Evolution of Volatile Sulfur Compounds during Wine Fermentation

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Evolution of Volatile Sulfur Compounds during Wine Fermentation Matias Ivan Kinzurik, Mandy Herbst-Johnstone, Richard C Gardner, and Bruno Fedrizzi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02984 • Publication Date (Web): 14 Aug 2015 Downloaded from http://pubs.acs.org on August 19, 2015

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Journal of Agricultural and Food Chemistry

Evolution of Volatile Sulfur Compounds during Wine Fermentation

Matias I. Kinzurik

§,*

, Mandy Herbst-Johnstone §, Richard C. Gardner †, and Bruno Fedrizzi

§,*

§

School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland, New

Zealand †

School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New

Zealand

* Author to whom correspondence should be addressed: Tel: + 64 9 9238473 Fax: + 64 9 3737422 E-mail: [email protected] (MIK); [email protected] (BF).

§ †

School of Chemical Sciences, University of Auckland School of Biological Sciences, University of Auckland 1 ACS Paragon Plus Environment


1

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Abstract

2 3

Volatile sulfur compounds (VSCs) play a significant role in the aroma of food and beverages.

4

With a very low sensory threshold and strong unpleasant aromas, most VSCs are considered

5

to have a negative impact on wine quality. In this study, headspace solid phase

6

microextraction coupled with gas chromatography mass spectrometry (HS-SPME/GC-MS)

7

was used to analyze the time course of the biosynthesis of 12 VSCs formed during wine

8

fermentation. Two different strains of Saccharomyces cerevisiae, the laboratory strain

9

BY4743 and a commercial strain F15 were assessed using two media: synthetic grape media

10

and Sauvignon Blanc juice. Seven VSCs were detected above background, with three rising

11

above their sensory thresholds. The data revealed remarkable differences in the timing and

12

evolution of production during fermentation, with a transient spike in methanethiol

13

production early during anaerobic growth. Heavier VSCs like benzothiazole and S-ethyl

14

thioacetate were produced at a steady rate throughout grape juice fermentation, while others,

15

like diethyl sulfide, appear towards the very end of the winemaking process. The results also

16

demonstrate significant differences between yeast strains and fermentation media.

17 18 19

Keywords: Volatile sulfur compounds, HS-SPME/GC-MS, yeast, wine fermentation

20 21

2 ACS Paragon Plus Environment

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22


Introduction

23 24

Volatile sulfur compounds (VSCs) are a family of small organic molecules that play an

25

important role in the aroma of food and beverages, determining the quality of the product and

26

leading to either consumer acceptance or rejection.1,2 They can interact with the different

27

flavors and contribute to aroma complexity

28

many foods including wines.2 VSCs have been shown to derive from the grape, microbial

29

fermentation, and chemical reactions.6 With a very low sensory threshold

30

flavor aromas, most are considered to have a negative impact. However, some compounds,

31

like dimethyl sulfide (DMS), are appreciated as adding to the overall bouquet and increasing

32

the red fruit aroma present in some red wines.7-9

33

VSCs in wine are separated into two categories: light (bp < 90 °C) and heavy (bp > 90 °C).

34

Studies suggest that the type and quantity of VSCs can be used to discriminate different wine

35

varieties, oxidation levels, and aging times used in winemaking.10-12 Volatility also plays a

36

big part in the final concentrations of these compounds in the finished wine. Heavier VSCs in

37

particular, which mostly display higher sensory thresholds, present a problem, as they cannot

38

be easily eliminated.7 It is, therefore, highly desirable to prevent their formation in wines.

39

Analytical techniques to detect VSCs have come a long way in the past decade,13 overcoming

40

traditional issues for the detection of these compounds associated with the complexity of the

41

sample matrix, their high reactivity and their low concentration.2 However, there is a

42

remarkable information gap in the literature, currently, regarding the concentration evolution

43

with which VSCs are produced in yeast during winemaking. Equally important, the effect of

44

different strains and media in the fermentation on said concentration evolution could pave the

45

way for a more refined tailoring of the wine, producing more of those VSCs that are coveted

46

by the consumer, while suppressing the formation of the rest.

3-5

participating in the quality and uniqueness of

2,6

and strong off-



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47

In this work we present a time course analysis of the VSCs that arise during fermentation of

48

synthetic and Sauvignon Blanc grape juice, using either a laboratory or a commercial yeast

49

strain. To the best of our knowledge, this is the first report on the kinetics of VSC formation

50

during winemaking, with the exception of H2S.14-16

51 52

Methods

53 54

Chemicals

55 56

The twelve sulfur compounds studied (Figure 1) were hydrogen sulfide (1), methanethiol (2),

57

ethanethiol (3), dimethyl sulfide (4), diethyl sulfide (5), dimethyl disulfide (6), diethyl

58

disulfide (7), carbon disulfide (8), methyl thioacetate (9), S-ethyl thioacetate (10), dimethyl

59

trisulfide (11) and benzothiazole (12). Dimethyl-d6 sulfide (d6-DMS), dipropyl disulfide

60

(DPDS), and 3-(methylthio)-1-hexanol (MTH), were used as internal standards (IS). All of

61

the purchased analytes had a purity of 98% and were supplied by Sigma-Aldrich (Sigma-

62

Aldrich, Germany), except for MTA and ETA (Alfa Aesa, USA). Absolute ethanol was of

63

analytical grade and purchased from Ajax Finechem (Mt. Wellington, Auckland, New

64

Zealand). Tartaric acid was obtained from Sigma-Aldrich (Sigma-Aldrich, Germany).

65 66

Yeast strains and culture

67 68

The study involved two different strains of yeast, the diploid laboratory strain BY4743

69

(MATa/α his3∆1/his3∆1 leu2∆0/leu2∆0 LYS2/lys2∆0 met15∆0/MET15 ura3∆0/ura3∆0) and

70

the commercial wine strain F15 (wild-type diploid; homosporic derivative of Zymaflore F15


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71

obtained from Laffort NZ Ltd, Auckland, New Zealand; originally isolated in Bordeaux,

72

France 17).

73

YPD media (10 g/L yeast extract, 20 g/L casein peptone and 20 g/L glucose) was used for

74

standard yeast pre-culture at 28°C. After pre-cultures reached saturation, cells were pelleted

75

by centrifugation (10 min at 3000 g) and washed twice with 20 mL of sterile water. Then

76

2x106 cells/mL were inoculated into 100-mL cultures of either Synthetic Grape Media (SGM)

77

[pH 3.2, total YAN: 300 mg N/L] (Table 1) or Sauvignon Blanc grape juice (SB Juice) from

78

the Rapaura region of Marlborough in New Zealand [21.4 °Brix (measured at 22°C), pH

79

3.14, total YAN: 206 mg N/L] (kindly supplied by Andy Frost of Pernod Ricard NZ Ltd).

80 81

Fermentation and H2S quantitation

82 83

Fermentations were carried out in triplicate in 250-mL conical flasks containing 100 mL of

84

SGM or Sauvignon blanc grape juice. Flasks were sealed with rubber stoppers fitted with

85

H2S-detecting, silver nitrate tubes (25-2000 ppm Komyo Kitagawa, Tokyo, Japan).

86

Fermentation was performed at 28 °C, with rotary shaking at 100 rpm and was monitored by

87

daily weighing. The concentrations of 1 were recorded by visual reading from the tubes daily.

88

Ferments were considered finished when weight loss was ≤ 0.1 g per 24 h. Wine was

89

separated from yeast cells and solids by centrifugation at 6,000 g for 10 min and was stored

90

frozen at −20 °C. Dimethyldicarbonate (DMDC) 0.2 µL/L was used to sterilize the SB Juice

91

prior to inoculation, and both media after the wine was collected.

92

Cultures were harvested at time zero and at five different stages of fermentation completion,

93

as estimated by percentage of weight loss: 2%, 25%, 50%, 70% and 100%.

94

Control ferments of both media with no cells were also analyzed, as well as media harvested

95

immediately after inoculation with yeast.



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Samples were kept in 50-mL containers with limited head space and frozen before analysis,

97

remaining frozen for no longer than 14 days as this has been observed to minimize VSC

98

loss.18

99 100

Quantitation of VSCs

101 102

All of the HS-SPME/GC-MS analyses were performed in triplicate. The sample preparation

103

procedure and the analytical method used for quantitation of the sulfur compounds have been

104

described elsewhere.19-20

105

Either the wine samples, or serial dilutions in model wine (12% ethanol, 5 g/L tartaric acid,

106

pH 3.2) containing the pure analytes that would make up the calibration curves were

107

separately prepared to a final volume of 10 mL, and placed in amber screw-top 20-mL vials.

108

Fifty µL of the IS mix (30 µg/L d6-DMS, 2 µg/L DPDS and 547 µg/L MTH), magnesium

109

sulfate salt, and magnetic stir bars were introduced as described before.19 To prevent

110

degradation/oxidation reactions from taking place, each sample was purged with argon gas

111

before analysis.

112

In the cases of 1 and 2, which are gaseous at room temperature, standards were prepared fresh

113

using sodium sulfide nonahydrate (Sigma) or sodium thiomethoxide (Sigma) in a solution

114

with model wine.

115

Analysis was carried out on an Agilent Technologies 7890 GC system coupled with a 5975C

116

inert XL MSD (Agilent, USA). The fiber composition was Divinylbenzene/Carboxen-

117

Polydimethylsiloxane (DVB/CAR-PDMS; 50/30 µm x 2 cm) (Product No. 57298U, Supelco,

118

Bellefonte, PA, USA). The separating column was composed of a 30 m x 0.320 mm x 0.25

119

µm HP-1MS coupled with a 30 m x 0.320 mm x 0.25 µm HP-Innowax fused silica capillary

120

column (Agilent, J&W Scientific, New Zealand). Agitation time and intensity was automated


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121

using magnetic stirrer bars in a Gerstel Agitator/Stirrer controlled by MAESTRO Software

122

(Version 1.2.0) (Gerstel, Mülheim an der Ruhr, Germany).

123

Analysis of the samples was performed using the MassHunter workstation software, Agilent

124

technologies (Agilent, USA). The ions used for the identification and quantitation of each

125

compound were chosen according to the literature and NIST library. The statistical software

126

used was SPSS (Version 22, IBM) using one way ANOVA (p < 0.05), and post hoc Tukey’s

127

HSD test (p < 0.05).

128

d6-DMS was used to quantify 1, 2, 3, 4, 5, 9, 10. Isopropyl disulfide was used for 6, 7, 8 and

129

11. 12 were quantified using 3-(methylthio)-1-hexanol as the internal standard.

130

In the cases for 1 and 12, the R2 of the calibration curves are not ideal. 1 and 12 are the two

131

extremes of the volatility/chromatographic space studied; the chromatography for 1 is not

132

ideal due to its high volatility, which we have overcome by using a second method of

133

detection (Kitagawa columns). In the case of 12, its absorption on the fiber does not show a

134

high reproducibility. Given the broad and novel scope of this paper and the attempt to

135

measure such a wide range of sulfur species some compromises had to be accepted.

136 137

Results and Discussion

138 139

The experiments used two yeast strains: BY4743, a diploid auxotrophic version of the widely

140

used laboratory strain S288c, and F15, a homozygous diploid, prototrophic, commercial wine

141

yeast strain whose sequence is known.21 The strains were grown both in synthetic medium

142

resembling grape juice (SGM) and in Sauvignon Blanc (SB) grape juice. Ferments were

143

sampled in triplicate for HS-SPME/GC-MS analysis of VSCs.

144

Both strains grew normally in the media provided (Figure 2A). In order to assess metabolite

145

production, five stages of fermentation were compared with the time zero stage (as well as



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un-inoculated media, see Methods). The five stages correlate with times during fermentation

147

during which groups of known fermentation genes are activated.22, 23 The stages 2% and 25%

148

weight loss correlate to early and late stages of exponential growth. By 50% weight loss,

149

most cell growth was completed, with the number of total cells reaching a plateau at around

150

7.5 x 107 cells for both strains (Figure 2A). From 50% to 100% of weight loss, the cells

151

fermented the remaining sugars producing increased levels of alcohol, but the total number of

152

cells in the culture did not change (Figure 2A).

153

In this work, of the 12 compounds detected by HS-SPME/GC-MS, only seven (those whose

154

results are shown in Figure 3) were found at concentrations above the background level,

155

which is defined as the concentration found at time zero, which for all compounds was

156

indistinguishable from the concentration found in media without cells.

157

compounds that were not detected, the ‘background level’ for three of them was low (7: 0.52

158

± 0.01 ng/mL, 11: 2.4 ± 0.2 ng/mL, 6: 16 ± 1 ng/mL). There was however, significant

159

background detected for both 8 (0.164 ± 0.001 µg/mL) and 4 (1.341 ± 0.002 µg/mL). These

160

high backgrounds possibly originated as contaminants of these compounds in the internal

161

standard mix injected into the sample.

162

Arguably the most studied and the most common VSC to arise during winemaking is 1. It is

163

also the best understood VSC in terms of yeast genetics, with a number of known mutations

164

in the sulfate assimilation pathway that can influence its production during wine making.21, 24,

165

26

166

of the most sought after traits in a winemaking yeast strain.24, 25 In this study, production of 1

167

was measured daily using detection tubes27 (Figure 2B), as well as by HS-SPME/GC-MS

168

(Figure 3A). By both measurements, the laboratory strain BY4743 did not produce detectable

169

1 during fermentation in either media. Both methods, however, did detect 1 from both F15

170

ferments, and both indicated that the majority of production of 1 by F15 occurred close to the

Of the five

Its rotten egg aroma also makes it most undesirable. Low production of 1 is therefore one


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171

time cell growth was completed (around 50% weight loss, see Figure 2A), although some

172

production was initially detected at 25% weight loss in some samples. By HS-SPME/GC-

173

MS, the final concentrations of 1 by F15 in both media stabilized well above the 1 ng/L

174

sensory threshold,28 with production in SB Juice being higher than in SGM for this strain

175

(Table 2).

176

The actual concentrations of 1 measured by the two approaches used here differed by two

177

orders of magnitude. This difference is perhaps not surprising since the methods measured

178

very different parameters: the tubes measured total accumulated production of 1 from a 100-

179

mL sample over the course of the fermentation, while the HS-SPME/GC-MS detection

180

approach measured steady state concentration of 1 that can be removed from the solution into

181

the head space at each time point. In addition to differences in the sensitivity of both

182

detection methods, the volatility of 1 could have played a role in the different concentrations

183

seen; there may also be effects associated with sample handling when breaking the anaerobic

184

confinement of the culture and harvesting the wine. Recently, it has been revealed that the

185

partition coefficients of VSCs, specifically those of 1 in grape juice media, are dependent on

186

the temperature of the fermentation and the composition of the media, specially its sugar

187

content.29 The same was observed for the partitioning of higher alcohols and esters,30 and

188

may

189

1 is known to be highly reactive and can combine with other compounds to form more

190

complex VSCs.31 For example, 3 can be formed in vitro by the reaction of 1 with ethanol or

191

acetaldehyde32, and can combine to form 5.33 Interestingly, in this study 3 presented steady

192

state patterns that were generally similar to 1. It was not detected in either ferment of strain

193

BY4743, and yet appeared high in F15, generally rising throughout the course of

194

fermentation (Figure 3B). However, the kinetics were different since the concentration rose

195

early with clear production even at 2% weight loss and achieved its peak of accumulation at

well

be

the

case

for

heavier

VSCs.



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70% weight loss. In the case of SB Juice this was followed by a drop at the end of

197

fermentation, indicating that the compound may either be consumed by yeast or act as the

198

substrate for a chemical reaction near the end of fermentation (Table 2).

199

Following a similar trend, 9 was also not produced by BY4743 in either media, but was

200

produced by F15. In SGM, production had started by 25% weight loss and increased steadily

201

until the end of fermentation; in SB Juice, however, it was found only at the final stages of

202

fermentation (Figure 3C).

203

Previously, the biosynthesis of 9 has been linked to the availability of a 2 donor in yeast34,

204

however this correlation in the timing of production could not be observed in this study.

205

Previous findings, however, are consistent with genetic strain differences playing a key role

206

in affecting the composition of volatiles in wine.35, 36 Given that formation of 2 has been

207

linked to 1 32, it may be that the biosynthesis of 1, 3 and 9 are associated with each other and

208

the latter two derive from high production of 1 by F15. To date, genetic differences in three

209

genes have been related to differential production of 1 by yeast strains: MET5, MET10 and

210

MET2.21, 26, 37 Of these, the R310G amino acid difference in MET2 stands out in a comparison

211

of F15 and BY4743, since the same genetic difference has already been shown to be

212

responsible for productivity of 1 in a cross between F15 and another wine yeast M2.21 The

213

F15 MET2 R310 allele is less efficient at producing O-acetylhomoserine, the nitrogen-carbon

214

skeleton into which 1 is incorporated to form homocysteine. Further experiments are needed

215

to confirm this role for MET2 in production of 1 by F15, and to determine whether or how

216

this difference in the production of 1 also affects 3 and 9 concentrations.

217

The production of 2 had the most distinct kinetics, as shown in Figure 3E. This compound

218

was found as a discrete pulse early during fermentation, with the pulse occurring earlier in

219

SGM than in grape juice. In SGM, 2 increased dramatically at 2% weight loss, an early

220

exponential stage of growth at which transcription is particularly active.23 It then dropped just


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as dramatically by 25% during fermentation in SGM, and remained at background levels

222

throughout the remainder of the fermentation. This early peak in the biosynthesis of 2 was

223

conserved for both strains and in both media. Interestingly, there was a delay for this peak in

224

SB Juice as opposed to synthetic media for both yeast strains. There was also a strain-

225

specific media response, with F15 more productive in SGM than in SB Juice for this

226

metabolite, whereas the opposite was true for BY4743 (Table 2).

227

2 was found recently to be formed by a methionine gamma lyase in Brevibacterium linens 38

228

hinting that a possible enzymatic origin could be found also in yeast. Together with our

229

finding, this could be interpreted if 2 is the product of early, rapid catalysis/assimilation of

230

sulfur-containing nutrients during the first stages of cellular growth. Such sources may be

231

more readily available in SGM (containing methionine, cysteine, glutathione, sulfate, biotin

232

and thiamine) than those found in SB juice, whose sulfur sources include peptides and

233

sulfurylated organic molecules as well as those in SGM. Alternative explanations for the

234

difference might include an unknown competitive inhibitor suppressing production of 2 that

235

is present in SB Juice, and missing in SGM. The subsequent disappearance of 2 during the

236

latter stages of yeast growth could be explained by its being utilized as a substrate for the

237

biosynthesis of more complex sulfur-containing compounds, either via chemical reactions or

238

by a yeast biosynthetic pathway. A detailed experimental procedure will be needed to further

239

test these hypotheses.

240

Heavier VSCs like 5, 10 and 12 have also been detected in wine,10 yet to date their origin

241

remains unknown. Firstly, the evolution of 12 production followed a steady increase

242

throughout most of the fermentation (Figure 3D). In the case of BY4743, production in both

243

media was very similar, peaking at 70% weight loss. In both media this strain produced 12 at

244

slightly above the perception threshold (50 µg/L), but concentrations decreased by the end of

245

fermentation. In F15 both media presented similar production curves, but at generally lower



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246

concentrations than BY4743. At the end of fermentation, F15 in both media and BY4743 in

247

SB Juice attained similar final concentrations with BY4743 in SGM showing slight, but

248

significant differences (p < 0.05).

249

Secondly, 5 was not detected until the end of fermentation where a marked rise in production

250

was observed. For both strains, this late burst was more pronounced in SB Juice than SGM,

251

achieving values for this metabolite an order of magnitude greater than the sensory threshold

252

of 0.93 μg/L.28 Significant strain differences were seen, with BY4743 showing higher

253

concentrations than F15 (Figure 3F) (Table 2), which would translate to a large difference in

254

taste for these wines.

255

Interestingly, together with 1, both 12 and 5 were produced at concentrations above their

256

perception threshold. This result does not necessarily associate this set of compounds to the

257

overall off-flavors detected in these wines, since the interaction with other compounds

258

produced during fermentation is known to produce a synergistic effect that lowers the

259

perception limits for some VSCs.39 Conversely, some compounds that are present well below

260

threshold values are known to influence overall aroma perception (e.g. β-damascenone).39

261

Finally, 10 followed a steadily increasing concentration pattern that was generally similar for

262

both strains and media. This metabolite’s biosynthesis was detected early in the fermentation,

263

rising steadily and plateauing at 70% weight loss. Strain differences were evident, with F15

264

producing twice as much as BY4743 (Figure 3G). For BY4743, production in SGM was

265

higher than in juice (Table 2).

266

In fact, overall we observed most compounds increased slowly during fermentation, with

267

some plateauing and some peaking at 70% weight loss. None of the compounds clearly

268

followed the cell growth curve (Figure 2A); this is perhaps surprising, since during

269

fermentation of wine yeast in synthetic media, transcription of genes in the sulfur assimilation

270

pathway was induced during the cell growth period and down-regulated subsequently.

22


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271

However, it has been observed that there is sustained induction of the Met4p transcription

272

factor late in fermentation of Riesling grape juice,

273

our own previous data in SB Juice 41, 42 which shows induced transcription of many genes in

274

the sulfur assimilation pathway late in fermentation (at 70% weight loss).

275

In summary, we have observed strain differences in either final concentration or production

276

during growth in the biosynthesis of 1, 2, 3, 5, 9, 10 and 12. Progeny studies using these

277

strains or a detailed screening of candidate gene deletions with possible involvement in

278

sulfur-carbon chemical reactions seems to be the best approach to shed light on the origin of

279

these compounds.

280

Different media also impacted the production of some VSCs. In particular, the production of

281

5 was up to 17 times higher in SB juice fermentation compared to that in SGM, whereas for

282

2, SB Juice produced a delay in this gas’s biosynthetic peak. While there may be unidentified

283

compounds in juice that are important for these differences, it may also be possible to identify

284

constituents of defined media that affect these parameters.43

285

Within this context, yeast assimilable nitrogen (YAN) supply in grape juice has been well

286

documented to influence yeast growth and metabolism during wine making.44 Often

287

observed, a low YAN can lead to stuck or sluggish fermentations. This deficiency, however,

288

can be overcome by supplementing with di-ammonium phosphate (DAP), an ammonium

289

source. In addition, high nitrogen (e.g. 480 mg N/L) can result in undesirable aromas

290

produced45, including, but not limiting, the accumulation of 1. In our study, however, F15

291

produced significantly more of 1 fermenting SB Juice (YAN: 206 mg N/L) than SGM (300

292

mg N/L) (Table 2). The complexity and diversity of nitrogen containing metabolites within

293

SB Juice as compared to that of SGM makes it difficult to predict if this difference in YAN

294

can be solely attributed for the difference in VSC production in our study. A more thorough

295

and comprehensive study is needed.

40

and this in turn, has been confirmed by



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296

Because we measured steady state levels, we cannot distinguish between several alternative

297

explanations for the temporal changes: they may result from differences in production of the

298

compounds, differences in their degradation or their use in subsequent pathways or chemical

299

reactions, or they may even occur as the result of changes in the rate of their transport outside

300

the cell. However, the knowledge of the approximate timing of their peak concentration

301

detailed here will allow these options to be evaluated more fully. The use of precursors with

302

labelling on different atoms and positions will be critical in such work, along with careful

303

selection of optimal sampling times. 46

304

Wine quality can be affected dramatically by the production of VSCs, and understanding the

305

parameters and conditions in which they are produced could have profound applications in

306

the wine industry. The differences in the timing of production by yeast, for the seven VSCs

307

presented here will facilitate detailed analysis of the biochemical pathways involved, about

308

which very little is currently known. The strain and media differences noted here should also

309

provide an initial basis for determining some of the genetic and physiological influences on

310

this class of wine aroma molecules.

311 312

Acknowledgements

313 314

We would like to thank Andy Frost (Pernod Ricard NZ Ltd) for supplying grape juice, and

315

New Zealand Winegrowers and the Romeo Bragato Trust for funding to make this research

316

possible.

317 318

Supporting information

319


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Supporting Information Available: Time zero control graphs, calibration curves used,

321

composition of SGM, and ion list used for identification and quantitation of VSCs. This

322

material is available free of charge via the Internet at http://pubs.acs.org.

323 324



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471

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Caption to figures

472 473

Figure 1. Chemical structures of the VSCs analyzed in this work. Hydrogen sulfide (1),

474

methanethiol (2), ethanethiol (3), dimethyl sulfide (4), diethyl sulfide (5), dimethyl disulfide

475

(6), diethyl disulfide (7), carbon disulfide (8), methyl thioacetate (9), S-ethyl thioacetate (10),

476

dimethyl trisulfide (11) and benzothiazole (12).

477 478

Figure 2. Fermentation progress assessed by (A) Growth curves as estimated by colony

479

counts plated, and (B) Production of 1 measured using detection columns. The data is shown

480

for BY4743 in SGM (full line green), BY4743 in SB Juice (dash dot line blue), F15 in SGM

481

(dash line, orange) and F15 in SB Juice (dotted line, red). Error bars show standard deviation

482

between replicates (n=3).

483 484

Figure 3. The cumulative concentrations of VSCs produced at 0%, 2%, 25%, 50%, 70% and

485

100% of fermentation for BY4743 in SGM (full line, green), BY4743 in SB Juice (dash dot

486

line, blue), F15 in SGM (dash line orange) and F15 in SB Juice (dotted line red). A: 1, B: 3,

487

C: 9, D: 12, E: 2, F: 5, G: 10. Error bars show standard deviation between replicates (n=3).

488 489 490 491 492 493 494 495


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Table 1. SGM composition as adapted from Henschke & Jiranek (Wine Microbiology and Biotechnology: 77-164 Table 4.4 (pg. 92), 1993). The amino acids and di-ammonium phosphate ((NH4)2HPO4), i.e. the nitrogen sources, were modified to reflect Marlborough Sauvignon Blanc (2004, 2005 & 2006) juice and winemaking practice. Amt: amount. Carbon Source, Salts, Minerals Amt Amt Nitrogen Source & Vitamins (mg/L) Glucose Fructose

105 g/L 105 g/L

L-alanine L-arginine-HCl

5 g/L 3 g/L

L-aspartic acid L-cysteine

Citric Acid K2HPO4

200 mg/L 1.14 g/L

L-glutamic acid L-glutamine

MgSO4.7H2O CaCl2.2 H2O

1.23 g/L 440 mg/L

L-glycine L-histidine

MnCl2.4 H2O ZnSO4.7 H2O

198.2 mg/L 287.5 mg/L

L-isoleucine L-leucine

FeSO4.7 H2O CuSO4

70.1 mg/L 25.3 mg/L

L-lysine-HCl L-methionine

H3BO3 CoCl2.6H2O

5.7 mg/L 23.8 mg/L

L-phenylalanine L-proline

NaMoO4.2H2O KIO3

24.2 mg/L 10.8 mg/L

L-serine L-threonine

Myo-Inositol Pyridoxine hydrochloride

100 mg/L 2 mg/L

L-tryptophan L-tyrosine

2 mg/L 1 mg/L

L-valine (NH4)2HPO4

Potassium tartrate Malic Acid

Nicotinic acid Ca-panthothenate

100 484 50 5 100 125 5 20 25 25 6 10 40 300 60 75 10 10 30 363

Thiamine hydrochloride

500 µg/L

p-amino benzoic acid

200 µg/L

Riboflavin

200 µg/L

Lipids and Glutathione Ergosterol

15 mg/L

Biotin Folic Acid

125 µg/L 200 µg/L

Tween 80 Glutathione

0.5 mL/L 50 mg/L

Amt



Page 24 of 28

Table 2. Statistical analysis of VSC concentrations (ug/L) using one way ANOVA (p < 0.05), and post hoc Tukey’s HSD test (p < 0.05). Statistically similar data points are grouped with similar characters. Small letters (Sig 1): Comparison of VSC concentrations at six different time points (0, 2, 25, 50, 70 and 100% weight loss) for each strain and media combination. Capital letters (Sig 2): Comparison of VSC concentrations within the four treatments of strain and media for each given time point. Aver.: average concentration of VSC (n=3 for each data point; ST Dev.: standard deviation; Sig.: significance group. All values are shown in ug/L of wine.

24

ACS Paragon Plus Environment

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Figure 1



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Figure 2


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Figure 3



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TOC Graphic


Evolution of Volatile Sulfur Compounds during Wine Fermentation

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