Characterization of Key Aroma Compounds in Raw and Thermally Processed Prawns and Thermally Processed Lobsters by Application of Aroma Extract Dilution Analysis

Article abstract of DOI:10.1021/acs.jafc.6b02728

Application of aroma extract dilution analysis (AEDA) to an aroma distillate of blanched prawn meat (Litopenaeus vannamei) (BPM) revealed 40 odorants in the flavor dilution (FD) factor range from 4 to 1024. The highest FD factors were assigned to 2-acetyl-1-pyrroline, 3-(methylthio)propanal, (Z)-1,5-octadien-3-one, trans-4,5-epoxy-(E)-2-decenal, (E)-3-heptenoic acid, and 2-aminoacetophenone. To understand the influence of different processing conditions on odorant formation, fried prawn meat was investigated by means of AEDA in the same way, revealing 31 odorants with FD factors between 4 and 2048. Also, the highest FD factors were determined for 2-acetyl-1-pyrroline, 3-(methylthio)propanal, and (Z)-1,5-octadien-3-one, followed by 4-hydroxy-2,5-dimethyl-3(2H)-furanone, (E)-3-heptenoic acid, and 2-aminoacetophenone. As a source of the typical marine, sea breeze-like odor attribute of the seafood, 2,4,6-tribromoanisole was identified in raw prawn meat as one of the contributors. Additionally, the aroma of blanched prawn meat was compared to that of blanched Norway and American lobster meat, respectively (Nephrops norvegicus and Homarus americanus). Identification experiments revealed the same set of odorants, however, with differing FD factors. In particular, 3-hydroxy-4,5-dimethyl-2(5H)-furanone was found as the key aroma compound in blanched Norway lobster, whereas American lobster contained 3-methylindole with a high FD factor.

Full text of DOI:10.1021/acs.jafc.6b02728

Article
pubs.acs.org/JAFC
Characterization of Key Aroma Compounds in Raw and Thermally
Processed Prawns and Thermally Processed Lobsters by Application
of Aroma Extract Dilution Analysis
Veronika Mall and Peter Schieberle*
Deutsche Forschungsanstalt fur Lebensmittelchemie Lise-Meitner-Strasse 34, 85354 Freising, Germany
̈
ABSTRACT: Application of aroma extract dilution analysis (AEDA) to an aroma distillate of blanched prawn meat (Litopenaeus
vannamei) (BPM) revealed 40 odorants in the flavor dilution (FD) factor range from 4 to 1024. The highest FD factors were
assigned to 2-acetyl-1-pyrroline, 3-(methylthio)propanal, (Z)-1,5-octadien-3-one, trans-4,5-epoxy-(E)-2-decenal, (E)-3-heptenoic
acid, and 2-aminoacetophenone. To understand the influence of different processing conditions on odorant formation, fried
prawn meat was investigated by means of AEDA in the same way, revealing 31 odorants with FD factors between 4 and 2048.
Also, the highest FD factors were determined for 2-acetyl-1-pyrroline, 3-(methylthio)propanal, and (Z)-1,5-octadien-3-one,
followed by 4-hydroxy-2,5-dimethyl-3(2H)-furanone, (E)-3-heptenoic acid, and 2-aminoacetophenone. As a source of the typical
marine, sea breeze-like odor attribute of the seafood, 2,4,6-tribromoanisole was identified in raw prawn meat as one of the
contributors. Additionally, the aroma of blanched prawn meat was compared to that of blanched Norway and American lobster
meat, respectively (Nephrops norvegicus and Homarus americanus). Identification experiments revealed the same set of odorants,
however, with differing FD factors. In particular, 3-hydroxy-4,5-dimethyl-2(5H)-furanone was found as the key aroma compound
in blanched Norway lobster, whereas American lobster contained 3-methylindole with a high FD factor.
KEYWORDS: aroma extract dilution analysis, blanched prawn meat, fried prawn meat, Norway lobster, American lobster
phenols might be responsible for the desirable brine-like, sea-
like flavor of fresh seafood.
INTRODUCTION
■
Over the past decades, crustaceans such as prawns, shrimp,
lobsters, crayfish, or crabs have become more and more popular
as healthy and tasty food. In particular, the characteristic fishy,
roasty aroma of processed crustacean meat is highly appreciated
by consumers all around the world. In former studies on
cooked crustaceans, more than 450 volatiles have been
identified. However, in earlier investigations, often a differ-
entiation of odor-active compounds from the bulk of odorless
volatiles, for example, by means of gas chromatography−
olfactometry (HRGC-O), was not carried out in the respective
food extract.1−6 Therefore, only some studies on the key
odorants of processed crustaceans such as lobster, crab, and
shrimp are available in which molecular data were correlated
with sensory perception.7−13 Baek and Cadwallader12 analyzed
the occurrence of key odorants in cooked meat of different
crustacean species and reported 2-acetyl-1-pyrroline and 3-
(methylthio)propanal to be the main contributors to the overall
aroma of cooked crustacean meat. In addition, they detected
trimethylamine, 2,3-butanedione, 3-methyl-1-butanol, (Z)-4-
heptenal, 1-octen-3-one, 2-methyl-3-furanthiol, 2-acetylthiazole,
and 2-acetyl-2-thiazoline as important odorants in cooked
prawn and shrimp meat.
In former studies on the aroma compounds of heated
crustacean meat, also marine, leather-like, and dry seaweed-like
odorants were detected, but their identification remained
uncertain.7−9,11,12 Whereas Whitefield et al.14,15 found 2,6-
dibromophenol to be the origin of the iodine-like off-flavor of
Australian prawns, Boyle et al.16,17 reported an abundant
occurrence of bromophenols in fish and crustaceans and,
contrary to former studies, they suggested that in low
concentrations of approximately 0.05 ppb these halogenated
For the overall aroma of lobster meat, a slightly different set
of key odorants was found. For example, different compounds,
such as 2-acetyl-3-methylpyrazine, 2-acetylpyridine, 3-methyl-
indole, (E)-2-octanal, and (E,E)-2,4-decadienal were re-
ported.8,9
In a study using aroma extract dilution analysis (AEDA), the
main odorants in roasted shrimp (Sergia lucens Hansen) were
reported to be methanethiol, 1-pyrroline, N-(2′-methylbutyl)-
pyrrolidine, N-(3′-methybutyl)-pyrrolidine, methyl isopropyl
disulfide, and 3-methylpyridine.13 Whereas the latter work was
carried out on sun-dried seafood, the aim of this investigation
was to characterize the key odorants in freshly prepared
blanched prawn meat (BMP) (Litopenaeus vannamei) by
application of AEDA followed by identification experiments
using HRGC/MS and two-dimensional HRGC-GC/MS. To
understand the influence of different processing methods on
the formation of aroma compounds, the odorants of raw and
fried prawn meat were compared to those in BMP. Finally, the
influence of species of crustaceans was investigated by
comparing the main odorants in blanched meat of two lobster
types (Nephrops norvegius and Homarus americanus) to that of
BMP.
Received: June 17, 2016
Revised: August 3, 2016
Accepted: August 3, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.6b02728
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Journal of Agricultural and Food Chemistry
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Extraction of Raw Prawn Meat with Water and Dichloro-
methane. Minced raw prawn meat (250 g) was mixed with 400 mL
of deionized water and stirred for 2 h at room temperature. This
mixture was directly subjected to SAFE distillation24 and then
extracted with dichloromethane (3 × 150 mL). After drying over
anhydrous sodium sulfate, the organic layer was concentrated using the
Vigreux column described above and subjected to HRGC-O and
HRGC-GC-O/MS.
EXPERIMENTAL PROCEDURES
■
Materials. Whiteleg shrimp (L. vannamei) (origin: aquaculture in
Thailand), Norway lobster (N. norvegicus) (captured in the northeast-
ern Atlantic (FAO-area 27) offshore Scotland), and American lobster
tails (H. americanus) (captured in the northwestern Atlantic (FAO-
area 21) offshore Canada) were purchased at a local supermarket. All
three products were frozen in the raw state with shell.
Chemicals. The reference compounds were obtained from the
following sources: 2, 4, and 36 were gifts from Symrise (Holzminden,
Germany); 5 (Alfa Aesar, Karlsruhe, Germany); 8−13, 15−17, 20−
23, 25−31, 34, 35, 38, 40−50, and U5 (Sigma-Aldrich Chemie,
Taufkirchen, Germany); 14, 18, 19, and 37 (Fluka, Neu-Ulm,
Germany); 24 (Chemos GmbH, Regenstauf, Germany); and 51
(Merck, Darmstadt, Germany). 2,4-Dibromophenol, dimethyl sulfate,
and 2,4,6-tribromoanisole were obtained from Sigma-Aldrich Chemie.
Reference solutions of (E,Z,Z)- and (Z,Z,Z)-5,8,11-tetradecatrien-2-
one were gifts from T. Hasegawa Co. (Tokyo, Japan).
Simultaneous Distillation/Extraction (SDE). To obtain better
yields, a SDE according to the method of Nickerson and Likens27 was
carried out additionally. For this extraction 200 g of powdered raw
prawn meat was mixed with distilled water (800 mL) and heated. On
the other side of the apparatus, dichloromethane was boiled at 45 °C.
The SDE procedure was maintained for 2 h, and the aroma extract
obtained was subsequently concentrated to ∼1 mL using Vigreux
column distillation and further to ∼200 μL via microdistillation.
HRGC-O, HRGC/MS, and HRGC-GC-O/MS. HRGC-O was
performed by means of a gas chromatograph 5160 Mega series
(Carlo Erba, Hofheim, Germany) with helium as carrier gas at a flow
rate of 2.2 mL/min. The samples were applied by the cold-on-column
technique onto the following fused silica capillaries: DB-FFAP (30 m
× 0.25 mm i.d.; 0.25 μm film thickness) (J&W Scientific, Folsom, CA,
USA) and DB-5 (30 m × 0.32; 0.25 μm film thickness) (Macherey-
The following compounds were synthesized according to the
literature cited: (Z)-3-methyl-1-butene-1-thiol (1) and (E)-2-methyl-
1-butene-1-thiol (3);18 2-acetyl-1-pyrroline (6);19 (Z)-1,5-octadien-3-
one (7),20 and 2,4,6-nonatrienal (32, 33);21 and (E)-3-heptenoic acid
(39).22
Diethyl ether and dichloromethane were freshly distilled prior to
Nagel, Duren, Germany). After injection of the sample at 40 °C, the
̈
use.
temperature was held for 2 min isothermally and then increased at 6
°C/min to 230 °C (DB-FFAP) or 240 °C (DB-5), respectively, and
held for 5 min. The effluent of the capillary column was split 1:1 by
volume and transferred to a flame ionization detector (FID) and a
heated (200 °C) sniffing port made of alumina, respectively, using a Y-
shaped quick-seal glass connector (Chrompack, Frankfurt, Germany)
and two deactivated fused silica capillaries (30 cm × 0.20 mm i.d.
each). Linear retention indices (RI) were calculated from the retention
times of n-alkanes as described previously.25 MS was performed by
means of a Mat 95 S mass spectrometer (Finnigan, Bremen, Germany)
connected to a 5160 Mega series gas chromatograph using the same
capillaries as described above. Mass spectra in the electron impact
mode (MS-EI) were generated at 70 eV. For two-dimensional
HRGC−HRGC/MS applications, a Mega 2 series gas chromatograph
(Fisons Instruments, Mainz-Kastel, Germany) was coupled to a CP
3800 gas chromatograph (Varian, Darmstadt, Germany) and a Saturn
2000 ion trap mass spectrometer (Varian) using the DB-FFAP
capillary in the first and the DB-5 column in the second oven. The
elution range selected was transferred into a cold trap (−100 °C) by
means of a moving column stream switching system (MCSS)
(Thermo, Dreieich, Germany) located in the first and a heated
transfer line (250 °C) (Horst GmbH, Frankfurt, Germany) connecting
both GCs. By heating the trap, the sample was transferred to the
second column. Simultaneous sniffing and FID detection at the first
HRGC or sniffing and MS detection at the second HRGC,
respectively, were done by splitting the effluent of the respective
capillary column as described above. Mass spectra in MS-EI mode
were generated at 70 eV.
Synthesis of 2,4-Dibromoanisole. Methylation of 2,4-dibromo-
phenol yielded the respective dibromoanisole as described by
McKillop et al.23 2,4-Dibromophenol (1 mmol, 0.25 g) was added
to an aqueous solution of sodium hydroxide (10%, 0.5 mL) with
stirring. After the addition of dimethyl sulfate (1 mmol, 0.126 g), the
solution was first stirred at room temperature for 10 min and then
refluxed for another 10 min to remove the remaining methylation
reagent. After cooling, the aqueous solution was extracted with diethyl
ether (total volume = 100 mL). The organic layer was washed with an
aqueous solution of sodium hydroxide (5%, total volume = 50 mL)
and water (total volume = 50 mL) and dried over anhydrous sodium
sulfate. Characterization of 2,4-dibromoanisole was carried out by GC/
MS.
MS-EI m/z (%): 63 (12), 75 (8), 170 (8), 172 (7), 221 (13), 223
(26), 225 (13), 249 (19), 251 (39), 253 (18), 264 (46), 266 (100),
268 (44).
Cooking Process of Crustaceans (Blanching). After thawing,
the crustaceans were cooked in unsalted boiling water (100 °C) for 1
min (whiteleg shrimp), 2 min (Norway lobster), or 4 min (American
lobster tails), respectively. The shell was removed, and the meat was
frozen with liquid nitrogen and minced to a fine powder.
Frying Process of Prawns. After defrosting, prawns were pan-
fried at 160 °C without using fat. After 6 min of evenly frying both
sides, the prawns were cooled and the shells removed. The meat was
frozen with liquid nitrogen and minced to a fine powder.
Isolation of Volatiles; Separation into Acid and Neutral/
Basic Compounds. Meat powder (100 g) was mixed with anhydrous
sodium sulfate (50 g) and extracted with diethyl ether (300 mL) with
stirring for 2 h. After filtration, the volatiles were isolated by solvent-
assisted flavor evaporation (SAFE).24 The aroma distillate obtained
was then dried over anhydrous sodium sulfate and concentrated to
∼50 mL using a Vigreux column (60 cm × 1 cm). For fractionation,
the distillate was extracted with an aqueous solution of sodium
bicarbonate (0.5 mol/L; total volume = 150 mL). The organic layer
was washed twice with brine (total volume = 150 mL) and dried over
anhydrous sodium sulfate, yielding the neutral/basic fraction (NBF).
The aqueous layers were recombined and adjusted to a pH of 2.5 using
hydrochloric acid (2 mol/L), and the acidic compounds were extracted
with diethyl ether (total volume = 300 mL). The combined ethereal
solutions were dried over anhydrous sodium sulfate to yield the acidic
fraction (AF). Both fractions were finally concentrated to 200 μL each
using the Vigreux column described above followed by micro-
distillation at 40 °C.25
AEDA. Three panelists performed HRGC-O with the undiluted
aroma extract to confirm the perception of the whole set of aroma
compounds within the sample and to minimize problems possibly
caused by anosmia. Then, the AEDA was performed by one person to
determine the flavor dilution (FD) factors of odor-active com-
pounds.28 The original aroma distillate (200 μL) obtained from 50 g of
crustacean meat was diluted stepwise using diethyl ether to obtain
dilutions of 1:1, 1:2, 1:4, 1:8, 1:16, ..., 1:2048 of the original extract.
Each dilution was analyzed by HRGC-O (injection volume = 1 μL)
until no odor was detectable during GC-O. The same GC conditions
and columns as described above were used.
RESULTS AND DISCUSSION
■
Key Odorants in BPM. Volatiles from freshly prepared
blanched prawns were extracted with diethyl ether and carefully
isolated using SAFE.24 A small drop of the distillate when
sniffed from a strip of filter paper evoked the characteristic
Isolation of Thiols. Thiols were isolated by means of mercurated
agarose gel as described recently.26
B
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Figure 1. (A) FID chromatogram obtained by GC-O of an aroma distillate of blanched prawn meat with odorant areas perceived (FD ≥ 128). (B)
FD chromatogram obtained by application of AEDA to an aroma distillate of blanched prawn meat (FD ≥ 64).
Figure 2. Structures of the most odor-active volatiles identified in blanched prawn meat (numbering refers to Table 1; FD factors in parentheses).
aroma of BPM, thus confirming that the entire set of aroma
compounds had been isolated. Application of GC-O and AEDA
to the distillate of BPM meat revealed 40 odor-active regions in
the FD factor range from 4 to 1024. The highest FD factor was
obtained for the roasted, popcorn-like-smelling compound 6,
followed by the cooked potato-like odorant 12 with an FD of
512 (Figure 1). Somewhat lower FD factors were found for
compounds 7 (metallic, geranium-like), 36 (metallic), 39 (sour,
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fecal, moldy), 43 (foxy, medicine-like), 11 (vinegar-like), 24
(fishy, green, straw-like), and 33 (fatty, oat-like) (Figure 1).
Identification of the odorants was performed following the
protocol described earlier.29 Comparison of retention indices
on at least two columns with different stationary phases (DB-
FFAP and DB-5) and of the odor quality with the data of an in-
house database containing ∼800 food aroma compounds gave a
first hint on possible structures of aroma compounds.
Recording mass spectra of the analytes and comparison to
retention indices, odor quality, and odor intensity of authentic
reference compounds completed the identification. Following
this approach, compound 6 was identified as 2-acetyl-1-
pyrroline and compound 12 as 3-(methylthio)propanal (Figure
2). Both compounds have also previously been described as
aroma compounds in heated crustaceans.7−12,30
Odorants 7, 36, 39, and 43 with FD factors of 256 were
identified as (Z)-1,5-octadien-3-one (metallic, geranium-like),
trans-4,5-epoxy-(E)-2-decenal (metallic), (E)-3-heptenoic acid
(sour, fecal, moldy), and 2-aminoacetophenone (foxy, medi-
cine-like), respectively (Figure 2). (Z)-1,5-Octadien-3-one and
2-aminoacetophenone were also described by Lee et al.9 in
lobster tail meat. Although (Z)-1,5-octadien-3-one was
determined as a key odorant in freshly boiled trout,31 the
foxy-smelling 2-aminoacetophenone was previously regarded to
have a negative impact on the overall odor in fermented tuna
sauce.32 trans-4,5-Epoxy-(E)-2-decenal and (E)-3-heptenoic
acid were identified for the first time in crustaceans. Because
(E)-3-heptenoic acid was not commercially available, it was
synthesized closely following a reported procedure,22 and its
mass spectrum is shown in Figure 3. To our knowledge this
total of 20 odorants are reported for the first time in
crustaceans among the 51 compounds listed in Table 1.
In earlier studies, Kubota et al.35 and Kobayashi et al.36 had
identified two new shrimp-like-smelling aroma compounds in
cooked shrimp (Euphausia superba, Euphausia pacifica, and
Sergia lucens Hansen) as stereoisomers of 5,8,11-tetradecatrien-
2-one. After synthesis of all eight possible isomers of the methyl
ketone, the unknown odorants were characterized as (E,Z,Z)-
and (Z,Z,Z)-5,8,11-tetradecatrien-2-one.35,36 When the prawn
meat was submitted to SDE, also in our study these isomers
could be found by means of two-dimensional HRGC-GC-O/
MS and were identified by means of authentic reference
compounds. However, no odor was perceivable at the sniffing
port at the respective areas, suggesting that the odor thresholds
of these methyl ketones were higher than their actual
concentration in the BPM. Therefore, subsequent quantitative
studies should evaluate their possible aroma contribution to the
aroma of BPM.
Influence of Different Thermal Processing Conditions
on the Aroma Compounds in Prawn Meat. Prior to
consumption, prawns are usually processed, and during heating
a desirable roasted, sweet, and fishy aroma is developed.
Although several odorants may be generated due to enzymatic
reactions, the thermal formation of aroma compounds from
odorless precursors plays an important role in the overall aroma
of processed prawn meat.12,34 These precursors are located in
the flesh as well as in the shell. To provide the full spectrum of
aroma precursors, the prawns were processed as such. Whereas
the aroma profile of the raw prawn meat was mainly dominated
by metallic, cucumber-like, green, and fishy notes, it shifted to
seasoning-like, fishy, and roasty odor notes after blanching (100
°C) (data not shown). During frying, the roasted, popcorn-like
odor note strongly increased, whereas the fishy odor note
somewhat decreased. To elucidate the reason for these sensory
changes on a molecular basis, an AEDA was next carried out on
the volatiles of fried prawn meat (FPM), using the same
amount of prawn meat and following exactly the same workup
procedure as for BPM.
In FPM, 40 odorants were detected with FD factors between
4 and 2048, 38 of which were identified. Again, the highest FD
factor was determined for 2-acetyl-1-pyrroline (6, roasted,
popcorn-like), followed by 3-(methylthio)propanal (12, cooked
potato-like) with an FD factor of 1024 and (Z)-1,5-octadien-3-
one (7, metallic, geranium-like) with an FD factor of 512
(Table 2). An FD factor of 256 was found for 4-hydroxy-2,5-
dimethyl-3(2H)-furanone (37, caramel-like, 4-HDF), (E)-3-
heptenoic acid (39, sour, fecal, moldy), and 2-amino-
acetophenone (43, foxy, medicine-like), respectively. A some-
what lower FD factor of 128 was assigned to 2,3-diethyl-5-
methylpyrazine (13, earthy) and linalool (14, citrus-like,
flowery). Among the compounds detected only in FPM, but
not in BPM, were two roasty-smelling compounds 2-
furanmethanethiol (10), eliciting a coffee-like, roasted smell,
and 2-acetylthiazole (21), with an earthy, roasted aroma note.
Apart from some odorants with relatively low FD factors,
basically the same set of odorants was identified in BPM and
FPM, respectively, however with different FD factors for some
compounds. Higher FD factors in FPM were determined for 4-
HDF, linalool, and 2,6-dimethoxyphenol, whereas the earthy-
smelling 2,3-diethyl-5-methylpyrazine (13, FD 128), γ-
octalactone (34, coconut-like, FD 64), γ-nonalactone (38,
coconut-like, FD 32), and 2,6-dimethoxyphenol (46, smoky,
FD 64) were not detected in BPM (Table 3). 2-Acetyl-1-
Figure 3. Mass spectrum (MS-EI) of (E)-3-heptenoic acid isolated
from blanched prawn meat.
compound was identified only in heated pork fat up to now and
was proposed to be a precursor for γ- and δ-lactones.33
Moreover, the identification experiments revealed various
compounds with FD factors of 128, for example, 1-octen-3-
one (5, mushroom-like), acetic acid (11, vinegar-like), 3-
methyl-2,4-nonanedione (24, fishy, green, straw-like), (E,E,Z)-
2,4,6-nonatrienal (33, fatty, oat-like), 3-hydroxy-4,5-dimethyl-
2(5H)-furanone (42, seasoning-like), decanoic acid (44,
musty), 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone (45, sea-
soning-like), and indole (48, mothball-like) (Table 1). Whereas
among the lipid-derived compounds, 1-octen-3-one34 was
previously reported in heated lobster and shrimp,8−10,12
a
D
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Table 1. Most Odor-Active Volatiles (FD ≥ 4) in Blanched Prawn Meat
d
RI on
a
b
c
e
f
no.
odorant
odor quality
fraction
FFAP
DB-5
FD factor
earlier identified in Crustaceae
1
(Z)-3-methyl-1-butene-1-thiol
(Z)-4-heptenal
roasted, sulfury
fishy
NBF
NBF
NBF
NBF
NBF
NBF
AF
1024
1232
1288
1325
1371
1417
1442
1446
1532
1558
1578
1596
1614
1640
1660
1693
1715
1726
1748
1755
1800
1815
1837
1858
1863
1877
1949
2003
2029
2039
2068
2149
2200
2222
2260
2265
2458
2506
2569
2583
765
902
975
922
981
8
16
4
1, 4, 6−12
8−10, 12
7−12, 30
9
5
1-octen-3-one
mushroom-like
128
1024
256
4
6
2-acetyl-1-pyrroline
(Z)-1,5-octadien-3-one
unknown
roasted, popcorn-like
metallic, geranium-like
citrus, green
7
g
9
nd
630
11
12
14
16
17
18
19
20
22
23
24
25
26
27
28
29
30
31
32
33
35
36
37
39
40
41
42
43
44
45
48
49
50
51
acetic acid
vinegar-like
128
512
4
30
3-(methylthio)propanal
linalool
cooked potato-like
citrus-like, flowery
sweaty, fruity
cucumber-like
popcorn-like
NBF
NBF
AF
907
7−9, 12
6
1098
792
2-methylpropanoic acid
(E,Z)-2,6-nonadienal
2-acetylpyridine
8
NBF
NBF
AF
1156
1031
825
32
8−10
64
butanoic acid
sweaty, cheese-like
flowery, honey-like
sweaty, fruity
fatty, green
32
3
phenylacetaldehyde
2- and 3-methylbutanoic acid
(E,E)-2,4-nonadienal
3-methyl-2,4-nonanedione
pentanoic acid
NBF
AF
1038
882
8
64
NBF
NBF
AF
1215
1240
920
32
fishy, green, straw-like
sweaty
128
4
3
(E,Z)-2,4-decadienal
2-acetyl-2-thiazoline
(E,E)-2,4-decadienal
geosmine
fatty, green
NBF
NBF
NBF
NBF
NBF
NBF
NBF
NBF
NBF
NBF
AF
1291
1091
1314
1404
nd
4
popcorn-like
64
10, 12
9, 10
fatty
64
beetroot-like
16
unknown
metallic, green, fruity
smoky, sweet
fatty, oat-like
8
2-methoxyphenol
1088
1262
1267
1219
1376
1067
1105
1073
1464
1104
1300
1376
1196
1290
1393
1268
1398
64
9
(E,Z,E)-2,4,6-nonatrienal
(E,E,Z)-2,4,6-nonatrienal
benzothiazole
4
fatty, oat-like
128
64
leather-like, phenolic
metallic
6, 9, 30
trans-4,5-epoxy-(E)-2-decenal
4-hydroxy-2,5-dimethyl-3(2H)-furanone
(E)-3-heptenoic acid
4-methylphenol
256
32
caramel-like
9
6
sour, fecal, moldy
horse stable-like
peach-like
AF
256
4
NBF
NBF
AF
γ-decalactone
32
3-hydroxy-4,5-dimethyl-2(5H)-furanone
2-aminoacetophenone
decanoic acid
seasoning-like
foxy, medicine-like
musty
128
256
128
128
128
16
NBF
AF
9
5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone
indole
seasoning-like
mothball-like
AF
NBF
NBF
NBF
NBF
1−3, 6
9
3-methylindole
mothball-like, fecal
honey-like, sweet
vanilla-like
2-phenylacetic acid
4-hydroxy-3-methoxybenzaldehyde
32
32
a
The odorant was identified by comparing the retention indices on two stationary phases, mass spectra (MS-EI and MS-CI), and the odor quality
b
with data of the respective reference compound. Odor quality detected at the sniffing port at a dilution factor 5 times above the odor threshold of
the reference compound. Fractions were obtained by separation of the neutral/basic and acidic volatiles. Retention index determined in
comparison to a homologous series of n-alkanes. Flavor dilution factor: last dilution of an extract in which an odorant was still detectable. Aroma
compound earlier identified in crustaceans according to the literature cited. nd, not determined.
c
d
e
f
g
pyrroline, 3-(methylthio)propanal, and (Z)-1,5-octadien-3-one
only showed any difference of one dilution step in their
respective FD factors, which does not indicate a difference
between blanched and fried prawn meat. However, quantitative
data are necessary to provide evidence of their potential
influence on the overall aroma of thermally processed prawn
meat.
On the other hand, for several compounds somewhat lower
FD factors were determined in FPM as compared to BPM, for
example, for the mushroom-like 1-octen-3-one (5), acetic acid
(11, vinegar-like), 3-methyl-2,4-nonanedione (24, fishy, green,
straw-like), 3-hydroxy-4,5-dimethyl-2(5H)-furanone (42, sea-
soning-like), and 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone
(45, seasoning-like) (Table 3).
Comparison with Raw Prawn Meat (RPM). To obtain
information on thermal formation pathways of certain odorants,
an additional AEDA was subsequently carried out on RPM,
using the same amount of prawn meat and following the same
workup procedure as for BPM and FPM. Whereas among the
thermally generated aroma compounds, the amino acid
degradation product 3-(methylthio)propanal showed the
second highest FD factor in both thermally processed samples,
it was not detectable in the raw prawn meat (Table 3). For 2-
acetyl-1-pyrroline, which is known to be generated by reaction
of proline or ornithine with carbohydrates via the intermediate
E
DOI: 10.1021/acs.jafc.6b02728
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Journal of Agricultural and Food Chemistry
Article
a
Table 2. Most Odor-Active Volatiles (FD ≥ 4) in Fried Prawn Meat
RI ond
no.
odoranta
odor qualityb
fractionc
FFAP
DB-5
FD factore
2
3-methyl-2-butene-1-thiol
(E)-2-methyl-1-butene-1-thiol
(Z)-4-heptenal
pungent, sulfury
roasted, sulfury
fishy
NBF
NBF
NBF
NBF
NBF
NBF
NBF
AF
1094
1105
1232
1288
1325
1371
1394
1428
1442
1446
1477
1532
1555
1578
1596
1614
1640
1643
1660
1693
1715
1726
1748
1755
1800
1858
1877
1918
1949
2029
2030
2039
2200
2222
2260
2275
2348
2506
2569
2583
821
819
32
32
16
16
2048
512
8
3
4
902
5
1-octen-3-one
mushroom-like
roasted, popcorn-like
975
6
2-acetyl-1-pyrroline
(Z)-1,5-octadien-3-one
trimethylpyrazine
922
7
metallic, geranium-like
earthy
981
8
1000
903
10
11
12
13
14
15
17
18
19
20
21
22
23
24
25
26
27
28
31
33
34
35
37
38
39
42
43
44
46
47
49
50
51
2-furanmethanethiol
acetic acid
coffee-like, roasted
vinegar-like
8
AF
630
32
1024
128
128
16
16
8
3-(methylthio)propanal
2,3-diethyl-5-methylpyrazine
linalool
cooked potato-like
earthy
NBF
NBF
NBF
NBF
NBF
NBF
AF
907
1156
1098
citrus-like, flowery
earthy, roasted
cucumber-like
popcorn-like
sweaty, cheese-like
flowery, honey-like
earthy, roasted
sweaty, fruity
fatty, green
f
unknown
nd
(E,Z)-2,6-nonadienal
2-acetylpyridine
1156
1031
825
butanoic acid
16
4
phenylacetaldehyde
2-acetylthiazole
NBF
NBF
AF
1038
1012
882
4
2- and 3-methylbutanoic acid
(E,E)-2,4-nonadienal
3-methyl-2,4-nonanedione
pentanoic acid
64
64
16
4
NBF
NBF
AF
1215
1240
920
fishy, green, straw-like
sweaty
(E,Z)-2,4-decadienal
2-acetyl-2-thiazoline
(E,E)-2,4-decadienal
2-methoxyphenol
fatty, green
NBF
NBF
NBF
NBF
NBF
NBF
NBF
AF
1291
1091
1314
1088
1267
1238
1219
1067
1359
1105
1104
1300
1376
1351
nd
64
4
popcorn-like
fatty
64
128
32
64
32
256
32
256
32
256
32
64
64
16
32
16
smoky, sweet
fatty, oat-like
coconut-like
(E,E,Z)-2,4,6-nonatrienal
γ-octalactone
benzothiazole
leather-like, phenolic
caramel-like
4-hydroxy-2,5-dimethyl-3(2H)-furanone
γ-nonalactone
coconut-like
NBF
AF
(E)-3-heptenoic acid
3-hydroxy-4,5-dimethyl-2(5H)-furanone
2-aminoacetophenone
decanoic acid
sour, fecal, moldy
seasoning-like
foxy, medicine-like
musty
AF
NBF
AF
2,6-dimethoxyphenol
unknown
smoky
NBF
NBF
NBF
NBF
NBF
metallic
3-methylindole
mothball-like, fecal
honey-like, sweet
vanilla-like
1393
1268
1398
2-phenylacetic acid
4-hydroxy-3-methoxybenzaldehyde
a
f
For footnotes a−e, see Table 1. nd, not determined.
1-pyrroline and 2-oxopropanal,37 interestingly, the highest FD
factor of 4096 was found in RPM (Table 3). Assuming that
solvent extraction with subsequent SAFE distillation represents
a very gentle method with only a minimum of thermal impact,
artifact formation during workup can be excluded as the origin
of this compound.24 However, Romanczyk et al.38 had shown
for cocoa that 2-acetyl-1-pyrroline can also be formed
microbiologically at low temperatures (35 °C) in the presence
of the required precursors. Thus, a microbiological formation of
this compound in RPM seems also likely.
Typical lipid degradation products such as (Z)-4-heptenal,
(E,E)-2,4-nonadienal, (E,Z)-2,6-nonadienal, and 1-octen-3-one
were also determined with somewhat higher FD factors in
prawn meat after heat treatment (Table 3).
The roasty-smelling compound 2-acetylthiazole, which had
been described as an important odorant in blanched
crustaceans before,12 was not detected in BPM, and only a
low FD factor of 4 was determined in FPM. Reasons might be
the different crustacean species and different processing
parameters used in the previous investigation.
Other odorants known to be generated by thermally induced
carbohydrate or protein degradation, such as 5-ethyl-3-hydroxy-
4-methyl-2(5H)-furanone, 4-HDF, or 3-hydroxy-4,5-dimethyl-
2(5H)-furanone, were found with higher FD factors in
blanched or fried prawn meat as compared to raw prawn
meat (Table 3). The caramel-like smelling 4-HDF had already
been reported before as an odorant in cooked lobster tail meat.9
Origin of the Typical Marine, Sea Breeze-like Odor of
RPM. Despite the distinct fresh, marine, and sea breeze-like
odor particularly recognized in the aroma profile of raw prawn
meat, AEDA and identification experiments did not reveal any
compounds covering this odor attribute. To further investigate
the origin of this typical odor, minced RPM was subjected to a
subsequent water and dichloromethane extraction followed by
F
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Article
Table 3. Comparison of the FD Factors of Selected Odor-Active Volatiles in Raw Prawn Meat (RPM) as well as in Blanched
(BPM) and Fried Prawn Meat (FPM)
c
FD factor in
a
b
no.
odorant
2-acetyl-1-pyrroline
odor quality
RPM
BPM
FPM
6
roasted, popcorn-like
cooked potato-like
metal, geranium-like
earthy
4096
<1
512
<1
<1
<1
<1
<1
<1
4
1024
512
256
<1
2048
1024
512
128
128
64
12
7
3-(methylthio)propanal
(Z)-1,5-octadien-3-one
13
14
34
46
38
45
4
2,3-diethyl-5-methylpyrazine
linalool
citrus-like, flowery
coconut-like
4
γ-octalactone
<1
2,6-dimethoxyphenol
γ-nonalactone
smoky
<1
64
coconut-like
<1
32
5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone
(Z)-4-heptenal
seasoning-like
fishy
128
16
2
16
23
37
17
5
(E,E)-2,4-nonadienal
4-hydroxy-2,5-dimethyl-3(2H)-furanone
(E,Z)-2,6-nonadienal
1-octen-3-one
fatty, green
4
32
64
caramel-like
4
32
256
16
cucumber-like
mushroom-like
fishy, green, straw-like
seasoning-like
vinegar-like
8
32
8
128
128
128
128
16
24
42
11
3-methyl-2,4-nonandione
3-hydroxy-4,5-dimethyl-2(5H)-furanone
acetic acid
<1
16
128
16
32
32
a
The odorant was identified by comparing the retention indices on two stationary phases, mass spectra (MS-EI and MS-CI), and the odor quality
b
with data of the respective reference compound. Odor quality detected at the sniffing port at a dilution factor 5 times above the odor threshold of
the reference compound. Flavor dilution factor: last dilution of an extract in which the odorant was still detectable.
c
HRGC-O analysis. Following this approach, three marine, sea
breeze-like-smelling odorants were detected among the entire
set of odorants (Table 4). However, even by application of two-
Table 4. Marine-like-Smelling Odorants Detected in Raw
Prawn Meat
RI on
compound
FFAP
DB-5
ΔRI
odor quality
Br1
Br2
Br3
2070
2100
2273
1415
1516
1612
655
584
661
marine, sea breeze-like
marine, sea breeze-like
marine, sea breeze-like
dimensional HRGC−HRGC-O/MS, no mass spectra could be
obtained, and comparison of RI and odor quality with data of
our in-house database did not give any results. Therefore, as a
next step, a SDE using a Likens−Nickerson apparatus27 was
carried out with RPM. Although this method should be used
with caution, because of its risk of generating thermal
artifacts,39 the advantage of this procedure is an increase in
the extraction yield as well as the easy enlargement of the
experimental scale. Thus, because the marine odorants were
first detected after gentle solvent extraction without thermal
impact, SDE was regarded as a suitable method to increase the
extraction yield of these odorants to obtain their mass spectra.
Finally, applying SDE to 200 g of RPM, concentrating the
extract to 20 μL, and subjecting this concentrate to two-
dimensional HRGC−HRGC-O/MS, yielded at least a mass
spectrum for compound Br3, which enabled us to link the
corresponding retention indices on two stationary phases of
different polarity and calculate the respective increments (Table
4). Thus, the marine-smelling odorant Br3 was identified as
2,4,6-tribromoanisole by comparison of its data with those of an
authentic reference compound (Figure 4A). With regard to its
low odor threshold of 0.047 ng/L air, a contribution of 2,4,6-
dibromoanisole to the fresh, marine, and sea breeze-like odor of
Figure 4. MS-EI of (A) 2,4,6-tribromoanisole and (B) 2,4-
dibromoanisole isolated from RPM using SDE.
RPM seems very likely. Its actual odor contribution, however,
will have to be clarified by subsequent quantitative studies.
Additionally, during the identification experiments, 2,4-
dibromoanisole, also featuring marine-like odor properties,
was identified in RPM (Figure 4B). However, as the compound
was not detectable during HRGC-O, its concentration in RPM
might well be below its odor threshold. Therefore, any
contribution of 2,4-dibromoanisole to the odor of RPM will
have to be clarified by subsequent quantitative studies.
G
DOI: 10.1021/acs.jafc.6b02728
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Table 5. Most Odor-Active Volatiles (FD ≥ 16) in Norway Lobster (NL) and American Lobster (AL) Meat
c
d
RI on
FD factor in
NL
<1
a
b
no.
odorant
odor quality
FFAP
DB-5
AL
e
U1
6
unknown
roasted, coffee-like
roasted, popcorn-like
metallic, geranium-like
coffee-like, roasted
vinegar-like
1060
1325
1371
1428
1442
1446
1536
1558
1578
1614
1660
1693
1715
1726
1800
1858
1874
1877
1949
1994
2003
2029
2039
2149
2200
2222
2260
2458
2485
2506
2569
2583
nd
922
32
64
2-acetyl-1-pyrroline
(Z)-1,5-octadien-3-one
2-furanmethanethiol
acetic acid
1024
512
4
7
981
1024
64
10
11
12
U2
16
17
19
22
23
24
25
28
31
U3
33
35
U4
36
37
39
41
42
43
44
48
U5
49
50
51
903
630
32
128
1024
32
3-(methylthio)propanal
unknown
cooked potato-like
green, moldy
sweaty, fruity
cucumber-like
sweaty, cheese-like
sweaty
907
32
nd
<1
2-methylpropanoic acid
(E,Z)-2,6-nonadienal
butanoic acid
792
16
128
16
1156
825
16
128
2048
128
128
64
256
1024
64
2- and 3-methyl butanoic acid
(E,E)-2,4-nonadienal
3-methyl-2,4-nonanedione
pentanoic acid
882
fatty, green
1215
1240
920
fishy, green, straw-like
sweaty
<1
<1
(E,E)-2,4-decadienal
2-methoxyphenol
fatty
1314
1088
nd
2048
32
4
smoky, sweet
metallic
256
64
unknown
16
(E,E,Z)-2,4,6-nonatrienal
benzothiazole
fatty, oat-like
leather-like, phenolic
sour, fecal, moldy
metallic
1267
1219
nd
32
64
32
64
unknown
64
<1
trans-4,5-epoxy-(E)-2-decenal
4-hydroxy-2,5-dimethyl-3(2H)-furanone
(E)-3-heptenoic acid
γ-decalactone
1376
1067
1105
1464
1104
1300
1376
1290
1575
1393
1268
1398
32
<1
caramel-like
64
4
sour, fecal, moldy
peach-like
1024
<1
256
32
3-hydroxy-4,5-dimethyl-2(5H)-furanone
2-aminoacetophenone
decanoic acid
seasoning-like
foxy, medicine-like
musty
2048
1024
512
32
256
512
256
64
indole
mothball-like
musty
dodecanoic acid
128
4
<1
3-methylindole
mothball-like, fecal
honey-like, sweet
vanilla-like
2048
256
16
2-phenylacetic acid
4-hydroxy-3-methoxybenzaldehyde
512
256
a
The odorant was identified by comparing the retention indices on two stationary phases, mass spectra (MS-EI and MS-CI) and the odor quality
b
with data of the respective reference compound. Odor quality detected at the sniffing port at a dilution factor 5 times above the odor threshold of
the reference compound. Retention index determined in comparison to a homologous series of n-alkanes. Flavor dilution factor: last dilution of an
extract in which the odorant was still detectable. nd, not determined.
c
d
e
The concentrations of the marine, sea-breeze-like-smelling
unknown compounds Br1 and Br2, however, were still too low
to obtain unequivocal mass spectra, even after the application of
SDE and HRGC−HRGC-O/MS. Furthermore, comparison of
their retention indices to those of various bromophenols with
marine odor qualities discussed in the literature (data not
shown) did not show any match.14−17 Thus, the identity of
these two compounds remains unclear, and their identification
will be the subject of further studies.
The occurrence of bromine-substituted organic compounds
in fish and seafood has been the subject of many previous
studies.14−17,40 Higher concentrations of bromophenols were
apparent in marine fish, and especially in wild-catches,
suggesting that fish and crustaceans assimilate these com-
pounds from their diet.16,17,40 Crustaceans live on marine algae
and microflora, in which the general existence of halogenated
compounds is already known.40−44 With the help of a bromine-
specific enzyme, bromoperoxidase, algae can take up free
bromine from the marine environment (65 mg/kg seawater)45
in their metabolism in the presence of hydroperoxides. The
halogenated phenols fulfill the function of defense against
bacterial and fungal infections.40 An O-biomethylation by algae,
accompanied bacteria or fungi, may then lead to the formation
of bromoanisoles.43
Differences in the Composition of Key Odorants
Compared to Lobster. To investigate the key odorants of
processed crustacean meat of various species, American lobster
tails (H. americanus) and Norway lobster (N. norvegicus) were
cooked, and the volatiles were isolated according to the
procedure described above for prawn meat. The two distillates
were then subjected to a comparative AEDA. To allow a direct
comparison of the FD factors in American lobster and Norway
lobster, as well as a comparison to the results obtained for the
BPM, the same portions of meat (100 g each) were taken for
workup and finally concentrated to the same volume of 200 μL.
Altogether, 27 or 25 odorants, respectively, with FD factors
≥16 were detected in the aroma distillates (Table 5).
In the aroma distillate of Norway lobster, the odorants with
the highest FD factor (2048) were identified as 2- and 3-
methylbutanoic acid (22, sweaty), (E,E)-2,4-decadienal (28,
fatty), and 3-hydroxy-4,5-dimethylfuran-2(5H)-furanone (42,
seasoning-like). 2- and 3-methylbutanoic acid were coeluted,
but mass spectra proved the existence of both isomers.
Furthermore, relatively high FD factors were determined for
H
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2-acetyl-1-pyrroline (6, roasted, popcorn-like), (E)-3-heptenoic
acid (39, sour, fecal, moldy), 2-aminoacetophenone (43, foxy,
medicine-like), (Z)-1,5-octadien-3-one (7, metallic, geranium-
like), decanoic acid (44, musty), and 2-phenylacetic acid (50,
honey-like, sweet). All compounds were also detected in the
aroma extract of BPM, however with different FD factors (cf.
Tables 1 and 5). Norway lobster was the only thermally treated
crustacean meat showing a low FD factor of 32 for 3-
(methylthio)propanal, whereas the Strecker aldehyde was
detected with high FD factors of 1024 and 512 in BPM and
FPM, respectively.
AUTHOR INFORMATION
■
Corresponding Author
*(P.S.) Phone: +49 8161 71 2932. Fax: +49 8161 71 2970. E-
mail: peter.schieberle@ch.tum.de.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Petra Steinhaus for careful revision of the
manuscript.
In the aroma distillate of processed American lobster meat,
the highest FD factor of 2048 was assigned to 3-methylindole
(49), eliciting a mothball-like odor. A slightly lower FD factor
of 1024 was found for (Z)-1,5-octadien-3-one (7), 3-
(methylthio)propanal (12), and 2- and 3-methylbutanoic acid
(22), respectively. FD factors from 512 to 256 were determined
for 2-aminoacetophenone (43), butanoic acid (19), 2-
methoxyphenol (31), (E)-3-heptenoic acid (39), 3-hydroxy-
4,5-dimethyl-2(5H)-furanone (42), and 2-phenylacetic acid
(50). The seasoning-like-smelling 3-hydroxy-4,5-dimethyl-
2(5H)-furanone was detected in the BPM (Table 1), but it
was not reported in heated crustaceans before. The roasted,
popcorn-like-smelling 2-acetyl-1-pyrroline reached only a
relatively low FD factor of 64 in the aroma distillate of
American lobster. Thus, American lobster meat represented the
only crustacean meat sample investigated in our studies in
which 2-acetyl-1-pyrroline was not found among the key aroma
compounds. In contrast, in their study on the aroma
components of cooked American lobster tail meat, Lee et al.9
had reported 2-acetyl-1-pyrroline to be among the most
important aroma compounds. However, in their study a high
FD factor for 2-acetyl-1-pyrroline was determined by
application of AEDA on an extract obtained by steam
distillation, whereas after application of AEDA on an aroma
extract obtained by direct solvent extraction and subsequent
high-vacuum distillation, only a relatively low FD factor was
found compared to the medium-high FD factor of 64 found in
our study. Thus, to clarify the actual aroma contribution of 2-
acetyl-1-pyrroline to the aroma of cooked American lobster
meat, quantitative data will have to be determined.
3-Methylindole (49, mothball-like, fecal) had also been
detected with a high FD factor in the studies of Lee et al.9 on
cooked American lobster tail meat, where it was, however,
suggested to have a negative influence on the overall aroma.
Compared to prawn meat, a total of five odorants with FD ≥
16 were detected only in the cooked lobster meat extracts.
Among them, the musty-smelling dodecanoic acid (U5) was
identified with an FD factor of 128 in the extract of Norway
lobster, whereas the identities of compounds U1−U4 remained
unclear.
Comparison of the odorants responsible for the overall
aroma of blanched Norwegian and American lobster with the
results of the BPM showed that most of the key odorants were
present in all three samples, however, with different FD factors.
Furthermore, the seasoning-like-smelling 3-hydroxy-4,5-di-
methyl-2(5H)-furanone was found as a key odorant only in
the extract of blanched Norway lobster, whereas the aroma
extract of American lobster featured a high FD factor for 3-
methylindole. Subsequent quantitative studies, calculation of
odor activity values, and aroma recombination experiments will
clarify the actual aroma contribution of single odorants to the
overall aroma of thermally processed crustaceans.
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■
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