Characterization of volatiles in unhopped wort

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ReleaseandEvaporationofVolatilesduring
BoilingofUnhoppedWort
ArticleinJournalofAgriculturalandFoodChemistry·August2008
ImpactFactor:2.91·DOI:10.1021/jf800610x·Source:PubMed

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1

Identification of Volatiles in Unhopped Wort

2

DAVID P. DE SCHUTTER1*, DAAN SAISON1, FILIP DELVAUX1, GUY

3

DERDELINCKX1,

4

DELVAUX1

JEAN-MARIE

ROCK²,

HEDWIG

NEVEN³,

FREDDY

R.

5
6

1

7

University of Leuven

8

Kasteelpark Arenberg 22 box 02463, 3001 Heverlee, Belgium

9

²Brasserie d’Orval S.A., 6823 Villers-devant-Orval, Belgium

10

Centre for Malting and Brewing Science, Faculty of Bioscience Engineering, Catholic

³Brewery Duvel Moortgat N.V., Breendonkdorp 58, 2870 Puurs, Belgium

11
12

* tel: +32 (0)16 321413; fax: +32 (0)16 321576;

13



14
15

TITLE RUNNING HEADER

16

Volatiles in unhopped wort

17

1


18

ABSTRACT

19

The volatile fraction of wort components was studied during boiling. Not less than 118

20

volatile compounds were identified when un-hopped pilsner wort was boiled and samples

21

of wort and condensed vapors were analyzed with headspace SPME-GC/MS, of which 54

22

were confirmed with reference compounds. The wort samples contained 61 identifiable

23

compounds, while the vapor condensate yielded 108 different compounds. Almost 30

24

known compounds were found that have not been described before in un-hopped pilsner

25

wort, while one previously unknown aldol reaction product was tentatively identified as

26

2-phenyl-2-octenal. The detection of branched 2-alkenals underlines the importance of

27

the aldol condensation in Maillard-type reactions, while the tentative identification of

28

alkyloxazoles and alkylthiazoles could once more accentuate the central role of α-

29

dicarbonyl compounds, aldehydes and amino acids in flavor generation. The

30

condensation of wort vapors joined with the SPME-GC/MS technique has proven to be a

31

useful tool in volatile analysis.

32

33

KEYWORDS

34

Keywords: wort boiling, volatiles, evaporation, condensate, aldehydes, ketones, 2-

35

alkenals, 2-phenyl-2-octenal, oxazoles, thiazoles, furans, Maillard reaction, flavor

36

compounds

37

2


38

INTRODUCTION

39

In the world of brewing technology, no topic is more controversial than wort boiling. The

40

reason for this is simple: wort is a complex mixture of sugars, amino acids, lipids,

41

proteins and polyphenols. Hundreds of reactions occur simultaneously, such as Maillard

42

reactions, lipid oxidation reactions, the thermal decomposition of S-methylmethionine to

43

dimethylsulfide and the isomerization of hop α-acids into iso-α-acids. Unfortunately, it is

44

not yet known to which extent every single reaction accounts for the quality of the final

45

product and the flavor stability of the beer during storage.

46

Brewing researchers agree that the reduction of thermal load during wort boiling

47

improves beer flavor stability (1,2). Apart from that, the evaporation of dimethylsulfide is

48

often the sole quality criterion used by suppliers to prove the value of their wort boiling

49

system. Progress in this field can only be made through a more profound knowledge of

50

the presence of the volatiles in wort and the chemical background of these compounds. In

51

the past, analysis of volatiles has already been carried out on malt extract (3), ale malt

52

(4), caramel malts (5), roasted malts (6), ale wort (7) and on pilsner-type beer (8,9). Other

53

papers focused primarily on odor-active compounds of hops in beer (10) or described

54

new techniques to identify more than 120 different compounds in hop oil, while probably

55

over 1,000 compounds are still waiting to be discovered in the hop oil fraction (11).

56

The presence of these abundant hop volatiles in wort could impede a proper detection and

57

identification of the malt-derived and boiling process related volatiles. Therefore, it was

58

decided to investigate the flavor profile of un-hopped pilsner wort together with the

59

condensate of the vapors generated while boiling. The advantage of the analysis of vapor

60

condensates is obvious: volatiles are generally enriched in the vapor phase, depending on

3

61

the volatility of the particular compound. If this flavor-rich mixture is combined with a

62

very sensitive analysis technique, such as solid-phase micro-extraction (SPME) in

63

combination with GC/MS (12,13), a powerful identification tool is created. The increased

64

sensitivity offers opportunities for the detection of new compounds in wort and

65

consequently, fur unraveling new reaction pathways that may occur during boiling. As

66

the volatility of every volatile compound is determined and expressed by its vapor-liquid

67

equilibrium (14), there must be a strict correlation between the presence of a compound

68

in the condensate and in the wort. Variations in wort will therefore be reflected in the

69

vapor condensate.

70

The purpose of the present study is the identification of a broad range of volatiles in order

71

to obtain a better understanding of the chemical background of unhopped wort. A crucial

72

factor in this identification is obtaining an increased sensitivity for the identification of

73

novel compounds. Therefore the SPME GC/MS technique is combined with the analysis

74

of wort vapor condensate, a combination that should greatly enhance the analysis

75

sensitivity.

76

In the present study, we used the SPME-GC/MS technique for the detection and

77

identification of volatiles in un-hopped pilsner wort and vapor condensates of the same

78

wort. The chemical background of some of the detected compounds is further

79

investigated and a comparison is made with data found in literature. New compounds

80

found in the un-hopped wort are discussed in detail.

81

MATERIALS AND METHODS

82

Chemicals

4

83

Methyl isobutyl ketone RECTAPURTM was bought from VWR International (West

84

Chester, PA). The compounds 2-pentylfuran (98 %) and (E,E)-2,4-decadienal (90 %)

85

were obtained from Alfa Aesar GmbH (Karlsruhe, Germany). All other chemicals were

86

acquired from Sigma-Aldrich (St. Louis, MO, USA): isobutyraldehyde, 2-phenyl-2-

87

butenal, (E)-2-nonenal, 5-hydroxymethyl-2-furfural (HMF), decanal, 5-methyl-2-furfural,

88

3-methylthiopropionaldehyde,

89

dimethyltrisulfide (DMTS), 2-ethyl-3,5(or 6)-dimethylpyrazine, 5-methyl-2-phenyl-2-

90

hexenal, 4-methyl-2-phenyl-2-pentenal, 2-acetylfuran, 2-isopropyl-5-methyl-2-hexenal,

91

2-methylpyrazine,

92

diacetyl, 2,3-pentanedione, β-myrcene, dimethylsulfide, β-damascenone, 4-vinylguaiacol,

93

hexanal, 2-heptanol, phenylethylalcohol, 3-methylbutanol, 1-hexanol, isovaleric acid, 2-

94

furfural, heptanal, propanal, furfuryl alcohol, pentanal, benzaldehyde, 2-methylbutanal,

95

octanal, butanal, isoamyl acetate and γ-nonalactone.

96

Wort production

97

Wort production was performed in the pilot scale (5 hl) brewery of the Centre for Malting

98

and Brewing Science (Heverlee, Belgium). An amount of 80 kg milled pilsner malt

99

(Dingemans, Stabroek, Belgium) was added to 220 liters of water at 55°C. The pH was

100

adjusted to 5.5 with lactic acid and the mash was subjected to a rest of 10 min. Next, the

101

temperature was raised to 63°C (35 min of rest) and the saccharification temperature of

102

72°C (20 min). After the saccharification rest, the mash was heated up to 78°C and

103

subsequently transferred to the lauter tun, in which the separation of spent grains and

104

sweet wort takes place. The filtered sweet wort was collected in the boiling kettle, where

105

it was heated up to 90°C. At 90°C, the wort was taken in 1.5L containers and

nonanal,

(E)-2-hexenal,

2,3-diethyl-5-methylpyrazine,

3-methylbutanal,

phenylacetaldehyde,

α-linalool,

5


106

immediately cooled down to room temperature. Hereafter the containers with sweet wort

107

were frozen at -25°C. Prior to the boiling experiments, the wort was defrosted; the

108

density was adjusted to 12°P and the pH was adjusted to 5.2 with sulfuric acid.

109

Lab-scale boiling

110

The boiling device consisted of a 6L stainless steel vessel, equipped with a wort sample

111

valve, a paddle stirrer and a temperature sensor, which was immersed in the wort. A glass

112

cover was used with inlets for the paddle stirrer, the temperature sensor and an outlet

113

chimney for wort vapors. A Liebig-cooler (cooling water at 2°C) was placed on the outlet

114

in order to condense the produced vapors. This condensate was collected in 50 ml tubes

115

on ice. The heating device consisted of a stainless steel plate with an integrated

116

temperature sensor and feed back temperature regulation was performed with the

117

immersed temperature sensor. While boiling, the heating plate temperature was kept

118

constant to guarantee uniform heating during the boiling experiment, while continuous

119

stirring ensured a homogenous heat distribution.

120

Sample preparation and Solid-Phase Micro-Extraction (SPME)

121

A 20 mL headspace vial was filled with a 10 mL aliquot of wort or vapor condensate,

122

together with 3.5 g NaCl (analytical grade) and 50 µL of a 200 ppm 2-heptanol solution,

123

used as an internal standard. The vial was placed in the tray of the CombiPAL (CTC

124

Analytics, Zwingen, Switzerland), where it was pre-incubated at 40°C for 10 minutes.

125

Hereafter a carboxen-polydimethylsiloxane-divinylbenzene fiber (CAR-PDMS-DVB,

126

Supelco, Bellefonte, PA) was used for extraction of volatiles in the headspace of the vial

127

during 20 min at 40°C.

128

GC/MS analysis

6


129

The injection of the CAR-PDMS-DVB fiber was performed in a split/splitless injector of

130

a Trace GC Ultra (Thermo, Waltham, MA) and desorption was executed for 2 minutes at

131

250°C in split mode (with a split ratio of 8). The used columns were of the type RTX-

132

200MS (30m, 0.25mm ID, 0.5µm df) and of the type RTX-5SilMS (60m, 0.25mm ID,

133

1.0µm df), both from Restek (Bellefonte, PA). A constant flow of 1.5 ml He per minute

134

was applied in both cases. The following temperature program was used with the RTX

135

200MS column: 30°C for 2 min, followed by a 3°C/min increase to 120°C, then a

136

10°C/min rise to 200°C, and finally with a 30°C/min rise to 260°C with a 1 min hold.

137

The following temperature program was used with the RTX 5SilMS column: 30°C for 2

138

min, then a rise to 70°C at a rate of 10°C/min with 1 min hold, followed by a rise up to

139

200°C at a rate of 4°C/min, and finally up to 270°C at a rate of 20°C/min, with a 2 min

140

hold. After separation, the volatiles were analyzed with a Dual Stage Quadrupole (DSQ)

141

MS (Thermo, Waltham, MA), which was set to detect positive ions with a mass to charge

142

ratio (m/z) of 33-260 in the electron-impact mode. The data were analyzed using

143

Xcalibur software (Thermo, Waltham, MA).

144

Identification of volatiles

145

The identification of wort volatiles was performed by GC-MS spectral analysis and their

146

Kovat’s retention indices were calculated for a RTX-200MS column and a RTX-5MS

147

column. Where possible, chemical standards were injected and compared with identified

148

compounds.

149

Aldol condensation reactions

150

De-ionized water was supplemented with 500 ppb of each reagent. The pH was adjusted

151

to pH 5.2 with lactic acid and 20 ml of the solution was incubated in sealed pyrex tubes

7


152

for 1 hour in a water bath at 100°C (in duplicate). After incubation, the samples were

153

cooled on ice and prepared for SPME-GC/MS analysis as described above.

154

RESULTS AND DISCUSSION

155

Headspace SPME GC/MS is a very sensitive technique for trace compound analysis in

156

the headspace of beverages, without the need for preliminary extraction and

157

consequently, without the risk of losing information with time-consuming extraction

158

procedures (13). The only consideration that has to be made is the choice of the

159

appropriate fiber. In this case, a 3-phase CAR-PDMS-DVB fiber was chosen in order to

160

obtain the broadest extraction spectrum of volatiles.

161

When wort is boiled, an important concern of the brewer is the quality of the wort after

162

boiling. The evaporated fraction of the wort is of minor importance, except for energy

163

recuperation by condensation. However, wort vapors contain high levels of volatiles due

164

to the higher volatility of those compounds in comparison with the volatility of water.

165

Therefore, the condensed vapors contain volatiles in levels that are related of the levels of

166

those volatiles in the remaining wort. Hertel and coworkers have determined the vapor-

167

liquid equilibrium of dimethylsulfide as 75.6, whereas the vapor-liquid equilibrium of

168

phenylacetaldehyde is no more than 5.6 (14). Hence analyzing the condensed vapor

169

fraction of the wort reveals more information than analysis of the same wort. The

170

difference in sensitivity is illustrated in Error! Reference source not found.1. While only

171

4 peaks have an intensity of more than 107 in wort (Chromatogram A), there are more

172

than 20 peaks with the same intensity in the condensate (Chromatogram B). Therefore it

173

was chosen to take samples from pilsner wort during boiling, as well as from the vapors

174

produced during the same wort boiling. The vapors were collected in subsequent

8


175

fractions, the first condensate fraction containing the highest total concentration of

176

volatiles as a mirror of the high content of volatiles in the un-boiled wort.

177

The volatiles that were identified in the wort and vapor condensates are shown in Table

178

1. In the un-hopped wort, it was possible to identify 61 volatile compounds: 6 sulfur

179

compounds, 10 furans, 11 linear aldehydes, 2 esters, 14 ketones, 8 alcohols, 4 branched

180

2-alkenals, 2 pyrazines, a terpenoid compound, an acid, a lactone and a phenolic

181

compound. On the other hand, analysis of the vapor condensate revealed 114 identifiable

182

compounds: 6 sulfur compounds, 14 furans, 13 linear aldehydes, 5 esters, 18 ketones, 14

183

alcohols, 19 2-alkenals, 13 pyrazines, 5 terpenoids, 2 oxazoles, 3 thiazoles, 1 lactone and

184

a phenolic compound. Some of these compounds have never been reported before as part

185

of the volatile fraction of un-hopped wort or even in beer. One previously unidentified

186

compound, 2-phenyl-2-octenal, was identified by mass spectral analysis and confirmed

187

by experiments with a model solution. This compound was never reported before in

188

literature, to the best of our knowledge.

189
190

Sulfur compounds. Methanethiol is generated from the degradation of methional or

191

methionine by a retro-Michael reaction and two molecules of methanethiol can further

192

associate to yield dimethyldisulfide (15). The compound dimethylsulfide is one of the

193

most important quality parameters for brewers while boiling. It is formed by the thermal

194

decomposition of S-methylmethionine (16). The flavor threshold of this compound is

195

around 50 ppb and in high concentrations it has an unpleasant, cooked cabbage flavor.

196

Thiophene is obtained as a result of the reaction of cysteine with Maillard reaction

197

products (17). Dimethyltrisulfide was easily detected in both wort and condensate,

9


198

despite of the general belief that this flavor compound belongs to the typical constituents

199

of the hop oil fraction (18). However, analogous to the production of dimethyldisulfide,

200

dimethyltrisulfide can also be formed from methional, as this process was observed

201

during beer aging as well (19). Dimethyltetrasulfide, a known compound in hops (20),

202

has never been detected before in non-hopped beer constituents. The level of

203

dimethyltetrasulfide in wort was very low as this compound was only detectable in the

204

vapor condensate.

205
206

Furans. Furans have always been associated with the Maillard reaction as this pathway is

207

one of the main furan-generating pathways. Furfuryl alcohol (and to a lesser extent 2-

208

furfural) is known as a precursor of furfuryl ethyl ether, an important beer staling

209

compound in beer (2,21). Furfural and 5-hydroxymethylfurfural are generally considered

210

as important beer aging markers (22). Acetylfuran and 5-methyl-2-furfural also tend to

211

accumulate during beer aging (23) and their formation is due to the Maillard reaction as

212

well. The alkylfurans 2-methylfuran, 2-ethylfuran and 2-pentylfuran have already been

213

observed in malt (4) and wort (7), while 2-butylfuran has only been reported in malt

214

extract (3). Pentylfuran, with a “cough syrup”-like flavor characteristic, is recognized as a

215

product of lipid oxidation and formed by a ring closure of 4-hydroxy-2-nonenal (24).

216

Subsequently 2-butylfuran could be formed in an analogous manner starting from 4-

217

hydroxy-2-octenal, and 2-ethylfuran could be formed starting from 4-hydroxy-2-hexenal

218

as well (Figure 1). Both possible precursors of 2-butylfuran and 2-ethylfuran have

219

previously been detected in vegetable oil (25). As these precursors have a polar nature,

10


220

they will not easily be evaporated. Only after ring-closure a more volatile compound is

221

generated, which can be evacuated by evaporation.

222

The compounds (E)- or (Z)-2-(2-pentenyl)-furan and 3-phenylfuran were tentatively

223

identified. While 2-(2-pentenyl)furan has only been reported in the volatile fraction of

224

cooked pork (26), the presence of 3-phenylfuran has already been observed in the volatile

225

fraction of chocolate and its flavor was described as cocoa, green or mint (27). The

226

tentatively identified 2-hexanoylfuran has never been described in food-related literature

227

so far.

228
229

Linear Alkanals and Strecker Aldehydes. The listed aldehydes were all identified and

230

compared with their reference compounds. The well-known Strecker aldehydes (2-

231

methylpropanal, 2-methylbutanal, 3-methylbutanal, methional, phenylacetaldehyde and

232

benzaldehyde) are considered as aging-relevant compounds and are frequently used as

233

indicators for wort boiling performance (1). The parent amino acids of the Strecker

234

aldehydes are easy to determine, except for benzaldehyde, as there is no direct correlation

235

between benzaldehyde and any available amino acid. Granvogl et al. (28) suggested an

236

imine-enamine

237

methylglyoxal, followed by the oxidation of the intermediate and a retro-aldol reaction,

238

generating benzaldehyde and formaldehyde. Phenylethylamine can be formed out of

239

phenylalanine. Consequently, direct formation of benzaldehyde from phenylalanine could

240

occur analogously with an additional decarboxylation step.

241

The linear alkanals have already been described many times in wort (29) and beer (30).

242

They originate from the enzymatic oxidation of fatty acids during the mashing process or

tautomerization

of

the

Schiff-base

of

phenylethylamine

and

11


243

from chemical autoxidation reactions during the wort boiling process. One of the most

244

flavor active compounds is (E,E)-2,4-decadienal, which is formed by oxidation of linoleic

245

acid and which is characterized by an unpleasant ‘fried oil’ flavor (31). The oxidized

246

form of (E,E)-2,4-decadienal, the 4,5-epoxy-2-decenal derivative, could also directly be

247

involved in a Strecker-type reaction with amino acids (32).

248
249

Esters. The identified esters probably originate from the barley malt. The tentatively

250

identified isopropyl laurate (characteristic ions 183, 200, 201) was never encountered

251

before in beer or wort. Strikingly this compound was not only detectable in the vapor

252

condensate, but in wort as well.

253
254

Ketones. Eighteen ketones were identified in the vapor fraction of the wort. Among them

255

β-damascenone was found, which could either be generated by hydrolysis of glycosides

256

(33) or by direct oxidation of carotenoids (34). Together with β-damascenone, its direct

257

precursor 3-hydroxy-β-damascone was tentatively identified for the first time in the wort

258

volatile fraction. Acetophenone was found in both wort and vapor fraction. Its presence

259

has already been observed in Maillard model systems (35). The presence of different

260

isomers of 3,5-octadien-2-one was also encountered for the first time in wort or beer.

261

Presumably this compound originates from lipid oxidation like most of the ketones listed.

262

The cyclic ketone 4-cyclopentene-1,3-dione was tentatively found for the first time in

263

wort and vapor condensate, since no reference compound is commercially available. The

264

compound has been described as quantitatively the most important carbonyl compound in

265

melanoidin degradation model systems (36) and its formation pathway has been proposed

12


266

by Yaylayan et al. (37). This pathway starts from a phosphorylated D-glucose, which is

267

converted to 3-deoxyglucosone-6-phosphate.

268

reaction a 1,2,4-tricarbonyl compound is generated.

269

undergoes cyclization via an intra-molecular aldol condensation. However it is not yet

270

known whether this compound could contribute to the overall flavor of beer.

Via a phosphate mediated retro-aldol
This tricarbonyl compound

271
272

Alcohols. The listed alcohols are known to be present in wort or beer. Some of them, like

273

3-methylbutanol and phenylethyl alcohol, are important flavor compounds of beer.

274
275

Alkenals. The linear 2-alkenals and 2,4-alkadienals have been described as important

276

off-flavors in aged beers (30). The most intriguing of these linear 2-alkenals is (E)-2-

277

nonenal, which is for decades held responsible for the aged flavor of beer, with a

278

cardboard-flavor kind of characteristic. An important route to (E)-2-nonenal formation is

279

the lipid oxidation pathway (38), but this 2-alkenal can could also be generated by the

280

aldol condensation of heptanal and acetaldehyde (39).

281

Remarkable in the group of 2-alkenals was the detection of the branched 2-alkenals 2-

282

isopropyl-5-methyl-2-hexenal, 2-phenyl-2-butenal, 4-methyl-2-phenyl-2-pentenal, 4-

283

methyl-2-phenyl-2-hexenal and 5-methyl-2-phenyl-2-hexenal. The phenyl-containing

284

compounds are each characterized by a cocoa-like flavor, and have been identified in

285

malt, cocoa and coffee according to Tressl (40). The compound 5-methyl-2-phenyl-2-

286

hexenal has also been found in ale wort (7) and malt extract (3) and is known as one of

287

the most important flavor compounds in cocoa (27,41). However it has not been

288

described before in pilsner wort, where pale malt is used. It was suspected that the

13


289

branched 2-alkenals would hardly be detected due the apparent correlation between the

290

concentration of these compounds and increasing malt color as stated by Coghe et al. (6).

291

It was suggested that the formation rate is higher in dark malts that was attributed to the

292

dehydrating conditions during malting and roasting. However, all known compounds

293

were easily detected in the vapor condensate, while 2-phenyl-2-butenal, 4-methyl-2-

294

phenyl-2-pentenal and 5-methyl-2-phenyl-2-hexenal were also found in the wort fraction.

295

Logically, these compounds are formed by aldol condensation of two Strecker aldehydes.

296

Coghe et al. (6) described the formation of 2-isopropyl-5-methyl-2-hexenal from 2

297

molecules of 3-methylbutanal. In the other cases phenylacetaldehyde seems to be the

298

central compound, which can react with acetaldehyde, 2-methylpropanal, 2-

299

methylbutanal and 3-methylbutanal to give rise to 2-phenyl-2-butenal, 4-methyl-2-

300

phenyl-2-pentenal,

301

respectively.

302

Another observation that has not been mentioned in literature so far was the presence of 2

303

isomers of each branched 2-alkenal in the vapor fraction (except for 4-methyl-2-phenyl-

304

2-pentenal, presumably because the isomer was under the detection level). Therefore, it is

305

likely that the two isomers are obtained after reaction of the parent molecules. However,

306

the ratio of isomers differs greatly. The isomers with the lowest RI (Table 1) displayed

307

the highest peak intensity: about 98 % of the sum of the two peak areas in case of 5-

308

methyl-2-phenyl-2-hexenal. Apparently the formation of every time the same isomer is

309

strongly favored during the reaction. This could be explained by sterical hindrance of the

310

bulky phenyl of isopropyl side chains, favoring either the cis- or the trans-configuration.

311

However, it is unclear whether the cis or the trans-configuration dominates.

4-methyl-2-phenyl-2-hexenal

and

5-methyl-2-phenyl-2-hexenal,

14

312

In addition, a previously unknown compound in food systems was tentatively identified

313

as 2-phenyl-2-octenal, which could be the product of the aldol condensation reaction

314

between hexanal and phenylacetaldehyde. When analyzing the mass spectrum (Figure 2),

315

the base structure was comparable to those of all branched 2-alkenals disposing of a

316

phenyl side group with characteristic peaks at m/z 115 and 117. A mass spectral

317

fragmentation pattern of 2-phenyl-2-octenal is proposed in Figure 3. After breakdown of

318

2-phenyl-2-octenal to the 2-phenyl-2-butenal ion during mass spectral fragmentation, the

319

smaller fragments are highly comparable to the fragments of 2-phenyl-2-butenal.

320
321

In order to investigate if the synthesis of branched 2-alkenals was possible under wort

322

boiling conditions, model experiments were performed with the addition of equal

323

amounts of Strecker aldehydes in a solution at pH 5.2. The following aldehydes were

324

incubated together with phenylacetaldehyde at 100°C: 3-methylbutanal, acetaldehyde, 2-

325

methylbutanal, 2-methylpropanal and hexanal. After 1 hour of boiling, the reaction

326

mixtures were prepared and analyzed with GC-MS. It was observed that the reaction

327

occurred in each sample, nevertheless in considerably lower amounts compared to the

328

concentration of the parent molecules (in the case of 5-methyl-2-phenyl-2-hexenal: peak

329

area 8.106 vs. 6.108 and 15.108 for 3-methylbutanal and phenylacetaldehyde respectively).

330

As the aforementioned reactions seem to occur in wort under boiling conditions, virtually

331

every aldehyde present in wort must be able to undergo an aldol condensation with

332

another aldehyde. These aldol condensations could give rise to a large group of merged

333

aldehydes with different flavor characteristics (e.g. the cocoa-flavor of 5-methyl-2-

15

334

phenyl-2-hexenal). An overview of the detected branched 2-alkenals together with their

335

parent compounds is depicted in Figure 4.

336
337

Pyrazines. The pyrazines were mostly found in the condensate fraction, although 2,5-

338

dimethylpyrazine and 3-ethyl-2,5(or 6)-dimethylpyrazine were detected in the pilsner

339

wort as well. Hence, the concentration of the pyrazines appeared to be low in most cases,

340

as the majority was not detectable in the pilsner wort. Pyrazines are characteristic

341

compounds of roasted cocoa beans (27,40,41), coffee (40,42) and roasted malt (4,40),

342

often characterized by nutty and roasted flavors. The presence of these pyrazines in

343

pilsner wort indicates that these compounds can be formed in more gentle conditions than

344

a roasting process, although in very low quantities.

345
346

Terpenoids. Linalool was easily detected in both wort and condensate. β-Myrcene, α-

347

terpineol, β-cyclocitral and α-cadinol (tentatively identified) were only detected in the

348

condensate. These compounds are typical constituents of hop essential oil (11,18) and

349

have never been associated with un-hopped wort before. Hence it seems that malt also

350

contains minimal amounts of terpenoids. The detection of these terpenoids demonstrates

351

once more the high sensitivity of this technique, as their concentration must be extremely

352

low.

353
354

Oxazoles.

The

oxazoles

4,5-dimethyl-2-isopropyloxazole

and

4,5-dimethyl-2-

355

isobutyloxazole were tentatively identified in the wort condensate. Oxazoles are formed

356

when an α-aminoketone and an aldehyde react together (15) or when a Strecker

16


357

degradation is not complete. They have also been described in model-systems (43). The

358

compound 4,5-dimethyl-2-isopropyloxazole probably originates from the reaction of the

359

α-aminoketone of diacetyl with 2-methylpropanal and consequently 4,5-dimethyl-2-

360

isobutyloxazole probably originates from the α-aminoketone of diacetyl with 3-

361

methylbutanal. Logically the compounds could also be formed by the immediate reaction

362

of diacetyl with valine and leucine, respectively. This oxazole formation could also be

363

obtained immediately from the Strecker degradation pathway, starting from

364

tautomerization of the imine intermediate. The hypothetical formation pathway of the

365

detected oxazoles is depicted in Figure 5. After the reaction of the Strecker aldehyde

366

with the α-aminoketone, a Schiff base is formed. This can undergo a cyclization,

367

followed by an oxidation, and finally yielding the respective oxazoles.

368
369

Thiazoles. Alkylthiazoles were tentatively identified in the wort vapor fraction. As these

370

compounds were only detected in the vapor condensate, the wort levels must have been

371

considerably low. The identified compounds were 2-isopropyl-4,5-dimethylthiazole, 2-

372

isopropyl-4-ethyl-5-methylthiazole and 2-isobutyl-4,5-dimethylthiazole. Alkylthiazoles

373

have already been found in the volatile fraction of cooked meat and are described as a

374

product of the reaction of α-dicarbonyl compounds with H2S, NH3 and virtually every

375

type of aldehyde (44,45). Possibly, the alkylthiazoles could also be formed, analogously

376

to the oxazole formation, directly from α-aminoketones, aldehydes and H2S or from the

377

reaction of an imine intermediate during Strecker degradation and H2S.

378

17

379

Other. The lactones γ-butyrolactone and γ-nonalactone were found in wort and vapor

380

condensate respectively. The peach-like flavored γ-nonalactone is known as an important

381

beer aging compound (33). The phenolic compound 4-vinylguaiacol (smoked, dentist-

382

like flavor), was detected in wort and vapor condensate with increasing intensity during

383

the wort boiling process. It is formed by thermal decarboxylation of ferulic acid, which is

384

released during the mashing process. However, the contribution of the thermal

385

decarboxylation to the total formation of 4-vinylguaiacol in beer is low compared with

386

the enzymatic production of 4-vinylguaiacol during fermentation (46).

387
388

Conclusion. The combination of Headspace SPME-GC/MS combined with vapor

389

condensate analysis was a successful tool for the detection of new volatile compounds.

390

More than 110 different volatile compounds were identified with this technique in un-

391

hopped wort, from which almost 30 compounds have never been described before in wort

392

originating from pilsner malt., from which 54 were confirmed with reference compounds.

393

The study on wort vapors has revealed the importance of the aldol condensation

394

mechanism between aldehydes for the generation of new compounds. Both isomers of the

395

unsaturated condensation product can be formed with this reaction. However, the bulky

396

side chains of the generated aldehyde seem to influence the balance between the two

397

isomers.

398

It was demonstrated that Strecker aldehydes could combine together to generate branched

399

2-alkenals. Heating hexanal and phenylacetaldehyde together in a watery solution

400

generated the previously unknown aldol condensation product 2-phenyl-2-octenal.

401

Hence, virtually every aldehyde present in wort could undergo the same reaction,

18


402

generating a large group of complex aldehydes with variable characteristics. Some of

403

these generated aldehydes could cause off-flavors, like the cardboard flavor of (E)-2-

404

nonenal, or they could be beneficial, like the cocoa flavor of 5-methyl-2-phenyl-2-

405

hexenal. Further research must elucidate the relevance of these compounds in beer, as no

406

information is present about actual concentrations and thresholds of these compounds.

407

The tentative discovery of alkyloxazoles and alkylthiazoles in the vapor fraction

408

highlights could indicate once more the central role of dicarbonyl compounds, aldehydes

409

and amino acids. At present, the flavor characteristics of some oxazoles have been

410

described as sweet, nutty or cocoa (47) and they may have a low odor threshold.

411

This paper focused primarily on the complete identification of the wort volatile fraction.

412

However, quantitative measures have not been taken, nor has there been a follow up op

413

volatiles during wort boiling. Further research will therefore pay attention at the

414

evolution of the identified compounds in the course of wort boiling.

415
416

ABBREVIATIONS

417

CAR-PDMS-DVB

Carboxen-polydimethylsiloxane-divinylbenzene

418

SPME

Solid Phase Micro-Extraction

419

420

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421

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