Download - Understanding Cap Extraction
6/9/2014
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Understanding Cap Extraction
Max Reichwage, Larry Lerno, Doug Adams, Ravi Ponangi, Cyd Yonker, Leanne Hearne, Anita Oberholster, and David Block
Driving innovation in grape growing and winemaking
Department of Viticulture and Enology
Understanding Cap Extraction in Red Wine Fermentors
• Motivation and key molecules• Methods and experimental design• Chemical gradients in red wine fermentors• Effects of cap and must temperature on phenolic
extraction
Department of Viticulture and Enology
Cap Extraction in Red Wine Fermentors
T
Cap/Skins
Juice
Add a second temp sensor
ColorMonomeric PhenolicsTanninPolymeric Pigment
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Department of Viticulture and Enology
Closer look at the molecules extracted
• Polymeric flavan-3-ols (tannins)• The most abundant class of phenolics in
grapes • Present in skins and seeds
• Anthocyanins• Malvidin-3-glucoside is the predominant
anthocyanin• Found in the skin
• Hydroxycinnamates• Ex: caftaric acid, caffeic acid, coumaric
acid• Found in the skin and pulp
(Adams 2006)
Department of Viticulture and Enology
Closer look at the molecules extracted
• Grape tannins-oligomers• catechin (C), epicatechin (EC),
epigallocatechin (EGC), and epicatechin gallate (ECG)
• Differences between skin and seed tannins
• Mean degree of polymerization (mDP) for skin tannin are ~30; seed tannins are ~10(Souquet et al. 1996)
• Proportion of ECG units is different in seeds (~30%) and skins (~5%) (Cheynier et al. 2006)
Hypothetical tannin tetramer(Adams 2006)
Department of Viticulture and Enology
A molecular mechanism for cap extraction
Epidermal Cell Layer
Hypodermal Cells
Mesocarp Cells
Structural Carbohydrate
Vacuole (containing phenolics)
Cell Nucleus
Release
Monomeric and Polymeric Phenolics
Reaction
Polymeric Phenolics
Readsorption
All steps are likely a function of temperature and EtOH
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Department of Viticulture and Enology
What temperature should we use: Temperature profiles in red wines complicate analysis
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Department of Viticulture and Enology
Cap temperature changes during fermentation and cannot be controlled with cooling jacket
Radial Distance (cm)
20 40 60 80 100 120 140 160 180 200
Dep
th (
cm)
0
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40
60
80
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120
140
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Is the temperature or temperature difference important in extraction?
Department of Viticulture and Enology
Experimental Design: UC Davis/E&J Gallo• Grapes: 2011 Cabernet
Sauvignon from Lodi, CA• 23.3˚Brix, pH of 3.41,
T.A. of 0.47 g/L• Hand-picked, destemmed and
crushed• Inoculated with S. cerevisiae strain
Lalvin D254®• YAN adjusted to 300 ppm, addition
of 50ppm SO2
• Pressed at dryness
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Department of Viticulture and Enology
Experimental Design: Are there chemical gradients too?
• Installed a “curtain” of 66 temperature sensors throughout the cross-section of a 2000 L tank
• 15 sample extraction points
• Fermented 2 Tons of Cabernet Sauvignon
• Pumped-over 1 tank volume 2x per day
• Peristaltic pump on catwalk for sample extraction
Department of Viticulture and Enology
Analyses Performed
1. Phloroglucinolysis of Isolated Tannins- Tannin concentration, mDP, skin/seed contributions
2. RP-HPLC Phenolic Assay- Monomeric phenolic concentrations
Department of Viticulture and Enology
Phloroglucinolysis• Method originally developed by
James Kennedy & Graham Jones (2001)
• Using heat and acidic conditions, the interflavonoid bonds of tannins molecules are cleaved, phloroglucinol acts as a nucleophile
• Allows for the characterization of the subunits that make-up condensed tannins
• Characterized via HPLC• Tannin from wine samples must
be isolated before the phloroglucinolysis reaction
- SPE (Solid Phase Extraction) using Toyopearl© HW-40 resin - SPE was performed in triplicate- Phloglucinolysis rxn was performed in duplicate- For every 1 sample = 6 reps
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Department of Viticulture and Enology
Phloroglucinolysis Reaction• Hypothetical reaction
for the acid catalyzed cleavage of tannin
• Following the phloroglucinolysis reaction, the mixture of C, EC, ECG, EGC, and their Phloroglucinol adducts can be measured via HPLC
HCl, Δ, 20 min. xs phloroglucinol
C-PEC-PEGC-PECG
What does Phloroglucinolysis tell us?1. Tannin concentration2. Average molecular weight3. Mean degree of polymerization (mDP)4. Percent Galloyation
Department of Viticulture and Enology
Fermentation Profile
Normalized y-axis : Brix (1.0 = 25 Brix), Free Anthocyanin (1.0 = 500 mg/L), Tannin (1.0 = 300 mg/L)
Department of Viticulture and Enology
0 100 200 300 400 500 600 700
24 hr 40 hr 48 hr 64 hr 72 hr 88 hr 96 hr 112 hr 120 hr 136 hr
Units = mg/L
Results – Free Anthocyanin
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Department of Viticulture and Enology
Free Anthocyanin Before and After A Pump-Over (Day 3)
100 200 300 400 500 600 700
Units = mg/L
8 am 10 am 11 am 12 pm 1 pm 2 pm 3 pm 4 pm
Department of Viticulture and Enology
Free Anthocyanin Before and After A Pump-Over (Day 5)
100 200 300 400 500 600 700
Units = mg/L
8 am 10 am 11 am 12 pm 1 pm 2 pm 3 pm 4 pm
Department of Viticulture and Enology
Units = mg/L
24 hr 40 hr 48 hr 64 hr 72 hr 88 hr 96 hr 112 hr 120 hr 136 hr
50 100 150 200 250
Tannin
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Department of Viticulture and Enology
100 125 150 175 200
8 am 10 am 11 am 12 pm 1 pm 2 pm 3 pm 4 pm
Units = mg/L
Tannin Before and After A Pump-Over (Day 3)
Department of Viticulture and Enology
100 125 150 175 200
8 am 10 am 11 am 12 pm 1 pm 2 pm 3 pm 4 pm
Units = mg/L
Tannin Before and After A Pump-Over (Day 5)
Department of Viticulture and Enology
• mDP determined by phloroglucinolysis
• Early in the fermentation mDPis higher in the cap
• After 64 hours the mDP is ~13.5 throughout the fermentor
• Later in the fermentation the mDP decreases in the cap
• The error bars represent the “pooled standard deviation” (considers 2 phloro reps x 3 SPE reps x 3 sampling points)
8 9 10 11 12 13 14
40 hr 64 hr 88 hr 112 hr 136 hr
2 possible explanations:1. Skin vs. seed tannin
contribution- Initially, the larger skin tannins are extracted in the cap- Later in the fermentation, the smaller seed tannins are extracted, bringing the mDP down
2. Readsorption of larger tannins to the skin cell wall material- Larger tannin molecules have more reaction sites for hydrogen bonding
Mean Degree of Polymerization (mDP)
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Department of Viticulture and Enology
• Percent galloyationis the percentage of ECG subunits of tannins (determinged with phloroglucinolysis)
• % galloyation is greater in the seeds than the skins
• Increasing percent galloylation in the cap suggests increased extraction of tannins from the seeds
40 hr 64 hr 88 hr 112 hr 136 hr
1 2 3 4 5 6
Percent Galloylation
Department of Viticulture and Enology
• The most prevalent of the hydroxycinnamicacids
• Present in the skins and pulp
• Forms a Caftaric acid gradient quickly (within 40 hours)
• Maximum cap concentration of ~30 mg/L then decreases (equilibrates) to same as bulk liquid
24 hr 40 hr 48 hr 64 hr 72 hr 88 hr 96 hr 112 hr 120 hr 136 hr
5 10 15 20 25 30 35
Units = mg/L
Phenolic Results – Caftaric Acid
Department of Viticulture and Enology
24 hr 40 hr 48 hr 64 hr 72 hr 88 hr 96 hr 112 hr 120 hr 136 hr
0 10 20 30 40 50 60
Units = mg/L
• Increases from ~8 mg/L to ~55 mg/L of catechin in the cap
• Relatively late to extract, could be a marker for seed extraction
Phenolic Results - Catechin
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Department of Viticulture and Enology
• Present in the skins, pulp, and seeds
• Fairly distinct gradient between cap and bulk liquid throughout the fermentation
24 hr 40 hr 48 hr 64 hr 72 hr 88 hr 96 hr 112 hr 120 hr 136 hr
0 4 8 12 16 20
Units = mg/L
Phenolic Results - Gallic Acid
Department of Viticulture and Enology
• Malvidin-3-glucoside (primary color component in red wine)
• Same extraction trend as free anthocyanin
• Small gradients can be noted towards the end of the fermentation
24 hr 40 hr 48 hr 64 hr 72 hr 88 hr 96 hr 112 hr 120 hr 136 hr
50 100 150 200 250 300
Units = mg/L
Phenolic Results – Malvidin-3-glucoside
Department of Viticulture and Enology
• Pigmented polymer = anthocyanin bound to tannin
0 5 10 15 20
Units = mg/L
24 hr 40 hr 48 hr 64 hr 72 hr 88 hr 96 hr 112 hr 120 hr 136 hr
Phenolic Results – Pigmented Polymer
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Department of Viticulture and Enology
Fermentation treatments (2012)
Set Treatment Liquid Temp Cap Temp Cap Management Regime
Control (0.5) 25°C n/a PO 0.5 volume, 2/dayControl (1) 25°C n/a PO 1 volume, 2/dayControl (2) 25°C n/a PO 2 volumes, 2/day
1 20 20
2 25 25
3 30 30
4 35 35
1 20 25
2 25 30
3 30 35
A
As needed to maintain tempB
C As needed to maintain temp
All treatments are in triplicate. Measured phenolic profile at various time points in each fermentation
Department of Viticulture and Enology
Wine production• Grapes: 2012 Cabernet Sauvignon
from Lodi, CA• 24.3°Brix• pH = 3.85• T.A. = 3.8 g/L (adjusted to 5.97
g/L)• YAN adjusted to 300 ppm, addition
of 50 ppm SO2• Inoculated with S. cerevisiae strain
Lalvin D254• Pressed after 14 days – 7-9 days
extended maceration• Sampling: AM & PM till dry, then AM
only• Fermentations performed in
triplicate using Cypress/UC Davis Research Fermentors (TJs)
Department of Viticulture and Enology
Controlling temperature in must and cap
Control the liquid temperature with the jacket and the cap temperature with pumpovers
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Department of Viticulture and Enology
“TJ” Fermentors
Water inlet
Water outlet
IFCS
Density meter
Cap temp probe
Must temp probe
Pump sha
Jacket temp probe
Screen
Department of Viticulture and Enology
Effect of Pumpover Volume
0
10
20
30
40
50
0 48 96 144 192 240 288 336
Ca
aric
acid (m
g/L)
Time (Hours)
Average Ca aric Acid Concentra on
0.5V2X
1V2X
2V2X
0
10
20
30
40
50
60
0 48 96 144 192 240 288 336
Catechin
(mg/L)
Time (Hours)
Average Catechin Concentra on
0.5V2X
1V2X
2V2X
Department of Viticulture and Enology
Effect of Pumpover Volume
0
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150
200
0 48 96 144 192 240 288 336
Mlv‐3‐O
‐glu
(mg/L)
Time (Hours)
Average Malvidin‐3‐O‐glucoside Concentra on
0.5V2X
1V2X
2V2X
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Department of Viticulture and Enology
Effect of Cap and Liquid Temperature
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Ca
aric
acid (m
g/L)
Time (Hours)
Average Ca aric Acid Concentra on
20M20C
25M25C
30M30C
35M35C
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Catechin
(mg/L)
Time (Hours)
Average Catechin Concentra on
20M20C
25M25C
30M30C
35M35C
Department of Viticulture and Enology
The effect of cap and liquid temperature
0 50
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0 48 96 144 192 240 288 336
Tan
nin
(mg/L)
Time (Hours)
Average Tannin Concentra on
20M20C
25M25C
30M30C
35M35C
0
10
20
30
40
50
0 48 96 144 192 240 288 336
Pigment.
Poly. (mg/L)
Time (Hours)
Average Pigmented Polymer Concentra on
20M20C
25M25C
30M30C
35M35C
Department of Viticulture and Enology
The effect of cap and liquid temperature
0
50
100
150
200
0 48 96 144 192 240 288 336
Mlv‐3‐O
‐glu
(mg/L)
Time (Hours)
Average Malvidin‐3‐O‐glucoside Concentra on
20M20C
25M25C
30M30C
35M35C
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Department of Viticulture and Enology
Is the main driver for extraction the liquid temperature or cap temperature?
0
10
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30
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70
0 48 96 144 192 240 288 336
Catechin
(mg/L)
Time (Hours)
Average Catechin Concentra on
20M20C
25M25C
30M30C
35M35C
Department of Viticulture and Enology
Is the main driver for extraction the liquid temperature or cap temperature?
0
10
20
30
40
50
60
70
0 48 96 144 192 240 288 336
Catechin
(mg/L)
Time (Hours)
Average Catechin Concentra on
20M20C
25M25C
30M30C
35M35C
20M25C
25M30C
30M35C
Department of Viticulture and Enology
Liquid temperature or cap temperature?
0
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150
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250
300
350
400
450
0 48 96 144 192 240 288 336
Tannin
(mg/L)
Time (Hours)
Average Tannin Concentra on
20M20C
25M25C
30M30C
35M35C
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Department of Viticulture and Enology
Liquid temperature or cap temerpature?
0
50
100
150
200
250
300
350
400
450
0 48 96 144 192 240 288 336
Tannin
(mg/L)
Time (Hours)
Average Tannin Concentra on
20M20C
25M25C
30M30C
35M35C
20M25C
25M30C
30M35C
Department of Viticulture and Enologya)
b)
c)
d)
Department of Viticulture and Enology
a)
b)
c)
d)
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Department of Viticulture and Enology
Summary• Chemical gradients were observed for a number of
important compounds • Extraction of compounds located in the skin appears
to occur early in fermentation• Could early cap tannin be a marker for final tannin levels?
• Extraction of compounds located in the seeds appears to occur later in the fermentation and is more temperature dependent
• Liquid temperature seems to have more effect than pumpover volume on extraction
Understanding the mechanism of extraction will allow better manipulation of phenolic profiles
Department of Viticulture and Enology
Fermentation treatments (2013)
Set Treatment Liquid Temp Cap Temp Cap Management Regime
1 25 NC 1/2 vol 8x per day2 25 NC 1 vol 4x per day3 25 NC 2 vol 2x per day4 25 NC 4 vol 1x per day1 1 day Cold Soak, 25 NC 2 vol 2x per day2 4 day Cold Soak, 25 NC 2 vol 2x per day3 7 day Cold Soak, 25 NC 2 vol 2x per day4 10 day Cold Soak, 25 NC 2 vol 2x per day
A
B
All treatments are in triplicate. Measured phenolic profile at various time points in each fermentation
Department of Viticulture and Enology
Acknowledgements• Dr. Roger Boulton• Chik Brenneman• Paul Green• Tim Jones• Dr. Cary Doyle• John Schadt• Tom Bell (Bainer Hall
Machine Shop)
• Collin Chew
• Dustin Owens
• The Adams Lab
• The Ebeler Lab
• E&J Gallo Winery:
– Dr. Leanne Hearne
– Dr. Ravi Ponagi
– Dr. Tom Pugh
– Dr. Nick Dokoozlian
– Cynthia Yonker
– Winery Interns: Daina, Jeff, Ben, Laura, and Quinton
Funding Sources:- E&J Gallo Winery- UC Davis Federal
Block Grant- Harry Baccigaluppi
Scholarship- Brad Webb
Memorial Scholarship
- Wine Spectator Scholarship