SYNTHÈSE DES TRAVAUX EN COURS SUR LA ROTUNDONE : un composé aromatique puissant responsable des notes poivrées dans les vins

MARKUS HERDERICH AWRI AUSTRALIAN

• Volume, Quality & Value 2012 crush 379,925 tonnes 45% of red ; 23% of total value 2011 approx. $1.8b 25.8% of domestic&exports

• Heritage Clones arrived 1832; no phylloxera 165 y old vines (Barossa Valley, SA) 149 y old vines (Victoria) 124-146 y old (Hunter Valley, NSW)

• Diversity of Styles > blends: Shiraz x Cab Sauv, x Shiraz x Mouvedre,… > co-ferments: Shiraz & Viognier > sparkling Shiraz => , regionality The Australian Australian cool climate Shiraz Research Institute Black pepper aroma in Shiraz

. A spicy, black pepper aroma is important to some key styles of high quality Australian Shiraz

. Described as ‘peppery’, ‘black pepper’, ‘white pepper’, ‘spicy’, ‘herbaceous’ . Shiraz . Cool climate

 Significant variation between and seasons

 No peppery impact aroma compound known not for grapes, wine or pepper Sensory analysis of Shiraz grapes

Axis 2: 25.5% Pepper . good correlation between Pepper aroma and flavour . Pepper related to Spice, Pepper fl not related to Green, Sweet or Fruit, or ripeness  volatile compound(s), stable in homogenates  differences among individual panel members

Spice fl Spicy Green Apple fl Axis 1: 43.1% Red Berry fl

Grassy Dark Berry fl Grassy fl Green Apple Red Berry Sweet fl Cooked Fruit Raisin

Cooked Fruit fl Identification of peppery grape volatiles

Optimised extraction, purification GC-MS chromatogram and enrichment Grape or wine extract

MS detector injector Retention time (min)

O Rotundone Sniff port

capillary column

GC Mass spectrum (-)-Rotundone identification

. Identity: confirmed with reference compound (Symrise) O 1H and 13C NMR, ORD GC-MS-O, coinjections . Quantification: multidimensional GC-MS & stable isotope dilution analysis; LOQ 0.5 ng/L

O

D D D D D

 Rotundone is the principal aroma impact compound for pepper aroma in grapes and wine. How potent is rotundone? The Research Institute

8 ng/L in water 16 ng/L in

20 to 25% of the panellists were anosmic to rotundone

Wood, C.; Siebert, T. E.; Parker, M. et al. J. Agric. Food Chem. 2008, 56, 3738-3744 How potent is Rotundone? aroma threshold in red wine is 16 ng/L

~ 20% of panellists could not smell rotundone at the highest level tested

10 9 8 7 6 5 4 3 Number of panellists of Number Number of panellists of Number 2 1 0 0.4 3.2 9.4 28.1 84.2 250 760 > 4000 The full story: Concentration (ng/L)

How potent is Rotundone? reconstitution model of Shiraz flavour

Cool climate Shiraz reconstitution model 42 volatiles; ethanol, water and tartaric acid; fructose, glucose, glycerol, succinic acid, malic acid, lactic acid, NaCl, K2PO4, K2S2O3

38 ng/L  0 ng/L rotundone (aroma threshold in red wine 16 ng/L) The Australian Wine Rotundone in Australian Research Institute

• We can now start to look at what influences rotundone levels • A large survey of commercial wines was undertaken to guide us

180 161 152 160 140 120 ng /L) 97 100 75 80 60 39 38 37 32 33 35 28 25 40 7 Rotundone ( 16 16 4 9 11 11 10 3 10 3 11 2 3 20 0 Hills Hills Vale River Valley Valley District Ranges Barossa Geelong McLaren Adelaide Adelaide Macedon Goulburn Canberra Tasmania Peninsula Frankland Hills/Yarra Clarendon Grampians Rutherglen Mornington Yarra Valley Yarra Yarra Valley Yarra Coonawarra

Region/Variety AWRI TR #180 (June 2009) Rotundone in commercially available The Australian Wine Shiraz wines; Duras from France Research Institute

260 240 220 200 180 161 152 160 140 120 ng /L) 97 100 75 80 60 39 37 32 33 28 25 40 7 Rotundone ( 16 16 11 11 10 20 0 Great District Hills Hills Vale Ranges Canberra Southern Geelong McLaren Adelaide Duras Macedon Clarendon Grampians France France Yarra Valley Yarra Coonawarra Hawke’s Bay Hawke’s New Zealand New

Region Rotundone in Duras, Graciano, & The Australian Wine Pineau d’Auris wine Research Institute Olivier Geffroy, IFV Sud-Ouest 2011 Graciano 17.5 & 11.3 ng/L 2011 Gamay 14.7 & 11.9 ng/L 2011 Pineau d'Aunis 65.6 & 200.0 ng/L 2012 Duras 26 ng/L

2011 Duras microvinification at 5 levels of maturity (rotundone ng/L) “Theorical” optimum picking date @day 45 after

days Where and when does rotundone The Australian Wine turn up in the berry? Research Institute

Adelaide Hills Shiraz berries during ripening NZ Hawke's Bay berries during ripening 70 30 Har 2009 AH 1127 60 ↓ 25 2009 NZ ME 2009 AH 2626 50 2010 NZ CR 20 40 15 30 10 Ver Mid 20 ↓ ↓ 5 10 Rotundone (ng/kg of of (ng/kg berries) Rotundone Rotundone (ng/kg of of (ng/kg berries) Rotundone

0 0 Veraison Midway 0 2 4 6 8 10 12 14 Ripening timepoint Sampling point (weeks)

 Vine management may impact rotundone concentration Where and when does rotundone The Australian Wine turn up in the berry? Research Institute

1127: 24.7 ng/kg

rotundone 2626: 49.5 ng/kg

not detected

not detected Concentration of rotundone and aroma The Australian Wine of Australian Shiraz grapes Research Institute

500 5

450 4.5

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350 3.5

300 3 ( ng /kg) 250 2.5

200 2 5 vineyards over 2 150 1.5 Rotundone Aroma Intensity Intensity Score Aroma 100 1 2003 50 0.5 2002

0 0 Aroma 2003

A 02 A 03 B 03 C 03 B 02 E 03 D 03 C 02 D 02 E 02 Aroma 2002 Rotundone extraction from berries The Australian Wine during Research Institute

AdelaideAdelaide Hills Hills Shiraz Shiraz commercial commercial fermentation fermentation 2009 2011

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ng /L) 30

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` 20 1127 Rotundone (ng/L) Rotundone ( 15 2626 - Tank 1

10 2626 - Tank 2 2626 - Tank 3 5 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 TimeFermentation from inoculation (Day) (days) Can yeast affect rotundone levels The Australian Wine during fermentation? Research Institute

Fermentation in defined juice medium 2011 4500 4000 3500 No yeast 3000 2500 2000 S. cerevisiae 1500

Rotundone (ng/L) Rotundone 1000 S. bayanus 500 0 Control 1631 796 1483 1555 1375 AWRI Yeast Stability and scalping of rotundone: no obvious effects

Scalping of Rotundone by Closures

100% 80% 60% 40%

Remaining 20% 0% % of Original Amount ROTE ROTE Cork Cork Cork Cork Natural Natural Glass ampoule Synthetic Synthetic

3 Months 6 Months Closure/Time Point

Variability across the vineyard & vintages: The Australian Wine Shiraz/Viognier wines - Canberra District Research Institute

same & style 

180 other

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Rotundone (ng/L) Rotundone 20

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Vintage Variability across the vineyard & vintages: The Australian Wine Great Southern Research Institute

WA Great Southern Shiraz berries 2009 80 70 60 50 40 30 20 Rotundone (ng/kg) Rotundone 10 0 Zone 1 Zone 2 Zone 3 Zone 4 Zone 5 Zone 6 Vine vigour and soil property Rotundone variability across a vineyard: The Australian Wine 2012 Grampians Shiraz Research Institute Nathan Scarlett, Mount Langi; Rob Bramley, CSIRO; Pangzhen Zhang, University of Melbourne rotundone in grapes 74 – 1081 ng/kg distinct spatial pattern < 200 ng/kg uniform TSS, TA, colour

>800 ng/kg Summary I: rotundone - the key to The Australian Wine peppery aromas in Duras and Syrah wine Research Institute

. vineyard site & vintage key to rotundone in grapes & wine . significant vine-to-vine variation in uniform sites

. Shiraz - & rotundone concentration:  ripening  clone, vigour, water stress, leaf removal & crop load (Gerard Logan, Uni Auckland)

. Duras - viticulture & rotundone concentration in wine:  Ripening

2012 – IFV trials (Olivier Geffroy):  irri / elicitor / crop thinning => 29-36 ng/L  control => 27 ng/L  leaf removal =>12 ng/L Summary II: rotundone - the key to The Australian Wine peppery aromas in Duras and Syrah wine Research Institute

 Limited options to lower rotundone & pepper aroma in the winery:  yeast?  skin contact and cap extraction? no effect: machine picked, crushed, de-stemmed & open fermentation vs hand picked & whole bunch open fermentation

 How can we manage our vineyards to increase or decrease rotundone in fruit, or to reduce variability?

 Why does rotundone occur in Shiraz and Duras compared to other cultivars, ie role of grapevine genome? what are the environmental factors that cause high rotundone in some grapevines & some seasons?

 What biological function does rotundone serve? The Australian Wine Acknowledgements Research Institute

Australian wine industry collaborators AWRI • Nathan Scarlett, Dan Buckle, Damien Sheehan (Mt • Tracey Siebert, Mango Parker & Claudia Wood Langi Ghiran) • Flavour & Sensory Teams (past & present members) • Darryl Catlin, Winemaker, and the winery and • Radka Kolouchova laboratory staff of Shaw and Smith Wines • Frank van de Loo, Mt Majura Vineyard New Zealand collaborators • Jim Lumbers, Lerida Estate • Gerard Logan, • Dr Ayalsew Zerihun, Curtin University of Technology The University of Auckland & EIT Hawke’s Bay • Allen and Andrea Hart (Fosters Group), Inca Pearce, Craggy Range Vineyards, Mission Estate Wines Martin Wirper (Orlando Wyndham), Sue Hodder (Wynns Coonawarra Estate) France Symrise, Germany • IFV Sud-Ouest Olivier Geffroy

The AWRI, a member of the Wine Innovation Cluster in Adelaide, research is financially supported by 's grape growers and winemakers through their investment body the Grape and Wine Research and Development Corporation, with matching funds from the Australian Government. Thank you!

O Stand by Concentration of rotundone and aroma The Australian Wine of Australian Shiraz grapes Research Institute

500 5

450 4.5

400 4

350 3.5

300 3 ( ng /kg) 250 2.5

200 2 5 vineyards over 2 vintages 150 1.5 Rotundone Intensity Score Aroma 100 1 2003 50 0.5 2002

0 0 Aroma 2003

A 02 A 03 B 03 C 03 B 02 E 03 D 03 C 02 D 02 E 02 Aroma 2002 Vineyard Variability across the vineyard & vintages: The Australian Wine Shiraz wine from Great Southern, WA Research Institute

WA Great Southern Shiraz wines 120

100

80 2006 - Site 1 60 2006 - Site 2 40 2006 - Site 3 Rotundone (ng/L) 20 2007 2009 0 Zone 1 Zone 2 Zone 3 Zone 4 Zone 5 Zone 6

Vine vigour and soil property Variability across the vineyard & vintages: The Australian Wine Great Southern Research Institute

WA Great Southern Shiraz wines 120

100

80 2006 - Site 1 60 2006 - Site 2 40 2006 - Site 3 Rotundone (ng/L) 20 2007 2009 0 Zone 1 Zone 2 Zone 3 Zone 4 Zone 5 Zone 6

Vine vigour and soil property Variability across the vineyard & vintages: The Australian Wine Great Southern Research Institute

WA Great Southern Shiraz wines 120

100

80 2006 - Site 1 60 2006 - Site 2 40 2006 - Site 3 Rotundone (ng/L) 20 2007 2009 0 Zone 1 Zone 2 Zone 3 Zone 4 Zone 5 Zone 6

Vine vigour and soil property A few facts about Syrah / Shiraz

• ancient variety Mondeuse blanche x Dureza Northern Rhone Valley, 100AD • 1980s only 10.000 ha • 140,000 ha in 2004/2005 one of world’s top six grape varieties along with , , , and

• World styles France: Côte-Rôtie, Hermitage : Boekenhoutskloof Syrah New Zealand: Craggy Range Le Sol Syrah Chile: Matetic Vineyards EQ Syrah Washington, USA: Gramercy Cellars “John Lewis” Walla Walla Valley Syrah California, USA: Saintsbury Rodgers Creek Carneros Syrah Rotundone is widely found in plants and plant products Comparison of grape volatiles by GCMS

High ‘pepper’ . differences, monoterpenes & sesquiterpenes . lots of variation, no pattern . no compound related to peppery aroma

Low ‘pepper’ The search for ‘pepper markers’: untargeted metabolomics experiment

THE IDEA: . compare quantitative sensory data (n=22) with comprehensive volatile profiles (entire headspace-GC-MS datasets, 12000 mass spectra/sample)

 to look for correlations among data sets  to identify marker substances in peppery grape samples  to identify key peppery aroma compounds The search for ‘pepper markers’: untargeted metabolomics experiment

PCA

PLS

mass spec versus pepper rating GC-MS data allow prediction of 'pepper' aroma new biomarker - sesquiterpene ylangene

Abundance

Ion 93.00 (92.70 to 93.70): 02MTBL2.D

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0 23.00 23.20 23.40 23.60 23.80 24.00 24.20 24.40 24.60 24.80 Time--> GC-MS data allow prediction of 'pepper' aroma

80

70 y = 0.9654x + 0.8957

60 R2 = 0.9825 MS (arbitrary units) - 50

40

30 2002 grapes

’ character by by character GC ’ 2003 grapes 20 Control grapes

epper (no pepper) ‘p 10

0 0 10 20 30 40 50 60 70 80 Predicted Predicted Measured ‘pepper’ character by sensory descriptive analysis (arbitrary units) The search for ‘pepper markers’: untargeted metabolomics experiment

. identified α-ylangene (a tricyclic sesquiterpene) as a new compound in grapes

. quantified α-ylangene with α-copaene as I.S. . good correlation of α-ylangene with ‘pepper’

H

 marker compound: α-ylangene has no peppery aroma H

Parker et al. (2007) JAFC, 55, 5948-5955 Identification of peppery grape volatiles: The Australian Wine GC-MS-Sniff in action Research Institute How potent is Rotundone? aroma threshold in red wine is 16 ng/L

1 drop of rotundone is enough to 1 g of rotundone is enough to make an Olympic size swimming make the entire Australian pool smell peppery! annual crush smell peppery!