Phenolic Assays: What do the Numbers Mean? James A. Kennedy Department of Viticulture and Enology California State University, Fresno Wine Flavor 101C The Impact of Phenolic Management on Wine Style Options February 15, 2013 Davis, CA

Outline Phenolic compounds Analyzing for phenolics General wine phenolic composition Specific compounds in grapes/wine and importance Analyzing for phenolics Non-specific Specific Combination Using analytical information Fruit development Fermentation/Maceration Aging Future trends

Wine Composition-Phenolics Waterhouse, A., Ann. N.Y. Acad. Sci. 2002, 957:21-36

Volatile Phenols Low molecular weight, volatile 4-ethylphenol Low molecular weight, volatile Associated with Brettanomyces sp. Infections 4-ethylphenol (4-EP), 4-ethylguaiacol (4-EG) Increase in proportion of 4-EG related to spiciness >500 g/L generally detectable, 3000 g/L high Brett. Smoke tainted wines Guaiacol, 4-methylguaiacol, 4-EP, 4-EG Glycosides (non-volatile) also present Analyzed by GC-MS or HPLC Concentrations Clean wine: not detectable Problematic wines: free guaiacol >200 g/L

Hydroxycinnamic Acids t-caftaric acid Present in all wines (present in the mesocarp) Produced as tartaric acid esters Major hydroxycinnamic acid ester is trans-caftaric acid ester Tartaric acid portion hydrolyzed over time Vicinal dihydroxy phenol susceptible to oxidation Glutathione adduct of caftaric acid (GRP) can provide information on oxidation of must/juice Useful for white wine production Involved in white wine color

Stilbenes t-resveratrol Belong to a group of compounds called phytoalexins (antimicrobials) Upregulated in grapes in response to fungal infection trans-resveratrol most abundant Glycosides (piceids) and polymers (viniferins) also found Provides indication of “rot” presence Some monitor for labeling purposes “wine and health”

Catechins Other names: Almost exclusively seed-derived flavan-3-ol monomers, (+)-catechin, (-)-epicatechin Almost exclusively seed-derived Ratio of [catechin] to [tannin] can provide information on relative seed extraction High concentrations suggestive of bitterness Concentration declines with fruit maturity Amount relative to extractable tannin concentration also declines (-)-epicatechin

Flavonols Present as glycosides in grape, partially cleaved in wine quercetin Present as glycosides in grape, partially cleaved in wine Concentration in grapes provides information on fruit exposure Indirectly related to exposure and flavor Concentration in wine indicates skin extraction (can be useful in white wine production). Poorly soluble in wine Low color and low tannin wines, with high flavonol concentrations are prone to physical instability (e.g.: Sangiovese)

Anthocyanins Responsible for initial color in red wine malvidin-3-glucoside Responsible for initial color in red wine Higher concentrations associated with darker red wine Several equilibrium forms pH sensitive Colored and non-colored forms Red form is not the major component

Tannins Other names: Responsible for astringency in red wine Proanthocyanidins, procyanidins, prodelphinidins, condensed tannins, leucoanthocyanidins Responsible for astringency in red wine Found in skin, seed, pulp and stem tissue Pulp tannin generally not present in wine Stem tannin present only when extracted Concentration related to perceived astringency Lower molecular weight tannins (less than 600 MW) considered bitter Considered to be essential for long term color stability in red wine. procyanidin B2

Pigmented Tannin Other names: Stable form of red wine color Pigmented polymer, polymeric color, polymeric anthocyanins Stable form of red wine color Generally thought to be a softening component of red wine astringency

Other Phenolic Products Generally of interest from mouthfeel and color stability standpoint. Active area of research Structure identification Sensory relevance oxidation products vitisin A acetaldehyde – vitisin B decarboxylated hydroxycinnamic acids – pinotins vinylcatechin-vitisin A - portisins

For phenolic analytical information to have value, results should relate (directly or indirectly) to perception.

Analyses and Maturity/Management Red Wines Grapes Catechin/tannin Flavonols Anthocyanins Phenolic extractability Smoke taint Fermentation/Maceration Anthocyanins/Pigmented tannin Tannin Aging Flavonols (phys. stability) Volatile phenols White Wines Grapes Flavonols Smoke taint Catechin/tannin Fermentation/Maceration GRP/caftaric Catechins (solids contact) Polymer at 360 nm Aging

Analytical Methodology - non specific - UV/Vis absorbance spectrophotometry Relates absorbance information to phenolic composition UV/Vis of wine at single wavelength (360, 420, 520, 620 nm) Folin Ciocalteu Disadvantages With exception of red color, measurements have historically lacked specificity Advantages Generally rapid and cheap Pretreatment can provide specificity (e.g.: protein precipitation) Subsequent direct UV/Vis measurement at multiple wavelengths can provide specificity Useful for color and tannin measurements

Red Wine Absorbance Somers, 1998

Analytical Methodology - specific - Specific phenolic compound(s) are separated from the matrix and quantified Volatile phenols: gas chromatography 4-EP, 4-EG . . . Non-volatile phenols: HPLC Hydroxycinnamic acids Flavonoids Advantages Excellent specificity Disadvantages Expensive

Phloroglucinol-Subunit Adduct Analysis Kennedy and Jones, J. Agric. Food Chem., 2001

Berry Integrity: Skin and Seed Tannin Extraction Cerpa-Calderon and Kennedy, J. Agric. Food Chem., 2008

Wine Analysis: Using HPLC-Based Data catechin tannin polymeric anthocyanins total anthocyanins catechin tannin ratio A 23 Oct 6 799 51 1097 0.01 B 25 Oct 7 621 32 1128 1 Nov 19 1072 944 0.02 C 24 Oct 35 390 23 925 0.09 26 Oct 70 888 39 1003 0.08 92 1313 58 809 0.07 10 Brix

Combination Combines simplicity of UV/Vis absorbance, with specificity Has been very useful in assessing tannins in red wine Measurement based upon precipitating tannins from solution and measuring absorbance of wine before and after precipitation Measurement of absorbance at multiple wavelengths Relating to precipitation has resulted in correlation of UV/Vis absorbance information to tannin concentration Enartis/Vinquiry: MCP Wine X-ray: A-H

Your samples Comparison samples

Tannin: Napa Valley Cabernet

Future Trends Phenolic structure-relationships are reasonably well developed Access to analytical information is increasing In house: UV/Vis spectrophotometry Analytical labs: Enartis/Vinquiry, ETS Labs, Wine Xray Challenges Reduce cost and increase throughput further Astringency quality in vineyard and winery

Tannin-Protein Interaction Most analytical methods measure tannin concentration/composition and use information to predict astringency

Tannin Response to Temperature Barak and Kennedy, Anal. Chem., In Review

Tannin Response to Temperature Barak and Kennedy, Anal. Chem., In Review

Tannin: Interaction vs. Size Increasing “grippiness” Decreasing Tannin Size Barak and Kennedy, Anal. Chem., In Review

Acknowledgements UC Davis Anita Oberholster Linda Bisson Kay Bogart Analytical Laboratories AWRI Paul Smith Enartis Vinquiry John Katchmer ETS Labs Gordon Burns Steve Price Funding American Vineyard Foundation