Characterization of Potent Aroma Compounds in Preserved Egg Yolk

Materials Science and Mechanical Engineering, Beijing Technology and Business University, Beijing 100048 , China ... Publication Date (Web): May 2...
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Article Cite This: J. Agric. Food Chem. 2018, 66, 6132−6141

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Characterization of 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⊥ †

Beijing Advanced Innovation Center for Food Nutrition and Human Health, ‡Beijing Laboratory for Food Quality and Safety, Beijing Key Laboratory of Flavor Chemistry, and ⊥School 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 (PEY), volatile compounds were isolated by headspace solid-phase microextraction and solvent-assisted flavor evaporation. Gas chromatography−olfactometry (GC−O) and gas chromatography−mass spectrometry (GC−MS) analyses identified a total of 53 odor-active compounds by comparing the odor characteristics, MS data, and retention indices with those of reference compounds. Twenty-seven odorants were detected in at least five isolates that were extracted and analyzed by the same method, and their flavor dilution (FD) factors, ranging from 1 to 2048, were measured by aroma extract dilution analysis (AEDA). To further determine their contribution to the overall aroma profile of PEY, 22 odorants with FD factors ≥16 and GC−MS responses were quantitated, and their odor activity values (OAVs) were calculated. According to the OAV results, 19 odorants with OAVs ≥ 1 are the potent odorants that greatly contribute to the characteristic aroma of PEY. Nine compounds were identified for the first time: (E,Z)-2,6-nonadienal, (E)-2-nonenal, 2methylbutanal, dimethyl disulfide, trimethylamine, methional, dimethyl trisulfide, diisopropyl disulfide, and diethyl disulfide. KEYWORDS: preserved egg yolk, GC−O, odor-active compounds, AEDA, quantitative measurements, OAV, potent aroma compounds

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INTRODUCTION Preserved egg (PE; also known as pidan, century egg, thousandyear egg, or songhuadan) is a unique Chinese food. In China, the history of manufacturing PE can be traced back two thousand years.1 Because PE has a special flavor, taste, and texture and promotes appetite, it is consumed by consumers in more than 30 countries around the world.2 PE is produced by pickling eggs in a mixture of water, sodium carbonate, calcium oxide, sodium chloride, and black tea for 4−6 weeks at room temperature.3 During pickling, calcium oxide reacts with water to form calcium hydroxide; thus, the resulting solution contains sodium, calcium, hydroxide, and carbonate ions. Some ions enter the egg and cause physical and chemical changes in the egg white and yolk, and these changes lead to the distinctive properties of PE such as its special flavor, amber and transparent egg whites, and dark green yolks.2 To preserve eggs, especially in the summer, one of the most popular egg processing routes produces PE. The eggs from hen, duck, and quail can be used to produce PE. However, duck eggs are more suitable than hen and quail eggs because their shells are thicker and not easily destroyed during production. Currently, research on PE has mainly focused on production technology,4 inorganic element determination in PE,2 microstructure changes,3 and analysis of nutritional ingredients.5,6 Flavor is an important sensory attribute of food; however, only a few studies exist about volatile compounds in PE. Chi-Tang Ho’s group first reported volatile compounds in PE. PE samples were smashed, and then, the volatile compounds in PE were isolated by simultaneous distillation extraction (SDE) and analyzed by gas chromatography−mass spectrometry (GC− © 2018 American Chemical Society

MS) with two different polarity columns. A total of 67 compounds was identified.7,8 Yan Zhao’s team separately isolated the volatile compounds in the yolk and egg white by SDE and analyzed the compounds by GC−MS. A total of 74 components in the yolk and 26 compounds in the egg white were identified.9,10 Huiping Liu’s group extracted the volatile constituents of PE from different sources by solid-phase microextraction (SPME); 50 compounds were identified.11,12 Chiu-Wen Lai reported differences in the volatile compounds from xiandan (egg pickled with salt) and PE; the volatile components were extracted by a vacuum distillation method and analyzed by GC−MS. The results showed 29 volatile components in PE, and 2,6-dimethylpyrazine was the most abundant.13 In these reports, SDE and SPME were used as the extraction methods. The volatile compounds in PE were identified only by MS, and the main aim of those studies was identification of the volatile components and not their contribution to the aroma of PE. Therefore, the most significant or unique aroma compounds in PE are still not known. Because the odor-active compounds are mainly present in preserved egg yolk (PEY), the main purposes of this study were (i) to identify the odoractive compounds and screen the main odorants in PEY by gas chromatography−olfactometry (GC−O), (ii) to quantitate the odor-active components, and (iii) to determine the potent Received: Revised: Accepted: Published: 6132

March 15, 2018 May 22, 2018 May 23, 2018 May 23, 2018 DOI: 10.1021/acs.jafc.8b01378 J. Agric. Food Chem. 2018, 66, 6132−6141

Article

Journal of Agricultural and Food Chemistry

prepared sample was placed into a thermostatic water bath and equilibrated for 20 min at 65 °C. A 2 cm, 50/30-μm divinylbenzene/ carboxen/polydimethylsiloxane fiber (DVB/CAR/PDMS; Supelco, Bellefonte, PA, USA) was exposed to the sample headspace for 30 min under the same conditions.17 After extraction, the fiber was transferred to the injector port and desorbed for 5 min at 250 °C for the GC−MS or GC−O analysis. Direct Solvent Extraction Combined with Solvent-Assisted Flavor Evaporation (DSE-SAFE). DSE-SAFE was used to extract the volatile compounds in PEY. The yolk was completely separated from the egg white and cut into small cubes of approximately 0.5 cm3 with a knife. The cubes were then frozen in liquid nitrogen for 5 min and finely ground with a blender for 30 s two times. The obtained PEY powder (40 g) was extracted with dichloromethane (DC) or a mixture of ethyl ether and pentane (EP) at a volume ratio of 1:1.2 (100 mL × 1, 50 mL × 3) by vigorous stirring for 0.5 h at room temperature. The mixture was centrifuged at 3810g (i.e., 8000 rpm) at 4 °C for 10 min before the solvent extract was collected. The extracts were combined and then the volatile compounds were isolated by means of the SAFE technique at 2.5 × 10−5 mbar (Edwards TIC Pumping Station from BOC Edwards, England).18 The distillate was dried over anhydrous sodium sulfate, concentrated to 5 mL with a Vigreux column (50 cm), and further concentrated under a gentle nitrogen stream to a final volume of approximately 200 μL. The concentrated fraction was stored at −20 °C prior to GC−MS and GC−O analysis. GC−MS Analysis. GC−MS analyses were performed on an Agilent 7890B GC equipped with an Agilent 5975 mass selective detector (MSD). The concentrated distillate (1 μL) was injected into the injection port, and splitless mode was used. Samples were analyzed on both a DB-Wax column (30 m × 0.25 mm i.d × 0.25 μm film, Agilent Technologies) and an HP-5 column (30 m × 0.25 mm i.d × 0.25 μm film, Agilent Technologies), and the injector port was held at 250 °C. The column carrier gas was helium at a constant flow rate of 1 mL/ min. The oven temperature was held at 33 °C for 2 min, increased to 100 °C at a rate of 4 °C/min, ramped to 230 °C at a rate of 10 °C/ min, and finally held at 230 °C for 10 min. Mass spectra in election ionization mode (MS-EI) were recorded with a 70 eV ionization energy, and the ion source temperature was set at 230 °C. Full-scan acquisition was used in the 33−350 amu range. GC−Olfactometry-FID Analysis. An Agilent 7890B series GC coupled with an olfactometer (ODP3 Gerstel, Germany) sniffing system (Gerstel GmbH) and a flame ionization detector (FID) (Agilent Technologies) operating as described for the GC−MS were used to locate odor-active components in the aroma isolates obtained by SPME and DSE-SAFE. Each concentrated fraction (1 μL) (or the isolates obtained by SPME) was injected in splitless mode. The GC effluent was split 1:2 between the FID (280 °C) and sniffing port with humidified air to maintain the nose sensitivity. The temperatures of the olfactory port and transfer line were kept at 220 and 250 °C, respectively. To avoid potentially not identifying odor-active compounds, a GC−O analysis of the concentrated distillate was carried out by three well-trained panelists on two columns with different polarities. All panelists were master candidates from the Beijing Key Laboratory of Flavor Chemistry at Beijing Technology and Business University. Before the GC−O analysis, the panelists were asked to smell the odors of propylene glycol solutions of reference compounds 2 h per day to recognize and describe their odor characteristics. The solutions contained different concentrations, and the training lasted for 3−4 months according to the familiarity of the panelists with the odors of these odorants. During the GC−O analysis, the panelists recorded the aroma descriptor and intensity value as well as the retention time. If two or more panelists detected the aroma, an odor-active location was identified. To identify the main odor-active compounds, 20 PEY samples were prepared. Ten samples were extracted by SPME, and five of them were analyzed by GC−O on an HP-5 column and five on a DB-Wax column. Five samples were extracted by dichloromethane combined with SAFE, and the isolates were analyzed by GC−O on HP-5 and DB-Wax columns. Five samples were treated with a mixture of EP at a

odorants contributing to the characteristic aroma of PEY by calculating the OAV values (the ratio of an odorant concentration to its odor threshold14) of the main odorants.

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MATERIALS AND METHODS

Materials. The PEs were purchased from Hubei Shendan Healthy Food Co., Ltd. (Xiaogan, China). The trademark name was Shendan. This sample was chosen for the experiment for three reasons: Shendan had the No. 1 market share in 2015 (issued by the China National Commercial Information Center in March 2016); the trademark of this sample was once the Chinese Well-known Mark (decided by the Trademark office of the State Administration for Industry and Commerce of the People’s Republic of China); and experts from this company took part in drafting Chinese National Standards for PE (GB/T 9694−2014). Before analysis, all preserved eggs were stored at 4 °C. Chemicals. The reference chemicals used for identification or quantitation were mainly obtained with purities over 95% (GC). 2Acetylthiazole (99%), decanal (97%), 2-decanone (98%), diethyl disulfide (99%), dimethyl trisulfide (98%), 2,6-dimethylpyrazine (98%), 3-ethyl-2,5-dimethylpyrazine (99%), 2-ethyl-3,5-dimethylpyrazine (99%), 2-heptanone (99%), indole (99.50%), (E,E)-2,4-nonadienal (85%), 3-methylbutanal (99%), and 2-methylpyrazine (98%) were supplied by J&K Chemical Ltd. (Beijing, China). Benzeneacetaldehyde (99%), benzyl acetate (99%), diallyl sulfide (98%), 2methylbutanal (98%), methyl phenylacetate (99%), 2-nonanone (99%), 1-octanol (99.50%), octanal (99%), (E)-2-octenal (95%), 1octen-3-one (95%), 1-pentanol (99.50%), 2-pentylfuran (98%), and trimethylamine (30% aqueous solution) were obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). Acetophenone (95%), Dlimonene (98%), heptanal (97%), undecanal (97%), D-carvone (99%), methional (96%), ethyl isobutanoate (98%), isopropyl isothiocyanate (98%), 1-octen-3-ol (98%), and 2-ethyl-6-methylpyrazine (98%) were purchased from Adamas Reagent Co., Ltd. (Shanghai, China). (E)-2Decenal (93%), dimethyl disulfide (98%), diisopropyl disulfide (95%), (E,Z)-2,6-nonadienal (98%), (E)-2-nonenal (95%), pentanal (95%), and 6-undecanone (98%) were supplied by TCI (Shanghai, China). Pyrazine (99%), (E,E)-2,4-decadienal (90%), and n-heptanol (99%) were obtained from Aladdin Reagents Co., Ltd. (Shanghai, China). αPinene (95%), benzaldehyde (95%), hexanal (95%), (E)-2-heptenal (95%), (E,E)-2,4-heptadienal (92%), and nonanal (96%) were purchased from Beijing Peking University Zoteq Co., Ltd. (Beijing, China). p-Cresol (99%), dichloromethane, ethyl ether, n-pentane, sodium chloride, and anhydrous sodium sulfate were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). Dichloromethane, ethyl ether, and n-pentane were freshly distilled before experiments. C7−C40 normal alkanes (solvent: hexane) used to calculate the retention indices (RIs) were purchased from O2si Smart Solutions (South Carolina, USA). Isopropyl isothiocyanate and isobutyl isothiocyanate were synthesized according to the literature.15 Sensory Aroma Profile Analysis. For the aroma profile analysis, 12 panelists (5 males and 7 females between the ages of 23 and 49) were recruited from the Beijing Key Laboratory of Flavor Chemistry at Beijing Technology and Business University. The panelists were trained in descriptive aroma profiling analysis (>10 h) and had participated in aroma profiling analyses of other food samples. They were trained for an additional 3 h to identify and define the descriptive terms for PEY. Five odor attributes (fishy, sulfurous, malty/nutty, earthy/mushroom, and fatty) were selected, and their intensities were rated on a ten-point linear scale from 0 (not perceivable) to 10 (strongly perceivable) by the panelists. The sensory analysis was conducted in a sensory room equipped with single booths at 21 ± 1 °C. The sample (5 g) was presented in 500 mL wash bottles with the siphon tubes removed from the caps. The bottles were covered with aluminum foil to ensure the panelists focused on the odor of the samples.16 SPME Extraction of Volatile Components. PEY (8 g) was frozen with liquid nitrogen for 5 min, finely ground with a blender, and quickly placed into a 40 mL glass vial with a silicon septum. The 6133

DOI: 10.1021/acs.jafc.8b01378 J. Agric. Food Chem. 2018, 66, 6132−6141

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Journal of Agricultural and Food Chemistry ratio of 1:1.2 combined with SAFE, and the isolates were also analyzed by GC−O on two columns. Aroma Extract Dilution Analysis. For AEDA, each concentrated isolate was diluted stepwise with dichloromethane in a series of dilutions, that is, 1:2, 1:4, 1:8...1:2048 (If the isolate was obtained by SPME, the dilution was conducted by adjusting the split ratio to 1:2, 1:4, 1:8...1:128.19). Each dilution was subjected to a GC−O analysis using a DB-Wax column under the same conditions described above until no odorant could be detected. To avoid potentially overlooking odor-active compounds, the GC−O analysis of the concentrated distillate was carried out by three well-trained panelists. Analyses were conducted three times by each panelist. The flavor dilution (FD) factor of each compound represents the maximum dilution at which the odorant can be perceived.14 Identification of each aroma compound was carried out by comparing their odors, RI, and mass spectra with those of reference standards. Quantitation of Selected Odor-Active Compounds. Unambiguously identified odor-active compounds were quantitated by constructing standard curves. Selective ion monitoring (SIM) MS was adopted. Volatile odorant isolation was carried out as previously described except that the PEY samples were first spiked with internal standards. To homogenize the internal standard in the samples, the mixtures were ground in a blender twice for 30 s each. Three compounds (trimethylamine, 2-methylbutanal, and 3methylbutanal) gave obvious responses only in the GC−MS chromatograms of the isolates obtained by SPME, and they were quantitated by SPME. Ethyl isobutanoate was used as the internal standard, and standard curves were prepared by analyzing the SPME extracts of standard solutions with different concentrations. To obtain reliable data, their isolation efficiency factors (IEFs) were measured. Because the three compounds were not identified in fresh duck egg yolk (FDEY), FDEY was used as matrix. Three reference compounds at certain concentrations were added the matrix, and they were quantitated by SPME and their standard curves. According to the quantitative results and the added amounts, their IEFs were determined. When the isolates obtained by DSE-SAFE were quantitated, ethyl isobutanoate and 6-undecanone were used as the internal standards. Three compounds (diisopropyl disulfide, E-2-octenal, and diethyl disulfide) had a high FD factor or were only identified in the EP extraction isolates; thus, they were quantitated by EP extraction combined with SAFE. The other compounds were quantitated by DC extraction combined with SAFE. The standard curves were prepared by analyzing standard solutions containing mixtures of internal standards and reference compounds with different concentrations. To measure their IEFs, the matrix was prepared. FDEY (40 g) was extracted with DC (100 mL × 1, 50 mL × 4) and EP (100 mL × 1, 50 mL × 4), respectively. The extracted FDEY was freeze-dried. After the extracts were combined, the volatiles were removed by SAFE, and the residue was obtained. The mixtures of the freeze-dried FDEY, the residue and water (62%) were used as matrix. The reference compounds at certain concentrations were added to the matrix, and they were quantitated by EP and DC extraction and their standard curves. Then their IEFs were determined as above. All standard curves were constructed by plotting the ratio of the peak area of the reference compound to that of the internal standard against their concentration ratio. All analyses were conducted in triplicate. Determination of Odor Detection Threshold in Water. To calculate the OAV values of diisopropyl disulfide and diethyl disulfide, their odor detection thresholds in water were determined according to procedure A reported in the literature.20

Figure 1. Aroma profiles of PEY.

contributing to the aroma attributes of PEY, the potent aroma compounds in PEY were investigated. Odor-Active Compounds Detected by GC−O. Because of the complexities of food matrices, the PEY volatile constituents among different PEs from the same manufacturer slightly vary. The detection frequencies (DFs) of odor-active compounds were measured in 20 samples, and the results are shown in Table 1. Table 1 showed that a total of 53 odor-active regions were detected by 30 GC−O runs of the isolates from 20 PEY samples on the HP-5 and DB-Wax columns. The structures of these compounds were identified by comparing MS data, RI values, and odor with those of reference compounds. Among the 53 odor-active compounds, 27 odorants (1−3, 7, 8, 13−15, 17, 22, 24−26, 28, 31, 33, 34, 36, 39−43, 46, 48, 51, and 52) had higher DFs in the samples, and nine odorants (8, 15, 24, 28, 33, 34, 39, 40, and 52) were detected in all 30 CG−O runs, that is, in all 20 samples. Moreover, trimethylamine was identified only in isolates obtained by SPME, and diethyl disulfide and diisopropyl disulfide were detected only in isolates obtained by extracting samples with a mixture of EP. These odorants cause PEY samples to have some common odor characteristics. The other 26 aroma compounds had lower DFs (10. Most of these compounds had been identified as volatile compounds in egg yolk,21 but only nine aldehydes, including 3-methylbutanal, hexanal, heptanal, octanal, benzeneacetaldehyde, (E)-2-octenal, nonanal, (E)-2-nonenal, and (E,E)-2,4-decadienal, had been identified as odor-active compounds.22 These aldehydes are thought to be a result of three main pathways. The first is autoxidation of unsaturated fatty acids (UFAs). PEY contains UFAs, and the mono-UFA and poly-UFA contents have been shown to decrease in PEY (42.069 mg/g and 44.703 mg/g) compared with that in fresh duck yolk (119.217 mg/g and

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RESULTS AND DISCUSSION To obtain an idea of the overall aroma of PEY, a descriptive sensory analysis was carried out. The results of the descriptive aroma analysis are summarized in Figure 1. The strongest intensity was noted for fishy note, followed by fatty, malty/ nutty, earthy, and sulfurous notes. To identify the odorants 6134

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Journal of Agricultural and Food Chemistry Table 1. Odor-Active Compounds Identified by GC−O in PEY number of samples in which odor-active compounds were detected SPMEc

RI a

no.

compound

HP-5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

trimethylamine 2-methylbutanal 3-methylbutanal pentanal pyrazine 1-pentanol dimethyl disulfide hexanal 2-methylpyrazine isopropyl isothiocyanate diallyl sulfide allyl isothiocyanate 2-heptanone heptanal methional 2,6-dimethylpyrazine diethyl disulfide α-pinene isobutyl isothiocyanate (E)-2-heptenal benzaldehyde dimethyl trisulfide 1-heptanol 1-octen-3-one 1-octen-3-ol 2-pentylfuran 2-ethyl-6-methylpyrazine octanal (E,E)-2,4-heptadienal 2-acetylthiazole diisopropyl disulfide D-limonene benzeneacetaldehyde (E)-2-octenal acetophenone 1-octanol 2-ethyl-3,5-dimethylpyrazine 3-ethyl-2,5-dimethylpyrazine 2-nonanone nonanal (E,Z)-2,6-nonadienal (E)-2-nonenal benzyl acetate methyl phenylacetate 2-decanone decanal (E,E)-2,4-nonadienal D-carvone (E)-2-decenal indole undecanal (E,E)-2,4-decadienal p-cresol

501 650 660 679 728 767 768 802 821 840 860 887 892 902 908 910 917 940 953 956 958 966 971 978 980 991 996 1003 1010 1017 1020 1027 1043 1057 1065 1071 1078 1083 1092 1104 1154 1164 1166 1179 1194 1206 1215 1263 1264 1296 1308 1318 1972

b

DC-SAFEd

EP-SAFEe

DB-Wax

odor quality

HP-5

DB-Wax

HP-5

DB-Wax

HP-5

DB-Wax

554 891 886 1016 1204 1248 1052 1065 1231 1155 1122 1321 1158 1161 1467 1292 1183 1040 1282 1288 1507 1335 1457 1271 1421 1207 1349 1260 1453 1595 1223 1175 1654 1417 1598 1524 1427 1409 1357 1361 1544 1528 1680 1709 1532 1468 1657 1799 1750 2388 1570 1657 2097

fish malty malty pungent, acrid roast fusel-like onion, radish green, fatty nutty mustard garlic-like mustard fruit, fatty fatty cooked potato roast alliaceous rosiny, pine needle-like mustard, radish fatty bitter almond-like sulfury, cabbage-like fatty mushroom mushroom green, sweet nutty, roasty citrus-like, green green roast, nutty sulfury, onion citrus floral fatty floral green roast roast fruity citrus-like, soap cucumber-like fatty, green floral floral floral fatty fatty, green caraway-like fruit, fatty fecal, jasmine aldehydic fatty, deep-fried phenolic

5 5 5 1 2 1 0 5 2 0 0 0 3 5 5 2 0 0 0 0 1 1 1 5 3 3 2 5 0 2 0 0 5 5 0 1 1 1 5 5 2 5 0 0 1 5 2 2 2 2 2 5 1

5 5 5 1 2 1 0 5 2 0 0 0 3 5 5 1 0 0 0 1 1 1 0 5 5 5 2 5 0 1 0 0 5 5 0 1 1 1 5 5 2 5 0 0 2 5 2 2 1 0 2 5 1

0 5 5 1 1 2 5 5 2 2 2 1 5 5 5 2 0 1 1 2 1 5 1 5 3 1 2 5 1 3 0 2 5 5 3 5 3 2 5 5 5 5 1 3 1 5 1 5 1 1 5 5 0

0 5 5 2 1 2 5 5 2 2 1 1 5 5 5 2 0 1 1 0 1 5 1 5 5 1 2 5 2 3 0 2 5 5 3 5 3 4 5 5 5 5 3 3 2 5 2 5 2 0 5 5 1

0 0 1 0 2 1 1 5 2 0 0 0 0 0 5 1 5 0 0 0 1 1 0 5 3 0 2 5 0 0 3 2 5 5 2 1 0 0 5 5 3 3 5 3 0 1 2 0 0 1 1 5 0

0 1 1 0 2 1 1 5 2 0 0 0 0 0 5 1 5 0 0 0 1 1 0 5 3 0 2 5 0 0 5 2 5 5 2 1 0 0 5 5 3 5 5 3 0 1 2 0 1 0 1 5 0

identificationf MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, RI, S, O MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, MS, RI, S, RI, S, O RI, S, O MS, RI, S, MS, RI, S, RI, S, O MS, RI, S, RI, S, O MS, RI, S, MS, RI, S, MS, RI, S,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O O O O O O

a Retention index of compounds on an HP-5 column. bRetention index of compounds on a DB-WAX column. cSPME means compounds isolated by solid-phase microextraction. dDC-SAFE means compounds isolated by dichloromethane combined with solvent-assisted flavor evaporation. eEPSAFE means compounds isolated by ethyl ether and pentane at a ratio of 1:1.2 combined with solvent-assisted flavor evaporation. fIdentification

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Journal of Agricultural and Food Chemistry Table 1. continued

methods: MS means identification by comparison with the NIST 11 mass spectra database; RI means confirmed by retention index; S means confirmed by authentic standards; O means confirmed by aroma descriptor.

Figure 2. Contrast in the retention time and mass spectra of diisopropyl disulfide and dipropyl disulfide.

126.284 mg/g).23 UFAs can react with oxygen to produce hydroperoxides that decompose to form linear aliphatic aldehydes (LAAs). Virtually all of these LAAs (saturated and unsaturated) were identified in the volatile components as autoxidation reaction products of oleic acid,24 linoleic acid,25 and arachidonic acid.26 The second pathway is thermal oxidation of saturated triacylglycerols. In the production of PE, calcium oxide and water react to form calcium hydroxide, and heat is released (temperature above 100 °C). Heat results in thermal oxidation of saturated triacylglycerols to produce some aldehydes.27,28 The third pathway is the Strecker degradation reaction. PEY contains amino acids;6 2-methylbutanal, 3-methylbutanal, and benzeneacetaldehyde could be the Strecker degradation products of leucine, isoleucine, and phenylalanine, respectively. In the presence of a base, the degradation product yields increased.29 Benzaldehyde can also be a degradation product of phenylalanine, but its formation is generally thought to be associated with that of phenylacetaldehyde.30 This pathway results in the concentrations of the 17 amino acids in FDEY decreasing in PE. Six ketones (13, 24, 35, 39, 45, and 48) were detected as odor-active compounds in PEY, and four ketones had high DFs (≥14). 1-Octen-3-one was also identified in PE13 and heated egg yolk;22 it was formed by autoxidation of UFAs.26 2Alkanones are derived from thermal oxidation of saturated triacylglycerols in PEY.27 D-Carvone may be from the tea used to produce PE because it has been identified as one of the flavor compounds in Jin Xuan oolong tea.31

Four alcohols (6, 23, 25, and 36) were identified as odoractive compounds in PEY, but only 1-octen-3-ol (25) and 1octanol (36) had high DFs (≥14). These alcohols were secondary products of the autoxidation of the lipids in PEY.27,32 Eight nitrogen-containing compounds (1, 5, 9, 16, 27, 37, 38, and 50), including amine, pyrazine and indole compounds, were detected as odor-active compounds, and four compounds (1, 5, 9, 27) had high DFs (≥10). Trimethylamine (TMA) was detected for the first time in PEY, and it had also been identified in fish and shrimp during storage.33 TMA results in a strong fishy smell at a low concentration, and the analysis of TMA has been used in the seafood industry to evaluate the freshness of seafood.34 TMA can be an off-flavor compound in PEY if its concentration exceeds a certain value. Pyrazine is mainly formed by Maillard reactions, and its formation is associated with α-amino acids, carbohydrates, and α-dicarbonyl compounds.35 PE is made by pickling duck eggs in an alkali solution; the pyrazine formation rates as well as the number of alkylpyrazines increase as the pH value and temperature increase.36 These pyrazines were identified by Chi-Tang Ho’s group,7,8 and Chiu-Wen Lai reported that 2,6-dimethylpyrazine was the most abundant volatile compound in PE.13 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 had been identified in scrambled, fresh, and whole eggs, egg yolk, egg white,21,37,38 spray-dried egg powder, accelerated freezedried egg powder,39 and fermented egg.40 Dimethyl trisulfide 6136

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Figure 3. Mass spectra of 1,2,3-trithiolane and 1,2,4-trithiolane. (a) Mass spectrum of 1,2,3-trithiolane identified in an isolate obtained by SDE. (b) Mass spectrum of 1,2,3-trithiolane in NIST11. (c) Mass spectrum of 1,2,4-trithiolane in NIST11.

increased when hens were fed feedstuff containing 10% rapeseed oil.45 Both diisopropyl disulfide and dipropyl disulfide had alliaceous, sulfurous and onion odors; their mass spectra were similar, but their retention times were different (Figure 2). The Chi-Tang Ho group identified 1,2,4-trithiolane compounds in PE,7,8 but those compounds were not detected in this experiment. The reason for this result might be that their concentrations were lower than the detection threshold or that these compounds were formed by heating during SDE. To investigate whether some sulfur-containing compounds could 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 had a sulfurous and onion

had been identified in egg yolk (not egg white) of cooked egg,41 and methional had 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 had been identified in egg yolk, and its concentration in yolk greatly 6137

DOI: 10.1021/acs.jafc.8b01378 J. Agric. Food Chem. 2018, 66, 6132−6141

Article

Journal of Agricultural and Food Chemistry

methylbutanal (malty), methional (cooked potato), octanal (citrus-like), and (E)-2-nonenal (fatty) with an FD factor of 32 and dimethyl trisulfide (sulfury) and benzeneacetaldehyde (rose, honey) with an FD factor of 16. In the isolates obtained by DC-SAFE, 1-octen-3-one had the highest FD factor of 2048, followed by hexanal and (E)-2nonenal with an FD factor of 512, 2-heptanone (fruit, fatty) with an FD factor of 128, and dimethyl trisulfide and benzeneacetaldehyde with an FD factor of 64. In the isolates obtained by EP-SAFE, 1-octen-3-one had the highest FD factor of 1024, followed by diisopropyl disulfide (alliaceous, sulfurous) with an FD factor of 512, hexanal with an FD factor of 128, and (E)-2-nonenal with an FD factor of 64. In addition, 1-octen-3-one had the highest FD factor in the three isolates, and aldehyde and sulfur-containing compounds also showed high factors. TMA (1) was easily detected in the SPME isolates; its FD factor was 32. Diethyl disulfide and diisopropyl disulfide (17 and 31) were identified in the isolate obtained using ethyl ether and n-pentane as the solvent; their FD factors were 32 and 512, respectively. (E)-2-octenal (34) was identified in three isolates, but its FD factor was higher when ethyl ether was used as solvent. Among the 27 aroma compounds, 17 odorants (7, 8, 13, 14, 22, 24, 25, 33, 36, 39, 40, 41, 42, 46, 48, 51, and 52) showed higher FD factors in the isolate obtained using dichloromethane as the solvent than that in the other isolates. Therefore, these odor-active compounds should be quantitated by different extraction methods. Quantitation of Odor-Active Compounds. To gain a deeper understanding of the aroma of PEY, a total of 22 aroma compounds with high DFs (>10) and FD factors ≥16 were quantified by constructing standard curves. The results are shown in Table 3. Among these odorants, TMA had the highest concentration (33,230 μg/kg), followed by diisopropyl disulfide (12,641 μg/ kg), 3-methylbutanal (8161 μg/kg), hexanal (5367 μg/kg), 2methylbutanal (3058 μg/kg), (E)-2-octenal (1845 μg/kg), and (E)-2-nonenal (1306 μg/kg). Because TMA had the highest concentration and a low boiling point, an obvious ammonia odor was smelled when PEY samples were cut into small cubes. TMA has not been identified as an important flavor compound in PE and PEY in published references, which may be because of two reasons. First, TMA was often eliminated during solvent removal when SDE and SAFE were used as the extraction methods. Second, TMA was overlooked when SPME was used and the initial oven temperature was at 40 °C.11,12 The concentration of diisopropyl disulfide was the second highest, which might be due to other formation pathways in addition to originating from duck feedstuff. The total concentration of aldehyde compounds was high, and the value exceeded 20 000 μg/kg. Among the aldehydes, the total concentration of saturated fatty aldehydes was greater than that of the unsaturated fatty aldehydes. The total concentration of sulfurcontaining compounds was the third highest. The quantitation results showed that the relative contents of five compounds (1, 31, 3, 8, and 2) accounted for a large proportion of the odoractive compounds (more than 92%). OAVs. To further investigate the contributions of the 22 odorants to the overall odor profile, their OAVs were calculated based on their concentrations and thresholds in water, and the results are listed in Table 4. Of the 22 odor-active compounds, 19 compounds yielded an OAV > 1, which indicated that these odorants contributed to

odor, was identified in the SDE isolate by GC−O and GC−MS (Figure 3). Four compounds containing nitrogen and sulfur were detected, that is, three isothiocyanate compounds and 2acetylthiazole with a low DF. Isothiocyanates in PEY were 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 was 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 was very high. After rapeseed oil was extracted from rapeseed, the obtained rapeseed cake was often used as an ingredient in duck feedstuff. Rapeseed cake contained several glucosinolates. Additionally, isothiocyanates degraded to give sulfide and disulfide,43 and the diisopropyl disulfide identified above might be related to the degradation of isopropyl isothiocyanate. The formation of 2-acetylthiazole was related to cysteine,46 and its concentration in duck yolk decreased in PE from that in FDEY. FD Factor of Odor-Active Compounds with Higher Frequency in PEY. To further determine the contributions of the 27 odor-active compounds with high DFs to the aroma profile of PEY, their FD factors were measured by GC−O, and the results were shown in Table 2. In the isolates obtained by SPME, hexanal (green) and 1octen-3-one (mushroom) had the highest FD factor of 64, followed by trimethylamine (fishy), 2-methylbutanal (malty), 3Table 2. FD Factors of Odor-Active Compounds with DFs ≥ 12 in PEY FD factora

a

no.

compound

SPME

1 2 3 7 8 13 14 15 17 22 24 25 26 28 31 33 34 36 39 40 41 42 43 46 48 51 52

trimethylamine 2-methylbutanal 3-methylbutanal dimethyl disulfide hexanal 2-heptanone heptanal methional diethyl disulfide dimethyl trisulfide 1-octen-3-one 1-octen-3-ol 2-pentylfuran octanal diisopropyl disulfide benzeneacetaldehyde (E)-2-octenal 1-octanol 2-nonanone nonanal (E,Z)-2,6-nonadienal (E)-2-nonenal benzyl acetate decanal D-carvone undecanal (E,E)-2,4-decadienal

32 32 32

DC-SAFE

64 4 2 32

32 32 32 512 128 8 32

16 64 4 1 32

64 2048 64 1 32

16 8

64 8 8 16 32 32 512 8 32 32 32 32

4 4 8 32 4 4 16

EP-SAFE

8 2 128

32 32 4 1024 32 8 512 32 32 1 4 8 8 64 8 4 1 16

FD factor, flavor dilution factor determined on a DB-Wax column. 6138

DOI: 10.1021/acs.jafc.8b01378 J. Agric. Food Chem. 2018, 66, 6132−6141

Article

Journal of Agricultural and Food Chemistry Table 3. Standard Curves and Concentrations of 22 Odor-Active Compounds in PEY no.

compound

extraction methoda

internal standardb

quantified ion

1 31 3 8 2 34 42 24 40 52 25 7 39 17 46 28 13 33 41 15 51 22

trimethylamine diisopropyl disulfide 3-methylbutanal hexanal 2-methylbutanal (E)-2-octenal (E)-2-nonenal 1-octen-3-one nonanal (E,E)-2,4-decadienal 1-octen-3-ol dimethyl disulfide 2-nonanone diethyl disulfide decanal octanal 2-heptanone benzeneacetaldehyde (E,Z)-2,6-nonadienal methional undecanal dimethyl trisulfide

SP EP SP DC SP EP DC DC DC DC DC DC DC EP DC DC DC DC DC DC DC DC

IS1 IS2 IS1 IS1 IS1 IS2 IS2 IS2 IS2 IS2 IS2 IS1 IS2 IS2 IS2 IS2 IS1 IS2 IS2 IS1 IS2 IS2

58 150 71 56 57 83 70 70 57 81 57 94 58 122 57 84 58 91 70 104 82 126

standard curve y y y y y y y y y y y y y y y y y y y y y y

= = = = = = = = = = = = = = = = = = = = = =

12.0850x + 0.3669 4.8390x − 14.7090 2.8147x − 0.1968 6.4797x − 1.9219 2.3431x − 0.1266 12.7000x − 0.0230 9.2276x + 0.0004 12.163x + 0.3911 0.9355x − 0.0757 3.2976x − 0.03900 1.2498x − 0.0556 1.0240x − 0.0045 0.8727x − 0.0240 1.2877x − 0.0189 1.3023x − 0.0137 1.2215x − 0.0327 0.7068x − 0.0122 1.0953x − 0.0253 6.2282x − 0.0213 1.2861x + 0.0045 0.7353x + 0.0212 1.3892x − 0.0273

R2

n

IEFc

conc. in PEYd (μg/kg)

0.9711 0.9936 0.9984 0.9921 0.9905 0.9915 0.9977 0.9980 0.9982 0.9939 0.9994 0.9595 0.9950 0.9940 0.9920 0.9946 0.9924 0.9957 0.9942 0.9963 0.9920 0.9982

5 7 5 7 5 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

0.44 0.87 0.38 0.78 0.31 0.90 0.94 0.83 0.94 0.88 0.93 0.65 0.82 0.89 1.01 0.93 0.80 0.85 0.91 0.88 1.04 0.76

33 230 12 641 8161 5367 3058 1845 1306 490 274 237 131 124 79.1 56.9 49.5 46.7 37.6 32.3 31.1 30.9 22.9 0.661

a SP means compounds extracted with SPME, DC means compounds extracted with dichloromethane combined with solvent-assisted flavor evaporation, EP means compounds extracted with ethyl ether and pentane at a ratio of 1:1.2 combined with solvent-assisted flavor evaporation. b Internal standard, IS1 refers to ethyl isobutanoate, and IS2 refers to 6-undecanone. cIEF means isolation efficiency factor. dAverage concentration of triplicate experiments revised by IEF.

the characteristic aroma of PEY. The results shown in Table 4confirmed that most of the aroma compounds with higher OAVs also had the high FD factors. 1-Octen-3-one had the highest OAV (30 625) and a high FD factor (2048). The OAV calculations showed that 1-octen-3-one, (E,Z)-2,6nonadienal, 3-methylbutanal, (E,E)-2,4-decadienal, (E)-2-nonenal, hexanal, and 2-methylbutanal were the most potent odorants contributing to the overall aroma of PEY, and their OAVs were >1000. Moderate potency odorants included (E)-2octenal, diisopropyl disulfide, diethyl disulfide, dimethyl disulfide, nonanal, trimethylamine, methional, and dimethyl trisulfide, and their OAVs ranged from 67 to 615. The other important potent odorants were decanal, octanal, 1-octen-3-ol, and benzeneacetaldehyde, and their OAVs were between 8 and 25. However, (E,Z)-2,6-nonadienal, (E)-2-nonenal, 2-methylbutanal, dimethyl disulfide, trimethylamine, methional, dimethyl trisulfide, diisopropyl disulfide, and diethyl disulfide were identified as volatile compounds and potent odorants in PEY for the first time. Comparing the OAV results with those from the aroma profile analyses reveals that the mushroom/earthy notes are from 1-octen-3-one, and 2(3)-methylbutanal imparts the malty/nutty note of PEY. The fatty note is from aldehyde compounds such as (E,E)-2,4-decadienal, (E)-2-nonenal, (E)-2octenal, and decanal. The sulfurous note is from dimethyl disulfide, diethyl disulfide, diisopropyl disulfide, and dimethyl trisulfide. Although the content of the sulfur-containing compounds was the third highest, the sulfurous note was not more intense in the descriptive sensory analyses, which may be because their total OAV is lower (