Control of hydrogen sulfide formation during fermentation

Transcription

1 II 1 ii d Cotrol of hydroge sulfide formatio durig fermetatio Ala T. Bakalisky, Olga Marti, ad Jim Keedy, Departmet of Food Sciece ad Techology, Orego State Uiversity INTRODUCTION Hydroge sulfide ad related sulfur compouds produced iterally by yeast durig fermetatio are cosidered highly udesirable if they are excreted ad exceed threshold cocetratios i the fiished wie, as they cause a upleasat "reduced character". The threshold for hydroge sulfide is o the order of parts per billio ad that for some of the other reduced sulfur compouds derived from hydroge sulfide rages from parts per millio to parts per billio. Factors which affect productio of these compouds iclude, but are ot limited to, the utritioal status of the grapes, yeast strai, ad fermetatio coditios (Rauhut, 1993). Formatio of excessive reduced character i table wies produced from soud grapes usig good wiemakig practices is a perplexig problem because it ca occur upredictably, ad spoil affected wies. Yeast produces this class of compouds iterally as a byproduct of the ormal ad ecessary sythesis of S-adeosyl-methioie ad the sulfur-cotaiig amio acids, methioie ad cysteie (Thomas ad Surdi-Kerja, 1997). Excessive sythesis leads to excretio of hydroge sulfide ito the wie. Although excess hydroge sulfide formatio has geerally bee correlated with itroge deficiecy (Jiraek et al., 1995a, 1995b), deficiecies i vitami B 6 ad patotheic acid, ad residual sulfur o grapes from the applicatio of fugicides have also bee implicated. I oe study, formatio of hydroge sulfide was foud ot to correlate with variatio i activity of the formative ezyme, sulfite reductase, which reduces sulfite to hydroge sulfide (Jiraek et al., 1996). This suggests that the amout of extracellular hydroge sulfide formed is cotrolled by other rate-limitig steps: its utilizatio i sulfur amio acid biosythesis, its active or passive trasport from the cell, or cosumptio through other biochemical pathways. Formatio still occurs uexpectedly, ad ofte eough to be cosidered a persistet problem (Boulto et al. 1996; Park et al. 2000). Oe complicatig factor i the study of hydroge sulfide productio is that formatio durig the early stages of fermetatio is ot always problematic, either because the yeast evetually takes up ad reuses the excreted sulfide or because the volatile sulfide is removed by etraimet i the stream of carbo dioxide that evolves durig the course of fermetatio. At the latter stages of fermetatio, whe excretio of the hydroge sulfide is believed to be most troublesome, the less vigorous yeast may o loger take it up, or evolutio of carbo dioxide may be so slowed as to o loger be a efficiet meas of removal. As a cosequece, the total amout of hydroge sulfide formed i a fermetatio is likely to be a poorer predictor of evetual reduced character tha measuremet at the ed of fermetatio. Because hydroge sulfide is produced withi the yeast cell directly from sulfite, its immediate biochemical precursor, we are testig the hypothesis that excretio of sulfite by yeast will miimize hydroge sulfide productio. Excretio of sulfite occurs due to the activity of the yeast protei, Ssu1 (Avram ad Bakalisky, 1997; Avram et al. 1999; Park et al. 1999; Park ad Bakalisky, 2000; Goto-Yamamoto et al. 1998), which has also bee implicated i seleium efflux (Piso et al. 2000). If the hypothesis is foud to be true, it will provide a simple meas of idetifyig wie yeast strais that ted to produce low levels of hydroge sulfide durig fermetatio. SPECIFIC AIM The aim of this year's experimets is to determie if icreased activity of the yeast sulfite pump (Ssu1) i laboratory strais ca miimize hydroge sulfide formatio. If so, experimets will be proposed i the secod year to determie Ssu1 activity i wie yeast strais that produce variable amouts of hydroge sulfide, ad to examie ways i which pump activity ca be icreased. 54

2 MATERIALS AND METHODS Yeast strais, media Three previously characterized laboratory strais of Saccharomcyes cerevisiae were used: d, MATa leu2-3, 112 ura3-52, d-T4-L1, MATa leu2-3, 112 ura3-52 ssu1l1, ad b, MATa leu2-3, 112 ura3-52 FZFJ-4 (Avram ad Bakalisky, 1997; Park ad Bakalisky, 2000) Coded wie strais 6040, 6494 ad 6012 were obtaied from Lallemad. Strais were grow i stadard rich medium (YEPD) or i a sythetic grape juice (Salmo ad Barre, 1998) supplemeted with uracil ad leucie, ad variable amouts of other amio acids ad ammoia. Sulfite efflux pump activity Activity of the sulfite efflux pump Ssu1 was measured as described (Park ad Bakalisky, 2000). Briefly, yeast cells were harvested i log phase durig growth i a rich laboratory medium (YEPD), washed i tartaric acid-based buffer supplemeted with glucose, ad resuspeded i the same buffer at about 3 mg dry weight cells per mi. Sulfite uptake was iitiated by additio of 1 mm sulfite at time zero. Cell samples were take at 30 secods ad at oe miute itervals from oe through 10 miutes. Total itracellular sulfite was measured based o the pararosailie colorimetric assay ad was expressed as a fuctio of dry weight cells. Hydroge sulfide Hydroge sulfide was measured at the ed of fermetatio i sythetic grape as a moobromobimae derivative by HPLC with fluorescece detectio (Newto et al., 1981). Samples of the fermeted sythetic juice were filtered to remove cells ad other isoluble material prior to reactio with moobrombimae. Sodium sulfide was used as a stadard. RESULTS ad DISCUSSION Sulfite accumulatio (Ssul activity) Sulfite accumulatio was measured i three laboratory strais ad three wie strais of the yeast S. cerevisiae. This measure reflected activity of the sulfite efflux pump, Ssul. The three laboratory strais were chose because they differ widely i pump activity. Strai b has a hyperactive pump, strai d has a ormal pump, ad strai d-T4-L1lacks a pump. The maximum level of itracellular total sulfite was foud to be about 28 moles/mg dry weight cells i d-T4-L1, about 18 moles/mg dry weight cells i d, ad about 3 moles/mg dry weight cells i b (Fig 1). Coded wie strais 6494 ad 6012 accumulated about 20 moles/mg dry weight cells ad wie strai 6040 accumulated about 2 moles/mg dry weight cells (Fig 2). Hydroge sulfide A represetative HPLC trace of hydroge sulfide as a bimae adduct is show i Figure 3. At least 5 other peaks are visible which correspod to breakdow products of moobromobimae. Figure 4 shows excellet correlatio betwee the peak area correspodig to the sulfide-bimae adduct ad added sulfide. This method of detectio has a sesitivity i the rage ofpicomoles (10-12 moles). We have sice modified the liquid chromatography gradiet so that the sulfide-bimae adduct elutes at about 18 miutes rather tha at 50 miutes (data ot show). We. have also modified the derivatizatio reactio to miimize formatio of the additioal fluorescet bimae breakdow products (data ot show). Prelimiary fermetatios I order to determie if a correlatio exists betwee activity of the sulfite efflux pump, Ssu1, ad hydroge sulfide excretio, trial fermetatios were performed. I the first model wies produced by the three laboratory strais, o hydroge sulfide was detected (data ot show). The model grape juice used had a high itroge cotet (300 mg!l as ammoia ad primary amio 55

3 II J i H 1l acids) ad this may have preveted excretio of hydroge sulfide by the yeast. Additioal fermetatios are curretly beig evaluated usig model grape juices cotaiig 60 to 100 mg!l itroge, ad variable amouts of the vitamis bioti ad patotheate. Our ratioale is that hydroge sulfide productio will be ehaced if itroge or these vitamis are decreased to suboptimal levels. LITERATURE CITED Avram, D. ad Bakalisky, A.T SSUJ ecodes a plasma membrae protei with a cetral role i a etwork of proteis coferrig sulfite tolerace i S. cerevisiae. J. Bact.179: Avrar, D., Leid, M., ad Bakalisky, A.T Fzflp of Saccharomyces cerevisiae is a positive regulator of SSUJ trascriptio ad its first zic figer regio is required for DNA bidig. Yeast 15: Boulto, R.B., Sigelto, V.L., Bisso, L.F., ad Kukee, R.E Priciples ad practices of wiemakig. Chapma ad Hall, NY. Goto-Yamamoto, N., Kitao, K., Shiki, K., Yoshida, Y., Suzuki, T., Iwata, T., Yamae, Y., Hara, S SSUJ-R, a sulfite resistace gee of wie yeast, is a allele of SSUJ with a differet 11 upstream sequece. J. Perm. Bioegieer. 86: Jiraek, V. Lagridge, P., ad Heschke, P.A. 1995a. Validatio of bismuth-cotaiig idicator media for predictig H 2 S-producig potetial of Saccharomyces cerevisiae wie yeasts }l uder eological coditios. Am. J. Eol. Vitic. 46: Jiraek, V. Lagridge, P., ad Heschke, P.A. 1995b. Determiatio of sulphite reductase activity ad its.respose to assimilable itroge status i a commercial Saccharomyces cerevisiae J wie yeast. J. Appl Bacteriol81: Jiraek V, Lagridge P, ad Heschke P.A Regulatio of hydroge sulfide liberatio i wie-producig Saccharomyces cerevisiae strais by assimilable itroge. Appl Eviro Microbial 61 :461-7.,.: l. t Newto, G.L., Doria, R., ad Fahey, R.C Aalysis of biological thiols: derivatizatio with moobromobimae ad separatio by reveresed-phase high-performace liquid. i chromatograpy. Aalyt. Biochem. 114: 'i Park, H. ad Bakalisky, A.T SSUJ mediates sulfite efflux is. cerevisiae. Yeast 16: Park, H., Lopez, N.J., ad Bakalisky, A.T Use of sulfite resistace i S. cerevisiae as a :! domiat selectable marker. Curr. Geet. 36: li Park, S.K., Boulto, R.B., ad Noble, A.C Formatio of hydroge sulfide ad glutathioe durig fermetatio of white grape musts. Am. J. Eol. Vitic. 51: Piso, B., Sagot, 1., ad Daiga-Fomier, B Idetificatio of gees affectig seleite toxicity ad resistace i Saccharomyces cerevisiae. Mol. Micro. 36: Rauhut, D Productio of sulfur compouds. I: Wie Microbiology ad Biotechology, ed. G.H. Fleet, p , Harwood Academic Publishers. Chur, Switzerlad. l1 Salmo, J.-M. ad Barre, P Improvemet of itroge assimilatio ad fermetatio kietics uder eological coditios by derepressio of alterate itroge-assimilatory pathways i a idustrial Saccharomcyes cerevisiae strai. Appl. Ev. Micro. 64: Thomas, D. ad Surdi-Kerja, Y Metabolism of sulfur amio acids i Saccharomcyes l I cerevisiae. Micro. Mol. Bioi. Rev. 61: ll i 1 j ACKNOWLEDGEMENTS We thak the Orego Wie Advisory Board ad the Pacific NW Ceter for Small Fruits Research for fiacial support.! 1 u 56

4 Figure 1. Sulfite accumulatio i three laboratory strais of Saccharomyces cerevisiae, b, d, ad d-T4-Ll. The data are meas of two replicates per strai. SULFITE ACCUMULATION IN cH4 L1.r 3o ) 25.; ;,1.lfl 20 e,o;; :. i Ill i I LAB YEASTS 309Q-9d Time (mi) Figure 2. Sulfite accumulatio i three wie strais of Saccharomyces cerevisiae. The data are meas of two replicates, except for strai 6040 which was ureplicated. The data for 6494 overlap that of SULFITE ACCUMULATION IN 3 WINE YEASTS 25 ~ )I Do,,. E --~~~. 15 "D. 0 II: 10 - I 0 D -lfl Time (mi) 57

5 I. ' ~ I Figure 3. Represetative reversed phase HPLC trace of sodium sulfide-bimae adduct elutig at 50.8 miutes. The peak correspods to 250 picomoles of added sodium sulfide detected by fluorescece (excitatio 360 m, emissio 480 m). The other peaks represet bimae breakdow products. LU 1J o-1--" ml Figure 4. Plot of peak area as a fuctio of icreasig amout of sodium sulfide-bimae adduct as detected by fluorescece followig separatio by reversed phase HPLC R 2 = !" 600 q < I 400 } !! d Sodium sulfide (moles)