Characterization of the Potent Aroma Compounds in Preserved Egg

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Chemistry and Biology of Aroma and Taste

Characterization of the Potent Aroma Compounds in Preserved Egg Yolk by Gas Chromatography-Olfactometry, Quantitative Measurements, and Odor Activity Value Yu Zhang, Yuping Liu, Wenxi Yang, Jia Huang, Yingqiao Liu, Mingquan Huang, Baoguo Sun, and Changlin Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01378 • Publication Date (Web): 23 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018

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Journal of Agricultural and Food Chemistry

Characterization of the Potent Aroma Compounds in Preserved Egg Yolk

by

Gas

Chromatography-Olfactometry,

Quantitative

Measurements, and Odor Activity Value Yu Zhang1, Yuping Liu1*, Wenxi Yang1,2, Jia Huang1,2, Yingqiao Liu1,3, Mingquan Huang3, Baoguo Sun1, Changlin Li4 1

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing

Technology and Business University; 2Beijing Laboratory for Food Quality and Safety, Beijing Technology and Business University; 3Beijing Key Laboratory of Flavor Chemistry, Beijing Technology and Business University; 4School of Materials Science and Mechanical Engineering, Beijing Technology and Business University, Beijing 100048, China.

1

ACS Paragon Plus Environment


1

ABSTRACT: To characterize potent odor-active compounds in preserved egg yolk

2

(PEY), volatile compounds were isolated by headspace solid-phase microextraction

3

and solvent-assisted flavor evaporation. Gas chromatography-olfactometry (GC-O)

4

and gas chromatography-mass spectrometry (GC-MS) analyses identified a total of

5

53 odor-active compounds by comparing the odor characteristics, MS data, and

6

retention indices with those of reference compounds. Twenty-seven odorants were

7

detected in at least 5 isolates that were extracted and analyzed by the same method,

8

and their flavor dilution (FD) factors, ranging from 1 to 2048, were measured by

9

aroma extract dilution analysis (AEDA). To further determine their contribution to

10

the overall aroma profile of PEY, 22 odorants with FD factors ≥16 and GC-MS

11

responses were quantitated, and their odor activity values (OAVs) were calculated.

12

According to the OAV results, 19 odorants with OAVs ≥1 are the potent odorants

13

that greatly contribute to the characteristic aroma of PEY. Nine compounds were

14

identified for the first time: (E,Z)-2,6-nonadienal, (E)-2-nonenal, 2-methylbutanal,

15

dimethyl disulfide, trimethylamine, methional, dimethyl trisulfide, diisopropyl

16

disulfide and diethyl disulfide.

17

Keywords: preserved egg yolk; GC-O; odor-active compounds; AEDA; quantitative

18

measurements; OAV; potent aroma compounds

19 20

2


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21

INTRODUCTION

22

Preserved egg (PE; also known as pidan, century egg, thousand-year egg, or

23

songhuadan) is a unique Chinese food. In China, the history of manufacturing PE can

24

be traced back two thousand years.1 Because PE has a special flavor, taste, and texture

25

and promotes appetite, it is consumed by consumers in more than 30 countries around

26

the world.2 PE is produced by pickling eggs in a mixture of water, sodium carbonate,

27

calcium oxide, sodium chloride, and black tea for 4-6 weeks at room temperature.3

28

During pickling, calcium oxide reacts with water to form calcium hydroxide; thus, the

29

resulting solution contains sodium, calcium, hydroxide and carbonate ions. Some ions

30

enter the egg and cause physical and chemical changes in the egg white and yolk, and

31

these changes lead to the distinctive properties of PE, such as its special flavor, amber

32

and transparent egg whites and dark green yolks.2 To preserve eggs, especially in the

33

summer, one of the most popular egg processing routes produces PE. The eggs from

34

hen, duck and quail can be used to produce PE. However, duck eggs are more suitable

35

than hen and quail eggs because their shells are thicker and not easily destroyed

36

during production.

37

Currently, research on PE has mainly focused on production technology,4

38

inorganic element determination in PE,2 microstructure changes,3 and analysis of

39

nutritional ingredients.5,6 Flavor is an important sensory attribute of food; however,

40

only a few studies exist about volatile compounds in PE. Chi-Tang Ho’s group (1989)

41

first reported volatile compounds in PE. PE samples were smashed, and then, the

42

volatile compounds in PE were isolated by simultaneous distillation extraction (SDE) 3



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43

and analyzed by gas chromatography-mass spectrometry (GC-MS) with two

44

different polarity columns. A total of 67 compounds was identified.7,8 Yan Zhao’s

45

team separately isolated the volatile compounds in the yolk and egg white by SDE

46

and analyzed the compounds by GC-MS. A total of 74 components in the yolk and

47

26 compounds in the egg white were identified.9,10 Huiping Liu’s group extracted the

48

volatile constituents of PE from different sources by solid-phase microextraction

49

(SPME); 50 compounds were identified.11,12 Chiu-Wen Lai reported differences in

50

the volatile compounds from xiandan (egg pickled with salt) and PE; the volatile

51

components were extracted by a vacuum distillation method and analyzed by

52

GC-MS.

53

2,6-dimethylpyrazine was the most abundant.13

The

results

showed

29

volatile

components

in

PE,

and

54

In these reports, SDE and SPME were used as the extraction methods. The

55

volatile compounds in PE were identified only by MS, and the main aim of those

56

studies was identification of the volatile components and not their contribution to the

57

aroma of PE. Therefore, the most significant or unique aroma compounds in PE are

58

still not known. Because the odor-active compounds are mainly present in preserved

59

egg yolk (PEY), the main purposes of this study were (i) to identify the odor-active

60

compounds

61

chromatography-olfactometry (GC-O), (ii) to quantitate the odor-active components,

62

and (iii) to determine the potent odorants contributing to the characteristic aroma of

63

PEY by calculating the OAV values (the ratio of an odorant concentration to its odor

64

threshold 14) of the main odorants.

and

screen

the

main

odorants

4


in

PEY

by

gas

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66

MATERIALS AND METHODS

67

Materials

68

The PEs were purchased from Hubei Shendan Healthy Food Co., Ltd. (Xiaogan,

69

China). The trademark name was Shendan. This sample was chosen for the

70

experiment for three reasons: Shendan had the No. 1 market share in 2015 (issued by

71

the China National Commercial Information Center in March 2016); the trademark of

72

this sample was once the Chinese Well-known Mark (decided by the Trademark office

73

of the State Administration for Industry and Commerce of the People’s Republic of

74

China); and experts from this company took part in drafting Chinese National

75

Standards for PE (GB/T 9694-2014). Before analysis, all preserved eggs were stored

76

at 4°C.

77

Chemicals

78

The reference chemicals used for identification and/or quantitation were mainly

79

obtained with purities over 95% (GC). 2-Acetylthiazole (99%), decanal (97%),

80

2-decanone

81

2,6-dimethylpyrazine

82

2-ethyl-3,5-dimethylpyrazine

83

(E,E)-2,4-nonadienal (85%), 3-methylbutanal (99%) and 2-methylpyrazine (98%)

84

were supplied by J&K Chemical Ltd. (Beijing, China). Benzeneacetaldehyde (99%),

85

benzyl acetate (99%), diallyl sulfide (98%), 2-methylbutanal (98%), methyl

86

phenylacetate (99%), 2-nonanone (99%), 1-octanol (99.50%), octanal (99%),

(98%),

diethyl

disulfide

(98%), (99%),

(99%),

dimethyl

trisulfide

3-ethyl-2,5-dimethylpyrazine 2-heptanone

(99%),

5


indole

(98%), (99%),

(99.50%),


87

(E)-2-octenal (95%), 1-octen-3-one (95%), 1-pentanol (99.50%), 2-pentylfuran

88

(98%), and trimethylamine (30% aqueous solution) were obtained from Macklin

89

Biochemical Co., Ltd. (Shanghai, China). Acetophenone (95%), D-limonene (98%),

90

heptanal (97%), undecanal (97%), D-carvone (99%), methional (96%), ethyl

91

isobutanoate (98%), isopropyl isothiocyanate (98%), 1-octen-3-ol (98%), and

92

2-ethyl-6-methylpyrazine (98%) were purchased from Adamas Reagent Co., Ltd.

93

(Shanghai, China). (E)-2-Decenal (93%), dimethyl disulfide (98%), diisopropyl

94

disulfide (95%), (E,Z)-2,6-nonadienal (98%), (E)-2-nonenal (95%), pentanal (95%),

95

and 6-undecanone (98%) were supplied by TCI (Shanghai, China). Pyrazine (99%),

96

(E,E)-2,4-decadienal (90%) and n-heptanol (99%) were obtained from Aladdin

97

Reagents Co., Ltd. (Shanghai, China). α-Pinene (95%), benzaldehyde (95%),

98

hexanal (95%), (E)-2-heptenal (95%), (E,E)-2,4-heptadienal (92%) and nonanal

99

(96%) were purchased from Beijing Peking University Zoteq Co., Ltd. (Beijing,

100

China). p-Cresol (99%), dichloromethane, ethyl ether, n-pentane, sodium chloride

101

and anhydrous sodium sulfate were obtained from Sinopharm Chemical Reagent Co.,

102

Ltd. (Beijing, China). Dichloromethane, ethyl ether, and n-pentane were freshly

103

distilled before experiments. C7-C40 normal alkanes (solvent: hexane) used to

104

calculate the retention indices (RIs) were purchased from O2si Smart Solutions

105

(South Carolina, USA). Isopropyl isothiocyanate and isobutyl isothiocyanate were

106

synthesized according to reference.15

107

Sensory Aroma Profile Analysis

108

For the aroma profile analysis, 12 panelists (5 males and 7 females between the 6


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109

ages of 23 and 49) were recruited from the Beijing Key Laboratory of Flavor

110

Chemistry at Beijing Technology & Business University. The panelists were trained

111

in descriptive aroma profiling analysis (>10 h) and had participated in aroma

112

profiling analyses of other food samples. They were trained for an additional 3 h to

113

identify and define the descriptive terms for PEY. Five odor attributes (fishy,

114

sulfurous, malty/nutty, earthy/mushroom, and fatty) were selected, and their

115

intensities were rated on a ten-point linear scale from 0 (not perceivable) to 10

116

(strongly perceivable) by the panelists. The sensory analysis was conducted in a

117

sensory room equipped with single booths at 21±1°C. The sample (5 g) was

118

presented in 500-mL wash bottles with the siphon tubes removed from the caps. The

119

bottles were covered with aluminum foil to ensure the panelists focused on the odor

120

of the samples.16

121

SPME Extraction of Volatile Components

122

PEY (8 g) was frozen with liquid nitrogen for 5 min, finely ground with a blender

123

and quickly placed into a 40-mL glass vial with a silicon septum. The prepared

124

sample was placed into a thermostatic water bath and equilibrated for 20 min at 65°C.

125

A

126

(DVB/CAR/PDMS; Supelco, Bellefonte, PA, USA) was exposed to the sample

127

headspace for 30 min under the same conditions.17 After extraction, the fiber was

128

transferred to the injector port and desorbed for 5 min at 250°C for the GC-MS or

129

GC-O analysis.

130

Direct Solvent Extraction Combined with Solvent-Assisted Flavor Evaporation

2-cm,

50/30-µm

divinylbenzene/carboxen/polydimethylsiloxane

7


fiber


131

(DSE-SAFE)

132

DSE-SAFE was used to extract the volatile compounds in PEY. The yolk was

133

completely separated from the egg white and cut into small cubes of approximately

134

0.5 cm3 with a knife. The cubes were then frozen in liquid nitrogen for 5 min and

135

finely ground with a blender for 30 s two times. The obtained PEY powder (40 g)

136

was extracted with dichloromethane (DC) or a mixture of ethyl ether and pentane

137

(EP) at a volume ratio of 1:1.2 (100 mL×1, 50 mL×3) by vigorous stirring for 0.5 h

138

at room temperature. The mixture was centrifuged at 3810 g (i.e., 8000 rpm) at 4°C

139

for 10 min before the solvent extract was collected. The extracts were combined, and

140

then, the volatile compounds were isolated by means of the SAFE technique at 2.5×

141

10-5 mbar (Edwards TIC Pumping Station from BOC Edwards, England).18 The

142

distillate was dried over anhydrous sodium sulfate, concentrated to 5 mL with a

143

Vigreux column (50 cm) and further concentrated under a gentle nitrogen stream to a

144

final volume of approximately 200 µL. The concentrated fraction was stored at

145

-20°C prior to GC-MS and GC-O analysis.

146 147

GC-MS Analysis

148

GC-MS analyses were performed on an Agilent 7890B GC equipped with an

149

Agilent 5975 mass selective detector (MSD). The concentrated distillate (1 µL) was

150

injected into the injection port, and splitless mode was used. Samples were analyzed

151

on both a DB-Wax column (30 m × 0.25 mm i.d × 0.25 µm film, Agilent

152

Technologies) and an HP-5 column (30 m × 0.25 mm i.d × 0.25 µm film, Agilent

153

Technologies), and the injector port was held at 250°C. The column carrier gas was 8


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154

helium at a constant flow rate of 1 mL/min. The oven temperature was held at 33°C

155

for 2 min, increased to 100°C at a rate of 4°C/min, ramped to 230°C at a rate of

156

10°C/min, and finally held at 230°C for 10 min. Mass spectra in election ionization

157

mode (MS-EI) were recorded with a 70 eV ionization energy, and the ion source

158

temperature was set at 230°C. Full-scan acquisition was used in the 33-350 amu

159

range.

160

GC-Olfactometry-FID Analysis

161

An Agilent 7890B series GC coupled with an olfactometer (ODP3 Gerstel,

162

Germany) sniffing system (Gerstel GmbH) and a flame ionization detector (FID)

163

(Agilent Technologies) operating as described for the GC-MS were used to locate

164

odor-active components in the aroma isolates obtained by SPME and DSE-SAFE.

165

Each concentrated fraction (1 µl) (or the isolates obtained by SPME) was injected in

166

splitless mode. The GC effluent was split 1:2 between the FID (280°C) and sniffing

167

port with humidified air to maintain the nose sensitivity. The temperatures of the

168

olfactory port and transfer line were kept at 220°C and 250°C, respectively. To avoid

169

potentially not identifying odor-active compounds, a GC-O analysis of the

170

concentrated distillate was carried out by three well-trained panelists on two columns

171

with different polarities. All panelists were master candidates from the Beijing Key

172

Laboratory of Flavor Chemistry at Beijing Technology & Business University. Before

173

the GC-O analysis, the panelists were asked to smell the odors of propylene glycol

174

solutions of reference compounds two hours per day to recognize and describe their

175

odor characteristics. The solutions contained different concentrations, and the training 9



176

lasted for 3-4 months according to the familiarity of the panelists with the odors of

177

these odorants. During the GC-O analysis, the panelists recorded the aroma descriptor

178

and intensity value as well as the retention time. If two or more panelists detected the

179

aroma, an odor-active location was identified.

180

To identify the main odor-active compounds, 20 PEY samples were prepared. Ten

181

samples were extracted by SPME, and 5 of them were analyzed by GC-O on an HP-5

182

column and 5 on a DB-Wax column. Five samples were extracted by dichloromethane

183

combined with SAFE, and the isolates were analyzed by GC-O on HP-5 and DB-Wax

184

columns. Five samples were treated with a mixture of EP at a ratio of 1:1.2 combined

185

with SAFE, and the isolates were also analyzed by GC-O on two columns.

186

Aroma Extract Dilution Analysis

187

For AEDA, each concentrated isolate was diluted stepwise with dichloromethane in

188

a series of dilutions, i.e., 1:2, 1:4, 1:8......1:2048 (If the isolate was obtained by SPME,

189

the dilution was conducted by adjusting the split ratio to 1:2, 1:4, 1:8......1:128.19).

190

Each dilution was subjected to a GC-O analysis using a DB-Wax column under the

191

same conditions described above until no odorant could be detected. To avoid

192

potentially overlooking odor-active compounds, the GC-O analysis of the

193

concentrated distillate was carried out by three well-trained panelists. Analyses were

194

conducted three times by each panelist. The flavor dilution (FD) factor of each

195

compound represents the maximum dilution at which the odorant can be perceived.14

196

Identification of each aroma compound was carried out by comparing their odors, RI,

197

and mass spectra with those of reference standards. 10


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198

Quantitation of Selected Odor-active Compounds

199

Unambiguously identified odor-active compounds were quantitated by

200

constructing standard curves. Selective ion monitoring (SIM) MS was adopted.

201

Volatile odorant isolation was carried out as previously described except that the PEY

202

samples were first spiked with internal standards. To homogenize the internal standard

203

in the samples, the mixtures were ground in a blender twice for 30 s each.

204

Three compounds (trimethylamine, 2-methylbutanal and 3-methylbutanal) gave

205

obvious responses only in the GC-MS chromatograms of the isolates obtained by

206

SPME, and they were quantitated by SPME. Ethyl isobutanoate was used as the

207

internal standard, and standard curves were prepared by analyzing the SPME extracts

208

of standard solutions with different concentrations. To obtain reliable data, their

209

isolation efficiency factors (IEFs) were measured. Because the three compounds were

210

not identified in fresh duck egg yolk (FDEY), FDEY was used as matrix. Three

211

reference compounds at certain concentrations were added the matrix, and they were

212

quantitated by SPME and their standard curves. According to the quantitative results

213

and the added amounts, their IEFs were determined.

214

When the isolates obtained by DSE-SAFE were quantitated, ethyl isobutanoate

215

and 6-undecanone were used as the internal standards. Three compounds (diisopropyl

216

disulfide, E-2-octenal and diethyl disulfide) had a high FD factor or were only

217

identified in the EP extraction isolates; thus, they were quantitated by EP extraction

218

combined with SAFE. The other compounds were quantitated by DC extraction

219

combined with SAFE. The standard curves were prepared by analyzing standard 11



220

solutions containing mixtures of internal standards and reference compounds with

221

different concentrations. To measure their IEFs, the matrix was prepared. FDEY (40 g)

222

was extracted with DC (100 mL × 1, 50 mL × 4) and EP (100 mL × 1, 50 mL × 4),

223

respectively. The extracted FDEY was freeze-dried. After the extracts were combined,

224

the volatiles were removed by SAFE, and the residue was obtained. The mixtures of

225

the freeze-dried FDEY, the residue and water (62%) were used as matrix. The

226

reference compounds at certain concentrations were added to the matrix, and they

227

were quantitated by EP and DC extraction and their standard curves. Then their IEFs

228

were determined as above.

229

All standard curves were constructed by plotting the ratio of the peak area of the

230

reference compound to that of the internal standard against their concentration ratio.

231

All analyses were conducted in triplicate.

232

Determination of the Odor Detection Threshold in Water

233

To calculate the OAV values of diisopropyl disulfide and diethyl disulfide, their

234

odor detection thresholds in water were determined according to procedure A reported

235

in the reference.20

236 237

RESULTS AND DISCUSSION

238

To obtain an idea of the overall aroma of PEY, a descriptive sensory analysis was

239

carried out. The results of the descriptive aroma analysis are summarized in Figure 1.

240

The strongest intensity was noted for fishy note, followed by fatty, malty/nutty,

241

earthy and sulfurous notes. To identify the odorants contributing to the aroma 12


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242

attributes of PEY, the potent aroma compounds in PEY were investigated.

243

Odor-Active Compounds Detected by GC-O

244

Because of the complexities of food matrices, the PEY volatile constituents

245

among different PEs from the same manufacturer slightly vary. The detection

246

frequencies (DFs) of odor-active compounds were measured in 20 samples, and the

247

results are shown in Table 1.

248

Table 1 shows that a total of 53 odor-active regions were detected by 30 GC-O

249

runs of the isolates from 20 PEY samples on the HP-5 and DB-Wax columns. The

250

structures of these compounds were identified by comparing MS data, RI values and

251

odor with those of reference compounds. Among the 53 odor-active compounds, 27

252

odorants (1-3, 7, 8, 13-15, 17, 22, 24-26, 28, 31, 33, 34, 36, 39-43, 46, 48, 51, and 52)

253

had higher DFs in the samples, and 9 odorants (8, 15, 24, 28, 33, 34, 39, 40, and 52)

254

were detected in all 30 CG-O runs, i.e., in all 20 samples. Moreover, trimethylamine

255

was identified only in isolates obtained by SPME, and diethyl disulfide and

256

diisopropyl disulfide were detected only in isolates obtained by extracting samples

257

with a mixture of EP. These odorants cause PEY samples to have some common

258

odor characteristics. The other 26 aroma compounds had lower DFs (10. Most of these compounds have been identified as volatile compounds in

267

egg yolk,21 but only 9 aldehydes, including 3-methylbutanal, hexanal, heptanal,

268

octanal,

269

(E,E)-2,4-decadienal, have been identified as odor-active compounds.22 These

270

aldehydes are thought to be a result of three main pathways. The first is autoxidation

271

of unsaturated fatty acids (UFAs). PEY contains UFAs, and the mono-UFA and

272

poly-UFA contents have been shown to decrease in PEY (42.069 mg/g and 44.703

273

mg/g) compared with that in fresh duck yolk (119.217 mg/g and 126.284 mg/g).23

274

UFAs can react with oxygen to produce hydroperoxides that decompose to form

275

linear aliphatic aldehydes (LAAs). Virtually all of these LAAs (saturated and

276

unsaturated) were identified in the volatile components as autoxidation reaction

277

products of oleic acid,24 linoleic acid,25 and arachidonic acid.26 The second pathway is

278

thermal oxidation of saturated triacylglycerols. In the production of PE, calcium oxide

279

and water react to form calcium hydroxide, and heat is released (temperature above

280

100°C). Heat results in thermal oxidation of saturated triacylglycerols to produce

281

some aldehydes.27,28 The third pathway is the Strecker degradation reaction. PEY

282

contains amino acids;6 2-methylbutanal, 3-methylbutanal and benzeneacetaldehyde

283

could be the Strecker degradation products of leucine, isoleucine and phenylalanine,

284

respectively. In the presence of a base, the degradation product yields increased.29

285

Benzaldehyde can also be a degradation product of phenylalanine, but its formation is

benzeneacetaldehyde,

(E)-2-octenal,

nonanal,

14


(E)-2-nonenal,

and

Page 15 of 41


286

generally thought to be associated with that of phenylacetaldehyde.30 This pathway

287

results in the concentrations of the 17 amino acids in FDEY decreasing in PE.

288

Six ketones (13, 24, 35, 39, 45, and 48) were detected as odor-active compounds

289

in PEY, and four ketones had high DFs (≥14). 1-Octen-3-one was also identified in

290

PE13 and heated egg yolk;22 it is formed by autoxidation of UFAs.26 2-Alkanones are

291

derived from thermal oxidation of saturated triacylglycerols in PEY.27 D-carvone may

292

be from the tea used to produce PE because it has been identified as one of the flavor

293

compounds in Jin Xuan oolong tea.31

294

Four alcohols (6, 23, 25, and 36) were identified as odor-active compounds in

295

PEY, but only 1-octen-3-ol (25) and 1-octanol (36) had high DFs (≥14). These

296

alcohols were secondary products of the autoxidation of the lipids in PEY.27,32

297

Eight nitrogen-containing compounds (1, 5, 9, 16, 27, 37, 38, and 50), including

298

amine, pyrazine and indole compounds, were detected as odor-active compounds, and

299

four compounds (1, 5, 9, 27) had high DFs (≥10). Trimethylamine (TMA) was

300

detected for the first time in PEY, and it has also been identified in fish and shrimp

301

during storage.33 TMA results in a strong fishy smell at a low concentration, and the

302

analysis of TMA has been used in the seafood industry to evaluate the freshness of

303

seafood.34 TMA can be an off-flavor compound in PEY if its concentration exceeds a

304

certain value. Pyrazine is mainly formed by Maillard reactions, and its formation is

305

associated with α-amino acids, carbohydrates, and α-dicarbonyl compounds.35 PE is

306

made by pickling duck eggs in an alkali solution; the pyrazine formation rates as well

307

as the number of alkylpyrazines increase as the pH value and temperature increase.36 15



308

These pyrazines were identified by Chi-Tang Ho’s group,7,8 and Chiu-Wen Lai

309

reported that 2,6-dimethylpyrazine is the most abundant volatile compound in PE.13

310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329

Six sulfur-containing odor-active compounds (7, 11, 15, 17, 22, and 31) were identified. Except for diallyl sulfide, all of the compounds had high DFs and had not been reported as volatile compounds in PE and PEY. However, dimethyl disulfide has been identified in scrambled, fresh, and whole eggs, egg yolk, egg white, 21,37,38 spray-dried egg powder, accelerated freeze-dried egg powder,39 and fermented egg.40 Dimethyl trisulfide has been identified in egg yolk (not egg white) of cooked egg,41 and methional has been identified in heated egg yolk by AEDA.22 The formation of dimethyl disulfide, methional and dimethyl trisulfide is associated with the degradation of methionine.42 When duck egg is processed into PE, the methionine concentration decreases. Diallyl sulfide is formed by thermal degradation of allyl isothiocyanate, and its yield increases with increasing pH value.43 The pH value of PE is higher than 9, which is beneficial to the formation of diallyl sulfide. Diethyl disulfide is generated via L-cysteine degradation because the degradation products of L-cysteine at pH 8 contain diethyl disulfide,44 and the cysteine content in yolk in PE decreases from that in FDEY. The diisopropyl disulfide source is probably related to duck feedstuff. Dipropyl disulfide has been identified in egg yolk, and its concentration in yolk greatly increased when hens were fed feedstuff containing 10% rapeseed oil.45 Both diisopropyl disulfide and dipropyl disulfide have alliaceous, sulfurous and onion odors; their mass spectra are similar, but their retention times are different (Figure 2). The Chi-Tang Ho group identified 1,2,4-trithiolane 16


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330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350

351

compounds in PE,7,8 but those compounds were not detected in this experiment. The reason for this result may be that their concentrations were lower than the detection threshold or that these compounds are formed by heating during SDE. To investigate whether some sulfur-containing compounds can be formed during SDE, two DSE-SAFE and SED isolates were separately analyzed by GC-MS and GC-O, and the results showed some differences in the constituents of the two isolates. Moreover, 1,2,3-trithiolane, which has a sulfurous and onion odor, was identified in the SDE isolate by GC-O and GC-MS (Figure 3). Four compounds containing nitrogen and sulfur were detected, i.e., three isothiocyanate compounds and 2-acetylthiazole with a low DF . Isothiocyanates in PEY are likely associated with duck feedstuff because allyl isothiocyanate (AI) was also identified in egg yolk when the feedstuff for hens contained 10% rapeseed oil, and glucosinolate in rapeseed is thought to be precursor of AI.45 The PE used in this experiment was produced by a manufacturer from Hubei province in China, and the annual production of rapeseed in Hubei province is very high. After rapeseed oil is extracted from rapeseed, the obtained rapeseed cake is often used as an ingredient in duck feedstuff. Rapeseed cake contained several glucosinolates. Additionally, isothiocyanates degrade to give sulfide and disulfide,43 and the diisopropyl disulfide identified above may be related to the degradation of isopropyl isothiocyanate. The formation of 2-acetylthiazole is related to cysteine,46 and its concentration in duck yolk decreased in PE from that in FDEY. The FD Factor of Odor-Active Compounds with a Higher Frequency in PEY 17



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352

To further determine the contributions of the 27 odor-active compounds with high

353

DFs to the aroma profile of PEY, their FD factors were measured by GC-O, and the

354

results are shown in Table 2.

355

In the isolates obtained by SPME, hexanal (green) and 1-octen-3-one (mushroom)

356

had the highest FD factor of 64, followed by trimethylamine (fishy),

357

2-methylbutanal (malty), 3-methylbutanal (malty), methional (cooked potato),

358

octanal (citrus-like) and (E)-2-nonenal (fatty) with an FD factor of 32 and dimethyl

359

trisulfide (sulfury) and benzeneacetaldehyde (rose, honey) with an FD factor of 16.

360

In the isolates obtained by DC-SAFE, 1-octen-3-one had the highest FD factor of

361

2048, followed by hexanal and (E)-2-nonenal with an FD factor of 512, 2-heptanone

362

(fruit, fatty) with

363

benzeneacetaldehyde with an FD factor of 64.

an FD factor of 128 and dimethyl

trisulfide and

364

In the isolates obtained by EP-SAFE, 1-octen-3-one had the highest FD factor of

365

1024, followed by diisopropyl disulfide (alliaceous, sulfurous) with an FD factor of

366

512, hexanal with an FD factor of 128 and (E)-2-nonenal with an FD factor of 64.

367

In addition, 1-octen-3-one had the highest FD factor in the three isolates, and

368

aldehyde and sulfur-containing compounds also showed high factors. TMA (1) was

369

easily detected in the SPME isolates; its FD factor was 32. Diethyl disulfide and

370

diisopropyl disulfide (17 and 31) were identified in the isolate obtained using ethyl

371

ether and n-pentane as the solvent; their FD factors were 32 and 512, respectively.

372

(E)-2-octenal (34) was identified in three isolates, but its FD factor was higher when

373

ethyl ether was used as solvent. Among the 27 aroma compounds, 17 odorants (7, 8, 18


Page 19 of 41


374

13, 14, 22, 24, 25, 33, 36, 39, 40, 41, 42, 46, 48, 51, and 52) showed higher FD

375

factors in the isolate obtained using dichloromethane as the solvent than that in the

376

other isolates. Therefore, these odor-active compounds should be quantitated by

377

different extraction methods.

378

Quantitation of the Odor-Active Compounds

379

To gain a deeper understanding of the aroma of PEY, a total of 22 aroma

380

compounds with high DFs (>10) and FD factors ≥16 were quantified by constructing

381

standard curves. The results are shown in Table 3.

382

Among these odorants, TMA had the highest concentration (33,230 µg/kg),

383

followed by diisopropyl disulfide (12,641 µg/kg), 3-methylbutanal (8161 µg/kg),

384

hexanal (5367 µg/kg), 2-methylbutanal (3058 µg/kg), (E)-2-octenal (1845 µg/kg) and

385

(E)-2-nonenal (1306 µg/kg). Because TMA had the highest concentration and a low

386

boiling point, an obvious ammonia odor was smelled when PEY samples were cut

387

into small cubes. TMA has not been identified as an important flavor compound in

388

PE and PEY in published references, which may be before of two reasons. First,

389

TMA is often eliminated during solvent removal when SDE and SAFE are used as

390

the extraction methods. Second, TMA was overlooked when SPME was used and

391

the initial oven temperature was at 40 °C.11,12. The concentration of diisopropyl

392

disulfide was the second highest, which may be due to other formation pathways in

393

addition to originating from duck feedstuff. The total concentration of aldehyde

394

compounds was high, and the value exceeded 20,000 µg/kg. Among the aldehydes,

395

the total concentration of saturated fatty aldehydes was greater than that of the 19



396

unsaturated fatty aldehydes. The total concentration of sulfur-containing compounds

397

was the third highest. The quantitation results show that the relative contents of 5

398

compounds (1, 31, 3, 8, and 2) account for a large proportion of the odor-active

399

compounds (more than 92%).

400

OAVs

401

To further investigate the contributions of the 22 odorants to the overall odor

402

profile, their OAVs were calculated based on their concentrations and thresholds in

403

water, and the results are listed in Table 4.

404

Of the 22 odor-active compounds, 19 compounds yielded an OAV >1, which

405

indicated that these odorants contribute to the characteristic aroma of PEY. The

406

results shown in Table 5 confirm that most of the aroma compounds with higher

407

OAVs also have a high FD factor. 1-Octen-3-one had the highest OAV (30,625) and

408

a high FD factor (2048).

409

The OAV calculations showed that 1-octen-3-one, (E,Z)-2,6-nonadienal,

410

3-methylbutanal, (E,E)-2,4-decadienal, (E)-2-nonenal, hexanal, and 2-methylbutanal

411

were the most potent odorants contributing to the overall aroma of PEY, and their

412

OAVs were >1000. Moderate potency odorants included (E)-2-octenal, diisopropyl

413

disulfide, diethyl disulfide, dimethyl disulfide, nonanal, trimethylamine, methional,

414

and dimethyl trisulfide, and their OAVs ranged from 67 to 615. The other important

415

potent odorants were decanal, octanal, 1-octen-3-ol, and benzeneacetaldehyde, and

416

their OAVs were between 8 and 25. However, (E,Z)-2,6-nonadienal, (E)-2-nonenal,

417

2-methylbutanal, dimethyl disulfide, trimethylamine, methional, dimethyl trisulfide, 20


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418

diisopropyl disulfide and diethyl disulfide were identified as volatile compounds and

419

potent odorants in PEY for the first time.

420

Comparing the OAV results with those from the aroma profile analyses reveals

421

that the mushroom/earthy notes are from 1-octen-3-one, and 2(3)-methylbutanal

422

imparts the malty/nutty note of PEY. The fatty note is from aldehyde compounds,

423

such as (E,E)-2,4-decadienal, (E)-2-nonenal, (E)-2-octenal, and decanal. The

424

sulfurous note is from dimethyl disulfide, diethyl disulfide, diisopropyl disulfide and

425

dimethyl trisulfide. Although the content of the sulfur-containing compounds was

426

the third highest, the sulfurous note was not more intense in the descriptive sensory

427

analyses, which may be because their total OAV is lower (

Characterization of the Potent Aroma Compounds in Preserved Egg

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