©Copyright 2013 All Rights Reserved 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

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

DOES THIS LOOK FAMILIAR? ©Copyright 2013 All Rights Reserved

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?

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%

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

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

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

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

REPRESENTATIVE LEVELS Total Anthos Free Anthos Bound Anthos Tannins Total IRPs 12CS ME PS ZI PN Current Vintage, Post-Fermentation ©Copyright 2013 All Rights Reserved

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

Gage R&R - REPEATABILITY Op/Tech Free Anthos SPPLPPTannins Total IRPs GC O/T O/T O/T O/T SIX WINES, 4 REPS, AVERAGE PERCENT ERROR ©Copyright 2013 All Rights Reserved

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

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

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

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

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

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

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

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

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

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

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

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

MACERATION ©Copyright 2013 All Rights Reserved

MACERATION ©Copyright 2013 All Rights Reserved

MACERATION ©Copyright 2013 All Rights Reserved

MACERATION ©Copyright 2013 All Rights Reserved

ANTHOCYANINS IN TIME ©Copyright 2013 All Rights Reserved

COMPARISON OF PEER GROUP 10 CS NAPA Total Anthos Free Anthos Bound Anthos Tannins Total IRPs Jones Bear Charlie Block Charlie Block Seven Oaks Paris Johnson ©Copyright 2013 All Rights Reserved * Assayed on 02/2011

Total Antho’s Free Antho’s Bound Antho’s Tannins Total IRPs 12 PN A Sonoma PN B Carneros CS A Napa CS B Napa PS A California PS B Mendocino CS “CULT” ©Copyright 2013 All Rights Reserved * Assayed on 02/09/2013 TASTING SESSION WINES

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

Total Antho’s Free Antho’s Bound Antho’s Tannins Total IRPs 12 PN A Sonoma PN B Carneros ‘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

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

- ‘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 CS B Napa * Assayed on 02/09/2013 ©Copyright 2013 All Rights Reserved

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

- ‘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 PS B Mendocino TASTING SESSION: 2011 & 2012 PS * Assayed on 02/09/2013

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

Total Antho’s Free Antho’s Bound Antho’s Tannins Total IRPs 2008 CS ‘CULT’ ©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’ * Assayed on 11/24/2008

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

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