2/22/2012

1

Methoxypyrazines, etc…

Gavin L. Sacks*, Justine Vanden Heuvel, Bruce Pan, Justin

Scheiner, Imelda Ryona, Sarah Harris

* Cornell University, Department of Food Science, NYS Agricultural

Experiment Station, Geneva, New York 14456

2012 Washington Association of Wine Grape Growers Meeting

Acknowledgements

Sacks Lab

Imelda Ryona (right)

Dr. Bruce Pan (left)

Sarah Harris

Dr. Justine Vanden Heuvel

Justin Scheiner

Dr. Alan Lakso

Dr. Diego Intrigliolo Projects were supported by:

New York Wine and Grape Foundation

Viticulture Consortium East

USDA - Federal Formula Funds

Kaplan Research Fund

Oren Kaye and John Thorngate

(Constellation Brands)

Fundamental Theorem of Wine Aroma (FTWA)

“Vegetal”, “Herbaceous” “Fruity”, “Sweet”

Unripe aromas and ripe aromas mask each other

(and, excessive amounts of either is a problem)

Masking and wine

2/22/2012

2

“Vegetal”, “Herbaceous” “Fruity”, “Sweet”

Balanced wines lie along this fruity, vegetal

continuum

Sauvignon

Blanc

Muscat

Most Dry Reds Carmenere

Excessive green more likely to be cited as a

problem, at least for East coast reds

Vegetal Herbal Earthy

Fractional usage of

term in tasting notes

Adapted from data compiled by Larry Perrine at Channing Daughters Winery

75-85 points (182 wines)

85-90 points (85 wines)

.09

.00

.21

.07 .07 .04

Low scoring wines more than

3x likely to have earthy,

vegetal, herbal descriptors

Wine Spectator scores and tasting notes for Long

Island red wines

Wine Spectator scores: continued

Ripe

Fractional usage of

term in tasting notes

Adapted from data compiled by Larry Perrine at Channing Daughters Winery

75-85 points (182 wines)

85-90 points (85 wines)

.16

.00

Fruit

1.32

.58 High scoring wines nearly 3x

more likely to have ripe, fruit

descriptors

2/22/2012

3

The most notorious contributor to green

3-Alkyl-2-methoxypyrazines (MPs)

Detection threshold for in wine

IBMP: 5-15 ng/L

IPMP: 0.5-2 ng/L

Most closely associated with “Bordeaux” wine

grapes: Cabernet, Merlot, Sauv blanc

N

N O C H 3

R

R Abbr Typical conc. in

Cabernet (ng/L) Aroma

isobutyl IBMP 5-20 Bell pepper, vegetal Predominant MP in

immature grapes

sec-butyl sBMP n.d. - 1 Peas, potatoes

isopropyl IPMP n.d. – 2 Asparagus, peas Predominant MP in

Asian lady beetle

R = alkyl group

Overview of talk today

Overview of strategies in the winery

Previous attempts

Something new: juice silicone fining

Controlling MPs in the vineyard

Empirical studies of accumulation and degradation

Moving towards mechanistic explanations (relating MP

concentrations to precursors)

Future directions

MPs and winemaking: extraction during maceration

0

2

4

6

8

10

0 20 40 60

Fermentation time (hours)

IBMP

(ng/L) MPs mostly in skins,

extraction kinetics

comparable to or slightly

faster than anthocyanins

Adapted from Roujou de Boubee 2001 thesis

2/22/2012

4

Good correlation between IBMP in whole grapes and

skin fermented wines

Ryona, Pan, Sacks (JAFC, 2009)

R2 = 0.96

MPs are stable in the bottle

(n=12 wines)

IBMP in wine vs.

IBMP in same wine

14 months later

(Pan and Sacks, unpublished)

Several ways to remove MPs from wines,

but none have selectivity

EXAMPLES:

Activated charcoal

Distillation/thermovinification

- MPs are readily extracted during fermentation, stable in

the bottle, challenging to remove selectively

- End result: attempts to achieve MP remediation on

finished wines have been unsuccessful

2/22/2012

5

Approach Matrix Reduction Caveats

Fluorescent & UV lights Wine ns No effect

Yeast selection Juice ns No effect

Oak sawdust, gallic acid,

epicathechin

Model

Juice

ns No effect

Synthetic closures Wine 70 - 80% Lack of selectivity (?)

Packaging – Tetrapack Wine 26 - 45% Lack of selectivity (?)

Activated charcoal,

deodorized oak chips,

bentonite

Wine ns – 34% Lack of selectivity

Thermovinification Juice 29-67% Sensory changes

Selectively reducing MPs after harvest: an

exercise in futility?

Data collected from several publications

Other recent ideas for MP removal

Flash détente - for grapes

Almost certainly removes C6 aldehydes

Data for MPs?

Odorant Binding Protein (OBP) – for wine or juice

Pickering et al 2010 patent

No literature reports yet

Silicone fining of grapes

Our group

Reminder: Classifications of Wine Flavors

Primary – Derived from grape (“foxy” aromas, MPs)

Secondary – Produced during alcoholic or malolactic

fermentations

• Byproduct of yeast biosynthesis

• Enzymatic transformation of non-volatile precursors from

substrate (e.g. terpenes)

Tertiary – Develop post-fermentation

• Oak

• Abiotic transformations during cellaring

2/22/2012

6

Could we use a not-so-selective sorbent to remove MPs

before fermentation without removing other volatiles?

Wine by GC-MS (>1000 detectable

compounds)

Grape Juice by

GC-MS (~100 compounds)

Idea: Fine juice/must with food grade silicone (PDMS)

prior to fermentation to remove MPs

Trials

2007 Cabernet Franc Rose

2008 Cabernet Franc Red

2008 Chardonnay White

2010 Riesling White

1. Add chopped silicone tubing to

juice (up to 40 g/L)

2. Inoculate, add nutrients

3. Remove silicone

4. End of Fermentation, <1 g/L RS

Standard Sequence

Why it works: polyphenolics & most grape aroma

precursors are semi-polar; MPs are non-polar

Example results. Ryona, Reinhardt and Sacks, Food Res. Int., 2012 in press

0

1

2

3

4

5

6

7

8

9

0 5 10 15 20 25 30 35 40 45

IBM

P c

on

ce

ntr

ati

on

(n

g/L

)

Silicone contact (hour)

No Silicone (15 L)

Silicone (15 L)

No Silicone (45 L)

Silicone (45 L)

2007 Cabernet Franc Rosé

Generally, 50-90% reduction in MPs; no significant reduction of

other wine volatiles

>90%

reduction

2/22/2012

7

Results from 4 trials over 3 years: Silicone treatment of

must reduces MPs, does not reduce most other volatiles

0

20

40

60

80

100

120

140

0 g/L 10 g/L 20 g/L 40 g/L

Nor

mal

ized

val

ue (%

)

nsns ns ns* * * *

A

BC

D

ABBAB

A

A

B AB

AB

AB

AB

B

A

Silicone added to must

>70 volatiles

profiled by SPE-

GC-MS

MPs reduced in

dose-response

fashion

Most secondary

volatiles

(fermentation

esters, some

terpenes) not

affected

Data for 2010 Riesling with “ladybug taint” shown above

3

4

5

6

7

8

0 2 4 6 8 10 12

0 g/L

4.4g g/L

13.3 g/L

40.0 g/L

0 2 4 5 6 7 11 * ns ns ns * * ns

Results for red winemaking

Still works, but more complicated

With reds, MPs

must first be

extracted from

skins into liquid

phase

(left) Must held

cold, then warmed

and inoculated

after 48 hours.

Silicone removed

after Day 5

2007 Cabernet Franc Rosé

(native IBMP)

> 90% Informal evaluation, less fresh green note on

treated wine

2010 Riesling

(IPMP from MALB)

56% 90% of ~100 wine industry workshop

participants select silicone treatment as less

affected by MALB taint.

Formal sensory trial: forced choice test for

‘greenness’ showed significant effect

2008 Cabernet Franc Red

(Native IBMP)

53% Informal evaluation, less green note

2008 Chardonnay white

(Spiked in IPMP)

93% Informal evaluation, less MALB taint

MP reduction and sensory evaluation

2/22/2012

8

Issues with silicone fining

Not approved for commercial wine production by TTB

Currently, using chopped pieces of food grade tubing.

Want to identify a higher surface area, more convenient

form (but still food-grade!).

Not appropriate for wines that rely on primary aromas

e.g. rotundone (“black pepper”) Labrusca-type, Muscat

Other polymers more effective?

Any health concerns?

Fermentation kinetics and color are not affected

Alternative to control in the winery:

Controlling MPs in the vineyard

General observations regarding MPs at harvest

and growing conditions (from the last two decades)

Lower concentrations with higher growing temperatures

Lower concentrations with higher cluster light exposure

Lower concentrations at less vigorous sites

Bogart and Bisson, 2006

Mechanistic explanations for these events were

scattered or non-existent

MP research at Cornell: distinguishing

accumulation from degradation

Time

IBM

Pconcentr

ation

Veraison

Accumulation

Degradation

Project 1: Role of

cluster exposure?

(“burn off the MPs?”)

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9

Variably shoot-thinned vine experiments

(2007 and 2008)

- Compared shaded and unshaded

berries within the same vine

- Accounts for vine-to-vine variation

Block I Block II Block III

Exposed Exposed ExposedExposed Shaded ShadedShaded

Unshaded Shaded

Two years of study in Finger Lakes

(2007 and 2008)

Evidence that cluster sun exposure pre-veraison will

inhibit MP accumulation

Little evidence that cluster exposure post-veraison

impacts MP degradation

Exposed fruits

accumulate

less IBMP 0

50

100

150

200

250

300

350

400

10 20 30 40 50 60 70 80 90 100 110 120

IBM

P c

on

cen

trat

ion

(p

pt)

Days post-bloom

Shaded

Exposed

Days From Bloom

IBM

P (

pp

t)

Adapted from Lakso and Sacks, Kliewer Symposium, 2009

2008 Shaded

2008 Exposed

0

50

100

150

200

250

5 15 25 35 45 55 65 75 85 95 105 115 125 135 145

IBM

P (

pp

t )

Days post-bloom Ryona, et.al. J. Ag Food Chem, 2008

2007 Shaded

2007 Exposed

Cluster exposure

to sunlight

2/22/2012

10

2007: Constant difference in MP between

exposed and shaded clusters during season

**P<0.05

*P<0.08

veraison

y = 0.1081x - 0.572

R2 = 0.9367

0

5

10

15

20

25

0 50 100 150 200 250

IBMP concentration on Day 47 (picogram/gram)

IB

MP

co

ncen

tra

tio

n a

t h

arv

est

(pic

og

ra

m/g

ra

m)

The importance of accumulation: Within a region, at same

maturity, MP at harvest reflects MP at veraison

Pre-veraison IBMP concentration (ppt)

Harv

est IB

MP

concentr

ation (

ppt)

13 sites on Seneca Lake

Ryona, et.al. J. Ag Food Chem, 2008

Experiment: Variable timing of leaf removal at two sites

- Cabernet Franc (Finger Lakes), 2007 and 2008

- Merlot (Long Island), 2008

J. Scheiner, S. Ennahli, A Wise, L Tarleton, B. Pan, G. Sacks, and J.

Vanden Heuvel; AJEV, 2010

Importance of exposure in pre-veraison period:

Corroboration from field studies

Summary of results: Reduction in IBMP at harvest

consistently observed only with early season interventions

- at berry set

- 30 days after berry set

{Recent corroborating results from UC Davis using shadecloth:

A. Koch, et al, Phys. Plantarum, 2012}

2/22/2012

11

The problem with the “Cluster Shading is the Lone

Gunman” explanation for differences in MPs

Pre-veraison IBMP (ppt)

13 sites on Seneca Lake

y = 0.1081x - 0.572

R2 = 0.9367

0

5

10

15

20

25

0 50 100 150 200 250

IBMP concentration on Day 47 (picogram/gram)

IB

MP

co

ncen

tra

tio

n a

t h

arv

est

(pic

og

ra

m/g

ra

m)

0 250

25

0

Harv

est IB

MP

(ppt)

125

Within a site, cluster

shading results in in

MPs

But, within a region,

we see nearly an

order of magnitude of

range in pre-veraison

and harvest IBMP!

Multivariate Field Study

“What variables really matter?”

Along with:

Justine Vanden Heuvel

Justin Scheiner

10 sites in NYS

2 Long Island

2 Lake Erie

6 Finger Lakes

10 vines at each site

2 x 5 vine panels

Scheiner, et al. AJEV 2012

Over 100 viticultural & environmental factors

characterized for each vine (e.g. light, water status,

cropload, etc.. at multiple time points)

MPs measured at harvest and 2x pre-veraison

Data analyzed by multivariate statistics (PLSR)

2/22/2012

12

IBMP accumulation pre-veraison independently

associated with

• higher temperature(!)

• greater water availability

• and to a lesser extent, cluster shading

IBMP degradation post-veraison correlates with

• maturity (sugar accumulation, etc)

• and not much else

Summary of 2008-09 Multivariate Studies

Scheiner, Vanden Heuvel, Pan, and Sacks. AJEV 2012

IBM

P c

once

ntr

ation (

pg

/g)

2010

very warm, wet 2009

cool & wet

Peak

Harvest

warm & dry warm & wet

Single site Multiple NY sites

0

100

200

300

400

500

600

2008 2007

Peak

Harvest

Surprise: warmer seasons = more IBMP

accumulation, although faster degradation

Highest pre-veraison IBMP our lab has ever seen: > 800 ppt

from Central Valley (CA) Merlot and Cabernet Sauvignon

MP in warm vs. cool climate, assuming

adequate water: a proposal

IBMP – warmer

sites

Time

IBMP –

cooler

sites IBM

P c

oncentr

ation

2/22/2012

13

If conditions that promote vine growth increase

MPs, can this inform viticultural strategies?

Idea 1: Shoot tipping on Cabernet franc

3 timings of shoot tipping

10 days before anthesis (10DBA)

anthesis (AN)

10 days after anthesis (10 DAA)

0

5

10

15

20

25

30

10 DBA

v 2009 2010

Harv

est IB

MP

(pg/g

)

Anthesis 10 DAA CON

Observations over two

years: treatment may

result in slight increase

pre-veraison, no sig

effect at harvest Scheiner, et al unpublished data

If conditions that promote vine growth increase

MPs, can this inform viticultural strategies?

Idea 2: Growth inhibitor (chlormequat)

2 application rates (400 and 800 mg/L)

3 application timings

7 days before anthesis (7 DBA)

anthesis (AN)

30 days after anthesis (30 DAA)

0

20

40

60

80

100

120 v 2010 – data from 7 DBA

50-D

AA

IB

MP

(pg/g

)

400 mg/L CON

Observations for one

year: pre-bloom

treatment increases

IBMP accumulation

Scheiner, et al unpublished data 800 mg/L

a

b

c

The problem: can we make rational

predictions about MP management without

understanding MPs at the molecular level?

2/22/2012

14

Putative MP formation/degradation

N

N OH

RN

N OCH3

R

Pre-veraison

Accumulation Degradation

OMT Branched

amino acid +

NH4+

+ α-dicarbonyl

Biosynthesis pathways suggested by Murray, et al J Sci Food Ag 1975

O-methyltransferase (OMT) capable of methylating hydroxyprazines (HP) to MP

purified by Hashizume, et al 2001, cloned by Dunlevy, et all 2010

“HP” “MP”

????

Proposed MP degradation during ripening

Converted back to odorless HP

N

N OH

RN

N OCH3

R

Pre-veraison

Accumulation Degradation

OMT Branched

amino acid +

NH4+

+ α-dicarbonyl

N

N OH

R

Ryona, Leclerc, and Sacks, J. Ag. Food Chem., 2010

Partial glycosylation

Some VERY recent data – Relationship of MP and HP

during the growing season

(left) IBHP and

IBMP in California

Merlot

Harris and Sacks,

unpublished 0

200

400

600

800

1000

5/2 6/21 8/10 9/29

Conc

ent

ration

(ng/L)

IBHP and IBMP (ng/L) v. Date

IBHP

IBMP

IBHP and IBMP are well correlated before veraison.

Potentially, MP biosynthesis is dependent on HP concentration If

so, what drives HP biosynthesis?

2/22/2012

15

One scary thought . . . .

Can IBHP be converted back to IBMP by yeast?

3-isobutyl-2-methoxypyrazine 3-isobutyl-2-hydroxypyrazine

(IBHP) (IBMP)

O-methylation

????

200 pg/mL or more ?????

Small scale fermentations to evaluate potential

of IBHP to be converted to IBMP

24 h at 25˚C 24 h at 25˚C 48-72 h at 25˚C

Yeast

Yeast Strains Used:

AMH

CY3079

EC1118

Vin 13

Kloeckera apiculata

1 ng/mL IBHP Juice Chemistry:

Brix = 20°

pH = 3.6

TA = 6.5 g/L

Harris, Ryona, and Sacks. Unpublished data

3-isobutyl-2-methoxypyrazine 3-isobutyl-2-hydroxypyrazine

(IBHP) (IBMP)

The short answer:

“no conversion of IBHP during fermentation”

O-methylation

No detectable IBMP (limit of detection ~ 1 ng/L)

If there’s conversion, its < 0.1%. Phew!

Harris and Sacks, unpublished

2/22/2012

16

Future directions for studying MPs

Moving beyond the empirical to the mechanistic

What explains increased MP accumulation in

conditions of high water availability, cluster shading,

etc…

Increased hydroxypyrazine (HP) production?

Or increased VvOMT activity?

And how do these relate to vine physiology?

What controls MP degradation?

Enzymatic? What enzyme?

Future directions:

genomics and grape breeding

Several groups (including ours) working on mapping genes

associated with high MPs

Possibility for marker assisted selection when using non-vinifera

species high in MPs (and other undesirable compounds)

0

200

400

600

800

IBM

P a

t 2

weeks

pre

-ve

rais

on

(ng

/L)

Individual in population

At left, IBMP in 168

individuals from

St Pepin X Cabernet franc

mapping population

Sacks and Owens,

unpublished

Acc. # IBMP

(ng/kg) IPMP

(ng/kg) TSS

( ͦ Brix) 1 13±1 10±3 20.5 2 110±9 7±2 20.3 3 143±8 17±3 18.0 4 17±1 1±0.4 15.0 5 33±5 8±2 21.2 6 286±12 16±4 23.5 7 251±10 31±5 20.6 8 52±6 n.d. 15.3

9 353±16 n.d. 20.4 10 18±2 n.d. 20.1

Something different:

IBMP and IPMP in

accessions of V. cinerea

(native species, potentially useful to

grape breeders)

V. cinerea

IBMP > 13 ng/kg in all accessions,

up to 353 ng/kg. Similar results for

V. riparia

No correlation between MPs and

soluble solids

2/22/2012

17

A final issue: how good a predictor of greenness

are MPs?

Roujou de Boubee,

et.al. (JAFC 2000)

Fifty Bordeaux and

Loire reds, judged

by expert panel

Exceptional

correlation between

IBMP and “green

pepper”

IBMP concentration (ng/L)

Gre

en

Pe

pp

er

Aro

ma

r2 = 0.74

Correlation of MPs and “bell pepper” more

modest around or below threshold (15 ng/L)

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25

IBMP (ng/L)

Bell

Pepper

Inte

nsity

r2 = 0.37

Zoom on Roujou data Weak or non-existent

correlations observed in other

reports

Chapman, et al (JAFC, 2004)

Falcao, et al (JAFC, 2007)

Preston, et al (AJEV 2008)

Scheiner, et.al. (AJEV, 2012)

Take home messages

MPs are an occasional fault (in NYS, ~5% of wines over 20 ppt).

Presence or absence of other compounds important to herbaceousness

IBMP in NYS wines (and elsewhere)

frequently between 5-15 ppt

Sacks, Pan; unpublished

2/22/2012

18

2009 2008

When MPs are < 15 ng/L, they are a lousy

predictor of “herbaceousness”

Data from NY State Cabernet franc multi-site study

Scheiner, et al AJEV 2012

IBMP (pg/mL) IBMP (pg/mL)

Inte

nsity o

f h

erb

ace

ou

sn

ess

Inte

nsity o

f h

erb

ace

ou

sn

ess

Summary

Veggie masks fruity and vice versa.

Selective removal of MPs from wine is hard. Removing MPs from

juice w/ non-polar sorbents may work

MP accumulation is increased in conditions that promote growth

(heat, water) and, to a lesser extent, in shaded clusters

MP degradation relates to maturity

Our molecular level understanding of “why” is still lousy

And, we cannot explain herbaceousness solely by MP concentrations

Wine is more than just ethanol, H2O, and MPs. Thankfully.