WAC 2014 - SESSION I : Active compounds ond enologicol processes
Oxldollve vs reduclive skln mqcerollon on fhlol precursors
Roberfo lorcher", Lorls Tonldondel", Giorgio Nicolini", Tomos R. vlllegos", Morlo Molocorneo, Bruno Fedrlzzib
" FEMJASMA Fondoùone Edmund Moch lstituto Agrorio di Son Michele ò 'Adige, via E. Moch l, 38010 Son Michele o 'Adige, TN, ltoly
b Wine Science Progrdmme, School of Chemicol Sciencet The University of Auckland, Prvote Bog 92019, Aucgond, New Zeolond
Keywords: skin mocerofion; fhiol precursors; LC-MS;
T. INTRODUCTION
Tropicol thiols ond their precursors is currently one of the most oppeoling reseorch topics in wine science [ ]. The recent finding thot pre-fermentotive processes could strongly influence the fìnol concentrotion of free thiols in wine [2] hos put further emphosis on the effect of technologicol steps, from grope horvest [3, 4] to grope pressing [5], on the content of these molecules.
Porticulorly interesting is the current dilemmo on the effect of oxidotive vs reductive conditions. Studies corried out in Austrolio showed how mecho nicol horvest (considered to be on oxidotive woy of hondling the gropes) olwoys coused o significont increose of-the S-glutothionyloted ond S-cysteinyloted precursors l4l. A following study tsl showed how the mechonisms involved in the production of pre-fermentotive thiol precursors is much more complex ond is depended on other unknown voriobles. In this porticulor study we ore presenting the thiol precursors voriobility ossocioted with two grope vorieties submitted to oxidotive ond reductive mocerotion.
2. MATERIATS AND METHODS
2.1 Chemicols qnd mqteriqls ( R/S) -3-S-cysteinylhexo n- I -ol ( R/S) -glutothionylhexo n- I -ol
(Cys-3MH), (GSH-3MH) ond their lobelled forms ds-(R/S)-3-S-cysteinylhexon-l -ol (d.-Cys-3MH), ond d3-(R/S)-glutothionylhexon-l -ol (d3-GSH-3MH) were supplied by Buchem B.V. (Apeldoorn, The Netherlonds). Formic qcid (FA) ond ocetonitrile (ACN) of HPLC grode, onhydrous ) 99% dimethyl corbonote, > 98% reduced glutothione (GSH) ,> 977" potossium metobisulfìte,
provided by Sigmo-Aldrich (Milon, ltoly). Six kg of sound ond technologicolly ripe gropes of Mueller-Thurgou (MT; N=19) ond Souvignon blonc (SB; 32) were horvested in Trentino (North ltoly) in2012.tor eoch somple the bunches were cut in 3-berries clusters, rondomly subdivided in two homogeneous subsomples of 2 kg, ond collected in polyethylene plostic bogs (40 x 40 cm, for domestic under-vocuum food pockoging). Eqch one of the two subsomples wos olternqtively submitted to o reductive (RD) or to on oxidotive (OX) protocol of skin-contoct mocerotion. In the RD treotment, potossium metobisulfite (320 mg), L-oscorbic ocid (1 ó0 mg), qnd dimethyl corbonote (400 mg) were odded to the berries, while in the OX protocol no odjuvonts were odded to the gropes. The RD bogs were seoled under portiol vocuum in order to remove the mojority of the oir; corefully ovoiding ony domoge of the berries, while the OX bogs were left open. Both the RD ond OX gropes were then monuqlly crushed ond let in skin-contoct mocerotion of 2O"C for 24 hours. Afterwords, 5 mL of must wos collected from eoch somple, if necessory piercing the close bogs with o lO-mL plostic medicol syringe. The must wos then ropidly diluted with 20 mL methonol chilled of - 20"C ond odded of d.-GSH-3MH ond d.-Cys-3MH (both ot 35 pg Lr) os internol stondords. The somple wos filtered on o O.22 pm PVDF syringe cortridge (Millex-GV; Millipore, Tullogreen, lrelond) ond stored in 2 mL gloss viol of -20"C until onolysis. 2.2 IC-MS
The precursors were onolysed using on UPLC Acquity (Woters Corporotion, Milford, US), connected with the Xevo TQ MS moss spectrometer (Woters). 5 pL somple wos
WAC 2014 - SESSION l: Active compounds ond enologicolprocesses
injected on qn Acquity UPLC HSS T3 C18 column (2.1 mm x .|00 mm, Woters), set ot 40oC, ond the flow rote wos set of 0.45 mL min-r. The eluents were woter {A) ond ACN (B), both odded of 0.1% formic ocid. The MS experiments were corried out in positive ion mode. Method lineority for the precursors wos studied between 0.I ond 30 Ug L-r ond the R2 for the deuteroted precursors were olwoys > 0.98.
3. RESUTIS AND DISCUSSION
3.1. Juice composilion
The juices were collected by monuolly squeezing the plostic bogs; their bosic composition for both MT ond SB juices showed lorge voriobility, nevertheless it wos generolly in ogreement with those found in literoture for the some cultivors. This voriobility is in ogreement with the remorkoble differences in sites oltitude (250-750 m obove seo level) ond in sunlight exposure in this mountoin region. Within-somple voriobility for totol soluble solids wqs obout 4.8oBrix for Ml ond obout 6.4"Brix for SB. Confirmotion of the effectiveness of the experimentol protocol in protecting mocerotion from oxygen, wos goined by meosuring GSH: the RD treotment produced o GSH concentrotion between l.ó ond 28.7 mg L-l in the MT juice ond 4.4-45.4 mg L-r in the SB juice. As expected, the GSH concentrotion wos signifìcontly (p < 0.00.|) impocted by the OX freotment, with o negligible content in the OX juices for MT ond below l0 mg L-r for SB.
3.2. 3-S-cysfeinyl qnd 3-S-glufolhionyl 3MH 3MH precursor contents in MT ond SB juices treoted occording to the RD ond
OX mocerotion protocols ore reported in f ì g u r e 1 .
The concentrotîon ronge (Ug L-'; os min, medion ond mox, respectively) for MT/OX juices wos: GSH-3MH = 48, 114, 290; CYs-3MH = I I , 28, 107, ond for MT/RD: GSH-3MH = 2 6 , 8 0 , 2 6 7 i C y s - 3 M H = 1 0 , 2 3 , l0 l . T h e s e contents ore in ogreement with those found by Rolond et ol. [ó] in the porent Riesling, ond similor to those of Souvignon Blonc juices. For SB/OX the concentrotion (pg L-r; min, medion ond mox, respectively) wqs: GSH-3MH = ,|43,
209, 577: Cys-3MH = 30, 82, 310, ond for SB/
RD: GSH-3MH = I I B, 201 , 5ó8; Cys-3MH = 28, 79, 273. These volues ore in ogreement with those of Copone ond Jeffery l4l but remorkobly higher thon those of Allen et ol.
[3] . Our results seem generqlly to confìrm whot wos observed by Rolond ond colleogues [ó] on the copobility of controlled oddition of oxygen into micro-fermentotions to increose the fìnol concentrotion of GSH-3MH. The OX treotment increosed GSH-3MH ond Cys-3MH in 1 ó ond 13, respeclively, of the 19 MT juice somples, ond in 23 ond 20, respectively, of the 32 SB juice somples. The effect of the OX treotment on juice composition wos verified using porometricol (Tukey's HSDT) ond non-porometricol {Wilcoxon motched-poirs test) tests. Signifìcont differences were found for GSH-3MH when using the non-pqrometricol CIpprooch, with meon volues higher for the OX treotment, whilst no differences were found for Cys-3MH. Even though most somples hod o higher GSH-3MH concentrotion under the OX protocol (84%in MT ond 72% in SB), other showed on opposite behovior. Similor evolution wos observed for Cys-3MH, with the percentoge equol Io 68% in MT ond 63% in SB. GSH ond either of the thiol precursors did not show ony correlotions for the triols; o more complicoted pothwoy could be hypothesised. These results ore similor to those reported by Rolond et ol. [ó] where o signifìcont increose for GSH-3MH wos meosured in oxidqtive environments even though lhot study considered only one somple per voriety.
The content rotio Cys-3MH/GSH-3MH (colculoted from the precursor meon concentrotions) for MT wos 0.25 ond 0.30 for OX ond RD, respectively; while for SB wos 0.43 ond 0.44, respectively. For SB the volues were in both OX ond RD treotments obout ten times lower thon those found by Rolond et ol.
[Z]. The OX ond RD treotments significontly (Tukey's HSDT; p
precursor concentrotion rotio only for MT. No correlotions between the bosic juice
composition ond the thiol precursor
obundonce for both reductive ond oxidotive treotments were observed for this study. Concluding, OX mocerotion promoted higher content of GSH-3MH, porticulorly in Ml whilst Cys-3MH showed to be independent on the oxygenotion of musts in this phose. This work confirms once more thot the de novo synthesis of the 3-MH precursors con
Oxidative vs reducfive skin mocerotion on thiol precursors
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reolly toke ploce during mocerotion or grope mochine horvesting, nonetheless no cleor strotegy con be suggested for o generol optimizotion of the winemoking processes.
References.
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n M€,úlan n26%"75% I i'londutlier Range ; outlierg ,,, Extrer!}eg
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6. A. Rolond, J. Violoret, A. Rozungles, P. Rigou, R. Schneider, Evolution of S-Cysteinyloted ond S-Glutothionyloted Thiol Precursors during Oxidotion of Melon B. ond Souvignon blonc Musts, J. Agric. Food Chem., 58 (20.|Ol 4406-4413.
Z. A. Rolond, R. SchneideL C.L. Guernevé, A. Rozungles, F. Covelier; ldentifìcotion ond quontificotion by LC-MS/MS of o new precursor of 3-mercoptohexon-l-ol (3MH) using stoble isotope dilution ossoy: Elements for understonding the 3MH production in wine, Food Chem., 121 (2010) 847-855. 100 Sauvignon Blanc
