Varietal thiols in wines : a review on their aromatic contribution and their liberation mechanisms from their precursors

Varietal thiols in wines : a review on their aromatic contribution and their liberation mechanisms from their precursors Dr. Rémi SCHNEIDER

Sulfur containing compounds a devilish universe? origin Mean level in wines Odor threshold Odor Carbon sulfide fermenta(on 2-2,5 30 µg/l sulfury H 2 S fermenta(on 0,3-17 µg/l 1 µg/l Ro0en egg 2-mercaptoethanol fermenta(on 70-125 µg/l 130 µg/l sewage methanethiol fermenta(on 0-5 µg/l 0,3 µg/l sewage éthanethiol fermenta(on 0-10 µg/l 0,1 µg/l sewage dimethyldisulfide fermenta(on/oxida(on 0-2,5 µg/l 2 µg/l Ro0en onion or cabbage

Some «positive» thiols in wines Origin Levels Odor threshold Odor Ethyl 3-methylthiopropanoate Fermenta(on 1-6 µg/l - Pineapple Dimethylsulfide Grape/ferm. 0-1 mg/l 30 µg/l Triffle/fruity enhancer 4-Methyl-4-mercaptopentan-2-one Grape 0-50 ng/l 0,8 ng/l Box Tree 3-Mercaptohexanol Grape 10-5000 ng/l 60 ng/l Grapefruit Mercaptohexyl acetate Grape 0-400 ng/l 4 ng/l Citrus, Passion fruit 2-Methyl-3-furanmethanthiol? > 100 ng/l 1 ng/l Roasted meat Furfurylthiol Ferm./Oak 0-50 ng/l 0,4 ng/l Coffee Benzylthiol? 30-400 ng/l 0,3 ng/l Toasted, smoke 3-Methyl-2-butenthiol? 0.5-2 ng/l 0.5-1 ng/l Cannabis,

Contribution of varietal thiols to wine aroma 3 varietal thiols have been identified as key aroma compounds H C 3 C H 3 S H S H H S C H 3 O C H 3 O H C H 3 OAc 4-mercapto-4-methylpentan-2-one 4MMP 0.8 ng/l 3-mercaptohexan-1-ol 3MH 60 ng/l 3-mercaptohexyl acetate A3MH 4 ng/l Occurrence in several white, rosé and some red wines : Sauvignon, Colombard, Petite Arvine, Manseng, Muscadet, Grenache, Syrah, Merlot and Cabernet,. 4

Which impact on Sauvignon sensorial profile example of NZ Sauvignon TROPICAL 3MHA PASSION FRUIT 3MH ASPARAGUS IBMP Lund et al., 2009

Which impact on rosé wine sensorial profiles THIOLS ESTERS THIOLS ESTERS THIOLS ESTERS Pouzalgues et al., 2013

State of the art : varietal thiols biogenesis in wine 7

Hypothesis of G3MH formation during winemaking O OH C H O O HN C O O HO C NH 2 C NH GSH extracted from berries at the beginning of the crushing SH HO O C O OH C HN O C C NH O [Hexenal] increases at the end of crushing NH 2 S CH 3 OH Roland., 2010

Grape skin tannins, a possible source? Botanic sources Nb samples G-3MH (mg/kg) Cys-3MH (mg/kg) Oak 16 < 0.3 < 0.2 Grape skin 9 0.2-138 0.3-200 Grape seeds 7 0.01-33 0.006-6 Gall 6 <0.003 < 0.003 Quebracho 4 <0.01 < 0.02 Chestnut 3 < 0.02 < 0.01 Fruit tree 4 < 0.1 < 0.03 Acaccia 2 <0.05 < 0.01 Tea 2 <0.02 < 0.01 Cherry tree/mimosa 1 0.5 0.2 Tara 1 0.2 < 0.01 Larcher et al., 2014; 2015

Grape skin tannins, a possible source? Botanic sources Nb samples G-3MH (mg/kg) Cys-3MH (mg/kg) Oak 16 < 0.3 < 0.2 Grape skin 9 0.2-138 0.3-200 Grape seeds 7 0.01-33 0.006-6 Technologically speaking Gall 6 <0.003 < 0.003 Quebracho 4 <0.01 < 0.02 Chestnut 3 < 0.02 < 0.01 200 mg/l of the richest tannin and conversion yield = 3% + 1 ppb of 3MH!! Fruit tree 4 < 0.1 < 0.03 Acaccia 2 <0.05 < 0.01 But addition before the fermentation! Tea 2 <0.02 < 0.01 Cherry tree/mimosa 1 0.5 0.2 Tara 1 0.2 < 0.01 Larcher et al., 2014; 2015

Subileau et al, 2008 Thibon et al., 2008 Cordente et al. 2015 Most of the sulfur compounds are considered as Cysteinylated and Glutathionylated conjugates Uptake into the yeast Cysteinylated conjugates Gap1 Glutathionylated conjugates Opt1 + other transporters Precursor

Cysteinylated and Glutathionylated conjugates Convertion by the yeast Cysteinylated conjugates Glutathionylated conjugates Precursor Thiols Tominaga et al., 1998 Subileau et al, 2008 Roland et al., 2010 Capone et al., 2010 Winter et al. 2011 Cordente et al 2015

The genes involved in the thiols revelation by yeast For cysteinylated conjugates Cys-4MMP To 1 to 4 genes involved 4MMP Howell et al.,2005 Thibon et al., 2007

The genes involved in the thiols revelation by yeast For cysteinylated conjugates Cys-3MH Very complex IRC7 and others. 3MH

And for glutathionylated precursors? Yeast cell Must Opt1 FOT 1/2? ptr2? DAL5? FOT 1/2? GAP1

What about the conversion yields? Conditions Yeast Molar conversion yields (%) Cys3MH Cys4MMP G3MH G4MMP Synthetic medium Natural must S. cerevisiae 0.2-0.4 (1,2) 0.06-0.8 (2) 0.6 (4) Inter sp. hydrids 3.5-10.9 (3) 0.5-0.8 (2) 4.4 (4) 0.3 (5) 1 Murat et al., 2001, Am. J. Enol. Vitic., 52, 136 4 Roland et al., 2010, Food Chem., 121, 847 2 Subileau et al., 2008. FEMS Yeast Res., 8, 771 5 Roland et al., 2010, J. Agric. Food Chem., 58, 1084 3 Masneuf et al., 2002,.J. Int. Sci. Vigne Vin., 36, 205

ü Vine water status 1200 Viticultural impact factors -27-25 -23-21 δ 13C Peyrot des Gachons et al., 2005 1000 800 600 400 200 0 Cys-4MMP (ng eq/l) 25 20 15 10 5 0 non irrigué irrigation FG-VER irrigation FG-post VER Thiols in the wines (nnmol/l) 2010 2011 2012 Latye harvest (> 50j aver veraison) Dufourcq, 2013

ü Nitrogen fertilization (in soil) % level increase due to fertilization 800% 600% Viticultural impact factors ü Nitrogen foliar spraying 400% 200% 0% Cys-3MH Cys-4MMP GSH Choné et al., 2006 OAV 80 60 40 20 0 A3MH Reference 3MH Foliar spraying

Less easy-to-use than ammonium must addition but more powerfull Fusel alcohols acetates gain Increase (%) 200 150 100 Isoamyl acetate Hexyl acetate Phenylethyl acetate 50 0 PulvérisaTon foliaire Nitrogen spraying Sels ammoniacaux Ammonium Dufourcq et al., 2009

ü Yeast strains 60 3-Mercaptohexanol ( OAV) 40 20 0 Yeast : ALS C19 KD NT116 VL1 VL3 Dufourcq et al., 2006 ü Fermentation temperature 3-mercaptohexanol (ng.l-1) 4000 3000 2000 1000 13 C 20 C 0 Yeast : VIN13 X5 Murat et al.., 2006 Enological impact factors ü Nitrogen composition of the must Cys-3MH Amino acid Gap1p NH 4 + excess NCR Subileau et al., 2008

ü Yeast strains 60 3-Mercaptohexanol ( OAV) 40 20 0 Yeast : ALS C19 KD NT116 VL1 VL3 Schneider and coll., 2006 ü Fermentation temperature 3-mercaptohexanol (ng.l-1) 4000 3000 2000 1000 13 C 20 C 0 Yeast : VIN13 X5 Dubourdieu and coll., 2006 Enological impact factors ü Nitrogen composition of the must ng L-1 600 500 400 300 200 100 0 7.7% Sauvignon Languedoc 2004 5.0% +NH 4 +

Adaptation of yeast nutrition The technological aspects Yest derivate (initial) + DAS (end) Yest derivate (initial) DAS (end) DAS (initial) Reference 0 5 10 15 20 25 30 3MH and A3MH (nmol/l) Yeast choice and nutrition strategy must be considered together and adapted to the nitrogen composition of the must

Conclusion : what are the new frontiers? G-3MH and G-4MMP cleavage mechanisms have to be clarifying ü In the must : enzymatic peptidic cleavage? ü In the yeast key role of the gamma-gt: (Cordente et al., 2015) According to Roland et al., 2010 and Böcker, 2014 35 to 80 % of the 3MH origins remain unknown Identification of the other 3MH origins is necessary

New precursors forms R 1 R HO CH 3 O CH 3 R1 = γglucys ou CysGly Degradation forms of Glutathione - harvest transportation (Capone et al., 2010) - Fermentation (Böcker, 2014) SO 2 Ethanal SO 3 - R CH - 3 O 3 S R = Glutathion, Böcker, 2014

New precursors forms R 1 R HO CH 3 O CH 3 R1 = γglucys ou CysGly Degradation forms of Glutathione - harvest transportation (Capone et al., 2010) - Fermentation (Böcker, 2014) SO 2 Ethanal SO 3 - R CH - 3 O 3 S R = Glutathion, Böcker, 2014

3MH origins in wine 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 80 10 Montpellier Roland et al, 2010 4 9 22 3 7 24 35 Hexenal 1 Bordeaux Böcker et al, 2014 Cys S-Conjugué GSH S-Conjugué G3MHAl G3MH-SO3 Inconnu

Further studies are needed To formely assess the precursor role and the conversion yield of the forms recently identified ² Transport system and its regulation ² Conversion yield To better understand the interconversion beetwen them in the must ² Enzymes involved ² Favorable or non favorable conditions To clarify the cleavage mechanisms of glutathionylated precursors in the yeast ² Biochemical and genoomic approaches To identify the forms responsible of the last 40% of 3MH ² Non targeted approaches