Antho’s Tannins Total IRPs 12 CS A Napa 7355891368181932 12 CS B Napa 48234912914922832 TASTING SESSION: 2012 CS * Assayed on 02/09/2013 ©Copyright 2013 All Rights Reserved

35. - ‘Total Anthocyanins’: ‘A’ 52% higher than ‘B’ - Relatively similar ‘Bound Anthocyanins’... Why? - ‘Tannins’ / ‘Total IRPs’: 42% and 53% respectively - ‘Total Anthocyanins’ and ‘Tannins”: ‘A’ ~ ‘B’ ; ‘A’ 32% of ‘B’ - Verdict : ‘A’ balanced, ‘B’ over-extracted TASTING SESSION: 2012 CS Total Antho’s Free Antho’s Bound Antho’s Tannins Total IRPs 12 CS A Napa 7355891368181932 12 CS B Napa 48234912914922832 * Assayed on 02/09/2013 ©Copyright 2013 All Rights Reserved

36. Total Antho’s Free Antho’s Bound Antho’s Tannins Total IRPs 11 PS A California 7055901095551621 12 PS B Mendocino 69651015713743061 ©Copyright 2013 All Rights Reserved TASTING SESSION: 2011 & 2012 PS * Assayed on 02/09/2013

37. - ‘Total Anthocyanins’: Higher or lower than ‘Tannins’? - ‘Bound Antho? Driver? Antho or Tannin? - ‘Tannins’ / ‘Total IRP’s’: 34% versus 45% - ‘Total IRP’s’ over 3,000: ‘very extracted’ style - Verdict: ‘A’ possibly under-extracted, ‘B’ over-extracted’ ©Copyright 2013 All Rights Reserved Total Antho’s Free Antho’s Bound Antho’s Tannins Total IRPs 11 PS A California 7055901095551621 12 PS B Mendocino 69651015713743061 TASTING SESSION: 2011 & 2012 PS * Assayed on 02/09/2013

38. Total Antho’s Free Antho’s Bound Antho’s Tannins Total IRPs 2008 CS ‘CULT’ 5262362759562137 ©Copyright 2013 All Rights Reserved TASTING SESSION: 2008 CS * Assayed on 02/09/2013

39. Total Antho’s Free Antho’s Bound Antho’s Tannins Total IRPs 2008 CS ‘CULT’ 5262362759562137 ©Copyright 2013 All Rights Reserved TASTING SESSION: 2008 CS * Assayed on 02/09/2013 - So, why is it ‘CULT?’ - ‘Tannins’ is 45% of ‘Total IRP’s’ - “Classic” - ‘Total Anthocyanins’: unusually high for its age - ‘Bound Anthocyanins’: “Rare” for the amount of ‘Tannins’ - Verdict, depending on preferred style: Balanced. Unusual wine.

40. Total Antho’s Free Antho’s Bound Antho’s Tannins Total IRPs 2008 CS ‘CULT’ 5262362759562137 ©Copyright 2013 All Rights Reserved TASTING SESSION: 2008 CS * Assayed on 02/09/2013 Total Antho’s Free Antho’s Bound Antho’s Tannins Total IRPs 2008 CS ‘CULT’ 107174632610672370 * Assayed on 11/24/2008

41. CONCLUSIONS Coffee, tea, wine or beer: they are all partial extractions. Vineyard performance can be measured by phenolic measurements rapidly and accurately. Differences in maceration profiles suggest what you do as a winemaker, greatly affects the final phenolic content of your wines. Evaluate maceration protocols: is it time, temperature, alcohol or movement? Knowledge of tannins and total phenolics amounts very useful in drain decision. Some basic ratios and general knowledge of relative amounts of phenolic materials (per variety and region) offer powerful insights into initial grape and wine quality. ©Copyright 2013 All Rights Reserved

42. We greatly appreciate the opportunity to share with you our excitement about Red Wine Functional Phenolics Profiling. Gianni Colantuoni & Scott McLeod Gianni@winexray.com Scott@winexray.com