Review of Phenolics in Grapes and Optimizing Their Extraction into Wine Doug Adams Department of Viticulture & Enology University of California, Davis Flavor 101C Impact of Phenolic Management on Wine Style Options February 15, 2013

Organization of the Talk 1.Major phenolics in the grape berry 2. Extraction during winemaking Review the phenolic classes and describe berry factors that complicate extraction.

BERRY STRUCTURE SEEDS tannins tannins SKIN pigments pigments tannins tannins PULP juice juice no pigment no pigment Not enough detail for a discussion of extraction

Tissue Types in the Grape (from a phenolics perspective) 1.Epidermis 2.Hypodermis 3.Outer mesocarp 4.Vascular tissue 5.Inner mesocarp } Skin } Pulp

Light micrograph of mature grape berry Scale Bar = 200 µm F. Famiani et al. J. Exp. Bot. 51: (2000)

Pericarp at Day 84 (‘Traminer’) (Hardie et al., 1996) Cross section of berry 84 days after flowering Note the polyphenolic deposits in the hypodermal cells Note the size and shape of the hypodermal and mesocarp cells.

Exocarp at Day 126 (‘Traminer’) (Hardie et al., 1996) Cross section of berry showing epidermal and hypodermal cells (the skin) Note the wall thickness of the cells with polyphenolic deposits

Tissue Types in the Grape (from a phenolics perspective) 6. Cuticle 7. Parenchyma 8. Testa 9. Aleurone? 10. Endosperm Seed

Polymeric Flavan-3-ols Condensed Tannins, Proanthocyanidins Four subunits In grape tannins Very diverse set of compounds

Simple Dimers of Catechin and Epicatechin Number of unique chemical structures is 2 n

Summary of Flavan-3-ol Composition of Skins and Seeds Skins have relatively low levels of free catechin and epicatechin. The size of the tannins in skin is much greater than in seeds and the polymers contain catechin, epicatechin and epigallocatechin. Seeds have much higher levels of free catechin and epicatechin. The size of the tannins in seeds is much smaller than in skins, and the extension units are mostly epicatechin. Some of the subunits contain gallate esters.

The Anthocyanins in Grape Berries

The Hydroxycinnamates of Grape This is the most abundant class of phenolics in white wines

Catechin and epicatechin are the most abundant flavan-3-ols in grape Most of the free catechin and epicatechin is found in the seeds.

The Flavonols Found in Grape Berries

Stilbenes are very minor phenolic components

Changes in the total amounts of the major individual anthocyanin species found in [Shiraz] grapes during development. From P.K.Boss, C.Davies and S.P. Robinson Plant Physiol. 111: (1996) Note that malvidin glucosides dominate the anthocyanin profile

Relative Amounts of Flavonoids in Skins of a Vitis Hybrid Total Anthocyanin 2.88 µmoles/g 42 % was Malvidin 3,5 diglucoside Total Hydroxycinnamic acid esters 0.98 µmoles/g 77% was Caftaric Acid Total Flavonol Glycosides 0.19 µmoles/g 93% were Quercetin glycosides Data from A.H Moskowitz and G.Hrazdina Plant Physiol 68: (1981)

Tannin in Syrah Fruit and Pomace from a Commercial Fermentation Example 1 Seeds – 4% Extracted Skins – 56% Extracted Wine Tannin 331 mg/L Average Syrah – 484 mg/L

Tannin in Syrah Fruit and Pomace from a Commercial Fermentation Example 2 Seeds – 56% Extracted Skins – 64% Extracted Wine Tannin 880 mg/L Average Syrah – 484 mg/L

Tannin Binding by Cabernet Sauvignon Cell Walls At Three Times During Ripening

Cell Wall Mass and Tannin Binding Capacity In Cabernet Sauvignon Fruit Cell Wall MassTannin Binding

Comparison of Total Fruit Tannin and Total Binding Capacity The insoluble Matrix could bind nearly 70% Of the tannin in the fruit.

We Must Be Cautious With Our Interpretation Because: Tannin binding is concentration dependent Tannin binding is reduced by ethanol Tannin binding is reduced at higher temperatures Measurements made prior to fermentation Nevertheless, the results indicate that the insoluble matrix constitutes a powerful fining agent, and plays an important role in tannin extraction during fermentation.

Missing Tannin 20%-60% So where is the missing tannin?

“Bound” Tannins (Proanthocyanidins): excess HCl in butanol with ferrous sulphate used as reaction mixture 95ºC for 1 hour Abs at 550nm, gives quantification in Absorbance Units (AU) Cons: Doesn’t measure terminal unit of proanthocyanidin Not directly quantifiable in common “tannin” terms (Ferric chloride positive)

9% extra tannin 7% tannin missing Estimating the amount of irreversibly bound tannin accounts for most of the ‘missing’ tannin.

Small Experimental Fermentation

Current Understanding of Tannin Extraction The tannins of the skin and seed are highly water soluble. The skin epidermis and the seed cuticle are effective extraction barriers. The cell walls of the berry constitute a powerful tannin fining agent. The insoluble matrix preferentially binds larger tannins. (larger tannins are more astringent on a weight basis) There are two classes of tannins on the insoluble matrix after fermentation. Loosely bound - extractable with 70% acetone Tightly bound - observable with acidic butanol and by NMR The amount of tightly bound tannin increases during fermentation.

Acknowledgements and Thanks Flavor 101 Organizers Funding Agencies American Vineyard Foundation Viticulture Consortium Participants