The Reducing Capacity of Thioredoxin on Oxidized Thiols in Boiled

Subscriber access provided by Stockholm University Library

Article

The Reducing capacity of thioredoxin on oxidized thiols in boiled wort Anne Murmann, Per Hägglund, Birte Svensson, and Marianne N. Lund J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04179 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 1, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

Journal of Agricultural and Food Chemistry

The Reducing Capacity of Thioredoxin on Oxidized Thiols in Boiled Wort

Anne N. Murmann1, Per Hägglund2,3, Birte Svensson2, Marianne N. Lund1,3*

1 Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26, DK1958 Frederiksberg C, Denmark 2 Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark 3 Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark

* Corresponding author: E-mail: [email protected], Phone: +45 3533 3547

1 ACS Paragon Plus Environment


Page 2 of 28

1

ABSTRACT

2

Free thiol-containing proteins are suggested to work as antioxidants in beer, but the majority of

3

thiols in wort are present in their oxidized form as disulfides and are therefore not active as

4

antioxidants. Thioredoxin, a disulfide-reducing protein, is released into the wort from some yeast

5

strains during fermentation. The capacity of the thioredoxin enzyme system (thioredoxin,

6

thioredoxin reductase, NADPH) to reduce oxidized thiols in boiled wort under fermentation-like

7

conditions were studied. Free thiols were quantitated in boiled wort samples by derivatization

8

with ThioGlo®1 and fluorescence detection of thiol-derivatives. When boiled wort was incubated

9

with all components of the thioredoxin system at pH 7.0 and 25 °C for 60 min under anaerobic

10

conditions, the free thiol concentration increased from 25 to 224 µM. At pH values similar to wort

11

(pH 5.7) and beer (pH 4.5), the thioredoxin system was also capable of increasing the free thiol

12

concentration, although with lower efficiency to 187 and 170 µM, respectively. The presence of

13

sulfite, an important antioxidant in beer secreted by the yeast during fermentation, was found to

14

inactivate thioredoxin by sulfitolysis. Reduction of oxidized thiols by the thioredoxin system was

15

therefore only found to be efficient in the absence of sulfite.

16 17 18 19

Key words: protein thiols, thioredoxin, sulfite, wort, oxidation, reducing capacity

20


Page 3 of 28


21

INTRODUCTION

22

Beer flavor stability is a critical quality problem faced by the brewing industry, where flavor

23

modifications may begin when the beer leaves the brewery. Thus, during shipment and storage,

24

flavor is compromised by elevated and fluctuating temperatures (up to 50-70 oC in containers) and

25

flavor stability is therefore difficult to control. The main reason for loss of positive flavoring

26

substances and development of aged flavors in beer is the unavoidable introduction of oxygen,

27

which ingresses through the crown cork. Beer oxidation starts by activation of atmospheric oxygen

28

facilitated by trace levels of iron and copper leading to the formation of reactive oxygen species,

29

such as hydrogen peroxide, superoxide and hydroxyl radicals.1-2 Sulfite is produced by yeast during

30

fermentation and is a well-established antioxidant in beer that quenches hydrogen peroxide.4, 5

31

However, after consumption of sulfite below a critical level, hydrogen peroxide reacts with iron

32

and copper, and generates highly reactive hydroxyl radicals via the Fenton reaction. Hydroxyl

33

radicals are highly reactive compounds and will react unspecifically with most beer components,

34

but due to the high abundance of ethanol, the majority of hydroxyl radicals will react with ethanol

35

to generate 1-hydroxyethyl radicals.3 Additionally, 1-hydroxyethyl radicals can either react fast

36

with bitter acids from hops leading to loss of desired bitter flavor4-6 or react further with oxygen,

37

leading to the formation of 1-hydroxyethyl peroxyl radicals, which decompose to form

38

acetaldehyde, an off-flavor compound, and hydroperoxyl radicals.6-8 These radicals will enter

39

another oxidation cycle as described above and cause further oxidative damage if no other

40

antioxidant defense system is present in the beer. Free thiols (R-SH) on peptides and proteins have

41

been suggested to act as antioxidants in beer9 due to their fast reaction with 1-hydroxyethyl

42

radicals, while disulfides are inactive towards the 1-hydroxyethyl radical.4-5, 10 The role of thiols

43

during radical reactions in beer is however not clear since some studies show prooxidative 3 ACS Paragon Plus Environment


Page 4 of 28

44

behavior of some thiol oxidation products.11-12 Nevertheless, thiols have also been reported to

45

bind aldehydes, which are generated during storage and known to create stale flavor in beer.13-15

46

The binding of staling aldehydes to thiols has been shown to positively affect flavor,16-17 so

47

increasing the thiol concentration may have multiple positive effects on flavor stability by binding

48

staling aldehydes and possibly by reacting with 1-hydroxyethyl radicals.

49

A minimum of 2/3 of the total thiol pool in wort and beer is present in a reversibly oxidized

50

form.18-19 Free thiols (R-SH) can be oxidized to among others disulfides (R-SS-R), sulfenic acid (R-

51

SOH), sulfinic acid (R-SO2H), or sulfonic acid (R-SO3H). Disulfides and sulfenic acids are readily

52

reduced back to the original thiol form by common reducing agents.20-21 These reversibly oxidized

53

thiols present in wort and beer therefore represent a potential and natural pool of antioxidants,

54

which may increase the antioxidant capacity of beer if they are reduced to free thiols. We have

55

previously tested the capacity of sulfite alone to reduce the pool of oxidized thiols in wort by

56

sulfitolysis (the reaction of sulfite with a disulfide bond to form one S-SO3- and one free SH group

57

for each disulfide bond cleaved reaction 1)22 but found that the concentration of sulfite typically

58

present in beer is too low to significantly increase the thiol concentration.23

59

− + ⇌ − +

(1)

60 61

The maximum concentration of sulfite in beer allowed by the current legislation in EU is 20 ppm

62

(equivalent to 312 µM SO2).24

63

Thioredoxin (Trx) is a small redox protein released into the wort from some yeast strains during

64

fermentation25-26 and capable of reducing disulfides in target proteins such as the lipid transfer

65

protein 1 (LTP1)27, which is the second most abundant protein in beer. Thioredoxin has been


Page 5 of 28


66

suggested to contribute with reducing capacity during fermentation of beer.9-10 The activity of Trx

67

is dependent on reduction of a redox dithiol motif. In living cells, such reduction is catalyzed by

68

thioredoxin reductase (TrxR) and requires NADPH (Figure 1)28, but it is unknown whether TrxR is

69

released from yeast cells together with Trx during beer fermentation.

70

The aim of the current study was therefore to examine the reducing capacity of Trx alone and in

71

combination with TrxR and NADPH on the pool of oxidized thiols in wort. The reducing capacity

72

was tested at pH 7.0 (optimum pH for Trx activity),29-31 pH 5.7 (pH of wort) and pH 4.5 (pH of

73

beer). Sulfitolysis has been found to occur in Escherichia coli Trx and plant Trx in the presence of

74

millimolar concentrations of sulfite at pH 7-8, and more pronounced in the presence of protein-

75

unfolding agents. Cleaving a disulfide bond in Trx by sulfitolysis, produces a thiosulfate group and

76

a thiol group, which has been found to inactivate Trx.32-33 Most yeast strains secrete sulfite and the

77

reducing capacity by the combination of sulfite and Trx in wort was therefore also investigated.

78

Employing and/or enhancing endogenous antioxidant defense systems from the raw material

79

applied during beer production such as barley, hops and yeast is desirable as it serves as a natural

80

solution. This is also in compliance with the Reinheitsgebot, the German purity law, that states

81

that only water, malted barley, hops and yeast must be used for beer brewing, and avoids the

82

addition of antioxidant ingredients, which would have to be labelled on the beer.34



Page 6 of 28

83

MATERIALS AND METHODS

84

Chemicals

85

ThioGlo 1 fluorescent thiol reagent was obtained from Berry and Associates (Dexter, MI).

86

Acetonitrile,

87

chloramphenicol, chlortetracycline, hydrogen chloride, sodium sulfite, nicotinamide adenine

88

dinucleotide phosphate (NADPH), ethylenediaminetetraacetic acid (EDTA) and acetic acid (≥99.7%)

89

were purchased from Sigma-Aldrich (St. Louis, MO). Tris(hydroxymethyl) amino methane (Tris),

90

trifluoroacetic acid (≥99.8%), disodium hydrogen phosphate dehydrate, sodium dihydrogen

91

phosphate dehydrate and sodium chloride were obtained from Merck (Darmstadt, Germany).

92

Bovine serum albumin (BSA) standard of 2.0 mg/mL was obtained from Thermo Fisher Scientific

93

(Rockford, IL). All chemicals were of analytical grade or the highest purity available. Water was

94

purified through a Milli-Q water purification system from Millipore (Billerica, MA).

glutathione,

N-acetyl-L-cysteine,

tris(2-carboxyethyl)phosphine

(TCEP),

95 96

Recombinant barley thioredoxin HvTrxh1 and thioredoxin reductase HvNTR2 (N139A) were

97

produced and purified as previously described.35-36 Enzyme concentration was determined by aid

98

of amino acid analysis.36

99 100

Reducing capacity of Trx in boiled wort

101

1 L of boiled unhopped wort (produced from 95 % pilsner malt + 5 % unmalted barley, pH 5.3) was

102

collected hot with minimal headspace and immediately cooled down to 5oC at the Carlsberg Group

103

R&D Center, Copenhagen, Denmark. On the same day the wort was aliquoted into individual 50

104

mL centrifuge tubes in an anaerobic chamber (Coy Lab, Grass Lake, USA) and kept at –20°C until

105

analysis. 6 ACS Paragon Plus Environment

Page 7 of 28


106

For the experiments, aliquots of 50 mL of boiled wort were thawed and filtered through a 0.45 μm

107

Minisart filter in an anaerobic chamber. To avoid microbial growth, 100 μL of chloramphenicol

108

dissolved in ethanol and 100 μL chlortetracycline dissolved in Milli-Q water were added to achieve

109

final concentrations of 0.2 µM and 0.4 nM, respectively. The reducing capacity of Trx in wort was

110

investigated at three different pH values; pH 7.0 (optimal pH for Trx activity), pH 5.7 (typical pH of

111

wort), and pH 4.5 (typical pH of beer). Trx (4 µM), TrxR (0.1 µM), NADPH (4 mM), EDTA (6 mM)

112

specified as final concentrations, wort (50 µL) and buffer (0.1 M phosphate, pH 7.0 or 5.7, or 0.1 M

113

acetate, pH 4.5) were added to Eppendorf tubes to a total volume of 125 µL and the pH values of

114

the samples were subsequently measured to confirm the desired pH values. Concentrations of Trx,

115

TrxR, NADPH and EDTA were chosen according to Jensen et al.37 Samples were incubated in an

116

anaerobic chamber for 10 min, 60 min and 24 h. The anaerobic chamber was used in order to

117

mimic the anaerobic conditions during fermentation and to avoid oxidation of Trx and thiols by

118

atmospheric oxygen during the incubation. Control samples only containing wort were included in

119

the experimental set-up. After incubation, samples were snap frozen in liquid nitrogen and kept at

120

–20oC until analysis. All samples were prepared in triplicates.

121 122

Stability of NADPH at pH 4.5—7.0

123

Stability of NADPH at pH 7.0, 5.7 and 4.5 was investigated by measuring at 340 nm, the

124

absorbance maximum of NADPH, on a Cary 100 Bio UV-Visible spectrophotometer. Samples

125

consisting of 0.4 mM NADPH in 0.1 M phosphate buffer (pH 7.0 or 5.7) or 0.1 M acetate buffer (pH

126

4.5) were incubated in an anaerobic chamber for 60 min followed by immediate absorbance

127

measurements. The pH-independent molar extinction coefficient of Ɛ340 = 6.22 mM-1cm-1 was

128

applied to calculate the concentration of NADPH.38-39 All samples were prepared in triplicates. 7 ACS Paragon Plus Environment


Page 8 of 28

129 130

Sulfitolysis of Trx

131

Sulfitolysis of Trx was investigated by incubating 4 µM Trx with 312 µM sulfite in 0.1 M phosphate

132

buffer (pH 7.0 or 5.7) in an anaerobic chamber for 2 min, 60 min and 24 hours. Control samples

133

without addition of sulfite were included. After incubation samples were snap frozen in liquid

134

nitrogen and kept at -20 oC until analysis. The first possible sampling was conducted 2 min after

135

start of incubation. All samples were prepared in triplicates.

136 137

Reducing capacity of Trx and sulfite in wort

138

Wort was incubated with 4 µM Trx, 312 µM sulfite and 6 mM EDTA in 0.1 M phosphate buffer (pH

139

7.0 or 5.7) in an anaerobic chamber for 2 min, 60 min and 24 hours. Control samples without

140

addition of sulfite and Trx were included. All samples were prepared in triplicates.

141 142

Quantitation of thiol (free and total) and sulfite

143

Sulfite and thiol concentrations were determined by derivatization with the fluorescent probe

144

ThioGlo1 and separation of sulfite and thiol adducts by HPLC followed by fluorescence detection

145

as described by Abrahamsson et al.40 and Hoff et al.41 Separation was performed on a Jupiter C18

146

column (150, 2.0 mm, 5 μm particle size, 300 Å pore size) (Phenomenex, Torrance, CA). Mobile

147

phase A (milli-q water) and mobile phase B (methanol) were acidified with trifluoroacetic acid (pH

148

2.0, 10 mM). The gradient was held at 25% B for 8 min (isocratic), instantly increased to 95% B and

149

kept at 95% B for 6 min, finally it was returned to starting conditions for 7 min. Thiols were

150

quantitated as free and total thiols, where total thiols were determined after reduction in boiled 8 ACS Paragon Plus Environment

Page 9 of 28


151

wort sample with varying TCEP concentrations for 5 min.18 Each sample was analyzed in technical

152

triplicates.

153

Samples not containing sulfite were analyzed for free thiol content as described by Lund and

154

Andersen20 by using ThioGlo1 as a thiol derivatizing agent and measurement of fluorescent

155

response with a plate reader (Fluoroskan Ascent, Thermo Scientific, Waltham, MA, USA).

156 157

Statistical analysis

158

Statistical analysis was performed by a paired-sample t test using IBM SPSS Statistics V22.0. Means

159

were used to compare differences, and least significant difference was applied to compare the

160

mean values. The significance level was P < 0.05.



Page 10 of 28

161

RESULTS AND DISCUSSION

162

Reduction of oxidized thiols in wort by Trx

163

The ability of Trx to reduce the pool of oxidized thiols in boiled wort was investigated by adding

164

different combinations of Trx, TrxR, NADPH, and EDTA to boiled wort adjusted to pH 7.0 and

165

incubating the samples in an anaerobic chamber to mimic fermentation conditions. Thiol oxidase

166

has been reported to be active in malt and thereby also in sweet, unboiled wort.42-43 Therefore,

167

boiled wort was chosen for the experiments to avoid possible interference from thiol oxidation

168

caused by thiol oxidase as previously observed.44

169

Free thiol concentration (R-SH) in wort without addition of any components was determined to be

170

25 ± 2 µM (Figure 2), which is comparable with a previous study where boiled wort was found to

171

contain 21 ± 5 µM free thiol.23 A free thiol concentration of 224 ± 8 µM was found for the wort

172

sample where all components from the thioredoxin system were added and incubated for 60 min

173

(Figure 2), while no significant increase occurred in free thiol concentration if one of the

174

components was excluded. This is evidence that Trx is able to reduce oxidized thiols in boiled wort,

175

but only in the presence of TrxR and NADPH.

176

The total concentration of thiols in the boiled wort was also determined by reduction with TCEP, a

177

chemical disulfide reducing agent, and found to be 241 ± 4 µM. Comparison of this thiol

178

concentration and that obtained with the thioredoxin system, shows that the thioredoxin system

179

very efficiently reduced disulfides in boiled wort under the conditions applied. The total thiol value

180

obtained by reduction with TCEP was higher compared to a previous study, where the total thiol

181

concentration in boiled wort was found to be ca. 170 µM.19 The boiled wort analyzed in the


Page 11 of 28


182

previous study was produced at a different brewery and by different raw materials, and variations

183

in total thiol concentrations between different studies are therefore not surprising.

184 185

Influence of pH on the reduction of the pool of oxidized thiols by Trx

186

Activity of the thioredoxin system is optimal around neutral pH.29-31, 45 The pH value of wort is

187

typically between 5.0 and 6.0 and pH of classical beers lies between 3.9 and 4.5.46 The influence of

188

pH on the reducing capacity of the thioredoxin system was therefore tested at pH values

189

representative to wort (pH 5.7) and beer (pH 4.5) in comparison to pH 7.0. Boiled wort samples

190

added Trx, TrxR, NADPH, EDTA, and phosphate buffer (pH 7.0 or 5.7) or acetate buffer (pH 4.5)

191

were incubated in an anaerobic chamber for 10, 30 and 60 min. After 10 min of incubation,

192

samples added all components from the thioredoxin system showed a significantly higher

193

concentration of free thiols compared to the control (Figure 3), but no significant difference in

194

thiol concentrations was found between wort samples with different pH incubated for 10 min.

195

With increasing incubation time, free thiol concentration significantly increased at all three pH

196

values, and this increase was most pronounced at pH 7.0. No significant difference was found

197

between samples at pH 4.5 and 5.7 at any of the analyzed incubation times. However, at pH 4.5

198

the increase in free thiol concentration seemed to level off suggesting lower reducing capacity of

199

the thioredoxin system at low pH values equivalent to beer pH, reminiscent of previous analysis of

200

pH activity dependence for this Trx-TrxR system.28

201

The lower reducing capacity at pH 5.7 and pH 4.5 could be caused either by lower activity of Trx

202

and TrxR, or instability of NADPH in this pH range, which has been reported previously.47 The

203

NADPH concentration was therefore determined spectrophotometrically at pH 7.0, 5.7 and 4.5



Page 12 of 28

204

(Table 1). The stability of NADPH was found to decrease at pH 4.5 and 5.7, but the NADPH was still

205

in large excess (10,000-fold) of TrxR. This observation suggests that the decreased reducing

206

capacity of Trx observed at pH 4.5 and 5.7 was due to lower Trx or TrxR activity and not due to loss

207

of NADPH under the conditions used in the present study.

208 209

Sulfitolysis of Trx

210

Anaerobic incubation of sulfite and Trx at pH 7.0 and pH 5.7 was conducted to investigate the

211

extent of sulfitolysis indirectly by quantitating the release of a free thiol group in Trx. The Trx used

212

in the present study contained three cysteine residues; the two cysteine residues in the active site

213

form a disulfide causing inactivation, and the third cysteine residue is situated in the N-terminal

214

part of the protein. Addition of sulfite to Trx at both pH values resulted in an increase in thiol

215

concentrations by factors of 1.8-2.2 (Table 2), indicating that sulfite was capable of reducing the

216

disulfide bond in Trx at least partly. Increasing the incubation time and pH value had a positive

217

effect on the concentration of free thiols, resulting in a 10-fold increase of free thiols when

218

incubating for 24 hours at pH 7.0. In the absence of sulfite an oxidation of the Trx samples

219

occurred, seen as a decrease of thiols over time. This may be explained by low levels of oxygen

220

present in the samples since these were not degassed prior to the experiments being conducted in

221

the anaerobic chamber.

222

The determined concentration of free thiols in Trx was lower than expected; in the current study

223

4 µM Trx was used, so without reduction a concentration of 4 µM free thiol was expected and the

224

fully reduced Trx would yield a free thiol concentration of 8 µM (equivalent to conversion of one

225

disulfide bond to one additional thiol group and one thiosulfate group).32-33 Our results showed


Page 13 of 28


226

only 0.2-1.0 µM free thiol in non-reduced Trx, which indicates that the Trx was already oxidized

227

quite extensively. Sulfitolysis studies of E. coli Trx and plant Trx also concluded that sulfite was

228

able to reduce the disulfide bond in Trx, but a complete reduction was not reached, even at sulfite

229

concentrations more than 10-fold higher than the sulfite concentration applied in the current

230

study.33 Our results indicate that sulfite is capable of reducing the disulfide bond in Trx (Table 2) in

231

agreement with results from studies of E. coli Trx and plant Trx sulfitolysis.32-33

232 233

Reducing capacity of Trx and sulfite in boiled wort

234

It is known that Trx and sulfite is released from some yeast strains,25 but whether or not TrxR and

235

NADPH is present in beer after fermentation is unknown. We therefore examined the reducing

236

capacity of the combination of Trx and sulfite in boiled wort that was incubated anaerobically for 2

237

min, 60 min and 24 h at pH 5.7 and 7.0. Samples containing only wort exhibited an overall

238

significant decrease in the concentration of free thiols over time (pH 7.0: P = 0.0002, pH 5.7: P =

239

0.0009), indicating that oxidation of the wort took place even though the incubation was

240

performed in an anaerobic chamber (Figure 4). This could be due to residual atmospheric oxygen

241

present in the wort since the wort was not degassed before it was transferred to the anaerobic

242

chamber. Boiled wort samples added sulfite alone and the combination of sulfite and Trx exhibited

243

a significant increase in free thiol concentration, but no additional effect was observed when Trx

244

was added together with sulfite compared to addition of sulfite alone. It was therefore concluded

245

that the observed increase in thiol concentration in wort was caused only by direct sulfitolysis of

246

the disulfides in wort and was not catalyzed by Trx. The lack of reducing capacity of Trx in the

247

presence of sulfite is in agreement with the previous study by Würfel et al.,32-33 who found that



Page 14 of 28

248

sulfite caused inactivation of Trx. The observed reducing effect of sulfite alone in wort is in

249

agreement with our previous study.23 Increasing the incubation time from 60 min to 24 h caused a

250

significant increase in free thiol concentration (Figure 4), which is also in agreement with our

251

previous study,23 where an interaction between sulfite concentration and incubation time was

252

observed when incubation time was increased from 60 min to 24 h or longer. Increasing the pH

253

from 5.7 to 7.0 caused a significant increase in free thiol concentration in wort samples containing

254

sulfite (both with and without Trx). This could be explained by optimum pH at 7.0 for sulfitolysis.48

255

Although sulfite was capable of reducing Trx at pH representative of wort, the combination of

256

sulfite and Trx did not provide any reducing capacity towards the pool of oxidized thiols in boiled

257

wort, indicating an inactivation of Trx. When Trx was present in boiled wort in combination with

258

TrxR and NADPH (without presence of sulfite), significant increase in free thiol concentration was

259

observed at pH values representative to both wort and beer. These results suggest that access to a

260

complete thioredoxin system is important for efficient reduction of the pool of oxidized thiols

261

during beer fermentation. Additionally, our results indicate that sulfitolysis inactivated Trx in

262

agreement with Würfel et al.,32-33 thereby preventing Trx from reducing oxidized thiols in wort.

263

The current results suggest that sulfite must be avoided in beer in order to achieve any reducing

264

capacity of the thioredoxin system, e.g. by choosing a yeast strain that does not produce any

265

sulfite during fermentation. However, it must be considered that excluding sulfite, the primary

266

antioxidant in beer,49 would most likely result in a decrease of the antioxidative defence system in

267

the beer.


Page 15 of 28


268

ACKNOWLEDGEMENT

269

Aida Curovic and Anne Blicher are acknowledged for technical assistance with production and

270

purification of recombinant Trx and TrxR, and amino acid analysis, respectively. Also we appreciate

271

the kind donation of boiled wort from the Carlsberg Group.

272

FUNDING

273

This work was funded by the Independent Research Fund Denmark for Technology and Production

274

through the project entitled “New Defence Systems Against Beer Oxidation” (DFF-1335-00337B).



Page 16 of 28

275 276 277 278

(1) Vanderhaegen, B.; Neven, H.; Verachtert, H.; Derdelinckx, G. The chemistry of beer aging - a critical review. Food Chem. 2006, 95, 357-381.

279 280

(2) Kaneda, H.; Kano, Y.; Koshino, S.; Onley-Watson, K. Behavior and role of iron ions in beer deterioration. J. Agric. Food Chem. 1992, 40 (11), 2102-2107.

281 282

(3) Andersen, M. L.; Skibsted, L. H. Electron spin resonance spin trapping identification of radicals formed during aerobic forced aging of beer. J. Agric. Food Chem. 1998, 46, 1272-1275.

283 284 285 286

(4) de Almeida, N. E. C.; Homem-de-Mello, P.; De Keukeleire, D.; Cardoso, D. R. Reactivity of beer bitter acids toward the 1-hydroxyethyl radical as probed by spin-trapping electron paramagnetic resonance (EPR) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS). J. Agric. Food Chem. 2011, 59, 41834191.

287 288

(5) de Almeida, N. E. C.; do Nascimento, E. S. P.; Cardoso, D. R. On the reaction of lupulones, hops b-acids, with 1-hydroxyethyl radical. J. Agric. Food Chem. 2012, 60, 10649-10656.

289 290 291

(6) Intelmann, D.; Haseleu, G.; Dunkel, A.; Lagemann, A.; Stephan, A.; Hofmann, T. Comprehensive sensomics analysis of hop-derived bitter compounds in beer during storage. J. Agric. Food Chem. 2011, 59, 1939-1953.

292 293 294

(7) Bothe, E.; Schuchmann, M. N.; Schulte-Frohlinde, D.; von Sonntag, C. Hydroxyl radical-induced oxidation of ethanol in oxygenated aqueous solutions. A pulse radiolysis and product study. Z. Naturforsch 1983, 38 b, 212-219.

295 296

(8) Andersen, M. L.; Gundermann, M.; Danielsen, B.; Lund, M. N. Kinetic investigation of the role of protein thiols during oxidation in beer. J. Agric. Food Chem. SUBMITTED. Manuscript ID: jf-2017-03289m.

297 298 299

(9) Wu, M. J.; Clarke, F. M.; Rogers, P. J.; Young, P.; Sales, N.; O'Doherty, P. J.; Higgins, V. J. Identification of a protein with antioxidant activity that is important for the protection against beer ageing. Int. J. Mol. Sci. 2011, 12, 6089-6103.

300 301

(10) Wu, M. J.; Rogers, P. J.; Clarke, F. M. 125th anniversary review: The role of proteins in beer redox stability. J. Inst. Brew. 2012, 118, 1-11.

302 303

(11) Pecci, L.; Montefoschi, G.; Musci, G.; Cavallini, D. Novel findings on the copper catalysed oxidation of cysteine. Amino Acids 1997, 13, 355-367.

304 305 306

(12) Sagristá, M. L.; GarcÍa, A. F.; De Madariaga, M. A.; Mora, M. Antioxidant and pro-oxidant effect of the thiolic compounds N-acetyl-L-cysteine and glutathione against free radical-induced lipid peroxidation. Free Radical Research 2002, 36 (3), 329-340.

307 308 309

(13) Malfliet, S.; Van Opstaele, F.; De Clippeleer, J.; Syryn, E.; Goiris, K.; De Cooman, L.; Aerts, G. Flavour instability of pale lager beers: Determination of analytical markers in relation to sensory ageing. J. Inst. Brew. Distilling 2008, 114, 180-192.

310 311 312

(14) Saison, D.; Vanbeneden, N.; De Schutter, D. P.; Daenen, L.; Mertens, T.; Delvaux, F.; Delvaux, F. R. Characterisation of the flavour and the chemical composition of lager beer after ageing in varying conditions. Brew. Sci. 2010, 63, 41-53.

REFERENCES


Page 17 of 28


313 314

(15) Jaskula-Goiris, B.; De Causmaecker, B.; De Rouck, G.; De Cooman, L.; Aerts, G. Detailed multivariate modeling of beer staling in commercial pale lagers. Brew. Sci. 2011, 64, 119-139.

315 316

(16) Baert, J. J.; De Clippeleer, J.; De Cooman, L.; Aerts, G. Exploring the binding behavior of beer staling aldehydes in model systems. J. Am. Soc. Brew. Chem. 2015, 73 (1), 100-108.

317 318 319

(17) Baert, J. J.; De Clippeleer, J.; Jaskula-Goiris, B.; Van Opstaele, F.; De Rouck, G.; Aerts, G.; De Cooman, L. Further elucidation of beer flavor instability: The potential role of cysteine-bound aldehydes. J. Am. Soc. Brew. Chem. 2015, 73 (3), 243-252.

320 321 322

(18) de Almeida, N. E. C.; Lund, M. N.; Andersen, M. L.; Cardoso, D. R. Beer redox stability conferred by thiol-containing compounds: A kinetic study of 1-hydroxyethyl radical scavenging ability. J. Agric. Food Chem. 2013, 39, 9444-9452.

323 324 325

(19) Lund, M. N.; Petersen, M. A.; Andersen, M. L.; Lunde, C. Effect of protease treatment during mashing on protein-derived thiol content and flavor stability of beer during storage. J. Am. Soc. Brew. Chem. 2015, 73 (3), 287-295.

326 327

(20) Lund, M. N.; Andersen, M. L. Detection of thiol groups in beer and their correlation with oxidative stability. J. Am. Soc. Brew. Chem. 2011, 69 (3), 163-169.

328 329

(21) Rogers, P.; Clarke, F. M. Sustainable redox power from beer proteins, European Brewery Convention, Proceedings of the 31st Congress, Venice, Italy, 2007.

330 331

(22) Gonzalez, J. M.; Damodaran, S. Sulfitolysis of disulfide bonds in proteins using a solid-state copper carbonate catalyst. J. Agric. Food Chem. 1990, 38, 149-153.

332 333

(23) Murmann, A. N.; Andersen, P.; Mauch, A.; Lund, M. N. Quantification of protein-derived thiols during atmosphere-controlled brewing in laboratory scale. J. Am. Soc. Brew. Chem. 2016, 74 (1), 30-35.

334 335

(24) European Parliament and the Council of the European. Directive 95/2/ec. No L 61/22. Off. J. Eur. Union. 1995.

336 337

(25) Berner, T. S.; Arneborg, N. The role of lager beer yeast in oxidative stability of model beer. J. Appl. Microbiol. 2011, 54, 225-232.

338 339 340

(26) Swan, T.; Baldwin.B.L.; Clarke, F. M.; Tonissen, K. F.; Rogers, P. J. The development and application of natural reductants to the stabilisation of beer, European Brewery Convention, Proceedings of the 29th Congress, Nürnberg, Germany, 2003.

341 342 343

(27) Maeda, K.; Finnie, C.; Svensson, B. Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: Identification of seed proteins targeted by barley thioredoxin h isoforms. J. Biochem. 2004, 378, 497-507.

344 345 346

(28) Hägglund, P.; Bunkenborg, J.; Yang, F.; Harder, L. M.; Finnie, C.; Svensson, B. Identification of thioredoxin target disulfides in proteins released from barley aleurone layers. J. Proteomics 2010, 73, 11331136.

347 348

(29) Holmgren, A. Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide. J. Biol. Chem. 1979, 254 (19), 9627-9632.



Page 18 of 28

349 350 351

(30) Shahpiri, A.; Svensson, B.; Finnie, C. The NADPH-dependent thioredoxin reductase/thioredoxin system in germinating barley seeds: Gene expression, protein profiles, and interactions between isoforms of thioredoxin h and thioredoxin reductase. Plant Physiol. 2008, 146, 789-799.

352 353

(31) Maeda, K.; Hägglund, P.; Björnberg, O.; Winther, J. R.; Svensson, B. Kinetic and thermodynamic properties of two barley thioredoxin h isozymes, hvtrxh1 and hvtrxh2. FEBS Lett. 2010, 584 (15), 3376-3380.

354 355

(32) Würfel, M.; Häberlein, I.; Follmann, H. Inactivation of thioredoxin by sulfite ions. FEBS 1990, 268 (1), 146-148.

356 357

(33) Würfel, M.; Häberlein, I.; Follmann, H. Facile sulfitolysis of the disulfide bonds in oxidized thioredoxin and glutaredoxin. Eur. J. Biochem. 1992, 211, 609-614.

358

(34) Guido; L.F. Sulfites in beer: Reviewing regulation, analysis and role. Sci. Agric. 2016, 73 (2), 189-197.

359 360 361

(35) Kirkensgaard, K. G.; Hägglund, P.; Shahpiri, A.; Finnie, C.; Henriksen, A.; Svensson, B. A novel twist on molecular interactions between thioredoxin and nicotinamide adenine dinucleotide phosphate-dependent thioredoxin reductase. Proteins: Struct., Funct., Bioinf. 2014, 82 (4), 607-619.

362 363

(36) Maeda, K.; Hägglund, P.; Finnie, C.; Svensson, B.; Henriksen, A. Structural basis for target protein recognition by the protein disulfide reductase thioredoxin. Structure 2006, 14, 1701-1710.

364 365

(37) Jensen, J. M.; Hägglund, P.; Christensen, H. E. M.; Svensson, B. Inactivation of barley limit dextrinase inhibitor by thioredoxin-catalysed disulfide reduction. FEBS Lett. 2012, 586, 2479-2482.

366 367

(38) Horecker, B. L.; Kornberg, A. The extinction coefficients of the reduced band of pyridine nucleotides. J. Biol. Chem. 1948, 175 (1), 385-390.

368 369 370

(39) Trabelsi, N.; d´Estaintot, B. L.; Sigaud, G.; Gallois, B.; Chaudière, J. Kinetic and binding equilibrium studies of dihydroflavonol 4-reductase from vitis vinifera and its unusually strong substrate inhibition. J. Biophys. Chem. 2011, 2 (3), 332-344.

371 372 373

(40) Abrahamsson, V.; Hoff, S.; Nielsen, N. J.; Lund, M. N.; Andersen, M. L. Determination of sulfite in beer based on fluorescent derivatives and liquid chromatographic separation. J. Am. Soc. Brew. Chem. 2012, 70 (4), 296-302.

374 375

(41) Hoff, S.; Andersen, M. L.; Larsen, F. H.; Lund, M. N. Quantification of protein thiols using Thioglo 1 fluorescent derivatives and hplc separation. Analyst 2013, 138, 2096-2103.

376 377

(42) Hoff, S.; Damgaard, J.; Petersen, M. A.; Jespersen, B. M.; Andersen, M. L.; Lund, M. N. Influence of barley varieties on wort quality and performance. J. Agric. Food Chem. 2013, 61, 1968-1976.

378 379

(43) Bamforth, C. W.; Roza, J. R.; Kanauchi, M. Storage of malt, thiol oxidase, and brewhouse performance. J. Am. Soc. Brew. Chem. 2009, 67 (2), 89-94.

380 381

(44) Hoff, S.; Lund, M. N.; Petersen, M. A.; Jespersen, B. M.; Andersen, M. L. Influence of malt roasting on the oxidative stability of sweet wort. J. Agric. Food Chem. 2012, 60, 5652-5659.

382 383 384

(45) Gorlatov, S. N.; Stadtman, T. C. Human thioredoxin reductase from hela cells: Selective alkylation of selenocysteine in the protein inhibits enzyme activity and reduction with NADPH influences affinity to heparin. Proc. Natl. Acad. Sci. USA 1998, 95, 8520-8525.


Page 19 of 28


385 386

(46) Hardwick, W. A. Properties of beer. In Handbook of brewing, 1 ed. Marcel Dekker, New York, 1995, p 578.

387 388

(47) Wu, J. T.; Wu, L. H.; Knight, J. A. Stability of NADPH: Effect of various factors on the kinetics of degradation. Am. Ass. Clin. Chem. 1986, 32 (2), 314-319.

389 390

(48) Kella, N. K. D.; Kinsella, J. E. A method for the controlled cleavage of disulfide bonds in proteins in the absence of denaturants. J. Biochem. Biophys. Methods 1985, 11, 251-263.

391 392

(49) Kaneda, H.; Takashio, M.; Osawa, T.; Kawakishi, S.; Tamaki, T. Behavior of sulfites during fermentation and storage of beer. J. Am. Soc. Brew. Chem. 1996, 54 (2), 115-120.



Page 20 of 28

FIGURE CAPTIONS

Figure 1. Reduction of protein-disulfide (protein-S2) to protein-thiols (protein-(SH)2 by thioredoxin (Trx-(SH)2), which has been activated by thioredoxin reductase (TrxR-(SH)2) and NADPH (Hägglund et al. 2010).28

Figure 2. Free thiol concentrations of boiled wort added different combinations of EDTA (6 mM), NADPH (4 mM), Trx (4 µM) and TrxR (0.1 µM) incubated for 60 min in an anaerobic chamber at pH 7.0 adjusted by using 0.1 M phosphate buffer. Values are presented as means ± standard deviation (n = 3). The different letters above the bars indicate significantly different values (p < 0.05).

Figure 3. Effect of pH on free thiol concentration of boiled wort incubated with Trx (4 µM), TrxR (0.1 µM), wort, NADPH (4 mM), and EDTA (6 mM) in an anaerobic chamber for 10, 30 and 60 min. pH was adjusted to desired pH by using 0.1 M phosphate buffer (pH 7.0 or 5.7) or 0.1 M acetate buffer (pH 4.5). Boiled wort without addition of any reducing agents after incubation for 10 min incubation is also shown in the figure. Values are presented as means ± standard deviation (n = 3).

Figure 4. Free thiol concentrations in boiled wort samples added different combinations of Trx (4 µM), sulfite (312 µM) at pH 7.0 and 5.7 incubated in an anaerobic chamber for 2 min, 60 min and 24 hours. Values are presented as means ± standard deviation (n = 3).


Page 21 of 28


TABLES

Table 1. Effect of pH on NADPH concentration in samples containing 0.4 mM NADPH and 0.1 M phosphate buffer (pH 7.0 or 5.7) or 0.1 M acetate buffer (pH 4.5). Samples were incubated for 60 min in an anaerobic chamber followed by immediate measurement at 340 nm. Values are presented as means ± standard deviations (n = 3). sample pH 7.0 pH 5.7 pH 4.5

NADPH (mM) 0.41 ± 0.02 0.32 ± 0.003 0.13 ± 0.01

loss of NADPH (%) 0 22.6 68.9



Page 22 of 28

Table 2. Reduction of the disulfide bond in thioredoxin by sulfite. Trx (4 µM) and sulfite (312 µM) were incubated for 2 min, 60 min and 24 hour in an anaerobic chamber at pH 7.0 and pH 5.7. Values are presented as means ± standard deviation (n = 3). Means with different letters within pH values are significantly different (p < 0.05). thiol (µM)

pH 7.0

pH 5.7

2 min 60 min 24 hours 2 min 60 min 24 hours

Trx

Trx + sulfite

1.05 a ± 0.05 0.58 b ± 0.08 0.23 c ± 0.01 0.76 a ± 0.32 0.61 a ± 0.05 0.45 b ± 0.05

3.01 d ± 0.27 2.57 d ± 0.26 2.54 d ± 0.33 2.45 c ± 0.12 2.64 c ± 0.17 2.87 c ± 0.35

fold thiol increase by addition of sulfite 1.8 3.4 10.8 2.2 3.3 5.5


Page 23 of 28


FIGURE GRAPHICS

Figure 1.



Page 24 of 28

Figure 2.

250

d

225

Free thiol (µM)

200 175 150 125 100 75 50 25

a

a

-

+ -

b

c

c

c

+ + + -

+ + + + -

+ + + +

c

0 EDTA Wort NADP Trx TrxR

+ + -

+ + + + +

+ + + +


Page 25 of 28


Figure 3.

250 225 200

Free thiol (µM)

175 150 125 100

pH 7.0 pH 5.7 pH 4.5 Boiled wort

75 50 25 0 10

20

30

40

50

60

Time (min)



Page 26 of 28

Figure 4.

2 min, pH 7 60 min, pH 7 24 hours, pH 7 2 min, pH 5.7 60 min, pH 5.7 24 hours, pH 5.7

140 120

Free thiol (µM)

100 80 60 40 20 0 wort

wort+sulfite

wort+sulfite+Trx


Page 27 of 28


GRAPHIC FOR TABLE OF CONTENTS



TOC figure 47x33mm (600 x 600 DPI)

ACS Paragon Plus Environment

Page 28 of 28

The Reducing Capacity of Thioredoxin on Oxidized Thiols in Boiled

Recommend Documents