1. Research and Development Phenolics and Tannin Assays for Practical Use in Winemaking Giovanni Colantuoni John Thorngate

2. Research and Development Outline Introduction  Grape and Wine Phenolics  Measuring Phenolics Adams-Harbertson Assays  Gage R&R Analysis  Creating a Standardized SOP The UV-Vis Predictive Model  Chemometrics — Model Calibration and Deployment  Comparison to Skogerson-Downey-Boulton  Using the Model Summary

3. Research and Development Chemists interested in polyphenols, in common with the majority of scientists, tackle today’s problems with yesterday’s tools, i.e., current problems are attacked with methods which are inadequate and to that extent are already out of date. The discovery and quick application of new methods or developments and extensions of existing methods is therefore of first importance. B.R.Brown, In Methods of Polyphenol Chemistry, 1964

4. Research and Development Introduction Why focus on phenolics?  Important for:  Color  Taste  Mouthfeel  Wine aging

5. Research and Development Introduction Why measure phenolics?  Identify higher quality lots more easily  Use phenolic data for:  Press decisions  Heavy press additions  Blend balancing  Evaluation of processing

6. Research and Development Grape and Wine Phenolics Phenolic compounds of interest to the winemaker: Phenolic acids Flavonoids Anthocyanins Tannins Polymeric Pigment J.A. Kennedy, Grape and wine phenolics: Observations and recent findings, Ciencia e Investigación Agraria 35:77-90, 2008

7. Research and Development Phenolic Acids Kennedy, 2008

8. Research and Development Flavonoids Quercetin A.L. Waterhouse, Wine Phenolics, Annals of the New York Academy of Sciences 957:21-36, 2002

9. Research and Development Anthocyanins Kennedy, 2008

10. Research and Development Tannins Schofield et al., Analysis of Condensed Tannins: A Review Animal Feed Science and Technology 91:21-40, 2001

11. Research and Development Polymeric Pigments Kennedy, 2008

12. Research and Development Phenolic Levels in Wine Waterhouse, 2002

13. Research and Development Measuring Phenolics Total Phenolics  A 280  Folin-Ciocalteu Tannins  Acid Butanolysis  Aldehyde Pigments Nota bene: unless you are chromatographically separating discrete compounds all measures of phenolics are methodologically defined

14. Research and Development Total Phenolics Absorbance at 280 nm Pro’s: Simple; just requires UV-transparent cuvette and a UV-capable spectrophotometer (express as A 280 in AU) Con’s: Subject to interferences from other aromatic ring containing compounds (e.g., nucleotides, aromatic amino acids) Nota bene...these are relatively small effects

15. Research and Development Total Phenolics Folin-Ciocalteu Pro’s: Measures all mono- and dihydroxylated phenolics; automatable Con’s: Subject to interferences from fructose and SO 2 ; spent reagent has to be disposed of as hazardous waste

16. Research and Development Tannins Acid Butanolysis Pro’s: Specific for tannins; anthocyanidin color measured with spectrophotometer (relative abundance) Con’s: Low reaction yields; highly dependent upon reaction conditions and the tannin structure

17. Research and Development Tannins Aldehydes (Vanillin, DMCA*) Pro’s: Measures flavan-3-ols and polymers (m-dihydroxy’s); color measured with spectrophotometer Con’s: Rate and extent of color development solvent dependent; vanillin adduct absorbs at 500 nm (problematic for red wines) *dimethylaminocinnamaldehyde

18. Research and Development Pigments Any number of spectrophotometric assays for pigments are available These procedures have been extensively researched by Chris Somers in Australia (e.g., The Wine Spectrum, Winetitles: Marleston, SA, 1998)  e.g., A 520, A 420 and all their permutations

19. Research and Development Adams-Harbertson Assays Functional assays providing quantitative information on various phenolic classes  Total iron-reactive phenols  Analogous to Folin-Ciocalteu  Caveat: doesn’t measure monohydroxylated phenols or anthocyanins  Protein (BSA) precipitable tannins  Tetrameric tannins and larger  Polymeric pigments  Non-SO 2 bleachable pigmented fractions  Non-protein precipitable: small polymeric pigment  Protein precipitable: large polymeric pigment  Free Anthocyanins

20. Research and Development Adams-Harbertson Assays Benefits  Can run the analyses in-house IF you have a Visible spectrophotometer, a microcentrifuge, a vortexer and the necessary micropipettes  The IRP is a measure of total phenolics (minus anthocyanins) and doesn’t generate hazardous waste  The protein-precipitable tannin is highly correlated to perceptual astringency

21. Research and Development Tannin vs. Astringency Kennedy et al., Analysis of Tannins in Red Wine Using Multiple Methods: Correlation with Perceived Astringency, AJEV 57:481-485, 2006

22. Research and Development  Sets of up to 24 samples  4/5 segments, 9 sets of readings, ~ 3 hours  5 results: anthocyanins, tannins, IRP, SPP, LPP Running the A-H Assay

23. Research and Development Gage R & R OBJECTIVE: Quantify Measurement Error in Measurement Systems  Integral Part of SIX SIGMA Methodology  Quality Systems… Zero Defects… ISO Standards…  Goal: less than 3.4 defects in a million opportunities  Early adapters: Motorola & Allied Signal (early 90’s)  General Electric Co. – most successful implementer  Two components  Standard Deviation of Measured Values  Assessment of Source of Variability  Contributors to Measurement Variation  Repeatability – Single Operator, Same Equipment  Reproducibility – Operators, Protocol, Equipment,…

24. Research and Development Gage R & R Study Conducted in April-June 2008  Design of Experiments - DOE  3 wineries, 5 wines, 4 technicians, 4 repetitions  full-factorial, randomized – 80 test results  Resulting Standard Deviations  (free-) Anthocyanins3.02%  SPP2.01%  LPP4.86%  Tannins2.79%  IRP3.78%  But… observed spikes of 7.6, 11.7,… 27.5%  ANOVA analysis needed – Used MINITAB

25. Research and Development Gage R & R Operator Contribution 3.3 %, # of Categories* 7 * Automotive Industry Action Group (AIAG) Measurement Systems Analysis (June 1998)

26. Research and Development Gage R & R Operator Contribution 34.4 %, # of Categories* 1 * Automotive Industry Action Group (AIAG) Measurement Systems Analysis (June 1998)

27. Research and Development Standard Procedure The Assay Protocol – Essential KEY to Repeatability & Reproducibility  Sources of Adams-Harbertson Assay Protocol  Technical literature and journals  UC Davis Department of Viticulture & Enology website  Trade publications  Individual laboratory adaptations  In practice… a multitude of ways of running the Assay  Consequently,  Large variations in reported results  And even declarations of intrinsic invalidity  Moreover,  A closer look at the assay reveals significant potential for improving its repeatability and reducing time of execution

28. Research and Development Standard Procedure Road to the Adams-Harbertson Assay SOP  Initial documented procedure in place at Rubicon Estate  Set up with the assistance of Dr. Harbertson & Dr. Adams  Base documents from UC Davis Department of V & E website  Modifications introduced and validated over time  Salient results shared with Dr. Adams  Jointly with Dr. Thorngate determined need for SOP  Now working with the Gold Standard Group  Created draft for the “Modified A&H Assay SOP”  Currently being cast in ISO format  Review and finalization to follow  Gage R&R planned for mid-year 2010  Expected SOP release date – Fall 2010  Preliminary results indicate reduction in error “spikes”, increased repeatability, and over 1/3 reduction in runtime

29. Research and Development UV-Vis Spectroscopy Early in Primary Fermentation

30. Research and Development UV-Vis Spectroscopy Later in Primary Fermentation

31. Research and Development Calibration / Modeling Linear Curve-fitting absorbance @ 520 nm anthocyanins * * * * * * A&H Assay Results – PredictedUV-Vis Spectrum MODEL

32. Research and Development UV-Vis Based A-H Assay Multivariate Modeling - Chemometrics  Openly-available, widely-used technology  Commercial software packages can be purchased  Implemented (and in use) in other process industries  Applications: lab, virtual sensors, process optimization  Expected Impact  Implemented locally in the winery laboratory  Once in place, no phenolics wet chemistry analyses  Essentially no sample preparation  Assay time of one-to-two minutes per sample  Ideal for real-time vinification decisions

33. Research and Development  Development Methodology UV-Vis Based A-H Assay PC / Notebook process analytical instrumentation (at-line or in-line; UV/Vis, IR, …) standardized measurements SAMPLE RESULTS CALIBRATION SAMPLES (training and testing) SPECTRA MEASURED VALUES laboratory analytical instrumentation (lab-based; HPLC, GC/MS, …) model building & deployment (multivariate; PCR, PLS, ANN,… ) MRSEC

34. Research and Development  Validation UV-Vis Based A-H Assay PC / Notebook standardized measurements SAMPLE RESULTS MEASURED VALUES model building & deployment (multivariate; PCR, PLS, ANN,… ) SPECTRA FIELD VALIDATION SAMPLES TEST SAMPLES process analytical instrumentation (at-line or in-line; UV/Vis, IR, …) laboratory analytical instrumentation (lab-based; HPLC, GC/MS, …) MRSEV or MRSEP

35. Research and Development  Deployment UV-Vis Based A-H Assay PC / Notebook SAMPLE RESULTS model building & deployment (multivariate; PCR, PLS, ANN,… ) SPECTRA TEST SAMPLES process analytical instrumentation (at-line or in-line; UV/Vis, IR, …)

36. Research and Development The Predictive Model (Ver. 4)

37. Research and Development Model Comparisons Data ranges of current data and Skogerson data CurrentSkogerson et al. 2007 MinMaxMinMax Anthocyanins a 0141901096 IRP b 72.6497919.82272 Tannins b 02667-8.1798 a mg/L malvidin-3-glucoside equivalents b mg/L catechin equivalents Prediction statistics for the Skogerson et al. (2007) model using our data RMSEPr pred 2 RPDCV pred Anthocyanins a 4660.200.5105.0 IRP b 9090.380.863.3 Tannins b 4060.331.070.3 NOTE: Skogerson data was for Australian wines; Current data was for domestic wines.

38. Research and Development That being said... Validation statistics for the prediction of phenolic components (n=248) RMSEPr pred 2 RPDCV pred Anthocyanins a 1490.531.433.0 IRP b 3830.762.125.6 Tannins b 2030.782.133.8 a mg/L malvidin-3-glucoside equivalents b mg/L catechin equivalents There is ample room for improvement! RMSEP: root mean square error of prediction r pred 2 : coefficient of determination of the prediction RPD: ratio of standard deviation to standard error of prediction CV pred : coefficient of variation of the prediction

39. Research and Development Summary  The Adams-Harbertson assays measure functional classes of phenolic compounds in wine  The Adams-Harbertson assays are repeatable and reproducible  The Adams-Harbertson assays SOP — a work in progress  The Predictive Model shows great promise — additional work is required

40. Research and Development Acknowledgments Dr. James Harbertson (Assoc. Prof.!) and his laboratory Dr. Douglas Adams Gold Standard Jordan Ferrier Dr. Roger Boulton, Dr. Mark Downey & Kirsten Skogerson Tondi Bolkan, Evan Schiff, Karen Moneymaker

41. Research and Development Acknowledgments