Quantitative analyses of key odorants and their precursors reveal

Subscriber access provided by Nottingham Trent University

Chemistry and Biology of Aroma and Taste

Quantitative analyses of key odorants and their precursors reveal differences In the aroma of gluten-free rice bread compared to wheat bread Anke R. Boeswetter, Katharina Anne Scherf, Peter Schieberle, and Peter Koehler J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04800 • Publication Date (Web): 05 Sep 2019 Downloaded from pubs.acs.org on September 5, 2019

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

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

Page 1 of 36

Journal of Agricultural and Food Chemistry

1

QUANTITATIVE ANALYSES OF KEY ODORANTS AND THEIR PRECURSORS

2

REVEAL DIFFERENCES IN THE AROMA OF GLUTEN-FREE RICE BREAD

3

COMPARED TO WHEAT BREAD

4

Anke R. Boeswetter1, Katharina A. Scherf1, Peter Schieberle2, Peter Koehler3*

5 6

1

7

Meitner-Str. 34, 85354 Freising, Germany

8

2

9

Garching, Germany

10

Leibniz-Institute for Food Systems Biology at the Technical University of Munich, Lise-

Department of Chemistry, Technical University of Munich, Lichtenbergstrasse 4, 85748

3 Biotask

AG, Schelztorstraße 54‑56, 73728 Esslingen, Germany

11 12 13 14 15 16 17 18 19

* To whom correspondence should be addressed

20

Phone: +49 711 31059068

21

Fax: +49 711 31059070

22

E-mail: [email protected]

-1-

ACS Paragon Plus Environment


23

ABSTRACT

24

Rice flour is one of the most important raw materials in gluten-free products. However, the

25

aroma of gluten-free rice bread is less accepted by consumers than that of commercial

26

wheat bread. Therefore, 18 selected aroma compounds were determined in rice and

27

wheat breads by stable isotope dilution assays (SIDA) to elucidate differences in the

28

sensory characteristics, concentrations and odor activity values (OAV). The OAVs of most

29

aroma compounds varied greatly between a rice and a wheat bread. In particular, 2-

30

aminoacetophenone with a grape-like, medicinal aroma was characteristic for rice bread

31

crumb and crust, while maltol was only relevant in wheat bread crust. Ehrlich pathway

32

products varied in their concentration between the bread crumbs and were correlated with

33

the contents of their corresponding free amino acid precursors in the flours and doughs.

34

The analysis of rice flour revealed that only few aroma compounds retained in the bread.

35

Consequently, the bread making process has a high relevance in aroma compound

36

formation. By comparison of breads prepared from fresh and stored rice flour, hexanal

37

was identified as an important indicator for ageing in rice bread and flour.

38 39

KEYWORDS

40

Stable isotope dilution analysis (SIDA); Odor activity values (OAV); Gluten-free; Rice

41

bread; Wheat bread; Free amino acids; Flour aging

42

-2-


Page 2 of 36

Page 3 of 36


43

INTRODUCTION

44

Due to its worldwide availability, low allergenicity and cheap price rice flour is one of the

45

most important raw materials for the production of gluten-free bread.1–5 Moreover the

46

production of gluten-free products has grown strongly in recent years.6 Not only patients

47

suffering from gluten- and wheat-dependent hypersensitivities, such as celiac disease,

48

wheat allergy or non-celiac gluten sensitivity (NCGS), but also healthy people consume

49

gluten-free products.7 However, one of the most important issues of gluten-free bread

50

making is the poor quality of the products in terms of aroma and texture compared to

51

wheat bread.8,9 A prerequisite for the improvement of the aroma of rice bread is (i) the

52

systematic characterization of its aroma compounds and (ii) the comparison with those of

53

wheat bread. The application of an aroma extract dilution analysis (AEDA) recently

54

resulted in the identification of the key aroma compounds in a rice bread and a comparison

55

to wheat bread aroma compounds.10. 2-Aminoacetophenone (medicinal, grape like) and

56

4-vinylphenol (phenolic) were highly relevant in the crumb and crust of rice bread, whereas

57

1-octene-3-one (mushroom-like) and (E)-2-nonenal (green, fatty) were important uniquely

58

in the crust of rice bread. Compared to wheat bread, in rice bread maltol (caramel-like)

59

and 2-methoxy-4-vinylphenol (smoky, clove-like) were absent. Yeast metabolites formed

60

via the Ehrlich pathway such as 3-methyl-1-butanol (malty), 2- and 3-methylbutanoic acid

61

(sweaty), 2-phenylethanol (flowery, honey-like) and phenylacetic acid (honey, bees-wax),

62

reached relevant FD factors in both bread types. Correlations between the presence of

63

aroma compounds and the texture of the breads were not established. Because AEDA

64

does not consider matrix effects and volatility it is necessary to quantitate aroma

65

compounds by stable isotope dilution analysis (SIDA) and calculate odor activity values

-3-



66

(OAV).11,12 However, comparative data on the concentrations and OAVs of relevant aroma

67

compounds in rice and wheat bread were not available at the beginning of the study.

68

Apart from quantitative data on aroma compounds, it is also important to study their

69

formation pathways. Specific odorants are already present in the flour, and their content

70

is affected by flour age, e.g. by lipid oxidation during long time storage.13 Other aroma

71

compounds are formed from odorless precursors during the bread making process, such

72

as free amino acids in the flour, which are important for the production of aroma active

73

yeast metabolites.14 Knowledge on sources and precursors of aroma compounds is a

74

prerequisite to modify or regulate aroma compound formation in gluten-free bread.

75

Therefore, the aim of this study was to compare the amounts and OAVs of 18 selected

76

aroma compounds in rice and wheat bread crumb and crust, respectively, and to elucidate

77

the formation of aroma compounds. For this purpose, the concentrations of the free amino

78

acids leucine, isoleucine and phenylalanine were monitored and compared to the contents

79

of the corresponding aroma-active metabolites. Additionally, concentrations and OAVs of

80

12 aroma compounds in rice flour were determined to estimate the impact of the flour on

81

the aroma of the bread. To verify the previously reported effect of flour age on rice bread

82

aroma, fresh and stored rice flour batches and the corresponding gluten-free breads were

83

analyzed.

84 85

MATERIALS AND METHODS

86

Raw materials and bread making. Two rice flour batches (batch R1: produced in

87

10/2017, 88.6 % flour dry matter (fdm); batch R2: produced in 10/2015, 89.5 % fdm;

88

Remyflo R7 90T LP; Beneo-Remy, Leuven-Wijgmaal, Belgium), wheat flour (W: 86.4 %

89

fdm; ash content 0.55 %; “Rosenmehl”, Rosenmuehle GmbH, Ergolding, Germany), fresh -4-


Page 4 of 36

Page 5 of 36


90

compressed baker’s yeast (F. X. Wieninger GmbH, Passau, Germany), sucrose (Alfa

91

Aesar, Karlsruhe, Germany) and sodium chloride (Merck, Darmstadt, Germany) were

92

used for bread making as previously described.10 After baking, breads were allowed to

93

cool at room temperature (RT ~22 °C) for 2 h prior to analysis.

94 95

Chemicals. All chemicals, solvents, reference aroma compounds and amino acids were

96

obtained from Merck (Darmstadt, Germany), Sigma Aldrich (Taufkirchen, Germany), Alfa

97

Aesar or Carl Roth GmbH (Karlsruhe, Germany) and were of analytical grade or higher.

98

Dichloromethane (technical grade; VWR, Darmstadt, Germany) was purified by distillation

99

before use. The isotopically labeled standards [d12]-hexanal, [d9]-3-methylbutanoic acid

100

and [d4,5]-2-phenylethanol were obtained from CDN isotopes (Quebec, Canada), [13C2]-

101

phenylacetic acid and [13C6]-vanillin from Sigma Aldrich, [13C6]-leucine and [13C6]-

102

isoleucine from Cambridge Isotope Laboratories (Andover, MA, USA) and [d5]-

103

phenylalanine from Euriso-top (Saarbrücken, Germany). The following labeled

104

compounds were synthesized as described before: [d2]-3-methyl-1-butanol,15 [d2,3]-1-

105

octene-3-one,16 [d2]-(E)-2-nonenal,17 [d4,5]-(E,E)-2,4-decadienal,18 [13C2]-maltol,19 [13C2]--

106

nonalactone,20 [13C6]-2-methoxy-4-vinylphenol,21 [d3]-2-aminoacetophenone22 and [d4]-4-

107

vinylphenol.22

108 109

Synthesis of [d20]-2-butyl-2-octenal. [d12]-Hexanal (1 mmol; 112 mg) was dissolved in

110

a mixture of ethanol and aqueous sodium hydroxide (1 mol/L)(1.4 mL;1/1, v/v) .The

111

solution was stirred overnight at 70 °C, extracted with dichloromethane (30 mL), and the

112

organic phase was dried over sodium sulfate. The purity was verified using two-

113

dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOF-MS), -5-



114

and the concentration was determined by gas chromatography coupled with a flame

115

ionization detector (FID) compared to the unlabeled substance as described below.

116 117

Quantitation of the synthesized isotopically labeled internal standards: To

118

determine the concentrations of isotopically labeled internal standards, peak intensities of

119

GC-FID-chromatograms were compared with those of unlabeled reference aroma

120

compound solutions using methyl octanoate as internal standard. Measurements were

121

performed on a Thermoquest (Egelsbach, Germany) Trace 2000 GC equipped with a PAL

122

autosampler (CTC-Analytics, Zwingen, Switzerland) and an FID. The response factor was

123

used for the quantitation of the labeled isotopologues present in solution.

124 125

Quantitation of aroma compounds.

126

Sample work up. Freshly baked bread samples were cooled for 2 h at RT and crumb and

127

crust were separated. For homogenization, crumb and crust were frozen with liquid

128

nitrogen and ground by a knife mill (GRINDOMIX GM 200; Retsch, Haan, Germany). Rice

129

flour was used directly. Samples (1-100 g, depending on the analyte concentration

130

determined in a preliminary analysis) were weighed into glass vessels. Dichloromethane

131

(25-400 mL), the isotopically labeled standards were added, and samples were stirred at

132

RT for 2 h. The volatile fraction was isolated by solvent-assisted flavor evaporation

133

(SAFE)23, dried over anhydrous sodium sulfate and concentrated to 200-1000 µL at 53 °C

134

by means of a Vigreux column (50 cm × 1 cm i.d.) followed by a micro distillation

135

apparatus. All analyses were performed in triplicate.

136

-6-


Page 6 of 36

Page 7 of 36


137

Quantitation by GC-MS. Samples containing known amounts of isotopically labeled

138

standard were analyzed using different GC-MS systems depending on the MS

139

fragmentation pattern and chromatographic separation needs (Table 1). The aroma

140

compound concentrations were calculated by the ratio of the mass signals of analyte and

141

standard and corrected with response calibration lines. These were determined by

142

analyzing mixtures of the labeled and the unlabeled compounds in 5 different mass ratios

143

(5:1 to 1:5) (Table 1). The lines were created by plotting the concentration ratios

144

(standard/analyte) on the x-axis against the area ratios (standard/analyte) on the y-axis.

145 146

Differentiation of geometric isomers. Mixtures of 2- and 3-methyl-1-butanol and 2-and 3-

147

methylbutanoic acid, respectively, were prepared in 5 different ratios (5:1 - 1:5) and

148

analyzed using an EI-GC-MS system. Calibration lines were created by plotting the

149

intensity ratios of characteristic mass signals on the x-axis against the percentage of the

150

respective 3-isomer in the mixture on the y-axis as follows: for the alcohols m/z 57 divided

151

by m/z 70 and for the acids m/z 60 divided by the sum of m/z 60 and m/z 74.

152 153

Quantitation of acetic acid. Acetic acid was quantitated by an enzymatic test kit (R-

154

Biopharm, Darmstadt, Germany) according to the manufacturer’s instruction. Bread

155

samples were homogenized as described above; flour was used directly. For the

156

extraction, the sample (4 g) and water (ca. 60 mL) were heated for 20 min at 50-60 °C in

157

a 100 mL flask. After cooling, the flask was filled up to the mark with water and the solution

158

was filtered.

159 160

Quantitation of free amino acids. -7-



161

Sampling. Flour-water suspensions and bread doughs made from wheat and rice flour

162

(R1), respectively, were prepared as previously described.10 Portions of 200 g suspension

163

or dough were taken directly after mixing and after 10, 20, 30 and 40 min fermentation

164

time (30 °C, 90 % relative humidity), respectively, frozen with liquid nitrogen, freeze dried

165

and ground by a knife mill. Fresh compressed yeast (100 g) was ground by a cryomill

166

(SPEX SamplePrep, Stanmore, United Kingdom) before freeze drying. Flour (100 g each)

167

was lyophilized directly.

168 169

Sample workup. Between 0.1 and 1 g sample (depending on the expected contents of

170

amino acids) were weighed into centrifuge tubes containing 3.5 mL trichloroacetic acid

171

(aq, 8.5 %) and the isotopically labeled internal standards (Table 2). After homogenization

172

with a mechanical blender (Ultra Turrax, IKA, Staufen, Germany) and an extraction time

173

of 3 min in an ultrasonic bath (Bandelin Electronic GmbH &Co. KG, Berlin, Germany), the

174

samples were centrifuged for 20 min with 4500 × g at 15 °C in a Heraeus Multifuge X3 FR

175

centrifuge (Thermo Fisher Scientific, Dreieich, Germany). The supernatant was poured

176

onto Amicon® centrifugal filters (15 kDa, Merck) and centrifuged up to 12 h at 3000 × g.

177

For derivatization, 2 mL of the filtrate was mixed with 2 mL aqueous sodium carbonate

178

(1 mol/l, pH 9.8) and 0.5 mL benzoyl chloride (1 mol/l) solved in acetonitrile and stirred for

179

30 min at RT. To stop the reaction, the pH was lowered to 2 using hydrochloric acid (32%,

180

w/w). The samples were used either directly or diluted with aqueous acetonitrile (10%,

181

v/v) for LC-MS/MS measurements. Amino acid concentrations were calculated from the

182

ratio of the mass signal of analyte and labeled internal standard and corrected with

183

response calibration lines (Table 2) as described for the quantitation of aroma compounds.

184 -8-


Page 8 of 36

Page 9 of 36


185

Gas Chromatography-Mass spectrometry (GC-MS) Systems:

186

High resolution (HR) GC-MS. An Agilent 7890B gas chromatograph (Waldbronn,

187

Germany) was equipped with a PAL Autosampler (CTC-Analytics) and an ion trap detector

188

type Saturn 2000 (Agilent). The samples were injected cold-on-column. Chromatography

189

was performed on an Agilent DB-FFAP capillary (30 m × 0.25 mm i.d.; 0.25 μm film

190

thickness) using helium as carrier gas and the following temperature program: 40 °C held

191

for 2 min, 6 °C/min up to 230 °C, held for 5 min. The characteristic ions were monitored in

192

the chemical ionization mode (MS-CI) with methanol as reactant gas.

193 194

Two-dimensional “heartcut”-GCxGC-MS. The system consisted of a Thermo Fisher

195

Scientific Trace 2000 series GC equipped with a PAL Autosampler (CTC-Analytics), an

196

FID, a sniffing port and a Thermo Fisher Scientific moving column stream switching

197

system (MCSS) connected to a Varian CP 3800 gas chromatograph (Waldbronn,

198

Germany) coupled to an Agilent Saturn 2000 mass spectrometer. Samples were injected

199

cold-on-column using helium as carrier gas, and after separation on an Agilent FFAP

200

capillary column (30 m × 0.32 mm i.d., 0.25 μm film thickness) in the first dimension the

201

substance of interest was transferred via MCSS into a cold trap (-100 °C) and via heating

202

to an Agilent DB5 capillary column (30 m x 0,25 mm i.d., 0,25 µm film thickness). The

203

temperature program of the first GC was as follows: 40 °C held for 2 min, 6 °C/min up to

204

230 °C, and finally held for 5 min. The second GC started the following temperature

205

program directly after the transfer of the effluent from the cooling trap at 40 °C, 6°C/min

206

to 240 °C, held for 3 min. The intervals of the cut times were set after an initial analysis of

207

reference compounds. The mass spectrometer was operated in the MS-CI mode using

208

methanol as reactant gas. -9-



Page 10 of 36

209 210

Comprehensive

two-dimensional

GCxGC-TOF-MS.

The

system

(LECO;

211

Mönchengladbach, Germany) consisted of a Gerstel PTV 4 injector (Mühlheim an der

212

Ruhr, Germany), a PAL autosampler (CTC-Analytics), an Agilent 6890N gas

213

chromatograph with a secondary oven mounted inside the primary GC oven and a

214

Pegasus 4D TOF-MS. The samples were injected cold-on-column (1-2 µL) and helium

215

served as carrier gas. The separation in the first dimension was performed with an Agilent

216

DB-FFAP capillary (30 m × 0.32 mm i.d.; 0.25 μm film thickness) using the following

217

temperature program: 40 °C held for 2 min, 6 °C/min up to 230 °C, held for 5 min. A two-

218

stage modulator for cryogenic temperatures with a 4 s modulation time transferred the

219

effluent continuously onto the second column, an Agilent DB17-MS-capillary (2.2 m x 0.18

220

mm i.d., 0.18 µm film) having permanently a 20 °C higher oven temperature compared to

221

the first column oven. The characteristic ions were monitored in the electron ionization

222

mode (MS-EI, 70 eV), measured with a frequency of 100 scans/s. Data obtained were

223

evaluated using the software GC-Image (version 2.1, Lincoln NE, USA).

224 225

LC-MS/MS measurements.

226

For amino acid quantitation, a Thermo Finnigan Surveyor HPLC-system (Egelsbach,

227

Germany) coupled to a triple-stage quadrupole mass spectrometer (TSQ Quantum,

228

Thermo Finnigan) was used. Chromatographic separation was performed on a Synergi

229

Hydro RP column (150 × 2 mm, 4 μm, 8 nm, Phenomenex, Aschaffenburg, Germany)

230

using the following conditions: solvent A, FA (0.1%, v/v) in water, solvent B, FA (0.1%,

231

v/v) in acetonitrile; gradient, 0−1 min 0% B, 1-1.1 min 0-15 % B, 1.1-20 min 15−35% B,

232

20−25 min 35-40% B, 25−29 min 40-90 % B, 39-36 min 90% B; flow rate, 0.2 mL/min; - 10 -


Page 11 of 36


233

injection volume, 10 μL; column temperature, RT. The ion source was operated in the ESI

234

positive mode using the following source parameters: spray voltage, 4000 V; sheath gas

235

pressure, 35 arbitrary units (au); aux gas pressure, 15 au; capillary temperature, 320 °C.

236

By means of multiple reaction monitoring (MRM), characteristic transitions from precursor

237

to product ions of each benzoylated amino acid were measured using experimentally

238

optimized collision energies (Table 2).

239 240

Sensory analysis. Sensory experiments were performed by a trained panel of 15-20

241

persons (male and female, aged 22 to 58 years) who had participated at weekly sensory

242

training sessions. Tests were organized in a sensory room with individual sections for

243

each panelist at RT and white light. Odor detection thresholds in starch were determined

244

as previously reported.24 Aroma compounds were added to 50 g of corn starch either

245

directly or dissolved in 50 µL water containing less than 10 mg/L ethanol and shaken in a

246

3D-shaker (Willy A. Bachofen AG, Muttenz, Switzerland) for 15 min. Reference starch

247

samples for the triangle tests were treated analogously by adding the same amount of

248

water.

249 250

Standard determinations. The water contents of the flours were determined thermo-

251

gravimetrically using an infrared moisture analyzer (MA35M, Sartorius AG, Goettingen,

252

Germany) according to the literature.25 Bread crumbs were homogenized as detailed in

253

the sample work up for aroma compound quantitation. The water content of the yeast was

254

determined by the mass difference before and after freeze drying.

255

- 11 -



256

Statistical data treatment. For the calculation of mean values and standard deviations

257

of triplicates Microsoft Office Excel 2013 (Microsoft Corporation, Seattle, Washington,

258

USA) was used.

259 260

RESULTS AND DISCUSSION

261

Differences in the aroma compounds of rice and wheat bread.

262

Recently,10 36 to 42 different aroma active compounds were identified by means of AEDA

263

in the crumb and crust of rice and wheat bread respectively. Among these, 18 odorants

264

were quantitated in samples prepared as recently reported. The selection criteria were

265

either a high FD factor (FD ≥1024) in one or both bread samples or an expected significant

266

difference of this aroma compound between wheat and rice bread (three or more FD

267

factors difference, FD ≥128). Additionally, acetic acid was analyzed because of its

268

difference in the FD factors between rice bread and rice flour. Hexanal and (Z)-2-butyl-2-

269

octenal were estimated to be important aging indicators. Some crust aroma compounds

270

reported in previous studies such as Strecker aldehydes and 2-acetyl-pyrroline didn’t meet

271

the criteria and were, therefore, not quantified.

272 273

In the wheat bread crumb and crust (Table 3) the highest contents were determined for

274

acetic acid (> 10 mg/kg), followed by 2-phenylethanol and 3-methyl-1-butanol (> 1 mg/kg),

275

2- and 3-methylbutanoic acid and 2-methyl-1-butanol (0.5-1 mg/kg). The contents of these

276

yeast metabolites and of (E,E)-2,4-decadienal were higher in wheat bread crumb

277

compared to the crust. However, maltol showed significantly higher concentrations in the

278

crust (7472 µg/kg) than in the crumb (45 µg/kg), as well as acetic acid, 2-methoxy-4-

279

vinylphenol and (E)-2-nonenal. In general the results were in agreement with the AEDA - 12 -


Page 12 of 36

Page 13 of 36


280

data of our previous study,10 and also agreed with previously published data on the aroma

281

of wheat bread.26–30

282 283

Next, OAVs were calculated for each compound. In wheat bread crumb and crust, 13 or

284

12 substances, respectively had an OAV of 1 or higher (Table 3). 3-Methylbutanoic acid

285

and 3-methyl-1-butanol were most relevant in the crumb, followed by 2-methoxy-4-

286

vinylphenol, phenylacetic acid and 2-methyl-1-butanol. The ranking of aroma compounds

287

for bread crumb and crust did not differ considerably, but the leucine metabolites were

288

more relevant in the crumb, while 2-methoxy-4-vinylphenol was of higher importance in

289

the crust. Furthermore, maltol with a caramel-like aroma reached an OAV of 5 in the wheat

290

bread crust.

291 292

Similar to wheat bread, acetic acid and the yeast metabolites also reached the highest

293

contents in the rice bread samples made from the fresh flour batch R1 (Table 4). The rice

294

bread crumb contained higher amounts of 3-methyl-1-butanol, 2-phenylethanol and 2-

295

methyl-1-butanol as well as (E,E)-2,4-decadienal than the crust. In contrast, higher

296

contents of 3-methylbutanoic acid, phenylacetic acid, maltol and of the lipid oxidation

297

products from linoleic acid31 (E)-2-nonenal and 1-octene-3-one, which are known to be

298

characteristic for the bread crust aroma,30 were present in rice bread crust.

299 300

Out of the 18 aroma compounds quantitated in the rice bread, 10 reached an OAV  1 in

301

the crumb and 9 in the crust (Table 4). In both, the crumb and the crust 3-methyl-1-butanol,

302

3-methylbutanoic acid, acetic acid, 2-methoxy-4-vinylphenol and phenylacetic acid

303

showed the highest OAVs. The OAVs confirmed the higher relevance of 3-methyl-1- 13 -



304

butanol and 2-phenylethanol for the aroma of rice bread crumb and of 1-octene-3-one,

305

acetic acid and phenylacetic acid for the aroma of the crust.

306 307

In general, rice bread samples differed from wheat bread samples due to significantly

308

higher contents of 2-aminoacetophenone, acetic acid (> 400 %) and 4- vinylphenol

309

(> 200 %) and significantly lower amounts of (E)-2-nonenal, hexanal, (E,E)-2,4-

310

decadienal, maltol (< 25 %) and -nonalactone (< 50 %) (Tables 3 and 4). Compared to

311

wheat, the rice bread crumb contained higher levels of alcohols known to be formed by

312

the metabolic activity of yeast, in particular 3-methyl-1-butanol, and lower concentrations

313

of 2- and 3-methylbutanoic acid. A partially opposite, but less pronounced difference was

314

found for the bread crusts. 1-Octene-3-one had higher and 2-methoxy-4-vinylphenol had

315

lower concentrations in the rice bread crust than in the wheat bread crust. The impact of

316

these differences on the overall aroma can be derived from a comparison of OAVs (Tables

317

3 and 4). Accordingly, 2-aminoacetophenone (grape-like, medicinal) is characteristic for

318

rice bread since its concentrations in wheat bread samples were below the odor detection

319

threshold. This odorant is known as a key aroma compound of cooked rice occurring in

320

concentrations of 0.8-1.5 µg/kg (up to 5 µg/kg dry matter).22 The concentrations in the rice

321

bread samples strongly exceeded the literature data. In addition, 3-methyl-1-butanol in

322

rice bread crumb, 3-methylbutanoic acid and 1-octene-3-one in rice bread crust as well

323

as acetic acid in both can be suggested to be responsible for the differences to wheat

324

bread. The grassy, green and fatty smelling aldehydes hexanal, (E)-2-nonenal and (E,E)-

325

2,4-decadienal only reached relevant OAVs in the wheat bread samples, the caramel-like

326

maltol and the smoky, clove-like 2-methoxy-4-vinylphenol reached much higher OAVs in

327

the wheat bread crust than in rice bread crust. - 14 -


Page 14 of 36

Page 15 of 36


328 329

Formation of aroma compounds by yeast metabolism.

330

Since the same type and amount of yeast was used for rice and wheat bread making, it

331

was assumed that differences in aroma compounds formed by yeast were caused by

332

differences in the amino acid composition of the raw materials. Therefore, the contents of

333

the free amino acids isoleucine, leucine and phenylalanine, the precursors of 2- and 3-

334

methylbutanol, 2-phenylethanol and their corresponding acids, were determined. The

335

contents differed significantly between rice flour, wheat flour and yeast (Figure 1). The

336

concentrations of amino acids in wheat flour confirmed previous results of Opperer32. But,

337

compared to wheat flour, rice flour contained only 3-13 % of each free amino acid, and in

338

particular, the isoleucine content was comparatively low. However, yeast could be

339

considered as an important source of amino acids even though its percentage in the

340

dough was very low (about 5.4 mg yeast dry matter/kg dough).

341 342

During soaking the free amino acid contents in the wheat flour suspension was increased

343

by 266%, 184% and 175% for leucine, phenylalanine and isoleucine (Figure 2 A). This

344

might have been the result of peptidases activated in the presence of water as described

345

for rice kernels by Saikusa et al..33 In contrast, no relevant increase of the free amino acid

346

content was observed for rice flour after soaking. Obviously, the enzymatic activity of the

347

rice flour was very low and indicated a low content of the enzyme-active pericarp fraction

348

in the flour.33

349 350

The amino acid contents of yeasted doughs as affected by the fermentation time are

351

shown in Figure 2 B. During mixing of wheat dough, the release of free amino acids in - 15 -



352

wheat flour exceeded the consumption by yeast so that the initial contents of leucine and

353

phenylalanine increased slightly. The isoleucine content remained constant. The higher

354

temperature during the subsequent fermentation increased the yeast activity and a decline

355

of all amino acids was observed. In contrast to wheat dough, the amino acid supply in the

356

rice dough was lower, and there was no release of free amino acids from the rice flour

357

during soaking. This resulted in a very fast decline of 50 % of the amino acids after mixing

358

and of 75 % after the first quarter of fermentation, but not in a total metabolization.

359 360

The data enabled the calculation of the total available free amino acid concentrations in

361

the doughs and the comparison with the content of aroma-active yeast metabolites in the

362

bread crumbs. The contents based on dry matter (wheat flour: 86.4%; rice flour: 88.6%,

363

wheat bread crumb: 56.5 %, rice bread crumb: 51.7 %) are shown in Figure 3. In wheat

364

dough (Figure 3 A) the availability of leucine was higher than of isoleucine and

365

phenylalanine resulting in higher amounts of leucine metabolites in the wheat bread

366

crumb. However, metabolic rates of all amino acids were below 50 %. As described in the

367

literature, a preferred formation of the alcohol was observed for all amino acids.14,32 The

368

rice-based model dough (Figure 3 B) gave opposite results. Although the contents of free

369

amino acids were similar, considerable differences in the concentrations of the

370

metabolites were determined. Especially high amounts of 3-methyl-1-butanol and 2-

371

phenylethanol were determined in the rice bread crumb exceeding the amount of available

372

precursors. Thus, alternative pathways of amino acid release might exist in the rice dough

373

model. Possibly, the nutrient deprivation in the rice dough system caused an increase of

374

the proteolytic activity of yeast enzymes.34,35 This might also explain the remaining free

375

amino acid concentrations in the rice dough at the end of fermentation (Figure 2 B). - 16 -


Page 16 of 36

Page 17 of 36


376 377

Retention of aroma compounds from rice flour in rice bread.

378

To investigate the retention of aroma compounds from rice flour to rice bread, the contents

379

of 12 selected substances were determined in the fresh rice flour R1 (Table 5). Compared

380

to the analysis of bread, the Ehrlich Pathway products that were formed during dough

381

fermentation were not considered in the flour. The overall aroma intensity of the rice flour

382

was low and only four compounds reached an OAV > 1. Acetic acid (OAV 42) occurred

383

with the highest concentration and OAV in the rice flour followed by hexanal (OAV 2). For

384

both compounds, and additionally for (E)-2-nonenal (OAV 1) and (Z)-2-butyl-2-octenal,

385

lower contents were determined in the rice bread samples (Table 4) so that a retention of

386

these aroma compounds can be assumed. 1-Octene-3-one (OAV 2) had a similar

387

concentration in the flour and the bread. Although the other odorants occurred in lower

388

concentrations in the flour than in the bread, all of them were detectable in the flour.

389 390

Influence of the rice flour age on the rice bread aroma compounds

391

In our previous study we have shown a high impact of storage of rice flour on the bread

392

aroma. To verify this result, 10 relevant bread aroma compounds were quantitated in the

393

stored rice flour R2 and the corresponding bread produced thereof (Table 6). In general,

394

all aroma compounds had higher concentrations in the stored flour R2 and in the bread

395

(Table 6) compared to the flour and bread from R1 (fresh) (Tables 4 and 5). The stored

396

rice flour R2 yielded a bread crumb with 40-66% higher concentrations of the Ehrlich

397

pathway products of leucine and isoleucine, i.e., 2- and 3-methyl-1-butanol, compared to

398

bread crumb prepared from flour R1. The alcohol levels were more than 100 % higher in

399

the crust of the R2 (stored) bread than in that of R1 (fresh) (Tables 4 and 6). This points - 17 -



400

to proteolytic activity in the flour that may lead to a degradation of the flour protein to

401

peptides and free amino acids during storage. This assumption is supported by data of

402

Dhalwali et al. who studied the storage behavior of milled rice and reported an increase

403

of proteolytic activity and free amino acid contents.36 In fact, the contents of free leucine

404

and isoleucine in the stored flour R2 were only slightly higher than in R1 (data not shown).

405

Upon storage of rice flour, lipid oxidation products, especially hexanal, -nonalactone and

406

(Z)-2-butyl-2-octenal showed the greatest changes. In particular (Z)-2-butyl-2-octenal had

407

a more than 90-fold higher content in the stored rice flour R2 than in the fresh flour R1.

408

The low retention rate of this compound from flour to bread was probably due to its higher

409

reactivity. Consequently, this substance can be seen as an important aging indicator.

410

Furthermore, (E,E)-2,4-decadienal and vanillin concentrations increased in flour upon

411

storage and can also be considered as indicators of storage. The higher concentrations

412

of these compounds in the stored flour R2 were retained in the respective breads.

413 414

Table 6 contains the OAVs of the aroma compounds in rice flour R2 and in the

415

corresponding bread crumb and crust. The data showed that not all substances formed

416

during storage had an influence on the overall aroma. 3-Methyl-1-butanol and 3-

417

methylbutanoic acid showed the highest OAVs in the crumb and crust from the stored rice

418

flour R2. However, the OAVs of the isoleucine yeast metabolites did not differ between

419

the rice breads made from different aged flour. (Tables 4 and 6). Hexanal, which had an

420

OAV 1 for (E,E)-2,4-decadienal (crumb and crust) and for (E)-2-nonenal (crumb) in the

426

rice bread samples of R2.

427 428

The data on the differences between the aroma compounds of rice and wheat bread

429

crumbs and crusts are the basis for the optimization of bread quality prepared from gluten-

430

free ingredients. Since the aroma of the rice flour itself was very weak, systematic changes

431

of the bread manufacturing process would be a promising tool to regulate the formation of

432

desired odorants and to avoid off flavors. Also, additives increasing the Maillard reaction

433

might improve the aroma of the rice bread crust. Furthermore, the flour age was found to

434

be an important factor for the sensory quality of rice bread. Because 2-

435

aminoacetophenone, a characteristic aroma compound of the rice bread, was not present

436

as odor-active compound in wheat bread, it can be assumed to be an off-flavor compound.

437

To confirm this assumption, further studies are underway.

438 439

ACKNOWLEDGMENT

440

This IGF project of the FEI was supported via AiF within the program for promoting the

441

Industrial Collective Research (IGF) of the German Ministry of Economics and Energy

442

(BMWi), based on a resolution of the German Parliament. Project CORNET AiF 147 EN

443

“GLUeLESS”. The authors wish to thank Stefanie Hackl for her excellent technical

444

assistance and Patrick Roehrl for carrying out the amino acid analyses.

445

- 19 -



446

ABBREVIATIONS USED

447

CI, chemical ionization; EI, electron ionization; FA, formic acid, FD, flavor dilution; FID,

448

flame ionization detector; OAV, odor activity value; RI, retention index; RT, room

449

temperature; SAFE, solvent assisted flavor evaporation; SIDA, stable isotope dilution

450

analysis

- 20 -


Page 20 of 36

Page 21 of 36


451

REFERENCES

452

(1)

Masure, H. G.; Fierens, E.; Delcour, J. A. Current and Forward Looking

453

Experimental Approaches in Gluten-Free Bread Making Research. J. Cereal Sci.

454

2015, 67, 92–111.

455

(2)

Yano, H.; Fukui, A.; Kajiwara, K.; Kobayashi, I.; Yoza, K. ichi; Satake, A.;

456

Villeneuve, M. Development of Gluten-Free Rice Bread: Pickering Stabilization as

457

a Possible Batter-Swelling Mechanism. LWT - Food Sci. Technol. 2017, 79, 632–

458

639.

459

(3)

Park, J. H.; Kim, D. C.; Lee, S. E.; Kim, O. W.; Kim, H.; Lim, S. T.; Kim, S. S.

460

Effects of Rice Flour Size Fractions on Gluten Free Rice Bread. Food Sci.

461

Biotechnol. 2014, 23 (6), 1875–1883.

462

(4)

463 464

Kim, M.; Yun, Y.; Jeong, Y. Effects of Corn, Potato, and Tapioca Starches on the Quality of Gluten-Free Rice Bread. Food Sci. Biotechnol. 2015, 24 (3), 913–919.

(5)

Feizollahi, E.; Mirmoghtadaie, L.; Mohammadifar, M. A.; Jazaeri, S.; Hadaegh, H.;

465

Nazari, B.; Lalegani, S. Sensory, Digestion, and Texture Quality of Commercial

466

Gluten-Free Bread: Impact of Broken Rice Flour Type. J. Texture Stud. 2018, 49

467

(4), 395–403.

468

(6)

Heller, L. Commercial Aspects of Gluten-Free Products. In Gluten-Free Food

469

Science and Technology; Gallagher, E., Ed.; Wiley-Blackwell: Oxford, UK, 2009;

470

Vol. 1, pp 90–106.

471

(7)

472 473 474

Scherf, K. A.; Koehler, P.; Wieser, H. Gluten and Wheat Sensitivities - An Overview. J. Cereal Sci. 2016, 67, 2–11.

(8)

Pico, J.; Bernal, J. L.; Gómez, M. Influence of Different Flours and Starches on Gluten-Free Bread Aroma. J. Food Sci. Technol. 2017, 54 (6), 1433–1441. - 21 -



475

(9)

Hager, A.-S.; Wolter, A.; Czerny, M.; Bez, J.; Zannini, E.; Arendt, E. K.; Czerny, M.

476

Investigation of Product Quality, Sensory Profile and Ultrastructure of Breads

477

Made from a Range of Commercial Gluten-Free Flours Compared to Their Wheat

478

Counterparts. Eur. Food Res. Technol. 2012, 235 (2), 333–344.

479

(10) Boeswetter, A. R.; Scherf, K. A.; Schieberle, P.; Koehler, P. Identification of the

480

Key Aroma Compounds in Gluten-Free Rice Bread. J. Agric. Food Chem. 2019,

481

67 (10), 2963–2972.

482

(11) Schieberle, P. New Developments in Methods for Analysis of Volatile Flavor

483

Compounds and Their Precursors. In Characterization of Food: Emerging

484

Methods; Gaonkar, A. H., Ed.; Elsevier: Amsterdam, the Netherlands, 1995; pp

485

403–431.

486

(12) Schieberle, P.; Hofmann, T. Auf Den Geschmack Gekommen: Die Molekulare

487

Welt Des Lebensmittelgenusses. Chemie Unserer Zeit 2003, 37 (6), 388–401.

488 489 490

(13) Zhou, Z.; Robards, K.; Helliwell, S.; Blanchard, C. Ageing of Stored Rice : Changes in Chemical and Physical Attributes. J. Cereal Sci. 2001, 33, 18–26. (14) Czerny, M.; Schieberle, P. Labelling Studies on Pathways of Amino Acid Related

491

Odorant Generation by Saccharomyces Cerevisiae in Wheat Bread Dough. In

492

Flavor Science: Recent advances and trends; 2006; pp 89–92.

493

(15) Gassenmeier, K.; Schieberle, P. Potent Aromatic Compounds in the Crumb of

494

Wheat Bread (French-Type) - Influence of Pre-Ferments and Studies on the

495

Formation of Key Odorants during Dough Processing. Z. Lebensm. Unters.

496

Forsch. 1995, 201 (3), 241–248.

497 498

(16) Kubícková, J.; Grosch, W. Quantification of Potent Odorants in Camembert Cheese and Calculation of Their Odour Activity Values. Int. Dairy J. 1998, 8 (1), - 22 -


Page 22 of 36

Page 23 of 36

499 500


17–23. (17) Lin, J.; Welti, D. H.; Arce Vera, F.; Fay, L. B.; Blank, I. Synthesis of Deuterated

501

Volatile Lipid Degradation Products to Be Used as Internal Standards in Isotope

502

Dilution Assays. 2. Vinyl Ketones. J. Agric. Food Chem. 1999, 47 (7), 2822–2829.

503

(18) Guth, H.; Grosch, W. Deterioration of Soya-Bean Oil : Quantification of Primary

504

Flavour Compounds Using a Stable Isotope Dilution Assay. Leb. + Technol. 1990,

505

23 (6), 513–522.

506 507 508

(19) Fickert, B. Untersuchungen zur Bildung von Aromastoffen bei der Mälzung von Getreide, PhD Thesis, Technische Universität München, 1999. (20) Schütt, J.; Schieberle, P. Quantitation of Nine Lactones in Dairy Cream by Stable

509

Isotope Dilution Assays Based on Novel Syntheses of Carbon-13-Labeled γ-

510

Lactones and Deuterium-Labeled δ-Lactones in Combination with Comprehensive

511

Two-Dimensional Gas Chromatography with Time-of-Flig. J. Agric. Food Chem.

512

2017, 65 (48), 10534–10541.

513

(21) Kiefl, J. Differentiation of Hazelnut Cultivars (Corylus Avellana L.) by

514

Metabolomics and Sensomics Approaches Using Comprehensive Two-

515

Dimensional Gas Chromatography Time-of-Flight Mass Spectrometry

516

(GCxGC/TOF-MS), Dissertation, Technische Universität München, 2013.

517

(22) Jezussek, M. Zur Aromabildung Beim Kochen von Naturreis (Oryza Sativa L.)

518

Sowie Blättern von Pandanus Amaryllifolius Roxb., Dissertation, Technische

519

Universität München, 2002.

520

(23) Engel, W.; Bahr, W.; Schieberle, P. Solvent Assisted Flavour Evaporation - a New

521

and Versatile Technique for the Careful and Direct Isolation of Aroma Compounds

522

from Complex Food Matrices. Eur. Food Res. Technol. 1999, 209 (3–4), 237–241. - 23 -



523

(24) Czerny, M.; Christlbauer, M.; Christlbauer, M.; Fischer, A.; Granvogl, M.; Hammer,

524

M.; Hartl, C.; Hernandez, N. M.; Schieberle, P. Re-Investigation on Odour

525

Thresholds of Key Food Aroma Compounds and Development of an Aroma

526

Language Based on Odour Qualities of Defined Aqueous Odorant Solutions. Eur.

527

Food Res. Technol. 2008, 228 (2), 265–273.

528

(25) Vogel, C.; Scherf, K. A.; Koehler, P. Effects of Thermal and Mechanical

529

Treatments on the Physicochemical Properties of Wheat Flour. Eur. Food Res.

530

Technol. 2018, 244 (8), 1367–1379.

531

(26) Rychlik, M.; Grosch, W. Identification and Quantification of Potent Odorants

532

Formed by Toasting of Wheat Bread. LWT - Food Sci. Technol. 1996, 29 (5–6),

533

515–525.

534

(27) Quílez, J.; Ruiz, J. a.; Romero, M. P. Relationships between Sensory Flavor

535

Evaluation and Volatile and Nonvolatile Compounds in Commercial Wheat Bread

536

Type Baguette. J. Food Sci. 2006, 71 (6).

537

(28) Kirchhoff, E.; Schieberle, P. Determination of Key Aroma Compounds in the

538

Crumb of a Three-Stage Sourdough Rye Bread by Stable Isotope Dilution Assays

539

and Sensory Studies. J. Agric. Food Chem. 2001, 49 (9), 4304–4311.

540 541 542

(29) Sahin, B. Charakterisierung von Schlüsselaromastoffen in Gegartem Hefegebäck, Dissertation, Technische Universität München, 2015. (30) Zehentbauer, G.; Grosch, W. Crust Aroma of Baguettes II. Dependence of the

543

Concentrations of Key Odorants on Yeast Level and Dough Processing. J. Cereal

544

Sci. 1998, 28 (1), 93–96.

545 546

(31) Ullrich, F.; Grosch, W. Identification of the Most Intense Volatile Flavour Compounds Formed during Autoxidation of Linoleic Acid. Z. Lebensm. Unters. - 24 -


Page 24 of 36

Page 25 of 36

547 548 549 550


Forsch. 1987, 184 (4), 277–282. (32) Opperer, C. Generierung Potenter Aromastoffe Aus Getreidemehlen Durch Mikrobielle Metabolisierung, Dissertation, Technische Universität München, 2014. (33) Saikusa, T.; Horino, T.; Mori, Y. Distribution of Free Amino Acids in the Rice

551

Kernel and Kernel Fractions and the Effect of Water Soaking on the Distribution. J.

552

Agric. Food Chem. 1994, 42 (5), 1122–1125.

553

(34) Teicherts, U.; Mechlere, B.; Muller, H.; Wolfll, D. H. Lysosomal (Vacuolar)

554

Proteinases of Yeast Are Essential Catalysts for Protein Degradation ,

555

Differentiation , and Cell Survival. 1989, 264 (27), 16037–16045.

556 557 558 559 560

(35) Bernreuther, D. Untersuchungen Zur Autophagocytose in Der Hefe Klonierung Und Charakterisierung Des SAI1 -Gens, Dissertation, Universität Stuttgart, 2002. (36) Dhaliwal, Y.; Sekhon, K.; Nagi, H. Enzymatic Activities and Rheological Properties of Stored Rice. Cereal Chemistry. 1991, pp 18–21. (37) Widjaja, R.; Craske, J. D.; Wootton, M. Changes in Volatile Components of Paddy,

561

Brown and White Fragrant Rice during Storage. J. Sci. Food Agric. 1996, 71 (2),

562

218–224.

563 564

(38) Bryant, R. J.; McClung, A. M. Volatile Profiles of Aromatic and Non-Aromatic Rice Cultivars Using SPME/GC-MS. Food Chem. 2011, 124 (2), 501–513.

565

- 25 -



566

FIGURE CAPTIONS

567

Figure 1 Contents of the free amino acids leucine (Leu), isoleucine (Ile) and phenylalanine

568

(Phe) in wheat flour (black), rice flour (R1, light grey) and yeast (dark grey) based on dry

569

matter. Standard deviations (n = 3) are given as error bars.

570 571

Figure 2 Relative changes of leucine (___), isoleucine (……) and phenylalanine (_ _)

572

contents of wheat (black) and rice flour R1 (light grey) during soaking (A) and of their

573

corresponding yeast doughs during fermentation (B). The timelines include the mixing

574

time (6 min), therefore the data points at 0, 10, 20, 30 and 40 min of fermentation are

575

shifted to the right by 6 min. Standard deviations (n = 3) are given as error bars.

576 577

Figure 3 Comparison of free amino acid contents of doughs (black) and of the resulting

578

contents of yeast metabolites (alcohols: light grey; acids: grey) in the crumb of wheat

579

bread (A) and rice bread (B) based on dry matter. Standard deviations (n = 3) are given

580

as error bars.

581

- 26 -


Page 26 of 36

Page 27 of 36


TABLES Table 1 Standard substances, labels, mass traces and calibration curves used in the stable isotope dilution assays for aroma compound quantitation substance

label Ionization mass trace (m/z) analyte standard a hexanal d12 CI 101 113 a 3-methyl-1-butanol d2 CI 71 73 b 1-octene-3-one d2,3 CI 127 129,130 b (E)-2-nonenal d2 CI 123 125 c (Z)-2-butyl-2-octenal d20 EI 139 152 a 3-methylbutanoic acid d9 CI 117 126 a (E,E)-2,4-decadienal d4,5 CI 153 157,158 b 2-phenylethanol d4,5 CI 105 109,110 13 a maltol C2 EI 126 128 13 c -nonalactone C2 EI 85 87 13 c 2-methoxy-4C6 EI 150 156 vinylphenol 2-aminoacetophenone d3 EIc 135 138 c 4-vinylphenol d4 EI 120 124 13 c phenylacetic acid C2 EI 136 138 13 c vanillin C6 EI 151 157

calibration curve slope intercept R2 0.965 -0.119 0.999 1.137 -0.023 1.000 0.723 -0.019 1.000 0.848 -0.008 1.000 0.898 -0.085 0.992 0.917 0.011 0.995 0.893 -0.051 1.000 0.873 0.029 0.999 1.489 -0.086 0.995 0.866 0.086 0.995 0.875 0.116 0.991 0.916 1.268 1.050 1.230

0.013 -0.078 -0.050 0.029

0.997 0.999 1.000 0.997

analysis using the HRGC-MS system; b analysis using the 2D “heartcut”-GCxGC-MS system; c analysis using the GCxGC-TOF-MS system; CI, chemical ionization using methanol as reaction gas; EI, electron ionization (70 eV) a

- 27 -



Page 28 of 36

Table 2 Multiple reaction monitoring (MRM) transitions and calibration curves for amino acid quantitation by LC-MS/MS amino acid leucine

label

MRM transitionsa

-

236.1 → 105.1 (18 V) 236.1 → 190.1 (8 V) 242.1 → 105.1 (18 V) 242.1 → 195.1 (8 V) 236.1 → 105.1 (18 V) 236.1 → 190.1 (8 V) 242.1 → 105.1 (18 V) 242.1 → 195.1 (8 V) 270.1 → 105.1 (18 V) 270.1 → 224.1 (8 V) 275.1 → 105.1 (18 V) 275.1 → 229.1 (8 V)

13C

isoleucine

6

13C 6 phenylalanine d5 a Characteristic

calibration curve slope intercept R2 0.783 0.038 1.000 0.820

0.045

1.000

1.061

0.053

1.000

MRM transitions, quantifiers are given in bold, fragmentation potentials are given in

parentheses

- 28 -


Page 29 of 36


Table 3 Concentrations, odor thresholds (OT) and odor activity values (OAV) of selected aroma compounds in wheat bread crumb (WBb) and crust (WBt) odorant 3-methylbutanoic acid 3-methyl-1-butanol 2-methoxy-4-vinylphenol phenylacetic acid 2-methylbutanoic acid 2-phenylethanol 1-octene-3-one acetic acid hexanal (E)-2-nonenal (E,E)-2,4-decadienal 2-methyl-1-butanol vanillin maltol 2-aminoacetophenone -nonalactone 4-vinylphenol (Z)-2-butyl-2-octenal

concentration [µg/kg]a OTb WBb WBt [µg/kg] 900.00 622.00 13.00 2711.00 1509.00 78.00 74.00 113.00 4.60 330.00 302.00 23.00 1016.00 638.00 77.00 2715.00 1712.00 470.00 0.90 0.99 0.23 97863.00 154199.00 30000.00 111.00 132.00 42.00 19.00 32.00 12.00 102.00 33.00 48.00 899.00 553.00 763.00 336.00 167.00 440.00 45.00 7472.00 1400.00 1.30 n.d. .0 2.90 96.00 72.00 3300.00 15.00 18.00 2100.00 d n.d. n.d.d 5400.00

a mean

OAVc WBb 69 35 16 14 13 6 4 3 3 2 2 1 1 .00

Quantitative analyses of key odorants and their precursors reveal

Recommend Documents