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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
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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
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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
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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
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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?”)
2/22/2012
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
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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}
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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?
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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.