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New insight into the role of sucrose in the generation of #-diketones upon coffee roasting Luigi Poisson, Noémie Auzanneau, Frédéric Mestdagh, Imre Blank, and Tomas Davidek J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04849 • Publication Date (Web): 25 Dec 2016 Downloaded from http://pubs.acs.org on December 27, 2016

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Journal of Agricultural and Food Chemistry

Original Research Article New Insight into the Role of Sucrose in the Generation of α-Diketones upon Coffee Roasting

Luigi Poissona, Noémie Auzanneaua, Frédéric Mestdagha, Imre Blankb and Tomas Davideka*

a

Nestlé Product Technology Centre Orbe, Nestec LTD., CH-1350 Orbe, Switzerland

b

Nestlé Research Centre, P.O. Box 44, CH-1000 Lausanne 26, Switzerland

*

Corresponding

author:

Phone:

+41244427342.

E-mail:

[email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

2

The origin and the formation pathways of the buttery smelling α-diketones 2,3-butanedione

3

and 2,3-pentanedione upon coffee roasting were studied by means of biomimetic in-bean

4

experiments combined with labeling experiments. For this purpose natural sucrose in the

5

coffee bean was replaced by fully or partially

6

unlabeled and fully

7

that sucrose contributes to both α-diketones, however its importance and reaction

8

pathways clearly differ. Whereas the major part of 2,3-pentanedione originates from

9

sucrose (about 76%), its contribution to 2,3-butanedione is much lower (about 35%).

10

Formation from intact sugar skeleton is the major pathway generating 2,3-pentanedione

11

from sucrose, while 2,3-butanedione is mainly generated by recombination of sucrose

12

fragments. The contribution of glucose and fructose moieties of sucrose to both α-

13

diketones is comparable. Finally, kinetic experiments with fully labeled sucrose showed that

14

contribution of sucrose changes during roasting.

13

13

C labeled sucrose or by a mixture of

C labeled sucrose (CAMOLA approach). The obtained data point out

15 16 17

Keywords. Coffee, flavor, precursor, sucrose, CAMOLA, roasting.

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INTRODUCTION

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The delightful aroma and taste of coffee is developed during coffee roasting at

20

temperatures higher than 200 °C, and it is generally accepted that the coffee’s intrinsic

21

quality is predetermined in the green bean by its precursor composition.

22

The main constituents in green coffee are carbohydrates, nitrogen-containing

23

compounds (mainly proteins, trigonelline and caffeine), lipids, organic acids, and water.1

24

The carbohydrates represent about half of the dry basis of green coffee beans.2,

25

main part of insoluble fraction form the structure of the cell walls consisting of cellulose,

26

galactomannans and arabinogalactan along with proteins and chlorogenic acids, all of

27

them showing complex structures.4 Nevertheless, it is the water soluble coffee fraction that

28

is considered as the more important precursor pool. Particularly the low molecular weight

29

constituents, comprising free sugars, amino acids, trigonelline and chlorogenic acids,4-6

30

rapidly degrade at the early stage of roasting, and instantly participate in manifold

31

reactions.7 Free sugars are almost exclusively represented by sucrose with about 8% of

32

dry matter in Arabica and 3-6% in Robusta. Its fast hydrolysis at the beginning of the

33

roasting process releases the reducing saccharides glucose and fructose, which hereupon

34

are strongly involved in caramelization and Maillard-type reactions. Arabinose was also

35

discussed as potential precursor, released from the arabinogalactans during roasting.7-10

36

Other free sugars like galactose, mannose or glucose are only present in trace amounts.

3

The

37

A large number of model studies were conducted to understand the mechanism

38

underlying the formation of different key coffee odorants such as thiols, α-diketones,

39

furanones and pyrazines under dry heating conditions.11-16 For example several

40

mechanism were proposed to explain formation of buttery smelling 2,3-butanedione and

41

2,3-pentanedione which were recently shown to render the harsh roasty / sulfury note of

42

2-furfurylthiol to pleasant coffee / mocha note.17 These mechanisms includes generation of 3 ACS Paragon Plus Environment


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2,3-butanedione from intact C4 hexose backbone, recombination of C1/C3 (formaldehyde

44

and 1-hydroxy-2-propanone) and C2/C2 (acetaldehyde and glycolaldehyde) sugar

45

fragments, and glycine mediated chain elongation of glyoxal and methylglyoxal.18-22

46

Similarly several mechanism were proposed to explain the formation of 2,3-

47

pentanedione. These includes recombination of C1/C4 sugar fragments (e.g. formaldehyde

48

and 2,3-butanedione), recombination of C2/C3 sugar fragments (e.g. acetaldehyde and 1-

49

hydroxy-2-propanone) or alanine mediated chain elongation of methylglyoxal. 15, 18, 23

50

In general, the number of precursors in model systems is strongly limited to reduce the

51

complexity. Hence, such systems cannot reproduce the chemical and physical

52

transformations of the coffee beans during roasting. To study the formation pathways of

53

coffee aroma compounds under more real conditions, the so-called biomimetic in-bean

54

experiments were developed, where the coffee bean itself is used as a pressurized

55

reaction vessel.24-26 The results revealed amongst others an important role of the soluble

56

saccharides in the formation of α-diketones, while free amino acids played only a minor

57

role. In addition, different formation pathways leading to 2,3-butanedione and 2,3-

58

pentanedione were highlighted by employing labeled precursors.26 The in-bean approach

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was also successfully applied to study the mechanism of coffee melanoidin formation,6

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and to investigate the presence and nature of thiol binding sites in raw coffee beans.24, 27

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Kinetic experiments applying time resolved sampling during roasting28 as well as on-line

62

mass spectrometric techniques such as proton-transfer-reaction mass spectrometry (PTR-

63

MS) or photon ionization mass-spectrometry were another approaches used to study

64

coffee aroma formation upon highly dynamic coffee roasting process.29-32

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Despite the significant effort devoted, the generation of aroma active compounds upon

66

coffee roasting is still not fully understood. The in-bean experiments were proven to be

67

very useful in providing insights into formation pathways, however the former study of


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Poisson et al.26 revealed also their limitations. Among others, the high cost of

69

precursors and relatively large batch size (125 g) enabled only partial replacement (17%)

70

of natural sucrose by [UL-13C6-fructose]sucrose. This consequently rendered the data

71

interpretation rather complex. In addition, the biomimetic reconstitution of the water

72

extractable fraction is still a challenge, and the incorporation efficiency of different

73

precursor classes is yet not sufficiently understood.

C labeled

74

Therefore, the present study aimed at substantiating the role of sucrose in the formation

75

of α-diketones upon coffee roasting by applying an improved extraction and incorporation

76

protocol combined with a down-sized roasting step. This allowed for an entire replacement

77

of the natural sucrose by fully or partially

78

with labeled sucrose and for CAMOLA study.

13

C labeled analogs, but also for kinetic studies

79 80 81 82 83

MATERIALS AND METHODS Raw material. Green coffee beans (Coffea arabica, Costa Rica, La Giorgia, wet processed) were used. Chemicals. The following chemicals were commercially available: Caffeine (99.5%),

84

copper(II) sulfate (99%), dichloromethane (99.8%), D-(-)-arabinose (99%),

85

galactose (99%), L-rhamnose (99%), D-(+)-mannose (99%), D-(+)-sucrose (99.5%), D-(-)-

86

quinic acid (98%), ethanol (99.5%), iron(III) chloride (97%), L-alanine (99.5%), L-arginine

87

(99.5%), L-asparagine (98.5%), L-aspartic acid (99.5%), L-cysteine (99.5%), L-glutamic

88

acid (99.5%), L-glutamine (99.9%), glycine (98.5%), L-histidine (99.5%), L-isoleucine

89

(99.5%), L-(+)-lactic acid (99%), L-leucine (99.5%), L-lysine (98%), L-methionine (99.5%),

90

L-phenylalanine (98%), L-proline (99.5%), L-serine (98.5%), L-threonine (98.5%), L-

91

tyrosine

92

manganese(II) chloride (98%), potassium acetate (99%), potassium hydroxide (85%),

(99%),

L-tryptophane

(99.5%),

L-valine

(99.5%),

malic

acid

D-(+)-

(99.5%),



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trigonelline hydrochloride (98%), zinc(II) sulfate heptahydrate (99%), chlorogenic acid

94

(95%), potassium citrate (99%) (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland), [UL-

95

13

96

[UL-13C5]arabinose

97

Biochemicals, Inc., IN, USA).

C6-fructose]sucrose (98%), [UL-13C6-glucose]sucrose (98%), [UL-13C12]sucrose (98%), D(98%),

D-[UL-13C6]galactose,

D-[UL-13C6]mannose

(Omicron

98

Water extraction of green coffee beans. Green coffee beans were extracted with hot

99

water applying following conditions: Ten kilograms of green coffee beans were mixed with

100

20 L of water in a Scanima Batch Mixer (Tetra Pak Scanima, Denmark) and heated to 60

101

°C for 1 h. The obtained extract was drained, and the coffee beans were extracted another

102

four times with 20 L of demineralized water at 60 °C for a total of 4 h to obtain the water

103

soluble substances (total 100 L of extract). The resulting extraction yield was found at

104

20.5% (23.2% on dry matter base). In a second step the exhausted green coffee beans

105

were dried in the Scanima mixer by increasing the temperature of the double-jacket to 110

106

°C and applying a vacuum of 150 mbar. During the first 2 h of drying the product

107

temperature raised from 60 °C to 80 °C, then the final temperature was held constant for

108

another 4 h (total 6 h drying time). In order to prevent bean breakage the agitator/mixer

109

was regulated at lowest possible rotation speed (24 rpm agitator, 100 rpm mixer). The

110

obtained green coffee extracts were combined and freeze-dried in a Lyobeta 35 freeze-

111

dryer (Telstar, Terrassa, Spain) and stored at -40 °C until use.

112

Incorporation of biomimetically recombined extract (Reference sample, BREB).

113

For the preparation of the biomimetically recombined extract (BRE; based on the

114

composition of water extract of green coffee beans, Table 1) the single components (see

115

Table 2) were dissolved in 2 g of demineralized water at 80 °C. The pH value of BRE

116

was adjusted to 5.5 (corresponding to the pH of the natural extract) with a 16.5% w/w

117

solution of KOH and water exhausted green coffee beans (EB, 5 g) were soaked with the


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BRE at 50 °C for 5 h and then overnight at room temperature. During soaking, the beans

119

were gently stirred using a Rotavapor (Büchi, Switzerland). To improve the incorporation of

120

BRE into EB, 1 ml of demineralized water was added into incorporated coffee beans and

121

absorbed using a Rotavapor (gentle stirring for 1 h at 50 °C then 5 h at room temperature).

122

After water absorption the treated beans were washed with the same mass of water as

123

beans during 10 seconds. The washing losses were controlled by analyzing the washing

124

waters with an ATAGO PAL3 pocket refractometer, which measures total solid content on

125

a Brix scale. The washed coffee beans were frozen to -80 °C, then freeze-dried for 24

126

hours at 0.1 mbar and about -80 °C on an Alpha 2-4 LSC freeze-dryer (Christ, Germany)

127

to reach a moisture content of 10 ± 0.5% (experiment 2 in

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Table 3). Finally the beans were roasted using procedure described below.

131

Labeling experiments. Similar procedure was applied to prepare green coffee beans

132

spiked with labeled precursors by replacing one or several free sugars in BRE with their

133

labeled analogs. The performed experiments are summarized in

134


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Table 3.

137

Labeled sucrose. The BRE was omitted in all sugars and spiked with [UL-13C12]sucrose

138

(500.6 mg; experiment 3), [UL-13C6-fructose]sucrose (500.6 mg; experiment 4), or [UL-

139

13

140

spiking level corresponded to the naturally occurring total sucrose content.

C6-glucose]sucrose (500.6 mg; experiment 5) prior its incorporation into 5 g of EB. The

141

Other labeled sugars. The BRE was omitted in all sugars except sucrose and spiked with

142

D-[UL-13C5]arabinose (0.4 mg), D-[UL-13C6]galactose (7.9 mg), and D-[UL-13C6]mannose

143

(1.2 mg) prior its incorporation into 5 g of EB (experiment 6).

144 145

Kinetic study. For the kinetic study with [UL-13C12]sucrose the setup of experiment 3 was scaled up by factor 6 (3004 mg [UL-13C12]sucrose per 30 g EB; experiment 8).

146

CAMOLA experiments. In order to reach a ratio of 1 : 1 of unlabeled and labeled

147

sucrose, the level of unlabeled sucrose in BRE was adjusted to compensate for the

148

residual level of sucrose in EB (49.9 mg per 5 g EB). Consequently, the BRE omitted in all

149

sugars was spiked with [UL-13C12]sucrose (250.3 mg) and unlabeled sucrose (200.4 mg)

150

prior its incorporation into 5 g of EB (experiment 7).

151

Laboratory roasting trials. A standard roasting profile assuring the same roasting

152

conditions (without considering the final color) was defined and applied to all coffee

153

samples. Consequently, the same thermal energy was provided to coffee beans, which

154

allowed for an appropriate comparison among all samples. Temperature and air flow

155

profile were optimized for the roasting of 15 g of green coffee. This profile led to a CTN

156

(color test number, Neuhaus-Neotec, Germany) of 86 for untreated coffee beans

157

(experiment 1 in

158



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Table 3). After freeze-drying 5 g of reincorporated green beans were counted, and

161

then mixed with 10 g of non-treated coffee beans to increase the batch size to a

162

critical amount needed for the roasting using a sample roaster.

163

batches (15 g) were roasted under same conditions on an IKAWA (Ikawa, London,

164

UK) sample roaster for 300 s (experiment 1 to 7) or for 330 s (experiment 8) until 203

165

°C. The roasting profiles are given in

166

Table 4 and

The individual

167

Table 5. The color of the roasted coffee samples was not measured. After roasting,

168

darker colored beans (reconstituted beans) were sorted out from the bulk beans (lighter

169

color) and counted to control their number against the initial number of provided beans.

170

Free sugar analysis. 2 g of cryo-ground (Kryomill, Retsch, Germany) green beans or

171

water extracted beans (EB), or 0.5 g of green coffee extract powder were weighed in a 20

172

mL volumetric flask. After addition of MilliQ water (Millipore, Zug, Switzerland), the slurries

173

were incubated for 1 h in a water bath at 100 °C, followed by centrifugation for 5 min at

174

10’000 G and filtration through a 0.2 µm filter (VWR International, Dietikon, Switzerland).

175

Further sample clean-up was done by passing 3 mL extract through a C18 cartridge (Sep-

176

Pak C18, Waters, Montreux-Chailly, Switzerland), which was previously conditioned with 2

177

mL methanol and 3 mL MilliQ water. Sugars were separated using high-performance

178

anion-exchange chromatography (10 µL injection) on a PA-100 column (ThermoFisher

179

Scientific, Ecublens, Switzerland), using the ICS-5000 system (ThermoFisher Scientific)

180

with a constant flow of 1 mL/min and following gradient: 100% eluent A (MilliQ water) until

181

55 min; at 65 min: 75% eluent A and 25% eluent B (1 M NaOH); at 70 min 50% eluent A

182

kept until 80 min; final equilibration at 100% eluent A until 95 min. A flow of 0.5 mL/min 0.3


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M NaOH was added post-column before the amperometric detector. Sugars were

184

quantified using external calibration curves.

185

Total coffee polyphenols. Total coffee polyphenols were measured using Folin-

186

Ciocalteu's phenol reagent by colorimetric detection according to the methodology

187

described by Georgé et al.33

188

Free amino acids. Free amino acids were analyzed after aqueous extraction of green

189

coffee samples by GC-MS using the Phenomenex EZfaast kit (Brechbuehler, Echallens,

190

Switzerland). Samples preparation was performed as follows: About 3.3 g of cryo-ground

191

green coffee beans or water extracted beans, or corresponding amount of green coffee

192

extract powder (0.75 g) were exactly weighed into a 50 ml volumetric flask and filled up

193

with water (Milli-Q). The extraction was performed for 1 hour during continuous stirring at

194

ambient temperature. After settling down of the solids, the supernatant was filtered using a

195

0.2 µm pore size syringe filter (SRI), and the amino acids were derivatized. The

196

derivatization was carried out according to the manufacturers’ manual (EZ:faast for free

197

amino acid analysis, Phenomenex). Therefore a defined amount of the sample extract

198

(100 µl; 50 µl for the abundant amino acids) was spiked with an internal standard solution

199

(norvaline, c = 200 nmol/l). The derivatization was followed by liquid injection and GC-MS

200

analysis. The standard solutions for the calibration curves (concentrations equivalent to

201

20-300 nmol/l) were spiked with 100 µl of internal standard solution and derivatized in the

202

same way as the samples. The sample (1 µl) was provided to the GC column by liquid

203

injection on split-splitless injector (in split mode; split of 60) at 250 °C. Separation was

204

performed on a 10 m x 0.25 mm Phenomenex Zebron-AAA column, (Brechbuehler,

205

Echallens, Switzerland) using an Agilent 7890A gas chromatograph (Agilent, Basel,

206

Switzerland). Helium was used as carrier gas with a constant flow of 3 mL/ min. Following

207

oven program was applied: initial temperature of 110 °C (0 min), then raised to 320 °C at



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208

32 °C/min, and final temperature held for 6.5 min. Mass spectrometry was performed on

209

an inert MSD 5975C quadrupole mass spectrometer (Agilent, Basel, Switzerland). Electron

210

impact ionization was applied, and the mass spectrometer was operated in the single ion

211

monitoring mode. Chromatograms were processed using the Agilent MassHunter

212

software.

213 214 215 216

Caffeine & trigonelline. Caffeine and trigonelline content were determined after aqueous extraction by HPLC-UV similar to Casal et al.34 Total fat. Total fat content was determined by the Weibull-Berntrop method based on ISO method 8262, involving a treatment with boiling hydrochloric acid.35

217

Organic acids. Organic acids were extracted from cryoground beans with water at 70

218

°C for 30 min. After filtration and C18 solid phase extraction (Sep Pak, Waters WAT

219

020515, Waters, Montreux-Chailly, Switzerland), the acids were analysed by high-

220

performance anion-exchange chromatography-conductometry using a Dionex IonPac

221

AS11-HC column (Thermo Fisher Scientific, Reinach, Switzerland).

222 223 224 225

Ash. Total ash content was determined after destruction of the organic matter at 550 °C in a muffle furnace, according to AOAC International method 920.93.36 Metals. Metal content was determined by ICP-OES according to AOAC International method 984.27.36.

226

Aroma Analysis by SPME-GC-MS. Roast and ground (R&G) coffee (0.5 g) was added

227

into a 20 mL headspace vial, and the sample was equilibrated for 10 min at 40 °C. Aroma

228

compounds were then extracted from the headspace by solid phase microextraction

229

(SPME) at 40 °C during 10 min (2 cm fiber, 50/30 µm StableFlex, coated with

230

PDMS/DVB/Carboxen; Supelco, Buchs, Switzerland), and thermally desorbed into the

231

split-splitless injector (in split-mode; split of 2) heated at 240 °C for 10 min. Separation was

232

carried out on a 60 m × 0.25 mm × 0.25 µm polar DB-624 column (Phenomenex,


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Brechbühler, Switzerland) using an Agilent 7890B gas chromatograph (Agilent, Basel,

234

Switzerland). Helium was used as carrier gas with a constant flow of 1.2 mL/ min.

235

Following oven program was applied: initial temperature of 40 °C was held for 2 min, then

236

raised to 240 °C at 5 °C/min, and final temperature held for 10 min. Mass spectrometry

237

was performed on a 7200 accurate mass Q-TOF mass spectrometer (Agilent, Basel,

238

Switzerland). Electron impact ionization was applied, and the mass spectrometer was

239

operated in the full scan mode (m/z 30-250) at a spectra acquisition rate of 5 spectra/s.

240

Chromatograms were processed using the Agilent MassHunter software. All results were

241

corrected for the

242

correction lower than 0.5% was set to 0% by definition.

13

C content of the natural isotope. The obtained percentage after

243 244

RESULTS AND DISCUSSION

245

Preparation of exhausted beans and incorporation of bio-mimetic recombinant. In

246

order to optimize the protocol for the water-extraction and the bio-mimetic recombinant

247

incorporation some modifications were developed and applied in the present study as

248

compared to the former one.26 Applying a hot water extraction temperature of 95 °C may

249

force the dissolution of non-soluble components into the green coffee extract. Therefore,

250

the extraction was completely revised in order to apply a moderate extraction temperature

251

of only 60 °C. Surprisingly, the extraction yield at 60 °C was not lower as compared to hot

252

extraction at 95 °C (20.5% vs. 19.4% soluble solids). Based on dry matter, the yields

253

reached 23.2% for the water extract and 76.8% for the exhausted coffee beans. The water

254

content was found to be 8.5% for the dried, exhausted beans and 3.0% for the freeze-

255

dried water extract, respectively.

256

The efficiency of the incorporation of the bio-mimetic recombinant into exhausted bean (1st

257

soaking step) could be significantly improved by introduction of a 2nd soaking step with



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water only, followed by rapid washing of the beans with water. The additional water

259

soaking seems to transport the precursors that were not entirely incorporated from the

260

surface towards the inner core of the bean. The washing procedure after water soaking

261

step aimed at removing the residual precursors from the bean surface. In average the

262

precursor losses were determined at 11%. The performed biomimetic in-bean as well as

263

spiking experiments are summarized in

264

Table 3.

265

Miniaturization of the roasting experiments. To reduce the need of the expensive

266

labeled precursors, the roasting experiments were down scaled as compared to previous

267

study.26 The employment of the small size IKAWA® fluidized bed roaster allowed to

268

decrease the roasting batch size to only 15 g of green coffee beans as compared to a

269

batch size of 125 g used in the previous study.26 The amount of the reincorporated coffee

270

beans could be decreased even further to only 5 g by blending of the reincorporated beans

271

with 10 g of the original non-treated green coffee beans. After roasting, the reincorporated

272

beans could be separated from non-treated beans, based on their darker color and darker

273

silver skin. In order to avoid inaccuracies, the incorporated beans were counted before and

274

after roasting.

275

Labeling Experiments. The total amount of sucrose in the green coffee beans was

276

determined at 8.6 g/100 g DM green beans. After water extraction the residual sucrose

277

accounted for 0.8 g/100 g DM exhausted beans, hence about 9% of the original level. For

278

the labeling experiment the total content of sucrose present in green coffee was replaced

279

by partially or fully labeled sucrose. Considering the residual sucrose level, the labeled

280

sucrose represented about 93% of the total sucrose level in the beans.


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281

Experiments with [UL-13C12]sucrose. The mass spectrum of 2,3-butanedione generated

282

in roasted coffee bean containing [UL-13C12]sucrose is shown in Figure 1 and the relative

283

distribution of the isotopologues in



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284 285

Table 6. The non-labeled isotopologue (m/z 86) represented the majority of 2,3-

286

butanedione formed (74%). The labeled isotopologues were formed in much lower

287

quantities. Apart of the fully labeled isotopologue ([M+4]+, m/z 90) formed at 14%, small

288

amounts of singly, doubly and triply labeled isotopologues were detected.

289

In contrast, the majority of 2,3-pentanedione (63%) was found labeled (Figure 2 and

290


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291 292

Table 7). The fully labeled isotopologue ([M+5]+, m/z 105) constituted the major part

293

(45%), followed by unlabeled (m/z 100; 37%) and triply labeled isotopologue ([M+3]+, m/z

294

103, 10%). Small amounts of singly, doubly and 4-fold labeled isotopologue were also

295

detected.

296

The detailed analysis of the mass spectrum of 2,3-pentanedione revealed that singly

297

labeled isotopologue integrated labeled carbon mainly in the propionyl residue as indicated

298

by the fragment m/z 58 corresponding to the fragments m/z 57 and m/z 60 of the

299

unlabeled and fully labeled isotopologue, respectively (Figure 2). The formation of such

300

isotopologue could be explained by the mechanism involving recombination of C1 and C4

301

sugar fragments as proposed by Weenen.18 On the other hand, the doubly labeled

302

isotopologue integrated labeled carbons mainly in acetyl residue, as only traces of the

303

fragment m/z 59 corresponding to doubly labeled propionyl residue were detected. These

304

data support the formation mechanism by recombination of C3 and C2 sugar fragments as

305

proposed by Hofmann.23

306

The presence of the partially labeled isotopologues of both α-diketones indicates that at

307

least part of α-diketones is formed by recombination of sucrose fragments. The small level

308

of the unlabeled residual sucrose present in exhausted beans, prevents to conclude

309

whether the partially labeled isotopologues were formed from sucrose fragments only or by

310

recombination of sucrose fragments with other fragments originated from coffee matrix.

311

Summarized, sucrose contributes to the formation of both studied α-diketones, however

312

its importance is not the same. Whereas sucrose is a major precursor of 2,3-

313

pentanedione, its contribution to 2,3-butanedione is only moderate. The results clearly

314

indicate that other green coffee constituents such as non-water soluble polysaccharides

315

play much more important role in the formation of 2,3-butanedione than the free sucrose. 17 ACS Paragon Plus Environment


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316

Experiments with [UL-13C6 - glucose]sucrose or [UL-13C6 - fructose]sucrose. The

317

contribution of the individual sugar moieties of sucrose (fructose and glucose) to the

318

formation of α-diketones was evaluated by replacing the natural sucrose content by either

319

[13C6]-glucose moiety labeled or [13C6]-fructose moiety labeled analog.

320 321

The isotopologue patterns determined from the cluster of the molecular ions of the evaluated α-diketones are given in


Page 19 of 54


322 323

Table 6 and

324



Page 20 of 54

325 326

Table 7. The labeled glucose moiety of sucrose generated 8% of fully labeled 2,3-

327

butanedione and 21% of fully labeled 2,3-pentanedione. Similarly, the labeled fructose

328

moiety generated 6% and 23% of fully labeled 2,3-butanedione and 2,3-pentanedione,

329

respectively. The sum of the fully labeled isotopologues obtained from the experiment

330

employing partially labeled sucrose analogs nearly corresponds to the level of these

331

isotopologues measured in the experiments with fully labeled sucrose (see above). In

332

addition, the experiments with partially labeled sucrose analogs showed similar isotopic

333

pattern. This indicates that glucose and fructose moiety not only contribute to the same

334

degree to the formation of both α-diketones, but also that their formation pathways from

335

both sugar moieties after cleavage of the disaccharide are similar.

336

The newly obtained results differ to those obtained in our former study, which suggested

337

that 2,3-butanedione is not formed from fructose moiety whereas 2.3-pentanedione is

338

formed mainly from this moiety.26 Due to much higher batch size of roasting experiments,

339

the former study permitted only partial replacement (16%) of sucrose by a labeled analog

340

([UL-13C6 - Frc]-sucrose). Consequently the data interpretation was rather challenging as

341

partial spiking generated only low levels of labeled compounds leading to the relatively low

342

signal-to-noise ratio of the labeled compounds. In addition, the various improvements to

343

the bean treatment protocols applied in this new study probably provided more accurate

344

results.

345

Experiments with [UL-13C6]-mannose, [UL-13C6]-galactose, [UL-13C6]-arabinose. Sucrose

346

is by far the most abundant free sugar in both Arabica and Robusta coffee species,

347

however it is not the only one. Therefore, the role of the other free sugars namely

348

arabinose, galactose and mannose was assessed by replacing the corresponding


Page 21 of 54


349

amounts of unlabeled sugars by their fully labeled analogs. The results revealed practically

350

no label incorporation into the assessed α-diketones (



Page 22 of 54

351 352

Table 6 and

353


Page 23 of 54


354 355

Table 7). Their isotopologue patterns were similar to the ones obtained in experiments

356

with BREB and contained only a non-significant amount (less than 1%) of doubly labeled

357

isotopologue. Hence, a significant contribution of free arabinose, galactose or mannose in

358

the formation of 2,3-butanedione and 2,3-pentanedione can be excluded. Their amounts

359

are probably just too low to influence the overall balance of different reactions occurring in

360

parallel.

361

This finding underpins the particular role of sucrose in the formation of α-diketones, but

362

also of other sugar sources, namely non-water soluble polysaccharides that seem to play

363

a more important role than expected.

364

CAMOLA experiments with sucrose. Based on the model systems several pathways

365

involving either intact sugar skeleton or recombination of sugar fragments have been

366

reported to explain the formation of 2,3-butanedione and 2,3-pentanedione.15,

367

However the results obtained from simplified model systems do not always reflect the

368

reality of the complex food system. Therefore, to gain deeper insight into reaction

369

mechanisms responsible for the formation of both α-diketones from sucrose upon coffee

370

roasting, the CAMOLA technique was employed.19 A mix of unlabeled and fully carbon

371

labeled sucrose ([UL-13C12]-sucrose) was applied into the recombinant and soaked into the

372

beans. Since the exhausted green coffee beans contained about 9% of residual sucrose,

373

the ratio of non-labeled sucrose in the CAMOLA mix was adjusted in such a way that an

374

exact 1:1 mix of non-labeled and labeled sucrose was obtained.

18, 19, 21, 23

375

2,3-Butanedione. The isotopologue distribution obtained for 2,3-butanedione at the end

376

of the CAMOLA experiment served to evaluate the relative contribution of different reaction

377

pathways. The interpretation of the obtained pattern is summarized in Table 8.



Page 24 of 54

378

The presence of singly labeled isotopologue (m/z 87) can only be explained by

379

recombination of a labeled C1 fragment with a non-labeled C3 fragment. Considering only

380

sucrose as key precursor such a C1/C3 recombination must also generate the triply labeled

381

(m/z 89), fully labeled (m/z 90) and unlabeled isotopologues (m/z 86). Statistically all these

382

isotopologues have to be formed at same level (i.e. at 3.6%). Consequently, the

383

recombination of C1 with C3 sucrose fragments, e.g. the aldol condensation of

384

formaldehyde with 1-hydroxy-2-propanone as proposed by Schieberle et.al.

385

to 14.4% of the total 2,3-butanedione formed (Figure 3., pathway a).

19

contributes

386

The recombination of two C2 fragments (aldol condensation of acetaldehyde with

387

hydroxyacetaldehyde) is another pathway that has been reported to contribute to 2,3-

388

butanedione in model systems.19 The doubly labeled isotopologue (m/z 88) clearly

389

indicates that this pathway is also active under coffee roasting conditions. Statistically this

390

isotopologue is formed in double amount as compared to unlabeled (m/z 86) and fully

391

labeled (m/z 90) isotopologues. As the doubly labeled isotopologue was formed at 5.6% it

392

can be concluded that C2/C2 recombination contributes to 11.2% of the total 2,3-

393

butanedione formed (Figure 3., pathway b). In reality the generation of 2,3-butanedione by

394

recombination of sucrose fragments (C1/C3 and C2/C2) may be a bit lower than shown in

395

Table 8 as the singly labeled isotopologue (m/z 87) could be also formed by recombination

396

of a labeled C1 fragment from sucrose with a non-labeled C3 fragment from other

397

precursors in the coffee matrix and the doubly labeled isotopologue (m/z 88) by

398

combination of a labeled C2 fragment from sucrose with a non-labeled C2 fragment from

399

the coffee matrix. However the obtained data do not allow more detailed calculations

400

unless speculation is done.

401

The remaining part of the triply labeled isotopologue (m/z 89), that cannot be explained

402

by C1/C3 recombination of sucrose fragments (6.2% - 3.6% = 2.6%) can only originate


Page 25 of 54


403

from a C3 labeled fragment of sucrose and an unlabeled C1 fragment, arisen from another

404

component present in the coffee matrix. This pathway must also form unlabeled

405

isotopologue at the same level and consequently contributes to 5.2% of the total 2,3-

406

butanedione formed (Figure 3., pathway c).

407

Similarly the remaining part of the fully labeled isotopologue (m/z 90) that does not stem

408

from a C1/C3 or C2/C2 recombination (8.6% - 3.6% - 2.8% = 2.2%) must be formed from

409

the intact skeleton of labeled sucrose, e.g. by the retro-aldol reaction of 1,4-

410

dideoxyhexosone, or from an isomerization product of 1-deoxyglycosone.18 As this

411

pathway forms only two isotopologues (unlabeled and fully labeled) in equal levels, it

412

contributes to 4.4% of the total 2,3-butanedione formed (Figure 3., pathway d).

413

The above described reaction pathways involving sucrose can explain only 11.2% of the

414

unlabeled isotopologue which was the major isotopologue formed at 76%. Consequently

415

the remaining 64.8% had to be formed form other green coffee constituents than sucrose.

416

These other constituents, most probably non-water-soluble polysaccharides, thus play a

417

much more important role in formation of 2,3-butanedione than sucrose (Figure 3.,

418

pathway e). The results also indicate that sucrose contributes mainly through the

419

fragmentation and recombination of its fragments. More specifically, 56% of 2,3-

420

butanedione originating from sucrose emerged from a C1/C3 recombination, around 32%

421

derived from a C2/C2 recombination, and only 12% stemmed from the intact sucrose

422

skeleton.

423

The obtained results differ a bit from the ones reported for model systems. Under dry

424

heating conditions at 135 °C, a model system of glucose and L-proline generated 2,3-

425

butanedione mainly from intact sugar skeleton (48% to 54%) followed by the

426

recombination of C1/C3 sugar fragments (36% to 44%). Only a minor part (8% to 10%) was

427

formed by recombination of C2/C2 sugar fragments.21 On the other hand, at 180 °C dry



Page 26 of 54

428

heating of glucose with proline generated 2,3-butanedione mainly by recombination of

429

C1/C3 fragments (87%). The remaining part (13%) stemmed from a C2/C2 recombination,

430

while no 2,3-butanedione was formed from the intact glucose skeleton.19 The higher

431

reaction temperature thus favors formation from fragments over intact sugar skeleton. The

432

different result obtained for coffee as compared to model systems, could be explained by

433

the complexity of the coffee matrix and by different reaction conditions (high temperature-

434

short time). At the beginning of the roasting cycle substantial part of 2,3-butanedione is

435

probably formed from the intact sucrose skeleton. As the roasting temperature increases

436

the fragmentation and recombination of the fragments gain importance and finally become

437

predominant at the end of roasting (203 °C).

438

2,3-pentanedione. The isotopologue distribution obtained for 2,3-pentanedione at the end

439

of the CAMOLA experiment permitted to calculate the contribution of individual reaction

440

pathways generating 2,3-pentanedione from sucrose (

441

Table 9). The presence of singly labeled isotopologue (m/z 101) at 1.6% indicates that

442

6.4% of 2,3-pentanedione was formed by recombination of C1 and C4 sucrose fragments

443

(Figure 4., pathway a). 4-Fold labeled isotopologue (m/z 104) was however formed at

444

higher level than singly labeled isotopologues. Therefore, the remaining part of the former

445

isotopologue must stem from recombination of C4 labeled sucrose fragments with C1

446

unlabeled fragment originating from other components of green the coffee matrix.

447

Consequently 1.6% of 2,3-pentanedione must be formed by recombination of C4 sucrose

448

fragment and C1 fragment from other sources (Figure 4., pathway d). The C1/C4

449

recombination can be theoretically explained by aldol condensation of 2,3-butanedione

450

with formaldehyde as proposed by Weenen.18 Nevertheless the labelling pattern obtained

451

for 2,3-butanedione does not support this hypothesis as the majority of the 2,3-

452

butanedione does not stem from sucrose. The majority of unlabeled 2,3-butanedione 26 ACS Paragon Plus Environment

Page 27 of 54


453

should lead to m/z 101 > m/z 104 which was not the case. Consequently, it can be

454

concluded that 2,3-pentanedione is not formed via 2,3-butanedione upon coffee roasting

455

or its formation is negligible. Yet unknown C4 fragment must be, therefore, involved in the

456

formation of 2,3-pentanedione via C1/C4 recombination.

457

The doubly labeled isotopologue (m/z 102, 4.7%) points out that 18.8% of 2,3-

458

pentanedione is formed by recombination of C2 and C3 sucrose fragments (Figure 4.,

459

pathway b), e.g. by aldol condensation of 1-hydroxy-2-propanone with acetaldehyde as

460

proposed by Hofmann.23 The triply labelled isotopologue (m/z 103, 9.1%) was, however,

461

formed at higher level as compared to doubly labeled one. Thus 4.4% of the former

462

isotopologue had to be formed from recombination of labeled C3 sucrose fragment with

463

unlabeled C2 fragment originating from the green coffee matrix. Such a recombination

464

contributed to 8.8% of 2,3-pentanedione (Figure 4., pathway c). The generation of 2,3-

465

pentanedione by recombination of sucrose fragments (C1/C4 and C2/C3) may be a bit lower

466

than shown in Table 9 as the singly labeled isotopologue (m/z 101) could be also formed

467

by recombination of a labeled C1 fragment from sucrose with a non-labeled C4 fragment

468

from other precursors in the coffee matrix and the doubly labeled isotopologue (m/z 102)

469

by combination of a labeled C2 fragment from sucrose with a non-labeled C3 fragment from

470

the coffee matrix. However similarly to 2,3-butanedione, the obtained data do not allow

471

more detailed calculations without speculation.

472

The fully labeled 2,3-pentanedione represented 26.4% of the total amount formed, but

473

recombination of the fully labeled sucrose fragments explained only 6.3%. So the

474

remaining 20.1% must derive from the intact sugar skeleton and consequently 40.2% of

475

2,3-pentanedione was formed from this pathway (Figure 4., pathway e).



Page 28 of 54

476

Finally, the remaining part of the unlabeled molecule that cannot be explained by

477

sucrose degradation (24.2%) must stem from other precursors present in coffee matrix

478

(Figure 4, pathway f).

479

In summary, the data clearly indicate that sucrose play a prominent role in the formation

480

of 2,3-pentanedione contributing to about three fourths of its level. Contrary to 2,3-

481

butanedione, where recombination of fragments was the key pathway from sucrose, 2,3-

482

pentanedione was mainly formed from intact sucrose skeleton (54% of the level generated

483

form sucrose). The possible reaction mechanism (Figure 4, pathway e) could involve

484

generation of 4-hydroxy-2-(hydroxymethyl)-5-methyl-3(2H)-furanone (2) via dehydration of

485

1-deoxyhexo-2,3-diulose (1). Retro-aldol reaction of this intermediate was shown to

486

generate 4-hydroxy-5-methyl-3(2H)-furanone (3).37 The latter compound may generate

487

2,3-pentanedione via reduction and acid catalyzed dehydration of 1-deoxypento-2,3-

488

diulose (4) as proposed by Whitfield and Mottram (Figure 5A).38 Alternatively, 4-hydroxy-5-

489

methyl-3(2H)-furanone may be transformed via Strecker degradation to 2-amino-4,5-

490

dihydroxy-3-pentanone (5). Loss of ammonia followed by the Strecker degradation of the

491

resulting 1-hydroxy-2,3-pentanedione may yield 2,3-pentanedione as proposed by Cerny

492

and Davidek.39

493

Kinetic study with [UL-13C12]sucrose. To get even deeper insight into the contribution

494

of sucrose into studied α-diketones, the evolution of the labeling pattern at different

495

roasting stages was measured. For this purpose, a larger batch of recombined green

496

coffee beans (30 g) containing fully labeled sucrose in place of unlabeled analog was

497

prepared. The batch was divided in 6 portions and roasted for different periods of time

498

varying between 80 s and 330 s.

499

The evolvement of the individual isotopologues of 2,3-butanedione as well as the total

500

amount of all isotopologues, normalized to their maximum level attained upon roasting, is


Page 29 of 54


501

shown in Figure 6. The total amount of generated 2,3-butanedione reached a maximum at

502

about 260 s, followed by a distinct decrease. Similarly, the fully labeled molecule reached

503

its maximum at 260 s, but the highest ratio of fully labeled isotopologue ([M+4]+, m/z 90) to

504

unlabeled isotopologue ([M]+, m/z 86) was reached between 150 s and 200 s. In this time

505

window fully labeled isotopologue predominated. From 210 s on the non-labeled

506

isotopologue strongly increased, whereas the fully labelled one declined as a

507

consequence of sucrose depletion (data not shown). Relatively high level of the unlabeled

508

isotopologue at early roasting stage (80 s) can be most probably explained by carry over

509

from the green coffee bean. The kinetics data points out that sucrose contribution to 2,3-

510

butanedione is substantial at the early roasting stages, however its contribution is

511

diminished at the later roasting stages in favor of other sources present in the green coffee

512

matrix, such as non-water-soluble polysaccharides.

513

The generation of different 2,3-pentanedione isotopologues is shown in Figure 7. The

514

maximum level of 2,3-pentanedione was reached between 260 s and 300 s, followed by a

515

slight decrease. The fully labeled isotopologue (m/z 105) was the most abundant along the

516

whole roasting cycle, however its share decreased in the advanced roasting stages. While

517

between 150 s and 210 s the unlabeled and fully labeled isotopologue were formed in the

518

ratio 1 to 4, their ratio decreased to only 1 to 1.6 at 330 s. The data clearly indicate that

519

upon coffee roasting 2,3-pentanedione is mainly formed from sucrose, however the

520

importance of sucrose diminishes at advanced roasting stages.

521

In summary, the combination of labeling experiments and kinetic studies was

522

demonstrated as a very powerful approach to gain a deeper insight into the role of

523

precursors in the formation of Maillard-derived aroma components upon food processing.

524

The results enabled to obtain deeper insight into role of sucrose to the formation of α-

525

diketones upon coffee roasting. Sucrose contributes to the formation of both diketones, but



Page 30 of 54

526

its importance and reaction pathways clearly differ. Whereas 2,3-pentanedione is mainly

527

formed from sucrose, 2,3-butanedione originates mainly from other precursors in green

528

coffee bean such as bound polysaccharides. Formation from intact sugar skeleton is the

529

major pathway generating 2,3-pentanedione from sucrose, while 2,3-butanedione is mainly

530

generated by recombination of sucrose fragments. On the other hand, the contribution of

531

glucose and fructose moieties of sucrose to both α-diketones is comparable and

532

contribution of other free sugars is negligible. Finally, the kinetic experiments with fully

533

labeled sucrose showed that contribution of sucrose to both α-diketones constantly

534

changes during the roasting. Therefore, the conclusions must be carefully drawn when

535

comparing different studies as the importance of free and bound precursors depends on

536

the roasting degree.

537

This newly obtained data will enable to better understand and control generation of α-

538

diketones upon roasting and to modulate the coffee aroma through molecularly guided

539

roasting profile. In addition the obtained results from labelling studies are being further

540

explored to get deeper insight into the contribution of sucrose to other key odorants

541

generated upon roasting and will be published later.

542 543

ABBREVIATIONS USED

544

EB – Exhausted beans

545

BRE – Biomimetically recombined extract

546

BREB – Exhausted beans reconstituted with the biomimetically recombined extract

547

CAMOLA – Carbon Module Labeling

548

ICP-OES - Inductively Coupled Plasma Optical Emission Spectrometry


Page 31 of 54


549

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37.

649

3(2H)furanone from sugar amino acid reaction mixtures. J. Agric. Food Chem. 1987, 35, 990-993.

650

38.

651

3(2H)-furanone and cysteine or hydrogen sulfide at pH 4.5. J. Agric. Food Chem. 1999, 47, 1626-

652

1634.

653

39.

654

Maillard reaction. J. Agric. Food Chem. 2003, 51, 2714-2721.

ISO 8262, Milk products and milk-based foods — Determination of fat content by the

AOAC, AOAC (Association of Official Analytical Chemists). Gaithersburg, Maryland,

Hiebl, J.; Ledl, F.; Severin, T., Isolation of 4-hydroxy-2-(hydroxymethyl)-5-methyl-

Whitfield, F. B.; Mottram, D. S., Investigation of the reaction between 4-hydroxy-5-methyl-

Cerny, C.; Davidek, T., Formation of aroma compounds from ribose and cysteine during the

655 656

FIGURE CAPTIONS

657

Figure 1. Mass spectrum of 2,3-butanedione in the experiment with [UL-13C12] sucrose

658

Figure 2. Mass spectrum of 2,3-pentanedione in the experiment with [UL-13C12] sucrose

659

Figure 3. Schematic presentation of different pathways contributing to 2,3-butanedione

660

upon coffee roasting. Recombination of sucrose fragments: 1-hydroxy-2-propanone and

661

formaldehyde (a) or acetaldehyde and glycolaldehyde as proposed by Schieberle et.al.19

662

(b). Recombination of 1-hydroxy-2-propanone from sucrose and formaldehyde from the

663

coffee matrix (c). Generation from intact sucrose backbone as proposed by Weenen18 (d)

664

and generation from other precursors in the coffee matrix (e) ACS Paragon Plus Environment

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665

Figure 4. Schematic presentation of different pathways contributing to 2,3-pentanedione

666

upon coffee roasting. Recombination of sucrose fragments: 1-hydroxy-2-propanone and

667

acetaldehyde as proposed by Hofmann23 (a) or C4-fragment and formaldehyde (b).

668

Recombination of 1-hydroxy-2-propanone from sucrose and acetaldehyde from the coffee

669

matrix (c). Recombination of 1 C4-fragment from sucrose and formaldehyde from the coffee

670

matrix (d). Generation from intact sucrose backbone (e) and generation from other

671

precursors in the coffee matrix (f)

672

Figure 5. Hypothetical formation pathways of 2,3-pentanedione from 4-hydroxy-5-methyl-

673

3(2H)-furanone: (A) via reduction and acid catalyzed dehydration of 1-deoxypento-2,3-

674

diulose as proposed by Whitfield and Mottram38 or (B) via successive Strecker

675

degradations as proposed by Cerny and Davidek39

676

Figure 6. Kinetics of formation of 2,3-butanedione isotopologues upon roasting of

677

recombined coffee beans containing [UL-13C12] sucrose

678

Figure 7. Kinetics of formation of 2,3-pentanedione isotopologues upon roasting of

679

recombined coffee beans containing [UL-13C12] sucrose

680


36

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TABLES Table 1: Composition of the green coffee extract in percent (expressed on dry matter base) Key component Lipids Ashes Total phenols Caffeine Trigonelline Total organic acids Free amino acids Free sugars Sucrose Metals

Amount (%/ DM) 0.0 4.1 5.6 0.71 0.13 2.7 0.56 8.1 7.9 0.013


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Table 2: Amounts of components used for the preparation of biomimetically recombined extract (BRE)a,b

Components

Amount

Components

(mg) Phenols

Amino acids (cont.)

Chlorogenic acid

324.7

L-Glutamine

0.6

Trigonelline

49.7

L-Tryptophane

1.3

Caffeine

41.2

L-Valine

0.6

L-Isoleucine

0.4

Organic acids D-(-)-Quinic acid

34.6

L-Leucine

0.6

L-(+)-Lactic acid

0.1

L-Phenylalanine

1.0

Potassium acetate

3.2

L-Lysine

0.7

Malic acid

15.9

Sugars

Potassium citrate

104.8

D-(-)-Arabinose

0.4

L-Rhamnose

0.1 7.9

Amino acids

a b

Amount (mg)

L-Alanine

1.9

D-(+)-Galactose

L-Threonine

1.6

D-(+)-Sucrose

500.6

L-Serine

1.3

D-(+)-Mannose

1.2

L-Aspartic acid

4.5

Metals

L-Glutamic acid

8.3

Iron(III) chloride

0.3

Glycine

0.2

Manganese(II) chloride

0.2

L-Histidine

0.7

Copper(II) sulfate

0.2

L-Arginine

3.0

Zinc(II) sulfate, heptahydrate

0.03

L-Proline

1.0

L-Tyrosine

0. 9

L-Asparagine

4.0

for 5 g exhausted beans (EB) composition of BRE was based on the composition of water extract of green coffee beans


38

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Table 3: Biomimetic in-bean experiments to study the formation of key aroma compounds from free sugars upon coffee roasting. Experiment Name 1

Description

Green beans

Original untreated green coffee beans

a

2

BREB (Ref)

Exhausted bean (EB) + biomimetically recombined extract (BRE)

3

BREB + [13C12]-SUCb

EB + BRE omitted in free sugars spiked with [UL-13C12]sucrose

4

BREB + [13C6Fru]SUC

EB + BRE omitted in free sugars spiked with [UL-13C6fructose]sucrose

5

BREB + [13C6Glc]SUC

EB + BRE omitted in free sugars spiked with [UL-13C6glucose]sucrose

6

BREB + other labeled sugars

EB + BRE omitted in free sugars but sucrose spiked with D[UL-13C5]arabinose, D-[UL-13C6]galactose, D-[UL13 C6]mannose

7

BREB + CAMOLAc

CAMOLA experiment: EB + BRE omitted in free sugars spiked sucrose and [UL-13C12]sucrose (50%/50%)

8

BREB + [13C12]-SUC kinetics

Kinetic study: EB + BRE omitted in free sugars spiked with [UL-13C12]sucrose

a

BREB coffee beans obtained by reincorporation of biomimetically recombined extract into water extracted b c beans, sucrose (SUC), Carbohydrate Module Labeling (CAMOLA)


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Table 4: Roasting profile for experiments 1 to 7 Time (s) Temp (°C)

0 148

42 168

95 183

176 196

300 203


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Table 5: Roasting profile for the kinetic study (experiment 8) Time (s) Temp (°C)

50 170

80 177

100 184

150 189

210 197

260 200

300 202

330 203


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Table 6: Relative distribution (%) of 2,3-butanedione isotopologues generated from

13

C

labeled sugars upon coffee roasting at 300 s. m/z

ion

[M]+

BREBa (Ref)

BREB+ [13C12]SUCb

BREB+ [13C6Fru]SUCc

BREB+ [13C6Glc]SUCd

BREB+ other labeled sugarse

86

99

74

81

79

99

[M+1]

+

87

0

2

3

3

0

[M+2]

+

88

1

5

5

5

1

[M+3]

+

89

0

5

5

5

0

[M+4]

+

90

0

14

6

8

0

a

BREB coffee beans obtained by reincorporation of biomimetically recombined extract into water extracted beans, b sucrose (SUC), c fructose (Fru), d glucose (SUC), e other labeled free sugars were : D-[UL13 13 13 C5]arabinose, D-[UL- C6]galactose, D-[UL- C6]mannose


42

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Table 7: Relative distribution (%) of 2,3-pentanedione isotopologues generated from

13

C

labeled sugars upon coffee roasting at 300 s. ion

[M]+

m/z

BREBa (Ref)

BREB+ [13C12]SUCb

BREB+ [13C6Fru]SUCc

BREB+ [13C6Glc]SUCd

BREB+ other labeled sugarse

100

99

37

63

65

99

[M+1]

+

101

0

1

1

1

0

[M+2]

+

102

1

4

4

4

1

[M+3]

+

103

0

10

7

7

0

[M+4]

+

104

0

3

2

2

0

[M+5]

+

105

0

45

23

21

0

a

BREB coffee beans obtained by reincorporation of biomimetically recombined extract into water extracted beans, b sucrose (SUC), c fructose (Fru), d glucose (SUC), e other labeled free sugars were : D-[UL13 13 13 C5]arabinose, D-[UL- C6]galactose, D-[UL- C6]mannose


43


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Table 8: Relative contribution (%) of different pathways generating 2,3-butanedione as calculated from isotopologue distribution of CAMOLA experiment Relative distribution of isotopologues (%) a

Measured m/z

Ion [M]+

Calculated contribution of individual pathways C1/C3b

C2/C2c

C1(o)/C3d

Intact skeletone

Other sourcesf

2.8

2.6

2.2

64.8

86

76

3.6

[M+1]

+

87

3.6

3.6

[M+2]

+

88

5.6

[M+3]

+

89

6.2

3.6

[M+4]

+

90

8.6

3.6

2.8

14.4

11.2

Total

5.6 2.6 2.2 5.2

4.4

64.8

a

isotopologue patterns determined from the cluster of the molecular ions of 2,3-butanedione, b recombination of C3 and C1 sucrose fragments (C1/C3), c recombination of two C2 sucrose fragments (C2/C2), d recombination e of C3 and sucrose fragments with C1 fragments origination from other precursors (C1(o)/C3), formation form f the intact C4 sugar backbone, formation form other precursors present in coffee bean than sucrose


44

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Table 9: Relative contribution (%) of different pathways generating 2,3-pentanedione as calculated from isotopologue distribution of CAMOLA experiment Relative distribution of isotopologues (%) Measured m/z

Ion [M]+

a

Calculated contribution of individual pathways C1/C4b

C1(o)/C4c

C2/C3d

C2(o)/C3e

Intact skeletonf

Other sourcesg

0.8

4.7

4.4

20.1

24.2

100

55.8

1.6

[M+1]

+

101

1.6

1.6

[M+2]

+

102

4.7

4.7

[M+3]

+

103

9.1

4.7

[M+4]

+

104

2.4

1.6

[M+5]

+

105

26.4

1.6

Total

6.4

4.4

0.8 4.7 1.6

18.8

a

20.1 8.8

40.2

24.2 b

isotopologue patterns determined from the cluster of the molecular ions of 2,3-pentanedione, recombination of C4 and C1 sucrose fragments (C1/C4), c recombination of C4 sucrose fragments with C1 fragments origination from other precursors (C1(o)/C4), d recombination of C2 and C3 sucrose fragments e (C2/C3), recombination of C3 sucrose fragments with C2 fragments origination from other precursors (C2(o)/C3) , f formation form the intact C4 sugar backbone, g formation form other precursors present in coffee bean than sucrose


45


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FIGURES GRAPHICS

Figure 1: Mass spectrum of 2,3-butanedione in the experiment with [UL-13C12] sucrose


46

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Figure 2: Mass spectrum of 2,3-pentanedione in the experiment with [UL-13C12] sucrose

-


47


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Figure 3. Schematic presentation of different pathways contributing to 2,3-butanedione upon coffee roasting. Recombination of sucrose fragments: 1-hydroxy-2-propanone and formaldehyde (a) or acetaldehyde and glycolaldehyde as proposed by Schieberle et.al.19 (b). Recombination of 1-hydroxy-2-propanone from sucrose and formaldehyde from the coffee matrix (c). Generation from intact sucrose backbone as proposed by Weenen18 (d) and generation from other precursors in the coffee matrix (e)


48

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Figure 4. Schematic presentation of different pathways contributing to 2,3-pentanedione upon coffee roasting. Recombination of sucrose fragments: 1-hydroxy-2-propanone and acetaldehyde as proposed by Hofmann23 (a) or C4-fragment and formaldehyde (b). Recombination of 1-hydroxy-2-propanone from sucrose and acetaldehyde from the coffee matrix (c). Recombination of 1 C4-fragment from sucrose and formaldehyde from the coffee matrix (d). Generation from intact sucrose backbone (e) and generation from other precursors in the coffee matrix (f)


49


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A

B

Figure 5. Hypothetical formation pathways of 2,3-pentanedione from 4-hydroxy-5-methyl3(2H)-furanone: (A) via reduction and acid catalyzed dehydration of 1-deoxypento-2,3diulose as proposed by Whitfield and Mottram38 or (B) via successive Strecker degradations as proposed by Cerny and Davidek39


50

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Figure 6: Kinetics of formation of 2,3-butanedione isotopologues upon roasting of recombined coffee beans containing [UL-13C12] sucrose


51


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Figure 7: Kinetics of formation of 2,3-pentanedione isotopologues upon roasting of recombined coffee beans containing [UL-13C12] sucrose


52

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TOC graphic


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