Flavor 101C
Impact of Phenolic Management on Wine Style Options
February 15, 2013
Review of Phenolics in Grapes and
Optimizing Their Extraction into Wine
Doug Adams
Department of Viticulture & Enology
University of California, Davis
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
SKIN
• pigments
• tannins
PULP
• juice
• no pigment
SEEDS
• tannins
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.
51:675-683 (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 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
OH
OH
HO
1
O
OH
4
OH
HO
8
OH
OH
2 Epicatechin
O
8
OH
OH
3
O
4
OH
HO
Four subunits
In grape tannins
OH
4
OH
HO
Catechin
8
OH
OH
OH
Epigallocatechin
OH
4
O
4
Epicatechin Gallate
O
OH
OH
O
OH
OH
Very diverse set
of compounds
Simple Dimers of Catechin and Epicatechin
Number of unique chemical structures is 2n
OH
OH
OH
OH
HO
HO
O
O
OH
OH
OH
OH
HO
OH
OH
OH
HO
O
B-1
O
B-2
OH
OH
OH
OH
OH
B-Tannins
OH
OH
OH
OH
HO
HO
O
O
OH
OH
OH
OH
HO
OH
O
B-3
OH
OH
OH
OH
HO
OH
O
B-4
OH
OH
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
R1
OH
HO
O
Anthocyanidin-3-Glucosides
R1
R2
Cyanidin
OH
H
Delphinidin
OH
OH
Peonidin
OCH3
H
Petunidin
OCH3
OH
Malvidin
OCH3
OCH3
R2
+
O-Glucose-R3
OH
O
O
O
OH
CH3
R3
-Acetyl
OH
-p-Coumaroyl
OH
-Caffeoyl
The Hydroxycinnamates of Grape
O
O
O
Trans-p-coumaroyltartaric acid (Coutaric acid)
HO
HO
OH
OH
O
O
O
O
Trans-caffeoyltartaric acid (Caftaric acid)
HO
HO
OH
OH
O
O
O
O
Trans-feruloyltartaric acid (Fertaric acid)
HO
H3C
HO
O
OH
OH
OH
O
This is the most abundant class of phenolics in white wines
Catechin and epicatechin are the
most abundant flavan-3-ols in grape
OH
OH
OH
HO
O 2
OH
HO
O 2
3 OH
OH
3 OH
OH
Catechin
Epicatechin
(2,3 Trans)
(2,3, Cis)
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
Three Stilbenes from Grape
OH
OH
OH
OH
OH
HO
1
OH
O
CH3
O
2
CH3
HO
3
O
1. 3,4',5-Trihydroxystilbene (Resveratrol)
2. 3,5-Dimethoxy-4'-hydroxystilbene
3. -Viniferin
Stilbenes are very minor phenolic components
OH
Seed and Skin Tannin in Cabernet
Berries in Two Consecutive Years
Tannin (mg/Berry) CE
Seed Tannin
Skin Tannin
1.5
1.5
1.0
1.0
0.5
0.5
Seed Tannin
Skin Tannin
8/3
8/17 8/31 9/14 9/28 10/12
Date 1998
7/12 7/26 8/9 8/23 9/6 9/20 10/4
Date 1999
Changes in the total amounts of the
major individual anthocyanin species
found in [Shiraz] grapes during
development.
Note that malvidin glucosides dominate
the anthocyanin profile
From P.K.Boss, C.Davies and S.P. Robinson
Plant Physiol. 111:1059-1066 (1996)
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: 686-692 (1981)
OH
OH
OH
HO
O
HO O
OH
OH
OH
HO
O
OH
O
HO
HO
OH
OH
B Tannin - Flavan-3-ol Dimer
OH
OH
OH
O
HO
O
Caftaric Acid - Cinnamate Ester
Resveratrol - Stilbene
OH
OH
OH
HO
O
OH
OH
OH
HO
HO
O
+
O
OH
OH
OH
O
OH
OH
O
Quercetin - Flavonol
OH
OH
Catechin - Flavan-3-ol
OH
O
Cyanidin-3-Glucoside - Anthocyanin
OH
Tannin in Syrah Fruit and Pomace
from a Commercial Fermentation
2.0
Seeds (4% Extracted)
Skins (56% Extracted)
Example 1
Fruit or Pomace Tannin (mg/Berry)
Wine Tannin = 331 mg/L
Seeds – 4% Extracted
Skins – 56% Extracted
1.5
1.0
Wine Tannin 331 mg/L
Average Syrah – 484 mg/L
0.5
0.0
Harvest
Pomace
Tannin in Syrah Fruit and Pomace
from a Commercial Fermentation
Example 2
Fruit or Pomace Tannin (mg / Berry)
Seeds ( 56% Extracted)
Skins (64% Extracted)
1.2
Wine Tannin 885 mg/L
Seeds – 56% Extracted
Skins – 64% Extracted
0.8
Wine Tannin 880 mg/L
Average Syrah – 484 mg/L
0.4
0.0
Harvest
Pomace
Tannin Binding by Cabernet Sauvignon Cell Walls
At Three Times During Ripening
% Tannin Removed
100
80
Mesocarp
60
40
Veraison
20 Days
40 Days
20
0
0
20
40
60
Cell Wall Added (mg)
140
Cell Wall Mass
120
Skin
Meso.
100
80
60
40
20
0
35
Tannin Binding Capacity (µg/mg CWM)
CellWall Material (mg/Berry)
Cell Wall Mass and Tannin Binding Capacity
In Cabernet Sauvignon Fruit
30
Tannin Binding
Skin
Meso.
25
20
15
10
5
0
Skin
Mesocarp
Skin
Mesocarp
Comparison of Total Fruit Tannin and Total Binding Capacity
Total Tannin = 7.9 mg/Berry
8
The insoluble Matrix could bind nearly 70%
Of the tannin in the fruit.
6
5
4
3
2
1
0
6
Skin
Seeds
Tannin Binding Potential (mg/Berry)
Tannin in Skin or Seeds (mg/Berry)
7
Total Binding Potential 5.48 mg/Berry
5
4
3
2
1
0
Skin
Meso.
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.
Pomace
Wine
Tank Lees
Tank Lees Seeds
Fruit Seeds
Fruit Skin
grams of Catechin Equivalents / Kg Fruit
5.0
4.5
4.0
Missing
Tannin
20%-60%
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Fruit Tannin
Post-Fermentation Tannin
So where is the missing tannin?
“Bound” Tannins
(Proanthocyanidins):
OH
OH
O
HO
OH
OH
OH
OH
HO
O
OH
OH
OH
OH
O
HO
OH
OH
R
• 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)
6.5
Fruit "Bound"
Fruit Skin
Fruit Seeds
Tank Lees Seeds
Tank Lees "Bound"
Tank Lees
Wine
Pomace "Bound"
Pomace
Lot 4
5.5
4.5
3.5
2.5
1.5
0.5
-0.5
Fruit Tannin
Post-Fermentation Tannin
9% extra tannin
grams of Catechin Equivalents / Kg Fresh Weight
grams of Catechin Equivalents / Kg Fresh Weight
Estimating the amount of irreversibly bound tannin
accounts for most of the ‘missing’ tannin.
6.5
Fruit "Bound"
Fruit Skin
Fruit Seeds
Tank Lees Seeds
Tank Lees "Bound"
Tank Lees
Wine
Pomace "Bound"
Pomace
Lot 4 WC
5.5
4.5
3.5
2.5
1.5
0.5
-0.5
Fruit Tannin
Post-Fermentation Tannin
7% tannin missing
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