WAC 2014 - SESSION l: Acfive compounds ond eno/ogicolprocesses
Oenologlcol fonnins os o source of thiol precursors
Roberfo Lorcher, Loris Tonidondel". Giorgio Nlcollnio, Tiziqno Nordln", Bruno Fedrizzib
"FEM-IASMA Fondozione Edmund Moch lstítuto Agrorio di Son Miche/e o,l'Adíge. vid E. Moch l, 38010 Son Míchele o 'Adige, TN, ltoly
bwine Science Progromme. School of Chemico, Sciences, The UnìversìIy of Auckland, Privote Bog 92019, Aucklond, New Zeolond
Ke),lvords: oenologicol fonnis; lhiol precursors; LC-MS * - , * - * - - _ - ' - * * - * - * * * l : l
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1 . I N T R O D U C T I O N
Oenologicol tonnins ore widely used os odjuvonts in winemoking. Their contribution to colour stobility, mouthfeel ond qromo profìle [ , 2] hos been studied in the post, while the possibility thot grope tonnins could olso provide vorietol oromo precursors, such os the S-g lutothionyloted o nd S-cysteinyloted precursors of thiols, wos only recently demonstroted [3]. In q second experiment, the effect of the pre-fermentqtive introduction of thiol precursors, vio tonnins qddition, on the 3-mercoptohexono-l-ol ond 3-mercoptohexyl ocetote levels in the fìnished wine wos verified [Lorcher 2014, submittedl. All the wines supplemented with grope tonnins showed higher level of free thiols thon the one without tqnnins. This evidence supports the hypothesis thqt oenologicol tqnnins could be used to odjust the fìnol level of tropicol thiols.
2. MATERIALS AND METHODS
2.1 Chemicqls ond mqteriols
Formic ocid (FA) ond ocetonitrile (ACN) of
HPLC grode were provided by Sigmo-Aldrich
(Milon, ltoly). (R/S)-3-S-cysteinylhexon-'l-ol ( C y s 3 M H ) , ( R / S ) 3 S g l u t o t h i o n y l h e x o n l
-ol (GSH-3MH) ond their lobelled forms
d3-(R/S)-3-S-cysteinylhexon-l -ol (d.-Cys-3MH),
o n d d 3 - ( R / S ) - 3 - g l u t o t h i o n y l h e x o n - l - o l ( d .
-GSH-3MH) were supplied by Buchem B.V.
(Apeldoorn, The Netherlonds).
4-S-Cysteinyl-4-methylpenton -2-one (Cys-4MMP),
4-S-g lutothionyl-4-methyl pe nto n-2-one ( GS H-4MMP) were synthesised following olreody published protocols 14, 51. Ultropure woter wos in lob-produced using on Ultropure Woter System (Arium Pro UV Dl; Sortorius, Góttingen, Germony). Fifty-eight tqnnin somples from on the ltolion morket were
collected of locol distributors ond wineries.
They were clossified occording to the lobel specificotions declored by producers or
distributors. Tonnin somple (50 mg) wos fìrst
moistened with some drops of methonol, dissolved in 45 mL methonol/woter (20:30)
s o l u t i o n , o n d o d d e d o f t h e i n t e r n o l s t o n d o r d s
d.-Cys-3MH ond d3-GSH-3MH. The solution,
fìltered through a 0.22-1tm PVDF syringe fìlter
{Millex-GV; Millipore, Tullogreen, lrelond), wos
tronsferred into a2mL gloss viol for the LC-MS
o no lysis. 2.2 LC-MS
The precursors were onolysed using on UPLC
Acquity (Woters Corporofion, Milford, US), coupled to o Xevo TQ MS mqss spectrometer {Woters) for oll the experiments. The chromqtogrophic module consisted of on
A c q u i t y U P L C H S S T 3 C I B c o l u m n { l . B U m ,
2 . 1 m m x 1 0 0 m m ; W o t e r s ) , s e t o f 4 0 "C. The
e l u e n t s , w o t e r ( A ) o n d A C N ( B ) b o t h o d d e d
of 0.1% f ormic ocid, were used of o flow rote o f 0 . 4 5 m l / m i n . T h e s o l v e n t B g r o d i e n t w o s :
5 % ( 2 m i n ) , l o I O 0 % ( i n 5 m i n ) , o t I O 0 % ( l m i n ) ,
t h e n t o 5 % ( i n 0 . 0 1 m i n ) o n d a t 5 % ( 2 m i n ) . The somple injection volume wos of 5 pl. MS experiments were performed in positive ion mode setting the copillory voltoge aI 2.5 kV. Desolvqtion ond collision gosses were
r e s p e c t i v e l y n i t r o g e n {1000 L h-l ) ond orgon
( 0 . 2 0 m L m i n - ' ) . D e s o l v q t i o n o n d s o u r c e
temperoture were set of 500 oC ond .|50 "C,
respectively. The experimentol conditions used for Product ion (Pl) experiment in the
identifìcotion of thiol precursors were: cone
voltoge potentiol ond collision energy were set respectively of I ó V ond I O eV for
Cys-3 M H ; 2 6 V o n d 1 2 e Y f o r G S H - Cys-3 M H ; I ó V o n d
I O eV for Cys-4M/tAP; ond I ó V ond l4 eV
for GSH-4MMP. For the method volidotion,
lineority wos studied of five colibrotion levels,
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wAc 2014 - sEssroN r : Acfive compounds ond eno/ogicolprocesses between 0.1 ond 30 pg/I, ond the correlotion
coeffìcients (R2) for the deuteroted thiol precursors were olwoys higher thon 0'98' 3. RESUTTS AND DISCUSSION
3.1 . ldentifìcotion of 3-S-cysfenylhexqn-1 -ol ond 3-S-glutothionylhexqn-1'ol in fonnin The LC-MS/MS experiments were orgonised i n t w o p h o s e s : t h e f i r s t , w i t h t h e d i r e c t infusion of the reference Cys-3MH, GSH-3MH' Cys-4MMP ond GSH-4MMP, to optimise the frogmentotion of the thiol precursors for the MRN^ experiments. The second, to record the Product lon (Pl) moss spectro (figure I ) ond optimise of the chromotogrophic seporotion of the lobelled ond unlobelled precursors. GSH-3MH wos present in oll of the onolysed somples, whilst Cys-3MH in most of them' No 4MMP precursors were obove the detection limit of the method (0.01 mg kg-' for both precursors).
F i g u r e 1 . P r o d u c t l o n M a s s S p e c t r a o f G S H - 3 M H a n d C Y s - 3 M H i n g r a P e t a n n i n '
3.2. Thiol precursors in commerciql lqnnins As reported in toble 1 , grope skin tonnins showed to be very rich of thiol precursors
( m i n = 0 . 2 3 2 , m e d i o n = l O - l o n d m o x = l 3 B mg kg I for GSH-3MH; O'33, 9'1' ond 200 mg tgi fór cys_3MH). Also the contents found in some grope seed tonnins (min = 0'0.|' medion =-0.ó1, ond mox = 33'ó mg kg-' for G S H - 3 M H ; 0 . 0 1 , 0 . 4 0 , o n d 6 . 2 m 9 k g - ' f o r C y s -3 M H ) w e r e o f t e c h n o l o g i c o l i n t e r e s t . T h e precursor contents found in the two grope p r o d u c t s w e r e s t o t i s t i c o | l y d i f f e r e n t ( M o n n -Whitney U-test, p < 0.05) ond consistent with the distribution of these molecules inside
the grope berry 16,71. Ook tonnins (lobelled os àtogic tonnins) were olso rich of thiot pt..rtórs with contents of 1.24 mg kg-' of GSH-3MH ond 0.246 mg kg-' of Cys-3MH' conversely, the other botonicol sources were generolly poor or obsent of these precursors, ond troces, when present, were reosonobly due to cross-contqminotions hoppened olong the production process. on the bosis of th;rs evidence we suggest on innovotive use of tonnin for supplementing thiol precursors ond in this woy possibly chonging ihe fr"" 3MH ond 3MHA content in wine ofter fermentotion. Moreover, other reseorches indicote o possible direct contribute of the precursor Cys-3MH in the retronosol sensory perception of 3MH [8]. This could prefìgure new scenorios for the oddition of precursors-rich tqnnins os tropicol-note flovouring ogents in unfermented food ond olcohol-free beveroges. Regording the obundonce of Cys-3MH ond GSH-3MH in grope products' their content rotio wos sig nifìco ntly depending on the specifìc origin. The volues ronged from 0.lB to 2.0 for grope seed tonnin ond from 0.23 to 'l .5 for grope skin tonnins' Finolly' if we hypothesise oltondord oddition (200 mg L') of o precursors rich tonnin (e.g' GSH-3MH =
l38 mg kg-'; Cys-3MH = 200 mg kg-'), ond we estimot e a 3% molor conversion of precursors to the free forms during fermentotion this would theoreliccrlly increose 3MH of up to
1 pg L-r, polentiolly impocting the sensory chorocteristics of wine.
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wAC 2014 - sEss/oN t : Active compounds ond eno/ogico/ processes Tannin {l'I} tntn mean max m r n T a b l e 1 ' GSH-3MH a n d C y s - 3 M H c o n t e n t i n 5 8 commercial g r o u p e d by botanical o r i g i n . Grape skin (9) Grape seeds (7} Oak {ellagic} (1) 4.232 0*008 0.s91 34"3 5 . 1 9 1.24 0"61S 0.170 0.053 0.048 CI.CI33 0"CI26 0.0f 5 4.a12 4.411 0"009 0.009 0.007 0.CI05 0"005 0.005 0.004 0"004 0.402 <0.00f 1 3 8 33.6 0"939 a32g 0.006 o.204 32.8 1 . 1 A 3 ú.248 0 . 3 1 S <0,00't a"aa7 4.ffis 0.035 <4.0a1 <0.00'l ú.aa4 0.006 4.027 0.0CI8 <0.001 0,003 <0,CICI1 ú.0a2 <0.CI01 <0.001 0.CI03 0.002 0.002 <0.001 200 6 . 1 7 o.426 0"135 8.4A2 CI"O't0 a_a17 CI.o19 0.0CI2 0.009 0.003 0.003 0.003 J. Agric. Food Grape skin (enociantn) (Z)
Tara (1| Acacia (tsrazittan) (1) Cherry tree/rnimosa 80:20 {1} Tree fruits (4) Tea (green) {1} Oak (Ameriean| {2) Chestnut {3} Quebrachl t4] Acacia (Gambier) {1) Oak {toasted} {5) Oak {French} {Z} oak {a) Gall (3)
Oak {French, toasted} (Z} Officinal plants (1)
Galf (Turky$h, Qfferc{rs infecioria! {1} Tea ('l)
Gall (ChineseRhus temialata
0.006 <0.001 ú"ú04 0.002 {0,001 0.003 0,002 <0.001 0.004 <0.001 0 . 1 0 5 0"029 0"023 4.0'tg ú.42 o"ú1 0,009 ú.ofi 0.005 <0.001 <0.001 <0.001 <a.oú1 <0.001 {0,001 <0.001 <ú.0a1 <0.001 References.
l. V. Kovoc, E. Alonso, M. Bouzeix, E. Revíllo, Effect of severol enorogicor proctices on the content of cotechins..crnd proonthocyonidins of red wines, Journol 9f Agricurturor ond Food' chemistry, 40 ttgó'zj r
9s3-1 9 5 7 .
?.. y.!. Cejudo-Bostonte, F. Sonni, F. Chinnici, A. Versori, M.s. Perez-coeilo,..c. Rponi, FermentotÍon of surphite-free whiîe musts with odbed rysozyme ond oenorogicol tonnins: .Nitrogen-consumption cind bioòenic ómines composition of fìnor wines, Lwt-Food 5cieÀce ond Technolo gy, 43 (201 0) I SOt -t SOZ.
3. R. Lorcher L. Tonidondel, G. Nicolini, B. tedrizzi, Firsl evidence of the presence of s-cvstèióvr,rted ond l-gtVlqll,'glyloted precursors in tonnins, robO óh"rn.,
1 4 1 ( 2 0 1 3 ) t I e 6 - 1 2 0 2 .
4. B. Fedrizzi, K.H. pordon, M.A. Sefton, G.M. Elsey, D.w. Jeffery First identificotion of a-5-grrîoihionyr-4-methylpenton-2-one, o potentiol pÉórìi"r of 4-mercopto-4-methylpenton -2_one, in Souvignon Plg: juice, J. Agric. Food Chem., 57 eAOn gll_ggS. 5. T. Tominogo, C. peyrot des Gochoni, D. Dubourdieu, I new type of flovor precursors in Vitis vinitéC L. cv. Souvignon Btonc:!19yst_ei1e conjugofes, J. Agri-. Food Chem., 46 (1998lr S215-5219.
I . y .Mog_qg, R. Winz, p.A. Kilmortin, M.C.T. Trought, L. Nicolou, Effect of skin contoct ond pressure ón the composition of Souvignon Blonc must,
C h e m . , 55 (2007) 10281-10288.
l. C.Peyrof des Gochons, T. Tominogo, D. Dubourdieu, Locolizotion of s-cysteine conjugóes it tÀ; -oerry: Effect of skin contoct on oromoic potentioi of vitis vinifero L. cv. souvignon Bronc must, Am. J. rÀor. Vitic., s3 (2002) 144-146.
8. C. Storkenmonn, B. Le Colve, y. Nicloss, l. Coyeux, s. Beccucci, M. Troccoz, olfoctory pèibéptùn of cysteine-s-conjugotes from Fruits onb vég-.tbótes, .1. Agric. Food Chem., 5ó (2008) 9575.
