phenolics and tannin assays for practical use in winemaking giovanni colantuoni john thorngate

Research and Development

Phenolics and Tannin Assays for Practical Use in

Winemaking

Giovanni ColantuoniJohn Thorngate


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


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yesterday’s tools, i.e., current problems are attacked with methods which are inadequate and to that extent are already out of date.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


Introduction

Why focus on phenolics?

Important for:ColorTasteMouthfeelWine aging


Introduction

Why measure phenolics?

Identify higher quality lots more easily

Use phenolic data for:Press decisionsHeavy press additionsBlend balancingEvaluation of processing


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


Phenolic Acids

Kennedy, 2008


Flavonoids

Quercetin

A.L. Waterhouse, Wine Phenolics, Annals of the New York Academy of Sciences 957:21-36, 2002


Anthocyanins

Kennedy, 2008


Tannins

Schofield et al., Analysis of Condensed Tannins: A ReviewAnimal Feed Science and Technology 91:21-40, 2001


Polymeric Pigments

Kennedy, 2008


Phenolic Levels in Wine

Waterhouse, 2002


Measuring Phenolics

Total PhenolicsA280

Folin-Ciocalteu

TanninsAcid ButanolysisAldehyde

Pigments

Nota bene: unless you are chromatographically separating discretecompounds all measures of phenolics are methodologically defined


Total Phenolics

Absorbance at 280 nm

Pro’s: Simple; just requires UV-transparent cuvette and a UV-capable spectrophotometer (express as A280 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


Total Phenolics

Folin-Ciocalteu

Pro’s: Measures all mono- and dihydroxylated phenolics; automatable

Con’s: Subject to interferences from fructose and SO2; spent reagent has to be disposed of as hazardous waste


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


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


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., A520, A420 and all their permutations


Adams-Harbertson Assays

Functional assays providing quantitative information on various phenolic classesTotal iron-reactive phenols

Analogous to Folin-CiocalteuCaveat: doesn’t measure monohydroxylated phenols

or anthocyaninsProtein (BSA) precipitable tannins

Tetrameric tannins and largerPolymeric pigments

Non-SO2 bleachable pigmented fractions Non-protein precipitable: small polymeric pigment Protein precipitable: large polymeric pigment

Free Anthocyanins


Adams-Harbertson Assays

BenefitsCan 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


Tannin vs. Astringency

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


Sets of up to 24 samples

4/5 segments, 9 sets of readings, ~ 3 hours5 results: anthocyanins, tannins, IRP, SPP, LPP

Running the A-H Assay


Gage R & R

OBJECTIVE: Quantify Measurement Error in Measurement Systems

Integral Part of SIX SIGMA MethodologyQuality Systems… Zero Defects… ISO Standards…Goal: less than 3.4 defects in a million opportunitiesEarly adapters: Motorola & Allied Signal (early 90’s)General Electric Co. – most successful implementer

Two componentsStandard Deviation of Measured ValuesAssessment of Source of Variability

Contributors to Measurement VariationRepeatability – Single Operator, Same EquipmentReproducibility – Operators, Protocol, Equipment,…


Gage R & R

Study Conducted in April-June 2008

Design of Experiments - DOE3 wineries, 5 wines, 4 technicians, 4 repetitionsfull-factorial, randomized – 80 test results

Resulting Standard Deviations(free-) Anthocyanins 3.02%SPP 2.01%LPP 4.86%Tannins 2.79% IRP 3.78%

But… observed spikes of 7.6, 11.7,… 27.5% ANOVA analysis needed – Used MINITAB


Gage R & R

Operator Contribution 3.3 %, # of Categories* 7

* Automotive Industry Action Group (AIAG) Measurement Systems Analysis (June 1998)


Gage R & R

Operator Contribution 34.4 %, # of Categories* 1

* Automotive Industry Action Group (AIAG) Measurement Systems Analysis (June 1998)


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


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


UV-Vis Spectroscopy

Early in Primary Fermentation


UV-Vis Spectroscopy

Later in Primary Fermentation


Calibration / ModelingCalibration / Modeling

Linear Curve-fitting

absorbance @ 520 nm

an

thocyan

ins *

*

*

*

**

A&H Assay Results – Predicted UV-Vis Spectrum

MODEL


UV-Vis Based A-H Assay

Multivariate Modeling - Chemometrics

Openly-available, widely-used technologyCommercial software packages can be purchased Implemented (and in use) in other process industriesApplications: lab, virtual sensors, process optimization

Expected Impact Implemented locally in the winery laboratoryOnce in place, no phenolics wet chemistry analysesEssentially no sample preparationAssay time of one-to-two minutes per sample Ideal for real-time vinification decisions


Development Methodology


PC / Notebook

process analytical instrumentation

(at-line or in-line; UV/Vis, IR, …)

standardized measurements

SAMPLERESULTS

CALIBRATIONSAMPLES

(training and testing)

SPECTRA

MEASURED VALUES

laboratory analytical instrumentation

(lab-based; HPLC, GC/MS, …)

model building & deployment(multivariate; PCR, PLS, ANN,…

)

MRSEC


Validation


PC / Notebook

standardized measurements

SAMPLERESULTS

MEASURED VALUES


)

SPECTRA

FIELDVALIDATION

SAMPLES

TEST SAMPLES



laboratory analytical instrumentation

(lab-based; HPLC, GC/MS, …)

MRSEVor

MRSEP


Deployment


PC / Notebook

SAMPLERESULTS


)

SPECTRA

TEST SAMPLES




The Predictive Model (Ver. 4)


Model Comparisons

Data ranges of current data and Skogerson data

Current Skogerson et al. 2007

Min Max Min Max

Anthocyaninsa 0 1419 0 1096

IRPb 72.6 4979 19.8 2272

Tanninsb 0 2667 -8.1 798

amg/L malvidin-3-glucoside equivalentsbmg/L catechin equivalents

Prediction statistics for the Skogerson et al. (2007) model using our data

RMSEP rpred2 RPD CVpred

Anthocyaninsa 466 0.20 0.5 105.0

IRPb 909 0.38 0.8 63.3

Tanninsb 406 0.33 1.0 70.3

NOTE: Skogerson data was for Australian wines;Current data was for domestic wines.


That being said. . .

Validation statistics for the prediction of phenolic components (n=248)

RMSEP rpred2 RPD CVpred

Anthocyaninsa 149 0.53 1.4 33.0

IRPb 383 0.76 2.1 25.6

Tanninsb 203 0.78 2.1 33.8

amg/L malvidin-3-glucoside equivalentsbmg/L catechin equivalents

There is ample room for improvement!RMSEP: root mean square error of predictionrpred

2: coefficient of determination of the predictionRPD: ratio of standard deviation to standard error of predictionCVpred: coefficient of variation of the prediction


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


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


Acknowledgments

phenolics and tannin assays for practical use in winemaking giovanni colantuoni john thorngate

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