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Evaluation of Key Aroma Compounds in Processed Prawns (Whiteleg Shrimp) by Quantitation and Aroma Recombination Experiments Veronika Mall, and Peter Schieberle J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00636 • Publication Date (Web): 10 Mar 2017 Downloaded from http://pubs.acs.org on March 13, 2017
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Journal of Agricultural and Food Chemistry
Evaluation of Key Aroma Compounds in Processed Prawns (Whiteleg Shrimp) by Quantitation and Aroma Recombination Experiments
Veronika Mall and Peter Schieberle#
Deutsche Forschungsanstalt für Lebensmittelchemie, Lise-Meitner-Straße 34, D85354 Freising, Germany
#
Corresponding Author Prof. Dr. Peter Schieberle Phone: +49 8161 71 2932 Fax: +49 8161 71 2970 E-mail:
[email protected] ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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2 1
ABSTRACT. In our previous study on the aroma compounds of heated prawn meat,
2
the main odorants in blanched (BPM) and fried prawn meat (FPM), respectively,
3
were characterized by means of gaschromatography/olfactometry and aroma extract
4
dilution analysis. In this follow-up study, these aroma compounds were quantified by
5
means of stable isotope dilution assays and odor activity values (OAV; ratio of
6
concentration to odor detection threshold) were calculated. Results revealed 2-
7
acetyl-1-pyrroline and (Z)-1,5-octadien-3-one as the most potent odor-active
8
compounds in both prawn samples. In FPM, as compared to BPM, higher OAVs
9
were determined for 2-acetyl-1-pyrroline, 2-acetyl-2-thiazoline, 3-methylbutanal, 3-
10
(methylthio)propanal, phenylacetaldehyde, 3-hydroxy-4,5-dimethyl-2(5H)-furanone,
11
4-hydroxy-2,5-dimethyl-3(2H)-furanone,
12
trimethylpyrazine. Aroma recombination experiments corroborated that the overall
13
aroma of the blanched as well as the fried prawn meat, respectively, could well be
14
mimicked by the set of key odorants quantitated in this study.
15
KEYWORDS. stable isotope dilution assay; odor activity value; aroma recombinate;
16
crustaceans,
prawns;
2,3-diethyl-5-methylpyrazine
litopenaeus
ACS Paragon Plus Environment
and
vannamei.
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Journal of Agricultural and Food Chemistry
3
17
INTRODUCTION
18
Crustaceans and among them, in particular prawns have become quite popular
19
in the last decades as healthy and delicious food. In order to characterize the aroma
20
compounds eliciting the desirable aroma of heated prawn meat, the main odorants of
21
blanched (BPM) and fried prawn meat (FPM) were recently identified by us based on
22
results from aroma extract dilution analysis (AEDA).1 Following this approach, forty-
23
three or forty-five odorant compounds respectively were found in BPM and FPM with
24
2-acetyl-1-pyrroline,
25
2-aminoacetophenone featuring the highest flavor dilution (FD) factors in both
26
samples. Among the set of fifteen compounds only occurring after heat treatment in
27
FPM were 2,3-diethyl-5-methylpyrazine and trimethylpyrazine, γ-octalactone and
28
γ-nonalactone as well as 2,6-dimethoxyphenol.1
(Z)-1,5-octadien-3-one,
3-(methylthio)propanal
and
29
AEDA is a useful screening method to locate aroma-active compounds in a bulk
30
of odorless volatiles. Ranking the odorants by means of FD factors gives an idea on
31
the impact of each aroma compound on the overall aroma of the investigated food
32
sample. However, as the odorants are completely vaporized during the procedure
33
and, thus, matrix effects are not taken into account, quantitation of the aroma
34
compounds and calculation of odor activity values should always follow this
35
screening approach.2,3
36
Until now, only few investigations exist aiming at quantitating the aroma
37
compounds of crustacean meat. In spiny lobster, American lobster and mangrove
38
crab, respectively, quantitation of odorants was carried out using internal standards,
39
and the OAVs of several odorants were calculated using odor thresholds available
40
from earlier publications.4-6 In these studies the highest OAVs were found for 2-
41
acetyl-1-pyrroline, 2,3-butanedione, trimethylamine, 3-(methylthio)propanal, (Z)-4-
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4 42
heptenal and 3-methylbutanal. However, exact quantitation methods using e.g. stable
43
isotope labeled isotopologues as internal standards have not yet been applied on the
44
aroma compounds of crustacean meat. Therefore, the aim of this study was to
45
continue our recent investigation1 on the key aroma compounds of blanched and
46
fried prawn meat with their quantitation by means of stable isotope dilution assays
47
and subsequent calculation of OAVs. Following, aroma recombination experiments3
48
should allow the unequivocal characterization of the main odorants provoking the
49
desired roasted and fishy odor of heated prawn meat.
50
MATERIALS AND METHODS
51
Material. Frozen whiteleg shrimp (Litopenaeus vannamei), raw with head and
52
carapace, were purchased at a local supermarket. After thawing, the prawns were
53
cooked in unsalted water for one min. The carapace was removed, the meat was
54
frozen with liquid nitrogen and minced to a fine powder. In a second set of
55
experiments, thawed prawns were pan-fried at 160 °C without addition of fat. After six
56
min of evenly frying both sides, the prawns were cooled and their carapace was
57
removed. Then, the meat was frozen with liquid nitrogen and minced to a fine
58
powder.
59
Chemicals. The following chemicals were obtained from the sources given in
60
parentheses. Dichloromethane, diethyl ether, ethanol, (99 %), sodium hydroxide,
61
anhydrous sodium sulfate (Merck, Darmstadt, Germany); thiourea (Fluka, Neu-Ulm,
62
Germany); [2H3]-methyl iodide, rhodium on alumina, methanol-d, deuterium oxide,
63
palladium on barium sulfate (5%) (Sigma-Aldrich Chemie, Taufkirchen, Germany).
64
Dichloromethane and diethyl ether were freshly distilled before use.
65 66
Reference Odorants. These were purchased from commercial sources or were synthesized as reported recently.1 ACS Paragon Plus Environment
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Isotopically Labeled Internal Standards. The isotopically labeled internal
68
standards, either labeled with deuterium or carbon-13, were synthesized as
69
previously described: [2H2–5]-2-acetyl-1-pyrroline,7 [2H4]-2-acetyl-2-thiazoline,8 [2H2-4]-
70
2-aminoaceto-phenone,9 [2H2]-butanoic acid,10 [2H2–7]-(E,E)-2,4-decadienal,11 [2H2]-γ-
71
decalactone,12 [2H2]-decanoic acid,13 [2H3–4]-2,3-diethyl-5-methylpyrazine,8 [2H3]-5-
72
ethyl-3-hydroxy-4-methyl-2(5H)-furanone,14
73
furanone,14
74
methoxybenzaldehyde,16
75
[2H2]-3-methylbutanoic
76
2,4-nonanedione,19
77
[2H2]-(E,Z)-2,6-nonadienal,11 [13C2]-(E,E,Z)-2,4,6-nonatrienal,22 [2H2]-(Z)-1,5-octadien-
78
3-one,11
79
acetaldehyde,24 [2H3]-trimethylpyrazine.25 [2H2]-(Z)-4-Heptenal was obtained from
80
Aromalab
81
[2H5-8]-2,6-dimethoxyphenol were made available by introducing deuterium via H/D
82
exchange to the unlabeled reference compound.26
[13C2]-3-hydroxy-4,5-dimethyl-2(5H)-
[13C2]-4-hydroxy-2,5-dimethyl-3(2H)-furanone,15 [2H3]-2-methoxyphenol,8 acid,17
[2H2]-1-octen-3-one,11
AG
(Planegg,
[2H2]-3-methyl-butanal,17
[2H6–8]-3-methylindole,18
[2H3]-3-(methylthio)propanal,20
[2H3]-3-methyl-
[2H3]-(E,E)-2,4-nonadienal,21
[13C2]-2,3-pentanedione,23
Germany).
[2H3]-4-hydroxy-3-
[13C2]-phenyl-
[2H1-3]-Benzothiazole
and
83
Synthesis of [2H3]-2-Acetylpyridine. [2H3]-2-Acetylpyridine was synthesized
84
according to the route outlined previously.27 2-Acetylpyridine (0.1 g; 0,83 mmol) was
85
added to a solution of 1,5,7-triazybicyclo[4.4.0]dec-5-ene (11.5 mg; 0083 mmol) in
86
chloroform-d (3 mL) which was let stir for 12 h at room temperature. The H-D-
87
exchange was quenched by the addition of hydrochloric acid (1 mL; 1 mol/L). The
88
organic layer was then washed with water (2 x 2 mL) and saturated brine (1 mL)
89
before drying over anhydrous sodium sulfate and purifying by means of SAFE-
90
distillation. Stabilization tests showed that this standard should only be used in
91
neutral conditions to avoid H-D-exchange.
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Synthesis of [2H1-3]-Benzothiazole. A mixture of benzothiazole (0,135 g; 1 mmol),
93
palladium on barium sulfate (5%; 0,25 g, equates 12,5 mg Pd) and deuterium oxide
94
(1,5 mL) were placed in a small autoclave, sealed and heated at 160 °C for seven d
95
in an aluminum heating block. Afterwards, the content of the autoclave tube was
96
filtered through a glass frit (G4), washed with deionized water (3 x 2 mL) followed by
97
diethyl ether (3 x 2 mL). After separation, the aqueous phase was extracted with
98
diethyl ether (3 x 25 mL) and the organic layers, containing the deuterated reaction
99
product were combined and dried over anhydrous sodium sulfate.27
100
Synthesis of [2H5–8]-2,6-Dimethoxyphenol. A mixture of 2,6-dimethoxyphenol
101
(0,154 g; 1 mmol), palladium on barium sulfate (5%; 0,25 g, equates 12,5 mg Pd)
102
and deuterium oxide (1,5 mL) were placed in a autoclave, sealed and heated at 160
103
°C for seven d in an aluminum heating block. Purification of the raw product was
104
carried out as described above.27
105
Synthesis of [2H3]-Methanethiol. A mixture of [2H3]-methyl iodide (2.68 g;
106
19 mmol) and thiourea (1.52 g; 20 mmol) was dissolved in ethanol/ water (1:1, by
107
vol.; 20 mL) and refluxed for 12 h. Afterwards, 100 µL of the reaction mixture was
108
added to an aqueous solution of sodium hydroxide (2 mol/L; 200 µL) in a gas tight
109
vessel sealed with a septum. After 20 min, defined headspace volumes were
110
withdrawn with a gas tight syringe and subjected to headspace-HRGC/MS (Figure 1).
111
Because of its high volatility, the isotopically labeled internal standard [2H3]-
112
methanethiol was synthesized freshly on the day of analysis.
113
Quantitation of Odorants. Freshly prepared, powdered fried or blanched prawn
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meat was mixed with diethyl ether and spiked with defined amounts of the labeled
115
internal standards depending on the respective amount of the analyte present in the
116
sample which had been estimated in a preliminary experiment. The mixture was
117
extracted for 2 h while stirring, before the extract was subjected to SAFE distillation28 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
7 118
and subsequent concentration via vigreux column and microdistillation to 200 µL.1
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For the quantitation of 3-(methylthio)propanal and 3-hydroxy-4,5-dimethyl-2(5H)-
120
furanone, the extract was divided into acidic and neutral/basic fraction prior to
121
analysis to avoid coelution of compounds.1
122
The samples were subjected to either HRGC/MS or two-dimensional HRGC-
123
HRGC/MS and the intensities of the respective ions given in Table 1 were monitored.
124
Concentrations were calculated from the relative abundances of ions selected for
125
analyte and internal standard. The data obtained were corrected by calibration
126
factors which were determined by analyzing mixtures of defined amounts of the
127
unlabeled aroma compound and the corresponding labeled standard in ratios ranging
128
from 5:1 to 1:5.
129
High Resolution Gas Chromatography/Mass Spectrometry (HRGC/MS). The
130
system described in the following was used for the quantitation of 2- and
131
3-methylbutanoic acid and decanoic acid. A Varian 3800 gas chromatograph
132
(Agilent, Waldbronn, Germany) was combined with an ion trap detector type Saturn
133
2000 (Agilent). The samples were injected cold-on-column on a DB-FFAP capillary
134
(30 m × 0.25 mm i.d.; 0.25 µm film thickness) (J&W Scientific, Folsom, CA) with a
135
starting temperature of 40 °C (held isothermally for 2 min), followed by a temperature
136
gradient of 6 °C/min up to 230 °C (held isothermally for 5 min). The characteristic
137
ions were monitored in the chemical ionization mode (MS-CI) with methanol as
138
reactant gas.
139
Two-Dimensional
High
Resolution
Gas
Chromatography/Mass
140
Spectrometry (HRGC-HRGC/MS). For the quantitation of all other odorants (except
141
methanethiol, trimethylamine and ammonia), two-dimensional HRGC-HRGC/MS was
142
applied using a Trace 2000 series gas chromatograph (Thermo Fisher Scientific,
143
Braunschweig, Germany) equipped with a moving capillary stream switching system ACS Paragon Plus Environment
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(MCSS) (Fisons Instruments, Mainz-Kastel, Germany) and linked to a second gas
145
chromatograph CP 3800 (Agilent) coupled with an ion trap mass spectrometer Saturn
146
2000 (Agilent). Samples were injected cold-on-column and after chromatography on
147
the first capillary, the analyte of interest and the internal standard were transferred
148
into a cold trap (-100 °C) by means of the MCSS system. By heating the trap, the
149
sample was transferred to the second capillary. Analytes were determined by means
150
of MS-CI with methanol as the reactant gas. In the first oven, a fused silica capillary
151
DB-FFAP (30 m × 0.32 mm i.d.; 0.25 µm film thickness) (J&W Scientific) was used in
152
combination with a DB-1701 (30 m × 0.25 mm i.d.; 0.25 µm film thickness) (J&W
153
Scientific) in the second oven. The oven temperature was held at 40 °C for 2 min,
154
then raised at 6°C/min to 230 °C and finally held for another 5 min. The cut time
155
intervals in the first dimension were determined by injection of the reference
156
compounds in a previous experiment.
157
Quantitation of Methanethiol by Means of Static Headspace Analysis.
158
Methanethiol was determined by means of SIDA of static headspace samples using
159
a headspace-HRGC/MS as described previously.29 The freshly prepared fried or
160
blanched prawn meat (20 g), was ground and put in a headspace vial (100 mL)
161
sealed with an airtight septum. The sample was spiked with a defined volume of the
162
labeled [2H3]-methanethiol (200 – 1000 µL= 0.04 – 0.21 µg) by injection through the
163
septum using a gastight syringe. After equilibration while stirring for 30 min at 40 °C
164
the sample was subjected to HS-HRGC/MS using a Trace GC Ultra (Thermo Fisher
165
Scientific) connected to an ion trap detector Varian 2100 T (Agilent). The headspace
166
sample was injected with a gas tight syringe. The gas chromatograph featured a Cold
167
Trap 915 (Thermo Fisher Scientific), cooled with liquid nitrogen, where the volatile
168
aroma compounds were trapped and co-injected air could be purged. A DB-5 thick
169
film capillary column (30 m × 0.25 mm i.d.; 1.0 µm film thickness) (J&W Scientific) ACS Paragon Plus Environment
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was held at a starting temperature of 0 °C for 2 min, before rising the temperature at
171
6 °C/min up to 100 °C, followed by a temperature gradient of 40 °C/min up to 240 °C
172
(held for 5 min).
173
Differentiation of 2- and 3-Methylbutanoic Acid. The differentiation of
174
2-methylbutanoic acid and 3-methylbutanoic acid was carried out by means of mass
175
spectrometry in the EI mode as described previously.30 Using [2H2]-3-methylbutanoic
176
acid as internal standard, the total of both isomers was quantitated by monitoring
177
mass trace m/z 85 and the standard ion m/z 87. Since 3-methylbutanoic acid
178
features a much lower odor threshold of 490 µg/kg compared to 2-methylbutanoic
179
acid (2200 µg/kg),31 a differentiation is crucial for their contribution to the overall
180
aroma. Thus, the isomer ratio was determined by means of HRGC/MS using the
181
characteristic fragments obtained by electron impact ionization (MS-EI; m/z 60 for
182
3-methylbutanoic acid and m/z 74 for 2-methylbutanoic acid, respectively) to be
183
45% 3-methylbutanoic acid and 55% 2-methylbutanoic acid.
184
Ion Chromatography. Determination of the amount of trimethylamine was
185
carried out by use of an ion chromatography system ICS-2000 with an AS
186
autosampler (Dionex, Sunnyvale, USA). Freshly prepared blanched or fried prawn
187
meat (50 g), respectively, was minced, mixed with double distilled water (80 mL),
188
stirred for 2 h and then subjected to a careful SAFE distillation.28 The aqueous
189
distillate obtained was then filled up to 100 mL in a measuring flask with doubly
190
distilled water and directly subjected to ion chromatography. The stationary phase
191
was a cation exchange column IonPac CS18 250 × 2 mm (Dionex) with a precolumn
192
IonPac CG18 50 × 2 mm (Dionex), heated to 40 °C. An aqueous solution of methane
193
sulfonic acid was generated by the eluent generator and used as mobile phase with a
194
flow rate of 0.3 mL/min. The following gradient was used for optimum detection of
195
trimethylamine and minimum matrix interferences: starting with a concentration of 3 ACS Paragon Plus Environment
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10 196
mM methanesulfonic acid for 10 min, raising the concentration to 10 mM in the
197
following 5 min, and again to 15 mM in the following 10 min. After the run, the
198
concentration was lowered to 3 mM to reach the starting conditions again. Analytes
199
were detected by means of suppressed conductivity detection (data collection rate
200
5.0 Hz) using the integrated conductivity detector and an electrolytic suppressor cell
201
(Dionex CSRS 300 2mm), running in auto-recycling mode with a suppression current
202
of 14 mA. Since ion chromatography featured good linearity, quantitation was carried
203
out by means of external calibration. Aqueous solutions of trimethylamine with
204
increasing concentrations (0.4 – 10 mg/L) were subjected to ion chromatography and
205
correlated to the peak areas.
206
Enzymatic Assay. For the determination of free ammonia in processed prawn
207
meat, an enzymatic bio analysis kit (Boehringer Mannheim/R-Biopharm, Darmstadt,
208
Germany) was used. The reaction of 2-oxogutarate and ammonia in the presence of
209
glutamate dehydrogenase (GDH) and reduced nicotine-amide-adenine dinucleotide
210
(NADH) was used for the assay. The amount of the oxidized NAD+, stoichiometric to
211
the amount of free ammonia was determined photometrically.
212
Sensory Evaluation. Sensory analyses were carried out in a sensory room
213
designed for this purpose with individual sections for each panelist. The room
214
temperature was adjusted to 20 – 25 °C and analyses were carried out in tinted light.
215
Sensory analyses were carried out by a trained panel consisting of 15 to 21 panelists
216
(male and female, age 24 to 41), who had participated in weekly sensory sessions to
217
train their ability to recognize and describe different aroma qualities.
218
Aroma Profile Analyses. The following reference substances were used as
219
aroma descriptors: metallic ((Z)-1,5-octadien-3-one), seasoning-like (3-hydroxy-4,5-
220
dimethyl-2(5H)-furanone), roasted, popcorn-like (2-acetyl-1-pyrroline), cucumber-like
221
((E,Z)-2,6-nonadienal), fishy ((Z)-4-heptenal), fatty ((E,E)-2,4-decadienal), caramelACS Paragon Plus Environment
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like
(4-hydroxy-2,5-dimethyl-3(2H)-furanone),
cooked
potato-like
223
(3-(methylthio)propanal), and fishy, rotten (trimethylamine). The odor attribute sea
224
breeze-like was rated without a reference substance. For the aroma evaluation of
225
fried and blanched prawn meat, respectively, the samples were freshly prepared (20
226
g) and kept in glass vessels at 60 °C in a water bath before evaluation. For aroma
227
profile analyses, the intensities of the respective aroma qualities were ranked on a
228
scale from 0 to 3 (0 = not perceivable, 3 = very high intensity) in steps of 0.1.
229
Determination of Odor Thresholds. Odor thresholds in water were determined
230
following a previously published protocol.31 For evaluation of the impact of the basic
231
compounds trimethylamine and ammonia, their thresholds were determined in a
232
phosphate buffer solution adjusted to the natural pH 6.9 of processed prawn meat
233
following the same protocol.
234
Aroma Recombination Experiments. Aroma models of blanched and fried prawn
235
meat, respectively, were prepared containing all odorants with odor activity values ≥
236
1 in their actual concentrations in an aqueous matrix adjusted to the natural pH 6.9 of
237
prawn meat by means of phosphate buffer. Exceptions were trimethylamine and
238
2-acetyl-1-pyrroline, which were added in their 3 fold and 5 fold concentration to the
239
aroma models, respectively, on the basis of preliminary sensory experiments (Tables
240
2 and 3).
241
The aroma model and the freshly prepared food sample were each placed in
242
closed glass vessels (20 g each) and presented to the panelists who were asked to
243
evaluate the recombinate in the same way as described above for the aroma profile
244
analysis. Additionally, the panelists were asked to rate the overall similarity of the
245
model to the original sample on a scale from 0 to 3 (0 = no similarity, 1 = slightly
246
similar, 2 = the food sample can be recognized, 3 = high similarity) in steps of 0.1.
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Omission Experiments. In further mixtures of blanched and fried prawn meat
248
odorants, several compounds were omitted and the models were again presented to
249
the sensory trained panel (Table 3).
250
251
Triangle Test. Triangle tests were carried out according to ISO 4120:2004.
RESULTS AND DISCUSSION
252
Quantitation of Key Aroma Compounds in Blanched (BPM) and Fried (FPM)
253
Prawn Meat. In our previous study,1 by application of aroma extract dilution analysis
254
(AEDA) and subsequent identification experiments 43 or 45 odor-active compounds,
255
respectively, were found in BPM and FPM. These odorants featuring high FD factors
256
in our previous study were now selected for stable isotope dilution assays (Table 1).
257
Additionally, an enzymatic assay to determine the content of free ammonia and an
258
ion chromatographic assay for the quantitation of trimethylamine were carried out.
259
Thus, 35 or 36 odorants, respectively, were quantitated in BPM and FPM.
260
Application of stabile isotope dilution assays to the processed prawn meat
261
samples revealed decanoic acid (BPM: 240 µg/kg; FPM: 810 µg/kg) and butanoic
262
acid (BPM: 450 µg/kg; FPM: 610 µg/kg) with the highest amounts in both samples
263
(Table 2). Compounds determined in concentrations ranging from 100 to 10 µg/kg
264
were 2- methylbutanoic acid (BPM: 34 µg/kg; FPM: 30 µg/kg) and 3-methylbutanoic
265
acid (BPM: 28 µg/kg; FPM: 50 µg/kg), 4-hydroxy-3-methoxybenzaldehyde (BPM: 28
266
µg/kg; FPM: 32 µg/kg), 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone (BMP: 17 µg/kg;
267
FPM: 9.8 µg/kg) and the isomers of methylbutanal (2-methylbutanal: BMP: 13 µg/kg;
268
FPM: 25 µg/kg; 3-methylbutanal: BMP: 12 µg/kg; FPM: 23 µg/kg).
269
On the other hand, some aroma-active compounds were found in very small
270
amounts (
