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


Cap Extraction in Red Wine Fermentors

T

Cap/Skins

Juice

Add a second temp sensor

ColorMonomeric PhenolicsTanninPolymeric Pigment

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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)


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)


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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What temperature should we use: Temperature profiles in red wines complicate analysis

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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

20

40

60

80

100

120

140

18 20 22 24 26 28

Is the temperature or temperature difference important in extraction?


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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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


Analyses Performed

1. Phloroglucinolysis of Isolated Tannins- Tannin concentration, mDP, skin/seed contributions

2. RP-HPLC Phenolic Assay- Monomeric phenolic concentrations


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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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


Fermentation Profile

Normalized y-axis : Brix (1.0 = 25 Brix), Free Anthocyanin (1.0 = 500 mg/L), Tannin (1.0 = 300 mg/L)


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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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


Free Anthocyanin Before and After A Pump-Over (Day 5)

100 200 300 400 500 600 700

Units = mg/L



Units = mg/L


50 100 150 200 250

Tannin

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100 125 150 175 200


Units = mg/L

Tannin Before and After A Pump-Over (Day 3)


100 125 150 175 200


Units = mg/L

Tannin Before and After A Pump-Over (Day 5)


• 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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• 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


• 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


5 10 15 20 25 30 35

Units = mg/L

Phenolic Results – Caftaric Acid



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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• Present in the skins, pulp, and seeds

• Fairly distinct gradient between cap and bulk liquid throughout the fermentation


0 4 8 12 16 20

Units = mg/L

Phenolic Results - Gallic Acid


• 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


50 100 150 200 250 300

Units = mg/L

Phenolic Results – Malvidin-3-glucoside


• Pigmented polymer = anthocyanin bound to tannin

0 5 10 15 20

Units = mg/L


Phenolic Results – Pigmented Polymer

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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


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)


Controlling temperature in must and cap

Control the liquid temperature with the jacket and the cap temperature with pumpovers

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“TJ” Fermentors

Water inlet

Water outlet

IFCS

Density meter

Cap temp probe

Must temp probe

Pump sha

Jacket temp probe

Screen


Effect of Pumpover Volume

0

10

20

30

40

50

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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


Effect of Pumpover Volume

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

0.5V2X

1V2X

2V2X

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Effect of Cap and Liquid Temperature

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

20M20C

25M25C

30M30C

35M35C

0

10

20

30

40

50

60

70

0 48 96 144 192 240 288 336

Catechin

(mg/L)

Time (Hours)


20M20C

25M25C

30M30C

35M35C


The effect of cap and liquid temperature

0 50

100 150 200 250 300 350 400 450

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


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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Is the main driver for extraction the liquid temperature or cap temperature?

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Catechin

(mg/L)

Time (Hours)


20M20C

25M25C

30M30C

35M35C


Is the main driver for extraction the liquid temperature or cap temperature?

0

10

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0 48 96 144 192 240 288 336

Catechin

(mg/L)

Time (Hours)


20M20C

25M25C

30M30C

35M35C

20M25C

25M30C

30M35C


Liquid temperature or cap temperature?

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450

0 48 96 144 192 240 288 336

Tannin

(mg/L)

Time (Hours)


20M20C

25M25C

30M30C

35M35C

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Liquid temperature or cap temerpature?

0

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100

150

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350

400

450

0 48 96 144 192 240 288 336

Tannin

(mg/L)

Time (Hours)


20M20C

25M25C

30M30C

35M35C

20M25C

25M30C

30M35C

Department of Viticulture and Enologya)

b)

c)

d)


a)

b)

c)

d)

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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


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


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

understanding cap extraction

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