Formation of Hydrogen Sulfide and Glutathione During Fermentation of White Grape Musts

Transcription

1 Frmatin f Hydrgen Sulfide and Glutathine During Fermentatin f White Grape Musts SEUNG K. PARK ~, ROGER B. BOULTON 2, and ANN C. NOBLE 3. Vlatile sulfur cmpunds (VSCs) and nnvlatile thil-cntaining cmpunds (NVTCs), which are ptential precursrs f VSCs, were mnitred by gas chrmatgraphy during fermentatin f eight white grape musts: ne Thmpsn Seedless, ne Palmin, ne Chenin blanc, tw Sauvignn blancs, and three Chardnnays. The assimilable amin acid (EAA) cncentratin f the musts ranged frm mg/l t 222 mg/l (mean = 3 mg/l). Using a GC-flame phtmetric detectr, three sulfur cntaining peaks were tentatively identified based n their retentin times: hydrgen sulfide (H2S), ethylmercaptan, dimethylsulfide, and ne unknwn peak (RT, 9.7 min) was detected. The predminant VSC, H2S, was cntinuusly prduced thrughut fermentatin and was highest during rapid grwth phase f yeast. All juices prduced H2S mst rapidly during rapid yeast grwth, but musts with lw EAA generally prduced higher levels f H2S thrughut the fermentatin. Ttal H2S, which ranged frm 2 t 5 mg/l, was inversely crrelated with cncentratin f assimilable amin acids (p <.) and with ttal nitrgen cntent (p <.). H2S at the end f fermentatin was negatively crrelated with EAA (p <.) and psitively with days t dryness (p <.5). Fr all musts, cncentratin f NVTCs, primarily glutathine (GSH), steadily increased twards the end f fermentatin. Final wine cncentratin f GSH (. t 5. mg/l) was crrelated with bth ttal N (p <.) and EAA (p <.5). KEY WORDS: vlatile sulfur cmpunds, hydrgen sulfide, thils, glutathine, grape juice, fermentatin, wine The mechanisms f frmatin f vlatile sulfur cmpunds (VSC) during wine fermentatin are nly partially understd due t the cmplex nature f the factrs invlved in winemaking. The prductin f VSCs during fermentatin has been studied in musts varying widely in cmpsitin as well as in mdel systems with nutrient deficiencies r with residual elemental sulfur. Thus, cnflicting results are reprted, which are ften valid nly under specific labratry cnditins r fr specific must cmpsitins. Previus studies indicated that factrs influencing frmatin f H2S during fermentatin include pantthenate deficiency, [,5,2], elemental sulfur [,5,2,23], yeast strain [7], free amin nitrgen level [,25], and fermentatin cnditins [5,,25]. Sme factrs, such as pantthenate deficiency, seldm cntribute t H2S frmatin in grape musts [7,5], while the cntributin f many ther variables can be reduced r eliminated. Residual sulfur can be eliminated by cntrl f vineyard applicatin f elemental sulfur. Deficiencies in nitrgen cntent can ften be remedied by additin f diammnium phsphate [,25]. Similarly, yeast strains which are lw prducers f H2S can be used fr fermentatin [7]. Despite this, H2S prblems still spradically ccur during wine fermentatins. Assciate Prfessr f Fd Chemistry, Kyung Hee University, Department f Fd Science & Technlgy, Yngin-Si, Kyungki-D, 9-7, Suth Krea; and 2.3 Prfessrs f Enlgy, Department f Viticulture and Enlgy, University f Califrnia, Davis, CA 95, USA. *Crrespnding authr ucdavis.cm]. This research was cnducted at the Department f Viticulture and Enlgy f the University f Califrnia, Davis, USA. Acknwledgement: Authrs gratefully acknwledge the financial supprt f the American Vineyard Fundatin. Manuscript submitted fr publicatin April 9 999; revised 3 Nvember 999. Cpyright 2 by the American Sciety fr Enlgy and Viticulture. All rights reserved. 9 Anther ptential surce f H2S culd be the tripeptide, glutathine (L-7-glutamyl-L-cysteinylglycine), which accunts fr % f the dry weight f the yeast S. cerevisiae []. One f its cmpnent amin acids, cysteine, has been implicated as a precursr in the prductin f H2S during yeast fermentatins [2,2]. Hwever, nthing is knwn abut the rle f glutathine (GSH) in prductin f H2S during fermentatin r aging f wines. In this study, frmatin f VSCs, including H2S, and f GSH and ther nnvlatile thil-cntaining cmpunds (NVTCs), was examined during fermentatin f eight white grape musts. Materials and Methds Cmpsitin f musts: Eight different musts were btained frm grapes grwn in the vineyards t which n sulfur had been applied within tw mnths f harvest. Grapes were crushed and 5 mg/l sulfur dixide added. After being settled at C, the racked musts were sterile-filtered thrugh.5-~tm membrane filter under nitrgen and stred in stainless steel drums at C until used. Brix, ph and titratable acidity fjuices were determined by standard prcedures [3] (Table ). Amin acid cntents were determined by a high perfrmance liquid chrmatgraphy equipped with a flurescence detectr (Hewlett Packard 9 HPLC system, Avndale, PA). Amin acids were derivatized with 2- mercaptethanl and -phthalaldehyde fllwed by the separatin n Cls reversed phase clumn (25 cm X mm, 5-mm packing, Waters, Milfrd, MA) as described elsewhere [2]. Ttal nitrgen cntent was determined by a Carlsn ammnia analyzer after digesting the juices [5]. Fermentatin: Musts (5 ml) were inculated

2 ~ 92-- PARK etal. Variety TA b Thmpsn Seedless Palmin Chenin blanc Sauvignn blanc Sauvignn blanc Chardnnay Chardnnay (Lw N) Chardnnay (High N) Table. Variety and cmpsitin a f eight grape musts. Cde adetermined by standard methds [3]. bexpressed as grams tartaric acid per ml. Origin (TSeed) Davis 23. (Pal) Davis 23.9 (Chenin) Sledad 9. (S.blanc-D) Davis 22.2 (S.blanc-O) Oakville 2.7 (Chard-L) Livermre 22. (Chard-L-O) Oakville 23.5 (Chard-H-O) Oakville 23.5 with. g f active dry Saccharmyces cerevisiae, Mntrachet (Universal Fds, Milwaukee, WI) after the yeast was rehydrated in water and incubated at 35 C fr 3 minutes. The inculated juices were sparged with 3 ml f sterile-filtered air. Fermentatins were cnducted at 23 C +. C and stirred at 2 rpm using a ne liter fermentr (Applikn, The Netherlands). Fermenting must (3 ml) was remved daily with a hypdermic syringe thrugh the Tefln-cated septum fr the analysis f sugar, yeast bimass and NVTCs. Fr the analysis f VSCs,.2 ml f headspace gas was withdrawn frm each fermentr daily. Juice analysis: A high perfrmance liquid chrmatgraphy system with a catin-exchange clumn (Bi-Rad, Hercules, CA) was used fr the analysis f sugars in fermenting juices. Glucse and fructse were quantified using standard curves. T estimate the yeast bimass, absrbance f fermenting medium was measured at 5 nm daily. Analysis f nnvlatile thil-cntaining cmpunds (NVTCs): The cncentratin f NVTCs was analyzed by HPLC (Hewlett Packard 9) fllwing pre-clumn derivatizatin using -phthalaldehyde and 2-aminethanl (Aldrich, Milwaukee, WI) as described elsewhere []. Analysis f VSCs: A Hewlett Packard 59 Series II gas chrmatgraph equipped with a flame phtmetric detectr (HP 925A) was used fr the detectin f VSCs which were separated n tw different capillary clumns cnnected in series. A nnplar capillary clumn (DB-, 3 m X.53 mm; film thickness,.5 mm, J & W Scientific, Flsm, CA) was serially cnnected t a plar capillary clumn (DB-Wax, 3 m X.53 mm; film thickness,. mm, J & W Scientific, Flsm, CA) using a direct capillary cnnectr frm Hewlett Packard (Avndale, PA) t minimize a dead vlume and maintain maximum inertness. Oven temperature was held at 27 C fr 3 minutes, then increased at 3 C per minute t 23 C. The injectr and detectr temperatures were 7 C and 2 C, respectively. Fr FPD, 72 ml/min hydrgen and 9 ml/min air were used. Carrier gas (He) flw rate was ml/min. Standard H2S (9.5 ppm) and SO 2 (.5 ppm) gases were purchased Brix frm Liquid Carbnic (Ls Angeles, CA). Authentic sulfur cmpunds, methanethil (MESH), ethanethil (EtSH), dimethylsulfide (DMS), ph ethylmethylsulfide (EMS), diethyl-.57 disulfide (DEDS) were purchased.59 frm Eastman Chemical (Rchester,.7 NY)..7 Headspace gas (.2 ml) was.9 withdrawn frm the fermentrs us-.5 ing a gas-tight syringe (Alltech,.5 Deerfield, IL) equipped with an n-.5 ff valve and injected directly int the splitless injectr f the GC. Detected sulfur vlatiles were quantified using ethylmethylsulfide (EMS) as an external standard. Headspace gas (.2 ml) cntaining mg/l EMS was injected between experimental headspace analysis runs. Cmpunds were identified by retentin times using authentic standards and quantified by develping the standard curves fr each cmpund. Cncentratins were expressed as mg/l headspace gas in the fermentr. Statistical analysis: Pearsn crrelatins were calculated between must and wine cmpsitinal variables and fermentatin respnses [9]. Where n detectable amunts f cmpnents were fund, values f. were assigned t permit calculatins. Results and Discussin Amin acid cmpsitin: The cncentratins f individual amin acids are shwn in Table 2, while must ttal nitrgen and the sum f the "easily assimilable" amin acids (EAA) are presented in Table 3. The "easily assimilable" amin acids were defined as the sum f amin acids which are first used in grape juice fermentatin [], and hence described as the ttal phase amin acids. Prline and GABA which can nt be metablized by yeast during fermentatin [,,3] were nt included in the EAA sums, but cntributed t the ttal nitrgen cntent (Table 3). EAA ranged frm mg/l (Chard-L-O) t 279 mg/l (Palmin), with crrespnding ttal nitrgen cntent frm t 9 mg N/L. Of the sulfur-cntaining amin acids, cysteine was nt detected, while methinine ranged frm belw detectin levels t 52 mg/l. Rate f fermentatin: The fermentatin rates measured by the decrease in glucse and fructse are shwn in Figure. Glucse was metablized at a slightly higher rate than fructse in all musts except S- Blanc-O. Fur musts (Palmin, C. Blanc-S, Chard-L- O, and Chard-L) shwed a rapid increase in yeast bimass with n lag time (Fig. 2). The ther fur (Thmpsn seedless, S. Blanc-O, S. blanc-d, and Chard-H-O) shwed a lag f tw t fur days befre yeast grwth and fermentatin were initiated. Althugh yeast grwth began immediately in Chard-L-O (lwest EAA), it had the lwest ppulatin f yeast and the slwest rate f fermentatin.

3 H2S and GLUTATHIONE- 93 Table 2. Cncentratin (mg/l) f amin acids and glutathine in eight grape juices. Pal - Palmin-Davis; TSeed - Thmpsn Seedless, Davis; Chenin - Chenin blanc, Sledad; S blanc O - Sauvignn blanc, Oakville; Chard-L - Chardnnay, Livermre (Wente); S blanc D - Sauvignn blanc, Davis; Chard-L-O - Chardnnay Lw n (Oakville). Cde ASP GLU ASN SET GLN HIS GLY THR ALA ARG TYR VAL MET TRP PHE ILE LEU PRO GABA GSH-m Amin acid Pal TSeed Chenin S blanc O Chard-L S blanc D Chard-H-O Chard-L-O Aspartic acid Glutamic acid nf Asparagine 2 nf 23 nf 3 nf 22 nf Serine G lutamin e 5 2 nf nf nf 5 Histidine nf 2 nf nf nf nf nf nf Glycine nf nf nf nf 55 nf nf nf Threnine nf Alanine Arginine Tyrsine 2 22 nf nf nf nf nf Valine Methinine nf nf Tryptphan Phenylalanine Islleucine Leucine P rline 'y-aminbutyric Glutathine (must) nf = nt fund The shrtest time f fermentatin t dryness was fund fr Chenin ( days) and the lngest was Chard-L- O ( days). Days t dryness were crrelated with EAA (r =.73, p <.5) (Table ) but nt with TOTN. Althugh a minimum f mg/l f assimilable nitrgen is reprted t be required fr cmplete fermentatin [2], all musts finished fermentatin under these labratry fermentatin cnditins. The absence f stuck fermentatins in these lw EAA and clear (sterile-filtered) juices may be attributed t the rapid stirring f the fermentatins as well as the relatively warm fermentatin temperature []. Prductin f VSCs: In additin t ne unknwn peak, peaks with retentin times crrespnding t thse f HzS, sulfur dixide (SO2), ethanethil (EtSH), Table 3. Days t dryness, cncentratins f must assimilable acids (EAA), must ttal nitrgen, and wine glutathine (mg/l), and f ttal hydrgen sulfide and H2S in headspace (pg/l), at the end f fermentatin. Days t Ttal Wine Ttal Must dryness EAA nitrgen GSH H2S Pal TSeed Chenin S blanc O Chard-L S blanc D Chard-H-O C hard- L-O. 5 and dimethylsulfide (DMS) were detected. Early in the fermentatins, EtSH and DMS were spradically present, but bth disappeared cmpletely by the end f fermentatin. Only H2S was prduced cntinuusly thrughut the fermentatin as shwn in Figure 2. Althugh ne juice with high EAA (S.Blanc-D) prduced high levels f H2S (Table 3), the ttal prductin f H2S was crrelated with EAA (r =-., df =, p <.) (Table ). In general, when there were enugh EAA, the highest cncentratins f H2S were reached during rapid fermentatin, and then prductin f H2S decreased tward the end f fermentatin cnsistent with ther reprts [9,25,2]. In lw EAA musts, H2S was als prduced in high cncentratins relatively early in the grwth phase, peaking when fermentatin rate was mst rapid, but this was fllwed by steady increase thrughut fermentatin (Fig. 2). H2S at end Actively grwing yeasts reduce sulfite t sulfide t bisynthesize the sulfur-cntaining amin acids. The prductin f sulfide at a higher rate than it can be metablized may re- 9 sult in yeast excreting sulfide int 9 the wine media [,22] accunting fr 9 the spike in H2S during the rapid 2 yeast grwth phase. Hwever, juices 3 with lw EAA and ttal nitrgen 7 (Chard-L-O and Chard-H-O) cntin- 2 ued t prduce higher levels f HzS thrughut fermentatin as bserved previusly [,9,25] perhaps

4 ~ 9-- PARK etal because f nitrgen r EAA deficiency. Fr the sulfur-cntaining EAA, methinine has been reprted t repress and inhibit enzymes invlved in the uptake f sulfate and in its cnversin t sulfide prir t cysteine bisynthesis [2,22,2]. Thus, presence f very lw levels f methinine culd result in relatively high H2S cncentratins during fermentatin []. With the exceptin f S Blanc-D, juices cntaining relatively high amunts f methinine ( 33 mg/ L t 52 mg/l) prduced lwer levels f ttal H2S than thse lw in methinine. Hwever, n H2S dr was detected at the end f the fermentatins, even in Chard-L-O which had the GLUCOSE I... <>"-- T, Seedles~ ~i Palmin i : ~-*--.---~... ~--" C,Blanc-S 2 t -~.~ ~"~--..~=.~ ~, "---~--" S,Blanc-D 't 2 ~ ~ - + : " 7:~", - i - ~"=~~ :-, ~--i =-~ ,5 highest cncentratin f H2S. The Mntrachet yeast strain used in this study typically is a high prducer f H2S []. Thus the absence f H2S dr in these wines may be the result f atypical fermentatin cnditins. These small-vlume fermentatins were cntinuusly stirred at a temperature higher than that encuntered in typical white fermentatins. In industrial scale fermentatins at lwer temperatures, the H2S and ther VSCs, such as methyl and ethyl mercaptans can physically be trapped and remained in finished wine. Althugh mre H2S is frmed in rapidly fermenting musts at higher temperatures [5], much f the H2S is lst by entrainment in the rapidly evlved carbn dixide. FRUCTOSE 2.~:~--~"'~, ~-.-. " T.Seedess... ~ Palmin...,... C.Blanc-S... S.Blanc-D 2 3 5; ~ =]: :-~":"... :"'"""... :::: : ' " '... " " " :- " "]... c,... S.Blanc-O Chard-L I ~ S.Blanc-O I Chard-L uj O ~ ~!! I! I!! T ~ "l: I!! I ~ T-'t- l I!! T " I! T -'T-r-V -'~ I t2!35 z "l......! t Chard-L-O,. ----'-- Chard-H-O l! l T T " I ~ T I 3 5 G 7 9 I35 FERMENTATION TIME (DAYS) " ~... =... Chard-L-O.t S 7 9 t35 FERMENTATION TIME (DAYS) Fig.. Decrease in glucse and fructse cncentratins during fermentatin.

5 2 i... ~ T, ~l~ ~ ~, -... =... Palmin... =.... C.Blanc-S " ~ ' " P " - " S.B]anc-D c ~ " z!..., YEAST BIOMASS HYDROGEN SULFIDE GLUTATHIONE 2 i~ ~ T.Seedess i i ----c... Palmin ]SO ~! "--~----- C.Bla:nc-S O % -'-"-'- S.Blanc-D T.S~~ ~~ii! Palmin C.Bi!anc-S 7 S, B I a n c:-d " 5 :3. ), 3 c_ m :3 m <,,.. J <._A z b,},2} - " I ' I " I"... I ' i " I ' I " I " l ' l " I " I ' I ~I 2 3 S S.Blanc-~ ~ Chard-L. - ) 2- :-' t 2 3 S ,-., 5 "-" (..) O tj ;SO I 2 3 S l 9 :235 ']: ~"" ~:...:..... :.:.:._,.,,~,.,,, ----,--- S.Blanc-O Chard-L T! I I! I i! I I I I I " r :. - v ~! I 2 3 S S ~, " - " "T I I l- I :: : ::::~:--... ::"::::::... " :... :::: :: ':" ';::::"": " ': ' :.-:-7-:~ S.Banc-O ~ Chard-L ~7 ~. ::.~u,~ 2 3 S ~ Chard-L-O Chard-H-O II I I I I I I! r ~! I l I I " I 3 S 7 B : S 7 9 IO:235 i 5 O SO '- =... Chard-L-O Chard-H-O FERMENTATION TIME (DAYS) 9 B 7 S 3 2.~. :... ~--- Chard-L--O Chard-H-O ' ,,,~,, ~... "It ~ T - Ir - "r " "IF '~* T - T ~ lr " "iv " I; " i "~! '~ I " T " I " I 2 3 S "" IX) O. G) r- C --I > "" ITI I c ' Fig 2. Increase in yeast bimass (estimated by absrbance at 5 nm) (left), prductin f hydrgen sulfide (~g/l headspace) (center), and glutathine (mg/l) (right) during fermentatin.

6 ~ 9-- PARK etal. Table. Significant Pearsn crrelatin cefficients between selected cmpsitin and fermentatin parameters (df = ). Parameter EAA Ttal N GSH-m GSH-w Aspartic. Threnine Arginine Valine P h enylalani n e Isleucine.7.73 Leucine.73. EAA Ttal N Must glutathine (GSH-m) Wine glutathine (GSH-w) Ttal H2S -. H2S at end -.3 Days t dryness (DRY) -.73 Ttal H2S End H2S Dry Significance f crrelatin cefficients: r >.7, p >.5; r >.79, p >.2; r >.3, p >. r >.92, p >.. Prductin f NVTCs: As shwn in Table 2, Palmin juice cntained the highest level f GSH (.2 mg/ L) whereas ther juices cntained nly trace amunts ( t.3 mg/l. Upn fermentatin, glutathine and a tentatively identified glutamyl-cysteine were detected in all juices with GSH the predminant NVTC bth during fermentatin and in finished wines. GSH levels increased tward the end f fermentatin suggesting that actively fermenting yeasts can prduce and release high amunts f GSH during fermentatin (Fig. 2). As shwn in Table, must and wine GSH levels were highly significantly crrelated with each ther and with bth ttal nitrgen (r =.9 and r =.7, p <., df =, respectively) and EAA (r =.79 and r =.7, p <., respectively). In the finished wines, Palmin juice, which had the highest juice GSH, ttal N and EAA, had the highest cncentratin in wine (5. mg/l) while GSH was belw. mg/l in the must with lwest EAA and ttal N (Chard-L-O). Althugh it is nt knwn if GSH is a precursr f H2S r mercaptans during aging, several factrs suggest it shuld be studied further. It is abundant in dry yeast [] and as demnstrated in this study is frmed during fermentatins. One f its cmpnents, cysteine, can be cnverted t H2S [2,2] and further it is pssible that the strng reducing agent GSH culd reduce elemental sulfur t H2S [27]. Relatinship between must cmpsitin and H2S prductin. Based n the EAA levels, juices can be categrized in fur grups: Extremely high: Palmin (279 mg/l); High (5-29 mg/l): T. Seedless, S. blanc-d, Chenin; Intermediate (9-73 mg/l): Chard-L, S. blanc-o, Chard-H-O; and Lw ( mg/l): Chard-L-O. Musts lw in EAA tk lnger t ferment and generally had higher H2S levels bth during and at the end f the fermentatins. Hwever the levels f H2S prduced are nt cnsistently inversely related t EAA. Of the amin acids quantified, seven were significantly crrelated with either r bth ttal and ending H2S. The crrelatins f these amin acids with H2S levels, GSH and days t dryness are shwn in Table. Aspartic acid and arginine were the nly amin acids significantly crrelated with bth H2S measures and days t dryness. S. blanc-d (high EAA) which prduced a high level f H2S, had average levels f these tw amin acids, but was lw in three which had significant inverse crrelatins with ttal HzS (phenylalanine, isleucine, and leucine). Frm the analysis f these eight musts, it is bvius that EAA is much mre strngly related t the prductin f H2S and slwed fermentatins mre than ttal nitrgen. But mre specific cnclusins cannt be drawn. Despite attempts t use partial least squares regressin t mdel prductin f HzS using the cmpsitin, nly pr slutins resulted. Similarly in principal cmpnent analysis f the data, the nly pattern that emerged was ne in which Palmin was separated frm the ther musts based n its higher level f EAA, TOTN and individual amin acids. Cnclusins Ttal prductin f H2S in eight grape juices was inversely related t the levels f EAA. In general, the highest cncentratins f HzS were prduced in the mst rapid phase f fermentatin. Althugh the maximum cncentratin was higher in musts high in EAA, the prductin f HzS decreased tward the end f fermentatin. In cntrast, must with lw nitrgen shwed prductin f H2S thrughut the fermentatin and had the highest cncentratin at the end f fermentatin. Deficiency in EAA may be a majr factr in the frmatin f H2S at later stages f fermentatin. The prductin f NVTCs, especially GSH was prprtinal t the levels f EAA, but its rle in frmatin f VSCs during strage and aging is unknwn.

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