1. 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

2. 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

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

4. 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

5. 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

6. 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”

7. 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

8. 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)

9. 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

10. 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

11. 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

12. 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

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

14. 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

15. 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

16. Red Wine Absorbance
Somers, 1998

17. 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

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

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

20. 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

21. 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

24. Your samples
Comparison samples

25. Tannin: Napa Valley Cabernet

26. 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

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

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

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

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

31. 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