Characterization of Potent Aroma Compounds in Preserved Egg Yolk

May 23, 2018 - Nine compounds were identified for the first time: (E,Z)-2,6-nonadienal, (E)-2-nonenal, 2-methylbutanal, dimethyl disulfide, trimethyla...
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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.

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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

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INTRODUCTION

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

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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

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

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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

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

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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

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

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

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

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

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

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during production.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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components were extracted by a vacuum distillation method and analyzed by

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GC-MS.

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2,6-dimethylpyrazine was the most abundant.13

The

results

showed

29

volatile

components

in

PE,

and

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

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

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

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

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

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

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compounds

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

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

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

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threshold 14) of the main odorants.

and

screen

the

main

odorants

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PEY

by

gas

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MATERIALS AND METHODS

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Materials

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

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

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

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

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

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

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

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

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at 4°C.

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Chemicals

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

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

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2-decanone

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2,6-dimethylpyrazine

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2-ethyl-3,5-dimethylpyrazine

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

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

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

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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%),

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indole

(98%), (99%),

(99.50%),

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

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

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

90

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

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

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

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

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

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

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

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

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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,

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

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

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

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

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

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

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synthesized according to reference.15

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Sensory Aroma Profile Analysis

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

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

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

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

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

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

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

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

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

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

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

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

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of the samples.16

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SPME Extraction of Volatile Components

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

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

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

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A

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

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

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

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GC-O analysis.

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Direct Solvent Extraction Combined with Solvent-Assisted Flavor Evaporation

2-cm,

50/30-µm

divinylbenzene/carboxen/polydimethylsiloxane

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fiber

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(DSE-SAFE)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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GC-MS Analysis

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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

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

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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

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

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

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

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

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

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

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range.

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GC-Olfactometry-FID Analysis

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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aroma, an odor-active location was identified.

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

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

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

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

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

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

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Aroma Extract Dilution Analysis

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

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

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

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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

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

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

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

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

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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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Quantitation of Selected Odor-active Compounds

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Unambiguously identified odor-active compounds were quantitated by

200

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

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

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

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

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

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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

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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

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

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

213

and the added amounts, their IEFs were determined.

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

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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

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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,

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

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

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

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

228

were determined as above.

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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.

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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

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

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

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Odor-Active Compounds Detected by GC-O

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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

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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

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(E)-2-nonenal,

and

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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

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

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

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results are shown in Table 2.

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

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

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

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

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

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

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

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(fruit, fatty) with

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benzeneacetaldehyde with an FD factor of 64.

an FD factor of 128 and dimethyl

trisulfide and

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

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

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

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

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

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

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

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

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

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

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

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

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

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different extraction methods.

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Quantitation of the Odor-Active Compounds

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

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

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standard curves. The results are shown in Table 3.

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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compounds (more than 92%).

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OAVs

401

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

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

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water, and the results are listed in Table 4.

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

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

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

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

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a high FD factor (2048).

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

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

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

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

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

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

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

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

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

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diisopropyl disulfide and diethyl disulfide were identified as volatile compounds and

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potent odorants in PEY for the first time.

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

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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

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

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

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

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