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The Bitter Chemodiversity of Hops (Humulus lupulus L.) Michael Dresel, Christian Vogt, Andreas Dunkel, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03933 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 9, 2016

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Page 1 of 41

Journal of Agricultural and Food Chemistry

1

The Bitter Chemodiversity of Hops (Humulus

2

lupulus L.)

3 Michael Dresel, Christian Vogt, Andreas Dunkel, and Thomas Hofmann*

4 5 6 7

Chair of Food Chemistry and Molecular Sensory Science, Technische Universität

8

München, Lise-Meitner-Str. 34, D-84354 Freising, Germany

9 10 11 12 13 14 15

*

16

PHONE

+49-8161/71-2902

17

FAX

+49-8161/71-2949

18

E-MAIL

[email protected]

To whom correspondence should be addressed

19 20

1 Environment ACS Paragon Plus


21

Abstract

22

To map the chemodiversity of key bitter compounds in hops, a total of 75 different

23

samples collected from the global hop market were analyzed for 117 key bitter

24

tastants by means of a multiparametric HPLC-MS/MSMRM method. Among the

25

compounds detected, 2′′,3′′-epoxyxanthohumol was detected for the first time in hops

26

and isoxanthohumol M was identified as a marker compound for varieties grown in

27

Germany. Hop ageing experiments in the absence and presence of air oxygen,

28

respectively, were conducted to address the stability of hop-derived compounds

29

during long-term storage.

30 31

Keywords:

32

Bitter taste, xanthohumol, prenylated flavanoids, hops, iso-α-acids, β-acids,

33

humulones, sensometabolites, Humulus lupulus L.

34 35


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Page 3 of 41

36


INTRODUCTION

37 38

For many centuries, hops (Humulus lupulus L.) has been used as an essential

39

ingredient in the brewing process to impart the alluring aroma and typical bitter taste

40

of the final beverage. Recently, craftbrewers are seeking new hop varieties to

41

produce beers with unique flavors, which led to the breeding of so-called flavor hop

42

varieties that form a new category besides the established categories bitter and

43

aroma hops.1 However, the hop industry faces a huge overproduction during the last

44

5 years which adds to over 10.000 tons α-acids and equals the amount of α-acids

45

needed for 1 year.1 Successful breeding of varieties suitable for storage with an

46

excellent flavor profile might prevent a massive financial impairment and overcome

47

supply shortenings in times when more hops is needed than produced. A new

48

approach toward felicitous breeding is the understanding of the molecular

49

mechanisms during storage and the identification of crucial precursors on a

50

molecular level.

51

Among the key taste compounds identified in the hop hard (1 - 40a/b, Figure 1)

52

and soft resin (41 - 65, Figure 2), the bitter compounds in hops are divided into two

53

groups

54

xanthohumol (1, Figure 1), as well as α-acids and β-acids.2,3 The α-acids and the β-

55

acids can be further divided regarding their alkanoyl side chain into the most

56

abundant co-, n- and ad-humulones and -lupulones (41, 42, Figure 2), respectively.

57

During the wort boiling process, α-acids are well-known to be converted into the

58

corresponding cis- and trans-iso-α-acids (43, 44, Figure 2), respectively,4 which have

59

been identified as the main contributors to the bitter taste of beer.5,6

of

prenylated

polyketides,

namely,

prenylated

chalcones

such

as

60

Within the past decades, various studies focused on volatile aroma

61

components in hops and beers,7-13 the transformation of hop α-acids and β-acids, as



Page 4 of 41

62

well as their isomerization and degradation upon wort boiling.4,14-17 Besides the beer

63

brewing process, aging of beer has been found to induce additional molecular

64

transformations of the hop-derived bitter molecules. Some of these reactions were

65

found to be oxygen dependent such as the formation of hydroperoxy-trans/cis-

66

alloisohumulones (51, 52, Figure 2) and hydroxy-trans/cis-alloisohumulones (48, 49,

67

Figure 2),18 as well as the formation of the humulinones (45, Figure 2)19 and

68

hulupones (58, Figure 2).20 Upon cleavage of the variable alkanoyl side chain, the

69

various hulupone congeners are further truncated to give hulupinic acid (59, Figure

70

2).21 Additionally, incubation experiments with hops revealed a series of oxidation

71

products,

72

oxycolupulones.24 Recently, the formation of tri- and tetracyclic degradation products

73

in beer (Figure 2)15-17 indicated the stability of hop-derived compounds om beer to be

74

dependent on the pH-value.

namely

lupoxes,

lupdoxes,

lupdeps,

lupdols21-23

and

tricyclo-

75

Besides α- and β-acid transformation, also xanthohumol (1) and related

76

flavonoids25 have been reported to undergo an isomerization during the kettle boiling

77

process, thus giving rise to the so-called isoxanthohumol derivatives.3 Just recently,

78

the bittering potential of the hop’s hard resin could be assigned to more than 40

79

compounds of beer26, amongst which 26 prenylated flavonoids and 10 other

80

polyphenols. Moreover, these compounds were shown to cause a pleasant bitter

81

sensation26. A similar observation has been reported by Stettner et al.27 and Biendl,28

82

showing that beers produced with a xanthohumol enriched extract exhibit a more fine

83

and pleasant bitterness than a traditionally produced beer.

84

Besides their contribution to aroma and taste, some of these hop ingredients

85

are known to enhance foam stability and, due to their antimicrobial activity,

86

increase the shelf-life of the final beer.29 Moreover, several hop compounds and, in

87

particular, the prenylated flavonoids xanthohumol (1) and 8-prenylnaringenin (4) have


to

Page 5 of 41


88

been demonstrated to exhibit various pharmacological activities such as, e.g. anti-

89

inflammatory, estrogenic, cancer chemopreventive, and anti-angiogenic properties,

90

and were reported to help counteracting obesity and diabetes type-2.30-36,42-44

91

In today’s brewing processes, various hop products are used to deliver one or

92

the other funactionality, e.g. hop cones, pellets, α- and β-acid rich carbondioxide and

93

ethanolic hop extracts, and special purpose materials like xanthohumol extracts or a

94

recently developed ε-extract, which is enriched in prenylated flavonoids without

95

containing large amounts of xanthohumol (1) and isoxanthohumol (2).26,

96

Depending on the hop product used, the combinatorial interplay of such hop derived

97

compounds has been recently reported to influence the taste perception and

98

modulate the pharmacological properties of the final product.46-48

28, 45

Better understanding such potential interactions and foreseeing adverse

99 100

effects

on

flavor

perception

requires

to

blueprint

101

sensometabolites49,50 in hop varieties that reflect the taste phenotype of the

102

products.50,51 Up to now, the knowledge on the composition of key taste compounds

103

in hops and hop products is rather fragmentary, e.g. previous studies have just been

104

focused on individual compound classes like flavonoids52 and quercetin as well as

105

kaempferol glycosides.53 Profiling of 13 European hop varieties by means of LC-MS,

106

FTICR-MS and NMR revealed chemical differences to be mainly due to bitter

107

compounds.54

108

The objective of the present study was therefore to quantitatively blueprint and

109

classify the key bitter sensometabolites in 75 hop varieties and to quantitatively

110

monitor their changes in the German hop varieties Hersbrucker spaet and Taurus

111

under anaerobic as well as aerobic conditions.

112


the

pattern

of

key


113

Page 6 of 41

MATERIALS AND METHODS

114 115

Chemicals. Formic acid, hydrogen peroxide (Merck, Darmstadt, Germany);

116

acetonitrile, methanol (Sigma Aldrich, Steinheim, Germany); DMSO-d6 (Euriso-Top,

117

Saarbrücken,

118

chromatography and LC-MS, respectively (J.T. Baker). Hop pellets (type 90) were

119

obtained from BierKulturHaus (Obertrum, Austria), Die Internationale Brau-

120

Manufakturen GmbH (Frankfurt am Main, Germany), de 'proef' brouwerij (Lochristi,

121

Belgium), Hallertauer Hopfenveredelungsgesellschaft mbH (Mainburg, Germany),

122

HVG Hopfenverwertungsgenossenschaft e.G. (Wolnzach, Germany), Joh. Barth &

123

Sohn GmbH & Co. KG (Nuremberg, Germany), Simon H. Steiner, Hopfen, GmbH

124

(Mainburg, Germany), SimplyHops (Kent, United Kingdom), and Yakima Chief

125

(Louvain-La-Neuve,

126

(http://www.craftbrewer.com.au/, Queensland, Australia). Pellets were vacuum-

127

packed and stored at 4°C until analysis. Unhopped beer was obtained from the Chair

128

of Brewing and Beverage Technology (Technische Universität München, Freising,

129

Germany).

Germany).

HPLC

Belgium),

and

or

MS

were

grade

solvents

purchased

were

from

used

for

CraftBrewer

130

Forced Aging of Hop Pellets. Hop pellets (harvest 2011) were ground and

131

stored in a Binder ED53 heating oven (Binder, Tuttlingen, Germany) at 50°C for 1 to

132

7 weeks. Thereafter, samples were vacuum-packed and stored at -20°C until

133

analysis.

134

Preparation of Reference Compounds. Compounds 41 - 65 were obtained

135

as reported earlier.15-17 Compounds 1 - 26 and 29 - 40 were isolated from spent hops

136

or synthesized from xanthohumol (1).26 After confirming their structural identity as

137

well as purity (> 98 %; HPLC/MS, 1H NMR), the bitter compounds were used as

138

external standards for the HPLC-MS/MS analysis of the sensometabolites in hops.


Page 7 of 41

139


Synthesis

of

Hop-Derived

Synthesis

Compounds.

of

2′′,3′′-

140

Epoxyxanthohumol (27). Xanthohumol (1; 500 mg) was solved in 50 mL of MeOH

141

and, after addition of 10 mL H2O2 (30%), was refluxed for 20 h at 65°C whilst stirring.

142

The reaction mixture was diluted with water and separated on a HPLC system

143

(Jasco, Great Dunmow, Great Britain) consisting of two Jasco PU-2087 pumps, a

144

7725i type Rheodyne injection valve (Rheodyne, Bensheim, Germany), and a Jasco

145

DAD MD-2010 diode array detector (λ = 220 - 400 nm). All separations were

146

performed on a 250 x 21.2 mm, 5 µm, Luna Phenyl-Hexyl column (Phenomenex)

147

using water (0.1 % formic acid) as solvent A and acetonitrile as solvent B (flow rate:

148

20 mL/min): 55% B kept for 1 min and, then, increasing B to 60 % within 1 min, to

149

75 % within 18 min, to 85 % within 2 min, and, finally to 100 % within 2 min. Solvent

150

B was kept for 2 min at 100 % and after 26 min decreased again to 55 % within 2

151

min. Compound 27 (Rt=5.68 min) was isolated in a purity of more than 98% and,

152

after the solvent was removed in vacuum and freeze-dried twice, was used for

153

spectroscopic structure determination. Compound 27 was identified as 2′′,3′′-

154

epoxyxanthohumol that has been proposed earlier as an intermediate of the

155

metabolism of xanthohumol (1).37, 58, 59

156

2′′,3′′-Epoxyxanthohumol, 27 (Figure 2). UV-Vis (ACN/water; 0.1% formic

157

acid): λmax = 372 nm; LC-MS (ESI-): m/z (%) 369 (100, [M - H]-), 119 (74), 113 (24);

158

LC-TOF-MS (ESI-): m/z 369.1344 ([M-H]- measured), m/z 369.1338 ([M-H]-, calcd. for

159

C21H21O6-); 1H NMR (400 MHz, DMSO; COSY): δ (ppm): 1.14 [s, 3H, H−C(4′′)] 1.15

160

[s, 3H, H-C(5′′)]; 2.97 [d, 2H, J = 8,7 Hz, H-C(1′′)]; 3.90 [s, 3H, H-C(1′′′)]; 4.71 [t, 1H, J

161

= 8.9 Hz, 17.5 Hz, H-C(2′′)]; 6.19 [s, 1H, H-C(6)]; 6.84 [d, 2H, 8.5 Hz, H-C(2′,6′)]; 7.53

162

[d, 2H, J = 8.6 Hz, H-C(3′, 5′)]; 7.68 [d, 2H, J = 15.5, H-C(2, 3)]; 8.21 [s, 1H, OH-

163

C(7)]; 10.28 [s, 1H, OH-C(4′)]; 14.19 [s, 1H, OH-C(9)];

164

HSQC, HMBC): δ (ppm): 25.22 [C(4′′)]; 26.32 [C(5′′)]; 26.81 [C(1′′)]; 56.88 [C(1′′′)];

13


C NMR (100 MHz, DMSO;


Page 8 of 41

165

70.49 [C(3′′)]; 86.91 [C(6)]; 91.89 [C(2′′)]; 105.8 [C(8)]; 106.19 [C(10)]; 116.44 [C(3′,

166

5′)]; 124.13 [C(3)]; 126.33 [C(1′)]; 131.02 [C(2′, 6′)]; 143.29 [C(2)]; 161.35 [C(4′)];

167

163.15 [C(9)]; 163.74 [C(5)]; 167.4 [C(7)]; 192.47 [C(4)].

168

Synthesis of Xanthohumol G (28). A solution of 2′′,3′′-epoxyxanthohumol (27;

169

50 mg) in MeOH/water (1/1, v/v; 100 mL) was stirred at room temperature for 20 h

170

whilest oxygen was bubbled through the solution. After dilution with water, the

171

reaction mixture was separated by HPLC using the same system and gradient as

172

reported above for compound 27. The target compound 28 eluting at 3.75 min was

173

isolated in a purity of more than 98% and, after the solvent was removed in vacuum

174

and freeze-dried twice, analysis by means of UV−Vis, LC-MS/MS, UPLC-TOF-MS,

175

and 1D/2D NMR demonstrated matching spectroscopic data with those reported

176

earlier.34

177

Liquid Chromatography/Mass Spectrometry (LC-MS/MS). LC-MS/MS

178

analyses were performed on an API 4000 Q-Trap system (Applied Biosystems,

179

Darmstadt, Germany) as reported earlier.15,26

180

UPLC/Time-of-Flight Mass Spectrometry (UPLC/TOF-MS). Mass spectra of

181

the compounds were measured on a Waters Synapt G2 HDMS system (Waters,

182

Manchester, UK) hyphenated with an Acquity UPLC core system (Waters) and a

183

BEH C18, 2 x 150 mm, 1.7 µm, column (Waters) using the parameters reported

184

recently.26

185

Nuclear

Magnetic

Resonance

Spectroscopy

(NMR).

1D/2D-NMR

186

experiments were performed on a 400 MHz DRX and a 500 MHz Avance III

187

spectrometer (Bruker, Rheinstetten, Germany) using the parameters reported

188

earlier.26

189

Quantitation of Bitter Compounds. Aliquots of hop pellets (1.5 g) were

190

extracted for 1h with methanol (100 mL) whilst ultrasonification. After filtration and


Page 9 of 41


191

1:100 dilution, the samples were membrane filtered (0.45 µm, Sartorius, Goettingen,

192

Germany) and aliquots (5 µL) were directly injected into a UltiMate 3000 series

193

UHPLC system (Dionex, Idstein, Germany) connected to the API 4000 Q-TRAP

194

system (MRM/ESI- mode) using optimized fragmentation parameters for compounds

195

1 - 26, 29 – 65 as reported recently.15-18, 26 After optimizing the declustering potential

196

(DP), the cell exit potential (CXP), and the collision energy (CE), compounds 27 (m/z

197

369.1 118.9; DP: -95; CE: -36; CXP: -7) and 28 (m/z 369.1 118.9; DP: -100;

198

CE: -38; CXP: -9) were quantified as well. Quantification of target compounds was

199

performed after 8-point external matrix calibration of every single compound in low

200

(2.5 nmol/L - 200 µmol/L) and high concentrations (0.2 mmol/L - 20 mmol/L). To

201

overcome matrix effects, hop-free beer was used as the matrix. To avoid negative or

202

exaggerated estimates at the low end of the concentration ranges, the functions were

203

forced through zero, thus leading to correlation coefficients of > 0.99 for all the

204

reference compounds. If necessary, concentrations were corrected for amounts

205

detected in a negative control (unhopped beer). Chromatography (flow rate: 0.6

206

mL/min) was done in a 150 x 2.0 mm Synergi, 4 µm, Hydro-RP column

207

(Phenomenex, Aschaffenburg, Germany) using acetonitrile (0.1 % formic acid) as

208

solvent A and water (0.1 % formic acid) as solvent B (Figure 3): solvent A: 10→40 %

209

within 15 min, 40→60 % within 13 min, 60→70 % within 20 min, 70→100 % within 2

210

min, 100→10 % within 5 min, followed by re-equilibration for 4 min prior to the next

211

run.

212

Multivariate Analysis. Data analysis was performed within the programming

213

and visualization environment R (version 2.13.2).55 The sensomics heatmap was

214

calculated using the heatmap.2 function of R based on the concentration data

215

(Tables 4-6, Supporting Information) after scaling the sum of all sensometabolites to

216

an equal value for each beer sample. The dendrograms were constructed by means



217

of an agglomerative linkage algorithm proposed by Ward56 specifying the distance

218

between two clusters as the increase in the error sum of squares after fusing two

219

clusters into a single cluster and seeking for a minimum distance at each clustering

220

step.57

221 222 223

RESULTS AND DISUSSIONS

224 225

In order to quantitatively analyse a total of 117 bitter compounds in 75 hop varieties,

226

collected from 12 different growing areas, by means of LC-MS/MS, first reference

227

material of compounds 1 - 26 and 28 - 65 was purified following protocols reported

228

earlier.15-17,26,34 As the structure of 2′′,3′′-epoxyxanthohumol (27) has been suggested

229

as a metabolic intermediate of xanthohumol (1),37,58,59 this compound was

230

synthesized by oxidation of xanthohumol (1) using hydrogenperoxide. Xanthohumol

231

G (28) was synthesized by oxidation of compound 27 as described earlier in this

232

manuscript.

233

Quantitation of Key Bitter Compounds and Hierarchical Cluster Analysis

234

of Hop Varieties. Hop-derived bitter compounds 1 - 65 (Figures 1 and 2) were

235

analyzed in hop pellets from year 2011 by multiparametric quantitation using a single

236

LC-MS/MS run with selective mass transitions as outlined in Figure 3. Humulones

237

(41) and lupulones (42) were found as the major sensometabolites in fresh hop

238

pellets, while only trace amounts of degradation and isomerization products like

239

compounds 43 - 65 were detectable (Table S1, Supporting Information). Although

240

suggested earlier as a reactive intermediate (bio-)transformation product of

241

xanthohumol (1),37,58,59 this is the first report on 2′′,3′′-epoxyxanthohumol (27) in hops.


Page 10 of 41

Page 11 of 41

242


To

examine

the

multivariate

distances

between

the

individual

243

sensometabolites throughout the different hop varieties, the concentrations

244

determined for each compound (Table S1, Supporting Information) were scaled and

245

a hierarchical cluster analysis was performed on the basis of these data (Figure 4).

246

The hierarchical analysis clustered the bitter compounds into the five

247

compound clusters 1 - 5, with cluster 2 devided into the two subclusters 2a and 2b,

248

and the hop varieties into the five clusters A - E (Table 1 and Figure 4). Cluster 1

249

comprises degradation products of α-acids (46a; conc. < 0.01%), β-acids (58a/b/c;

250

conc. < 0.3%), and iso-α-acids (53b, 60a, 61a/b/c; conc. < 0.1%). In addition,

251

xanthohumol D (15), xanthohumol B (16), xanthohumol O (23), xanthohumol M (25),

252

isoxanthohumol M (26), and 2′′,3′′-dehydrocyclohumulohydrochinon (29) are present

253

in this cluster. A unique behaviour was observed for xanthohumol O (23) and

254

isoxanthohumol

255

isoxanthohumol M (26), was only present at low levels in a few hop varieties, levels

256

of xanthohumol O (23) and isoxanthohumol M (26) were affected by the hop variety

257

and were found in European varieties as well as American varieties that are related

258

with the breed Brewers Gold.61 For a simplified overview on the pedigree of the hop

259

varieties62-70 see Supporting Information. Moreover, elevated concentrations of these

260

compounds (up to 72 mg/100g) were exclusively found in varieties grown in

261

Germany. Whenever the same variety was grown in other countries e.g. Belgium,

262

much lower concentrations were observed (Hallertauer Mittelfrüh - GER: 51.5

263

mg/100g, BG: 7.2 mg/100g; Magnum - GER: 71.8 mg/100g, BG: 4.9 mg/100g). As a

264

general rule, varieties with a low concentration of humulones (41) also featured a

265

lower concentration of isoxanthohumol M (26). In contrast, non-German varieties

266

contained at maximum of 0.05 % of isoxanthohumol M (26). A similar behavior was

267

observed for 2′′,3′′-dehydrocyclohumulohydrochinon (29). Although this compound

M (26). Whereas xanthohumol M (25), the precursor of



Page 12 of 41

268

was not always present in breeds containing detectable amounts of xanthohumol O

269

(23) or isoxanthohumol M (26), respectively, the occurrence of this compound was

270

limited to those varieties. The presence of single compounds in distinct varieties has

271

been previously observed for 4′-O-methylxanthohumol and flavokawin71 or β-

272

farnesene.62 Future research will have to investigate the impact of varying

273

gowth/harvest conditions on the observed regional differences in bitter tastant

274

chemodiversity of the same hop variety.

275

Next to the α-acids (41a/b/c; conc.: 5 - 15%), xanthohumol (1) and

276

xanthohumol derivatives (2, 7, 11 - 13) are grouped in subcluster 2a (Figure 4) and

277

are present in higher concentrations in hop samples of clusters C and D (up to 1.7%).

278

Lower concentrations of these sensometabolites are typically found in European hop

279

and aroma hop varieties grouped in clusters A, B and E, respectively. Although

280

xanthohumol (1) showed high abundance in the hard resin (0.3 - 2.6 %), no

281

significant correlation to the content of humulones (41) was observed.

282

Iso-α-acids

(43a/b/c,

44a/b/c),

humulinones

(45a/b/c),

the

iso-α-acid

283

degradation productcs 52a/b and 57a, desmethylxanthohumol (3), and 1′′,2′′-

284

dihydroxanthohumol K (10) were found in subcluster 2b. Similar to the bitter

285

compounds in subcluster 2a, the concentrations of these target molecules are higher

286

in hop cluster D and significantly lower in hop samples of clusters A, B and E,

287

respectively.

288

Compound cluster 3 comprises 4′-hydroxytunicatachalcon (6), xanthohumol I

289

(22), xanthohumol L (24), scorpiohumol (57b), and hulupinic acid (59) which are

290

typically found at higher concentrations in hop varieties grouped in cluster A. This

291

cluster contains mainly European varieties and also the American breed Vanguard,

292

whose parentage involved the German breed Hallertauer Mittelfrueh. Furthermore,


Page 13 of 41


293

the variety Tomahawk is found in this cluster, its genetic descent being rather

294

unclear.

295

The iso-α-acid degradation products 46b, 54a and 60a/c, 1-methoxy-4-

296

prenylphloroglucin (30), N-trans-feruloyltyramin (31), quercetin (32), p-coumaric acid

297

(38a/b), the xanthohumol derivatives 17, 18, 20, and 21, and the glycosides 33 -

298

36a/c are grouped in cluster 4. Next to the β-acids (42a/b/c; up to 7%), cluster 5

299

covers some xanthohumol derivatives (4, 5, 14), phloroisovalerophenon-3,5-Di-C-ß-

300

D-glucopyranoside (37b), and cis- and trans-p-coumaric acid methyl ester (39a/b).

301

Independent of the analyzed hop sample, a constant ratio of 1 : 2 to 1 : 3 was found

302

for 8-prenylnaringenin (4) to 6-prenylnaringenin (5), thus confirming earlier

303

observations.60 Compared to compound clusters 1-3, the sensometabolites in

304

clusters 4 and 5 are present in all hop varieties in comparable concentrations and do

305

not allow a clear clustering of hop varieties.

306

Affect of Cold-Storage on Concentrations of Bitter Compounds in Hops.

307

To gain insight into storage-induced alterations of the sensometabolome pattern of

308

hops, bitter compound analysis was performed in fresh and kilned hops (Hersbrucker

309

spaet), harvested in 2011, and hop pellets (Hersbrucker spaet), which were

310

harvested between 2003 and 2011 and kept at -20°C. Quantitative data were

311

normalized to their dry matter, determined by freeze-drying, and are shown in Table

312

S2 (Supporting Information). A hierarchical cluster analysis was performed for the

313

stored hop samples, and the results were visualized in a sensomics heatmap that

314

was combined with hierarchical agglomerative clustering of the sensometabolites 1 -

315

65 (Figure 5).

316

The hierarchical analysis arranged the sensometabolites into 4 large clusters,

317

that are labeled 1 - 4 in Figure 5, the second one consisting of 3 subclusters (2a -

318

2c). Cluster 1 consisted of the multifidol glycosides (36, 37) and the kaempferol-



319

malonyl-glycoside (35). Furthermore, p-cumaric acid (38), xanthohumol P (11) and

320

some isoxanthohumol derivatives (4, 5, 19) were found in this cluster. The

321

concentration of these compounds went through a maximum after the first and

322

second year of storage. Subcluster 2a contains hydroperoxylupones (62) and

323

desmethylxanthohumol (3) that were present in low concentrations and reached a

324

maximum concentration after 2 or 3 years of storage.

325

In comparison, subcluster 2b consisted only of the humulone congeners (41),

326

that increased in concentration during kilning and pelletizing and showed a slight

327

increase during the first 2 years of storage. Interestingly, the concentration of the

328

humulones (41) was found to be strongly increased during storage, while the

329

concentration of the lupulones (42) and other soft resin derived compounds seems to

330

be hardly affected. This might be explained by a release of humulones (41) from

331

glycosidically bound precursors during kilning as reported for other flavor compounds

332

in hops.73 While the concentrations of humulones (41) and lupulones (42) as well as

333

their degradation products (43, 45, 57, 58, 62) did not change during the first two

334

years of storage, humulones (41) and lupulones (42) decreased in concentration

335

upon further storage. The concentration of the degradation products 44, 46 - 56, 59 -

336

61, 63 - 65 was not influenced at all. Lupulone (42), N-trans-feruloyltyramine (31),

337

xanthohumol (1) and the xanthohumol derivatives 7, 13, 17, and 20 showed the

338

highest concentrations after 2 years of storage and were grouped in subcluster 2c.

339

Humolone and lupulone degradation products (43, 45b, 58, 59) that are

340

generated via an oxygen-independent pathway15-17 were found in cluster 3.

341

Furthermore, the flavonol glycosides 33 and 34 as well as two xanthohumol

342

degradation products 22 and 24 are grouped in the same cluster. Although a clear

343

distribution pattern is not visible within this cluster, the concentration of the flavonoid

344

glycosides (33 - 35) seem to decrease. Known to possess antioxidant potential,37,72


Page 14 of 41

Page 15 of 41


345

these flavonoids are probably degraded as a result of drying during the kilning

346

process. Interestingly, concentrations of flavonol glycosides (33 - 35) are declining

347

after more than 3 years of storage. Hydrolyses of the glycosidic bound can be

348

excluded since no increase was found for quercetin (32), the aglycon of glycoside 33.

349

The large cluster 4 summarizes most of the xanthohumol derivatives (6, 8, 10,

350

15, 16, 23, 25, 27), some isoxanthohumol derivatives (2, 9, 12) as well as compound

351

29, all of them have been recently discovered as key taste compounds in the hops

352

hard resin.26 In particular, oxidation products like 2′′,3′′-epoxyxanthohumol (27),

353

xanthohumol O (23) and 2′′,3′′-dehydrocyclohumulohydrochinon (29) were found to

354

rise steadily over time. Isoxanthohumol (2) shows a similar tendency, while amounts

355

of xanthohumol (1) are decreasing. Moreover, the cumulative amounts of the

356

degradation products cannot fully explain the loss of xanthohumol (1), thus indicating

357

an alternative degradation pathway for xanthohumol (1).

358

Affect of Oxygen on Hop Bitter Compounds. Using forced storage

359

conditions to accelerate natural aging,74,75 hop pellets of the variety Hersbrucker

360

spaet and Taurus, respectively, harvested in year 2011 were stored several weeks at

361

50°C in the presence of air and were then quantitatively analyzed for the bitter

362

compounds 1 – 65 (Table S3, see Supporting Information). Hierarchical cluster

363

analysis revealed two large clusters for the hop variety Hersbrucker spaet with the

364

second cluster devided into two subclusters (Figure 6).

365

The cluster 1 contains the α-acids (41), β-acids (42) and some xanthohumol

366

derivatives (1, 3 - 5) with xanthohumol (1) being the most predominant prenylated

367

flavonoid. As expected, the concentration of the humulones (41) and the lupulones

368

(42) dropped during aging, thus confirming earlier findings.74 Interestingly, only

369

hulupone (58b) and hulupinic acid (59) increased slightly during storage, whereas all

370

the other oxidation products (43 - 57, 60 - 65) found in beer15-17 were not detected in



Page 16 of 41

371

increasing amounts. These data indicate yet unknown transformation reactions of

372

humulones (41) and lupulones (42) in aerobic hop aging. In parallel to the decrease

373

in xanthohumol (1) concentration, the concentrations of its degradation products and

374

other hard resin derived compounds (2, 6, 13, 14, 16, 22 - 25, 27, 29, 30) decreased

375

strongly.

376

Xanthohumol

P

(11),

isoxanthohumol

H

(19),

xanthohumol N (20),

377

isoxanthohumol M (26), isoxantholupon (7), N-trans-feruloyltyramine (31) and

378

phloroisovalerophenon-3,5-di-C-β-D-glucopyranoside (37b), grouped in Cluster 2a,

379

are present in rather low concentrations in the unaged hop pellets. Although their

380

concentrations are slightly increasing during the aging process, those substances are

381

subject to a further degradation or transformation as their concentrations decrease

382

again. In comparison, cluster 2b features bitter compounds showing a significant

383

concentration decrease after the first week of storage, e.g. isoxanthohumol (2), 1′′,2′′-

384

dihydroxanthohumol C (8), 1′′,2′′-dihydroxanthohumol K (10), isoxanthohumol P (12),

385

5′-prenylxanthohumol (13), 1′′,2′′-dihydroxanthohumol F (14), xanthohumol D (15),

386

xanthohumol B (16), xanthohumol C (17), xanthohumol I (22), xanthohumol O (23),

387

xanthohumol L (24), and xanthohumol M (25). These compounds are oxidation and

388

transformation products of xanthohumol (1) formed during the accelerated aging

389

process. Furthermore all other sensometabolites of the hard resin are found in this

390

cluster: 4′-hydroxytunicatachalcone (6), dihydrocyclohumulohydrochinone (29), 1-

391

methoxy-4-prenylphloroglucin (30), cis-/trans-p-cumaric acid (38a/b), quercetin-3-O-

392

β-D-glucopyranoside (33), kaempferol-3-O-β-D-glucopyranoside (34), kaempferol-3-

393

O-β-D-(6′′-malonyl)-glucopyranoside

394

phloroglucinolglucopyranoside

395

phloroglucinolglucopyranoside (36c). Interestingly, the concentration of the flavonol

396

glycosides (33 - 35) is increasing during the first week of storage, before reaching a

(35), (36a),

1-O-β-D-(2-methyl-propanoyl)and


1-O-β-D-(2-methylbutyryl)-

Page 17 of 41


397

plateau. This observation has also been made during the first three years for the

398

samples aged without oxygen. However, it remains unclear why the concentrations of

399

these glycosides are increasing during storage.

400

The results of the forced aging experiment for the variety Taurus (Table S4;

401

Supporting Information) are visualized in Figure 7. As the variety Taurus contains

402

more soft and hard resin derived compounds than the variety Hersbrucker spaet,

403

quantitative changes are more pronounced and many parallels between the varieties

404

Taurus and Hersbrucker spaet can been detected during the forced aging

405

experiments. Therefore, similarities and differences will be examined at this point.

406

The heatmap for the variety Taurus (Figure 7) shows three clusters (1 - 3), of which

407

the last one can be divided into two subclusters (3a and 3b).

408

Cluster 1 contains desmethylxanthohumol (3) for which a concentration

409

decrease has been already described during aging for the breed Hersbrucker spaet.

410

Furthermore this cluster contains the compounds xanthohumol C (17), N-trans-

411

feruloyltyramine (31), co-multifidolglucoside (36a) and p-cumaric acid (38a/b).

412

Although, the concentrations of these substances are decreasing during the first

413

week, a constant concentration increase was observed afterwards as it was the case

414

for the aroma hop variety.

415

Cluster 2 comprises xanthohumol (1), some xanthohumol derivatives (7, 11,

416

12, 13, 15, 26), two glucosides (33, 36c), humulones (41), lupulones (42),

417

isohumulones (43, 44) as well as some degradation products of the isohumulones

418

(45) and lupulones (58, 61). Characteristic for those compounds is a significant

419

concentration decrease during the first week as found also for the variety

420

Hersbrucker spaet. In particular, the concentrations of the humulones (41), lupulones

421

(42) and cis-isohumulones (43) were significantly diminishing. The recently

422

discovered compound 7, that is present in the variety Taurus in larger amounts and



423

shows strong structural similarities to the lupulones (42),26 was found to be strongly

424

decreased as well during forced aging, thus suggesting a similar reaction mechanism

425

as for the lupulons (42). Again, no significant amounts of degradation or

426

transformation products (45 - 65) of humulones (41), lupulones (42) or the

427

isohumulones (43, 44) were detected, thus cleary indicating that molecular

428

transformation mechanisms of bitter compounds reported for beer might not apply to

429

hops. Furthermore, a concentration drop of xanthohumol (1) and a simultaneous rise

430

of almost all transformation and degradation products (6, 10, 14, 16, 22 - 24, 30)

431

were observed. Present in larger quantities in the variety Taurus, all other

432

xanthohumol derivatives (11, 13, 15, 18, 20, 21, 25) share the same fate as

433

xanthohumol (1) and are decreasing significantly.

434

Cluster 3a comprises the 6-prenylnaringenin (5), 1′′,2′′-dihydroxanthohumol C

435

(8), 1′′,2′′-dihydroisoxanthohumol C (9), 1′′,2′′-dihydroxanthohumol K (10), 1′′,2′′-

436

dihydroxanthohumol F (14), xanthohumol B (16), xanthohumol I (22), xanthohumol O

437

(23), xanthohumol L (24), kaempferol-3-O-β-D-glucopyranoside (34) and kaempferol-

438

3-O-β-D-(6′′-malonyl)-glucopyranoside (35). The concentration of these substances is

439

increasing during the forced aging experiments confirming the results obtained for

440

the variety Hersbrucker spaet. Cluster 3b shows compounds 2, 4, 6, 13, 20, 30, 37b,

441

43b, 57a, 58b and 59 whose concentrations increase during the first week as it was

442

also the case for the aroma hop variety. Afterwards the concentrions were

443

decreasing or reached a contstant value, respectively.

444

The data presented give a profund insight into molecular bitter compound

445

variability of commercially available hop varieties and might help to select appropriate

446

hop varieties for breeding programs in order to obtain offsprings that are better suited

447

for storage or contain larger amounts of desirable target compounds. Quantitative

448

analysis of hops stored with and without oxygen influence revealed that the decline of


Page 18 of 41

Page 19 of 41


449

distinct hop constituents can be not explained by an increase in transformation

450

products formed during beer aging, thus implying unkown degradation mechanisms

451

in hops. While the degradation of the soft resin-derived humulones (41) and

452

lupulones (42) seems not be influenced by oxygen, xanthohumol (1) is degraded to

453

give a series of transformation products recently reported as key bitter taste

454

compounds in hops.26 Therefore, further studies are necessary to elucidate the

455

aerobic transformation mechanisms of soft-resin components during hop aging.

456 457

Notes:

458

The Authors declare no competing financial interest.

459 460

Acknowledgement

461

We are grateful to Dr. Marc Rauschmann (Die Internationale Brau-Manufacturen

462

GmbH, Germany), Yvan Borremans (de 'proef'brouwerij, Belgium), Carlos Ruiz (HVG

463

Hopfenverwertungsgenossenschaft e.G, Germany), Dr. Christina Schönberger, Dr.

464

Elisabeth Wiesen (Joh. Barth & Sohn GmbH & Co. KG, Germany), Dr. Martin Biendl,

465

Sandro Cocuzza (Hopsteiner, Germany), Jack Teagle (SimplyHops, United Kingdom)

466

and Philippe Lefèvre (Yakima Chief, Belgium) for providing hop samples and to

467

Cynthia Almaguer (Chair of Brewing and Beverage Technology, Technische

468

Universität München, Germany) for providing unhopped beer.

469 470

Supporting Information Available

471

Quantitative data and a simplified hop pedigree. This material is available free of

472

charge via the Internet at http://pubs.acs.org.



473

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Deinzer,

M.L.;

Buhler,

D.R.,

In

vitro

632

biotransformation of xanthohumol, a flavonoid from hops (Humulus lupulus),

633

by rat liver microsomes. Drug Metab. Dispos. 2001, 29, 223-231.

634

59.

Nikolic, D.; Li, Y.; Chadwick, L.R.; Pauli, G.F.; van Breemen, R.B., Metabolism

635

of xanthohumol and isoxanthohumol, prenylated flavonoids from hops

636

(Humulus lupulus L.), by human liver microsomes. J. Mass Spectrom. 2005,

637

289 - 299.

638

60.

Stevens, J.F.; Taylor, A.W.; Deinzer, M.L., Quantitative analysis of

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xanthohumol and related prenylflavonoids in hops and beer by liquid

640

chromatography-tandem mass spectrometry. J. Chromatography A. 1999,

641

832, 97-107.

642

61.

Yakima Chief Inc. Hop varietal guide, 2012.

643

62.

Biendl, M.; Engelhard, B.; Forster, A.; Gahr, A.; Lutz, A.; Mitter, W.; Schmidt,

644

R.; Schönberger, C., Hopfen – Vom Anbau bis zum Bier. Fachverlag Hans

645

Carl. Nürnberg, 2012.

646

63.

Neve, R.A., Hops. Chapman & Hall, London. 1991.

647

64.

Seefelder, S.; Ehrmaier, H.; Schweizer, G.; Seigner, E., Genetic diversity and

648

phylogenetic relationship among accessions of hop, Humulus lupulus, as


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649

determined

650

compared with pedigree data. Plant Breeding. 2000, 119, 257-263.

651

65.

amplified

fragment

length

polymorphism

fingerprinting

Darby, P., The history of hop breeding and development. J. Brew. Hist. Soc. Online. 2005, 121, 94-112.

652 653

by

66.

Hieronymus, S., For the love of hops. The practical guide to aroma, bitterness and the culture of hops. Brewers Publications, Boulder, CA, USA, 2012.

654 655

67.

Hop Growers of America, Variety manual. Moxee, WA, USA. 2012.

656

68.

Lemmens,

G.W.C.,

The

breeding

and

parentage

657

http://brewerssupplygroup.com/FileContent/TheBreeding

658

_Varieties[1].pdf, downloaded 12.10.2012.

659

69.

of

hop

varieties.

Rohwer, C.; Fritz, V., Pedigrees of common hop varieties. University of

660

Minnesota. 2012, http://sroc.cfans.umn.edu/

661

People/Faculty/VinceFritz/Hops/index.htm, aufgerufen am: 13.10.2012.

662

70.

Personal communications: Dr. Seigner, Bavarian State Agency for Agriculture,

663

Freising; Dr. Beatson, The New Zealand Institute for Plant & Food Research

664

Ltd, Auckland, Neuseeland, to Hr. Lutz, Bavarian State Agency for Agriculture,

665

Freising; Dr. Darby, WYE Hops Ltd. Canterburry, Kent, UK; Dr. Biendl,

666

Hopsteiner, Mainburg; Dr. Whittock, Hop Products Australia, Barth-Haas-

667

Group, Bellerive, Australien

668

71.

Stevens, J.F.; Taylor, A.J.; Nickerson, G.B.; Ivancic, M.; Henning, J.; Haunold,

669

A.; Deinzer, M.L., Prenylfavonoid variation in Humulus lupulus: Distribution

670

and taxonomic significance of xanthogalenol and 4′-O-methylxanthohumol.

671

Phytochemistry. 2000, 53, 759-775.

672

72.

Cao, G.; Sofic, E.; Prior, R.L., Antioxidant and prooxidant behavior of

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flavonoids: Structure-activity relationships. Free Radical Biology & Medicine.

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1997, 22, 749–760.



675

73.

Kollmannsberger, H.; Biendl, M.; Nitz, S., Occurrence of glycosidically bound

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flavor compounds in hops, hop products and beer. BrewingScience. 2006, 59,

677

83-89.

678

74.

and changes upn beer brewin (in German). Brewing Science. 1985, 2, 86-96.

679 680

Narziss, L.; Scheller, L. On the bitter composition of hops and hop products

75.

Malfliet, S.; Van Opstaele, F.; De Clippeleer, J.; Syryn, E.; Goiris, K.; De

681

Cooman, L.; Aerts, G., Flavour instability of pale lager beers: determination of

682

analytical markers in relation to sensory ageing. J. Instit. Brew. 2008, 114, 180

683

- 192.

684 685


Page 28 of 41

Page 29 of 41


TABLES Table 1: Assignment of the single hop varietiesa to the clusters A - Eb

A-1 GB Challenger A-2 D-H Opal A-3 D-T Tettnanger A-4 US Tomahawk

A A-5 GB Goldings A-6 US Vanguard A-7 B Challenger A-8 B Tettnanger

B-1 US Super Galena B-2 D-H Tradition B-3 D-S Spalt B-4 CZ Saazer B-5 US Ahtanum B-6 US Glacier B-7 D-H Saphir

B B-8 B Hallertauer Mittelfrüh B-9 D-H Spalter Select B-10 US Bravo B-11 B East Kent Goldings B-12 US Cluster B-13 US Cascade B-14 US Willamette

C-1 US Chinook C-2 US Summit C-3 B Magnum C-4 US Millenium C-5 US Sterling

C C-10 US Centennial C-11 US Galena C-12 GB Sovereign C-13 US Mosaik C-14 SLO Celeila

C-6 GB Northdown

C-15 PL Marynka

C-7 US Simcoe C-8 US Zythos C-9 US Amarillo

C-16 D-H Merkur C-17 GB Phoenix C-18 AUS Pride of Ringwood

D-1 GB Pilgrim D-2 D-H Brewers Gold D-3 D-H Northern Brewer D-4 D-H Nugget D-5 D-H Taurus

D D-6 D-H Herkules D-7 US Zeus D-8 GB WGV D-9 US Columbus D-10 US Palisade

A-9 D-H Hallertauer Mittelfrüh A-10 F Strisselspalt

B-15 GB Bramling Cross B-16 GB Fuggles B-17 GB East Kent Goldings B-18 NZ Kohatu B-19 NZ Wai-iti B-20 D-H Smaragd B-21 SLO Styrian Goldings C-19 GB Admiral C-20 GB First Gold C-21 AUS Topaz C-22 AUS Galaxy C-23 NZ Stricklebract C-24 AUS Ella vormals „Stella“ C-25 NZ Nelson Sauvin

D-11 NZ Pacific Jade D-12 D-H Polaris D-13 NZ Pacific Gem D-14 US Sorachi Ace

E E-1 D-H Hallertau Blanc E-2 D-H Perle

686 687 688 689

E-3 D-H Magnum E-4 D-H Hersbrucker spät

E-5 D-H Mandarina Bavaria

a

Country abbreviations: GB – Great Britain, D-H – Germany (Hallertau), D-T – Germany (Tettnang), US – United States of America, B – Belgium (Poperinge), F – France, D-S – Germany (Spalt), CZ – a Czech Republic, NZ – New Zealand, SLO – Slovnia. PL – Poland, AUS – Australia. Numbering of the clusters referring to the letters given in Figure 4.



Page 30 of 41

Figure Captions 690

Figure 1: Chemical structures of key taste compounds of the hard resin of hops:

691

xanthohumol

(1),

692

prenylnaringenin (4), 6-prenylnaringenin (5), 4′-hydroxytunicatachalcone (6),

693

isoxantholupone (7), 1′′,2′′-dihydroxanthohumol C (8), 1′′,2′′-dihydroisoxanthohumol

694

C (9), 1′′,2′′-dihydroxanthohumol K (10), xanthohumol P (11), isoxanthohumol P

695

(12), 5′-prenylxanthohumol (13), 1′′,2′′-dihydroxanthohumol F (14), xanthohumol D

696

(15),

697

isoxanthohumol H (19), xanthohumol N (20), 2′′-hydroxy-xanthohumol M (21),

698

xanthohumol I (22), xanthohumol O (23), xanthohumol L (24), xanthohumol M (25),

699

isoxanthohumol M (26), 2′′,3′′-epoxyxanthohumol (27), xanthohumol G (28), 2′′,3′′-

700

dehydrocyclohumulohydrochinon (29), 1-methoxy-4-prenyl-phloroglucin (30), N-

701

trans-feruloyltyramine (31), quercetin (32), quercetin-3-O-ß-D-glucopyranoside (33),

702

kaempferol-3-O-ß-D-glucopyranoside

703

glucopyranoside (35), 1-O-ß-D-(2-methyl-propanoyl)-phloroglucinol-glucopyranoside

704

(co-multifidolglucoside)

705

glucopyranoside (ad-multifidolglucoside) (36c), phloroisovalerophenon-3,5-di-C-ß-

706

D-glucopyranoside

707

acid methyl ester (39a/b), cis-/trans-p-coumaric acid ethyl ester (40a/b).

xanthohumol

isoxanthohumol

B

(16),

(2),

xanthohumol

(36a),

(34),

desmethylxanthohumol

C

(17),

xanthohumol

(3),

H

8-

(18),

kaempferol-3-O-ß-D-(6′′-malonyl)-

1-O-ß-D-(2-methylbutyryl)-phloroglucinol-

(37b), cis-/trans-p-coumaric acid (38a/b), cis-/trans-p-coumaric

708

Figure 2: Chemical structures of α- and β-acids (41, 42) and their degradation products

709

(43 - 65): cohumulone (41a), humulone (41b), adhumulone (41c), colupulone (42a),

710

lupulone (42b), adlupulone (42c), cis-isocohumulone (43a), cis-isohumulone (43b),

711

cis-isoadhumulone (43c), trans-isocohumulone (44a), trans-isohumulone (44b),

712

trans-isoadhumulone (44c), cohumulinone (45a), humulinone (45b), adhumulinone

713

(45c), cis-cohumulinic acid (46a), cis-humulinic acid (46b), cis-adhumulinic acid

714

(46c), trans-cohumulinic acid (47a), trans-humulinic acid (47b), trans-adhumulinic 30

ACS Paragon Plus Environment

Page 31 of 41


alloisoadhumulone (49c), trans-alloisocohumulone (49a), trans-alloisohumulone

717

(49b),

718

hydroxy-cis-alloisohumulone (50b), hydroxy-cis-alloisoadhumulone (50c), hydroxy-

719

trans-alloisocohumulone (51a), hydroxy-trans-alloisohumulone (51b), hydroxy-

720

trans-alloisoadhumulone

721

hydroperoxy-cis-alloisohumulone (52b), hydroperoxy-cis-alloisoadhumulone (52c),

722

hydroperoxy-trans-alloisocohumulone

723

(53b),

724

tricyclohumol (54b), tricycloadhumol (54c), tricyclocohumene (55a), tricyclohumene

725

(55b), tricycloadhumene (55c), tricyclocohumulactol (56a), tricyclohumulactol (56b),

726

tricycloadhumulactol (56c), scorpiocohumol (57a), scorpiohumol (57b), cohulupone

727

(58a), hulupone (58b), adhulupone (58c), hulupinic acid (59), tetracyclocohumol

728

(60a), tetracyclohumol (60b), tetracycloadhumol (60c), hydroxytricyclocolupone

729

(61a),

730

hydroperoxytricyclocolupone

731

hydroperoxytricycloadlupone (62c), tricyclocolupone (63a), tricyclolupone (63b),

732

tricycloadlupone (63c), dehydrotricyclocolupone (64a), dehydrotricyclolupone (64b),

733

dehydrotricycloadlupone (64c), nortricyclocolupone (65a), nortricyclolupone (65b),

734

nortricycloadlupone (65c).

736

(49c),

(51c),

hydroxy-cis-alloisocohumulone

hydroperoxy-cis-alloisocohumulone

(53a),

hydroperoxy-trans-alloisoadhumulone

hydroxytricyclolupone

(48b),

cis-

716

trans-alloisoadhumulone

(48a),

cis-alloisohumulone

acid

735

(47c),

cis-alloisocohumulone

715

(61b), (62a),

(50a),

(52a),

hydroperoxy-trans-alloisohumulone (53c),

tricyclocohumol

hydroxytricycloadlupone hydroperoxytricyclolupone

(54a),

(61c), (62b),

Figure 3: RP-LC/MS-MS chromatograms of bitter tasting target molciles in hops. Chemical structures are given in Figure 1 and 2.

737

Figure 4: Sensomics heatmapping of bitter compound concentrations in different hop

738

varieties. Concentrations of compounds are given as Supporting Information. The

739

dendrogram is based on an agglomerative linkage algorithm.56 Chemical structures

740

of the individual compounds are given in Figures 1 and 2. 31



Page 32 of 41

741

Figure 5: Sensomics heatmapping of bitter compounds in fresh hops, kilned hops and hop

742

pellets of the variety Hersbrucker spaet stored in the absence of oxygen.

743

Concentrations of compounds are given as Supporting Information. The

744

dendrogram is based on an agglomerative linkage algorithm.56 Chemical structures

745

of the individual compounds are given in Figures 1 and 2.

746

Figure 6: Sensomics heatmapping of bitter compounds in fresh hops and pellets of the

747

variety Hersbrucker spaet stored under forced aging conditions. Concentrations of

748

compounds are given as Supporting Information. The dendrogram is based on an

749

agglomerative linkage algorithm.56 Chemical structures of the individual compounds

750

are given in Figures 1 and 2.

751

Figure 7: Sensomics heatmapping of bitter compounds in fresh hops and pellets of the

752

variety Taurus stored under forced aging conditions. Concentrations of compounds

753

are given as Supporting Information. The dendrogram is based on an agglomerative

754

linkage algorithm.56 Chemical structures of the individual compounds are given in

755

Figures 1 and 2.

32


Page 33 of 41


Figures Figure 1 (Dresel et al.)

R HO

OH

OH

1 R=

27 R =

O

OH

R HO

O

3 R1 = R3 = H, R2 =

R2 HO

O

OH

OH

2 R=

O

R1 O

15 R =

11 R =

O

OH

OH

28 R =

18 R =

HO

HO

OH

O

O

19 R =

12 R =

OH

R1

26 R =

HO

4 R2 = H, R1 =

O

O

O

20 R =

13 R3 = CH3, R1 = R2 =

OR3 O

O

25 R =

21 R =

R2

5 R1 = H, R2 =

OH O

HO

R OH O

O

OH

O

OH

8 R=H O

O

OH

O O

OH

16 R = OH O

OH

O

OH O

OH

O O

O

OH

O O

9

O

17

7

6

OH R1

10 R1 = R2 = H O

OH

O

HO

O

HO HO

HO O

R2

O O

O

24 R1 = OH, R2 = H

O

NH

HO

O

29

30

OH

OH HO

O

O O

OH OH O

OH

R

HO

31

OH O

23

OH

OH

HO

O

HO

O

22

OH

O O

OH

OH

14 R1 = H, R2 =

O

OH O

32

HO

OH

HO

33 R = OH 34 R = H

O O O

O

OH O HO

OH

OH

OH OH

OH

O

O

35

OH HO HO

R HO

O

HO

O OH

OH

OH

OH

36a =

HO HO

O OH

O

36c =

HO

O OH O OH

HO OH

HO

O

OH

38a R = H 39a R = CH3 40a R = CH2CH3

OH RO O

38b R = H 39b R = CH3 40b R = CH2CH3

37b

33



Page 34 of 41

Figure 2 (Dresel et al.) OH O

OH O

HO HO

O

HO

O

41

O

HO O

OH

O

O

O

OH OH

OH

O

OH OH

O

HO

54

HO

R OH O HO

HO

OH

60

OH

55

O

HO

O

61

OH OH

O

O

56

HOO

O

62

O

O

OH OOH

53

(a)

R

(b)

R

(c)

R

O

OH

O

O

HO R O

O

OH

59

58

O

63

OH

R O

H

O

O

OH

HO

R

47 O

OOH

57

HO

R

O

OH

R HO O

52 O

OH OH

O

OH

R

OH

O R

HO H

OH

R HO O

OH

R

OH

HO H

O

OH

O

R

O

46

O

O

51

O

O

O R

OH

R HO O

OH

R

O

45

O

50

49

O

HO O

44 O

OH

48

O

R

OH

R

R HO O

OH

HO O

OH

O

O

HO R

43

R HO O

HO O

O

O

O

R

42

O

O

O

R

R

O R O

64

HO

O

O

R O

65

34


Page 35 of 41


Figure 3 (Dresel et al.)

35



Page 36 of 41

Figure 4

36


Page 37 of 41


Figure 5

37



Page 38 of 41

Figure 6

38


Page 39 of 41


Figure 7

39



40


Page 40 of 41

Page 41 of 41


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The Bitter Chemodiversity of Hops (Humulus lupulus L.) - Journal of

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