Review of Phenolics in Grapes andOptimizing Their Extraction into Wine

Doug Adams

Department of Viticulture & Enology

University of California, Davis

Flavor 101CImpact 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 describeberry factors that complicate extraction.

BERRY STRUCTUREBERRY STRUCTURE

SEEDSSEEDS• tanninstannins

SKINSKIN• pigmentspigments• tanninstannins

PULPPULP• juicejuice• no pigmentno pigment

Not enough detail for a discussion of extraction

Tissue Types in the Grape (from a phenolics perspective)

1. Epidermis2. Hypodermis

3. Outer mesocarp4. Vascular tissue5. Inner mesocarp

} Skin

} Pulp

Light micrograph of maturegrape berry Scale Bar = 200 µm

F. Famiani et al. J. Exp. Bot.51:675-683 (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 cellsNote 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

OH

OH

OH

O

O

O

OH

OH

OH

OH

O

OH

OH

OH

OH

OH

OH

O

OH

OH

OH

OH

OH

O

OH

OH

OH

OH

OH

Catechin

Epicatechin

Epigallocatechin

Epicatechin Gallate

1

2

3

4

4

8

4

8

8

4

4

Polymeric Flavan-3-olsCondensed Tannins, Proanthocyanidins

Four subunitsIn grape tannins

Very diverse setof compounds

OOH

OH

OH

OH

OH

OOH

OH

OH

OH

OH

OOH

OH

OH

OH

OH

OOH

OH

OH

OH

OH

OOH

OH

OH

OH

OH

OOH

OH

OH

OH

OH

OOH

OH

OH

OH

OH

OOH

OH

OH

OH

OH

B-Tannins

B-1 B-2

B-3 B-4

Simple Dimers of Catechin and EpicatechinNumber of unique chemical structures is 2n

Summary of Flavan-3-ol Composition of Skins and Seeds

Skins have relatively low levels of free catechin andepicatechin. The size of the tannins in skin is muchgreater than in seeds and the polymers contain catechin, epicatechin and epigallocatechin.

Seeds have much higher levels of free catechin andepicatechin. The size of the tannins in seeds is muchsmaller than in skins, and the extension units are mostly epicatechin. Some of the subunits contain gallate esters.

The Anthocyanins in Grape Berries

Anthocyanidin-3-Glucosides R1 R2

Cyanidin OH H

Delphinidin OH OH

Peonidin OCH3 H

Petunidin OCH3 OH

Malvidin OCH3 OCH3

CH3

OO

OH

O

OH

OH

OOH

OH

R1

OH

O-Glucose-

R2+

R3

R3 -Acetyl -p-Coumaroyl -Caffeoyl

O

OH OH

O

OH

O

O

OH

O

OH

O

OH

O

OH

O

O

OH

CH3

O

OH

OH

OH

O

OH

O

O

OH

Trans-p-coumaroyltartaric acid (Coutaric acid)

Trans-caffeoyltartaric acid (Caftaric acid)

Trans-feruloyltartaric acid (Fertaric acid)

The Hydroxycinnamates of Grape

This is the most abundant class of phenolics in white wines

OOH

OH

OH

OH

OH

OOH

OH

OH

OH

OH

Catechin Epicatechin (2,3 Trans) (2,3, Cis)

2

3 3

2

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

R3

Flavonols R1 R2 R3

Kaempherol H H H

Quercetin OH H H

Myricetin OH OH H

Isorhamnetin OCH3 H H

Flavonols are present in the fruit as glucosides, galactosides and glucuronides

where R3 is glucose, galactose or glucuronic acid respectively

OH

O H O

O

R2

O H

R1

O

OH

OH OH

OH

O O

CH3CH3

OH

OH O

OH

OH

OH

Three Stilbenes from Grape

1. 3,4',5-Trihydroxystilbene (Resveratrol)2. 3,5-Dimethoxy-4'-hydroxystilbene3. -Viniferin

1 2 3

Stilbenes are very minor phenolic components

Date 19997/12 7/26 8/9 8/23 9/6 9/20 10/4

0.5

1.0

1.5

Seed TanninSkin Tannin

Date 19988/3 8/17 8/31 9/14 9/28 10/12

Tan

nin

(mg/

Ber

ry)

CE

0.5

1.0

1.5

Seed TanninSkin Tannin

Seed and Skin Tannin in Cabernet Berries in Two Consecutive Years

Changes in the total amounts of themajor individual anthocyanin speciesfound in [Shiraz] grapes during development.

From P.K.Boss, C.Davies and S.P. RobinsonPlant Physiol. 111:1059-1066 (1996)

Note that malvidin glucosides dominatethe anthocyanin profile

Relative Amounts of Flavonoids in Skinsof a Vitis Hybrid

Total Anthocyanin 2.88 µmoles/g 42 % was Malvidin 3,5 diglucoside

Total Hydroxycinnamic acid esters 0.98 µmoles/g77% was Caftaric Acid

Total Flavonol Glycosides 0.19 µmoles/g93% were Quercetin glycosides

Data from A.H Moskowitz and G.Hrazdina Plant Physiol 68: 686-692 (1981)

OH

OHOH

OH

OH

O OH

O

OH

OH O

O

O

OH

OH

OH

OH

OHO

O

OH

OH

OH

OH

OH

O

OH

OH

OH

OH

OH

O

OH

OH

OH

OH

OH

O

OHOH

OH

OH

O

OH

OH

OHO

OH

Resveratrol - Stilbene Caftaric Acid - Cinnamate Ester

Quercetin - Flavonol Catechin - Flavan-3-ol

B Tannin - Flavan-3-ol Dimer

+

Cyanidin-3-Glucoside - Anthocyanin

Fru

it or

Pom

ace

Tan

nin

(mg/

Ber

ry)

0.0

0.5

1.0

1.5

2.0Seeds (4% Extracted)Skins (56% Extracted)

Wine Tannin = 331 mg/L

Harvest Pomace

Tannin in Syrah Fruit and Pomace from a Commercial Fermentation Example 1

Seeds – 4% ExtractedSkins – 56% Extracted

Wine Tannin 331 mg/L

Average Syrah – 484 mg/L

Fru

it or

Pom

ace

Tan

nin

(mg

/ Ber

ry)

0.0

0.4

0.8

1.2

Seeds ( 56% Extracted) Skins (64% Extracted)

Wine Tannin 885 mg/L

Harvest Pomace

Tannin in Syrah Fruit and Pomace from a Commercial Fermentation Example 2

Seeds – 56% ExtractedSkins – 64% Extracted

Wine Tannin 880 mg/L

Average Syrah – 484 mg/L

Tannin Binding by Cabernet Sauvignon Cell WallsAt Three Times During Ripening

Tann

in B

indi

ng C

apac

ity (µ

g/m

g C

WM

)

0

5

10

15

20

25

30

35

SkinMeso.

Skin Mesocarp

Cel

lWal

l Mat

eria

l (m

g/B

erry

)

0

20

40

60

80

100

120

140

SkinMeso.

Skin Mesocarp

Cell Wall Mass and Tannin Binding CapacityIn Cabernet Sauvignon Fruit

Cell Wall Mass Tannin Binding

Ta

nn

in in

Ski

n or

Se

ed

s (m

g/B

err

y)

0

1

2

3

4

5

6

7

8

SkinSeeds

Total Tannin = 7.9 mg/Berry

Tan

nin

Bin

din

g P

oten

tial (

mg

/Ber

ry)

0

1

2

3

4

5

6

SkinMeso.

Total Binding Potential 5.48 mg/Berry

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 matrixconstitutes a powerful fining agent, and plays an important role in tannin extraction during fermentation.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Fruit Tannin Post-Fermentation Tannin

gra

ms

of

Cat

ech

in E

qu

ival

ents

/ K

g F

ruit

Pomace Wine Tank Lees

Tank Lees Seeds Fruit Seeds Fruit Skin

MissingTannin

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)

O

OH

OH

OH

OH

O

O

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

R

Lot 4

-0.5

0.5

1.5

2.5

3.5

4.5

5.5

6.5

Fruit Tannin Post-Fermentation Tannin

gra

ms

of

Cat

ech

in E

qu

ival

ents

/ K

g F

resh

Wei

gh

t

Fruit "Bound"Fruit SkinFruit SeedsTank Lees SeedsTank Lees "Bound"Tank LeesWinePomace "Bound"Pomace

9% extra tannin

Lot 4 WC

-0.5

0.5

1.5

2.5

3.5

4.5

5.5

6.5

Fruit Tannin Post-Fermentation Tanningra

ms

of C

ate

chin

Eq

uiv

ale

nts

/ K

g F

resh

We

igh

t

Fruit "Bound"Fruit SkinFruit SeedsTank Lees SeedsTank Lees "Bound"Tank LeesWinePomace "Bound"Pomace

7% tannin missing

Estimating the amount of irreversibly bound tanninaccounts 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% acetoneTightly bound - observable with acidic butanol and by NMR

The amount of tightly bound tannin increases during fermentation.

Acknowledgements and Thanks

Flavor 101 Organizers

Funding AgenciesAmerican Vineyard Foundation

Viticulture Consortium

Participants