Strecker-type degradation produced by the lipid oxidation products 4,5-epoxy-2-alkenals

Article abstract of DOI:10.1021/jf048883r

Strecker degradation is one of the most important reactions leading to final aroma compounds in the Maillard reaction. In an attempt to clarify whether lipid oxidation products may be contributing to the Strecker degradation of amino acids, this study analyzes the reaction of 4,5-epoxy-2-alkenals with phenylalanine. In addition to N-substituted 2-(1-hydroxyalkyl)pyrroles and N-substituted pyrroles, which are major products of the reaction, the formation of both the Strecker aldehyde phenylacetaldehyde and 2-alkylpyridines was also observed. The aldehyde, which was produced at 37°C - as could be determined by forming its corresponding thiazolidine with cysteamine - and pH 6-7, was not produced when the amino acid was esterified. This aldehyde is suggested to be produced through imine formation, which is then decarboxylated and hydrolyzed. This reaction also produces a hydroxyl amino derivative, which is the origin of the 2-alkylpyridines identified. All these data indicate that Strecker-type degradation of amino acids is produced at 37°C by some lipid oxidation products. This is a new proof of the interrelations between lipid oxidation and Maillard reaction, which are able to produce common products by analogue mechanisms.

Full text of DOI:10.1021/jf048883r

7126 J. Agric. Food Chem. 2004, 52, 7126−7131
Strecker-type Degradation Produced by the Lipid Oxidation
Products 4,5-Epoxy-2-Alkenals
FRANCISCO J. HIDALGO AND ROSARIO ZAMORA*
Instituto de la Grasa, Consejo Superior de Investigaciones Cient´ıficas,
Avenida Padrea Garc´ıa Tejero 4, 41012 Seville, Spain
Strecker degradation is one of the most important reactions leading to final aroma compounds in the
Maillard reaction. In an attempt to clarify whether lipid oxidation products may be contributing to the
Strecker degradation of amino acids, this study analyzes the reaction of 4,5-epoxy-2-alkenals with
phenylalanine. In addition to N-substituted 2-(1-hydroxyalkyl)pyrroles and N-substituted pyrroles, which
are major products of the reaction, the formation of both the Strecker aldehyde phenylacetaldehyde
and 2-alkylpyridines was also observed. The aldehyde, which was produced at 37 °Csas could be
determined by forming its corresponding thiazolidine with cysteaminesand pH 6-7, was not produced
when the amino acid was esterified. This aldehyde is suggested to be produced through imine
formation, which is then decarboxylated and hydrolyzed. This reaction also produces a hydroxyl amino
derivative, which is the origin of the 2-alkylpyridines identified. All these data indicate that Strecker-
type degradation of amino acids is produced at 37 °C by some lipid oxidation products. This is a new
proof of the interrelations between lipid oxidation and Maillard reaction, which are able to produce
common products by analogue mechanisms.
KEYWORDS: 2-Alkylpyridines; carbonyl-amine reactions; flavor production; lipid oxidation; Maillard
reaction; nonenzymatic browning; pyrroles; Strecker aldehydes
INTRODUCTION
Scheme 1. Strecker Degradation of Amino Acids Produced by
-Dicarbonyl Compounds
R
Strecker degradation is one of the most important reactions
leading to final aroma compounds in the Maillard reaction (1-
4). It involves the initial Schiff base formation of an R-dicar-
bonyl compound (1) with an amino acid (2). After rearrange-
ment, decarboxylation, and hydrolysis, an R-amino carbonyl
compound (3) and the corresponding Strecker aldehyde (4) are
generated (Scheme 1). R-Amino carbonyl compounds (3) are
precursors of pyrazines (5-7), and many Strecker aldehydes
(4) are significant flavor compounds (8).
Strecker degradation is supposed to be produced from
carbohydrate degradation products, despite the well-known
contribution of lipids to flavor formation in cooked foods (9,
10). This is likely a consequence of the lack of lipids to produce
significant amounts of R-dicarbonyl derivatives upon oxidation.
However, compounds that have a certain analogy to R-dicar-
bonyl compounds are common products of lipid oxidation, and
they might also produce Strecker-type degradation of amino
acids. This is the case of 4,5-epoxy-2-alkenals (5), which have
two conjugated oxygenated functions.
when starting from n - 6 polyunsaturated fatty acids, the
epoxyalkenal obtained is 4,5(E)-epoxy-2(E)-decenal (5b) by
decomposition of an intermediate 12,13(E)-epoxy-9-hydroper-
oxy-10-octadecenoic acid (11). Analogously, the 4,5(E)-epoxy-
2(E)-heptenal (5a) is the product of oxidation of the n - 3
polyunsaturated fatty acids (12). These epoxyalkenals have been
detected in many different food systems (13).
In an attempt to clarify whether lipid oxidation products may
be contributing to the Strecker degradation of amino acids, this
study analyzes the reaction of 4,5-epoxy-2-alkenals (5) with
phenylalanine (6). Phenylalanine (6) was selected because its
aldehyde derivative phenylacetaldehyde (14) has a high boiling
point (195 °C), can be easily determined by gas chromatogra-
4,5-Epoxy-2-alkenals (5) are secondary products of lipid
peroxidation. They are produced in the decomposition of
intermediate epoxyhydroperoxy fatty acids by a mechanism that
is common for the different polyunsaturated fatty acids. Thus,
* To whom correspondence should be addressed: tel +(34) 954 611
550; fax +(34) 954 616 790; e-mail rzamora@ig.csic.es.
10.1021/jf048883r CCC: $27.50
© 2004 American Chemical Society
Published on Web 10/15/2004

Strecker-type Degradation Produced by Epoxyalkenals
J. Agric. Food Chem., Vol. 52, No. 23, 2004 7127
to 240 ° C at 5 °C/min and then to 325 °C at 10 °C/min; transfer line
to MSD, 280 °C; ionization (EI), 70 eV.
phy-mass spectrometry (GC-MS), and is a very powerful
odorant (8). 4,5-Epoxy-2-alkenals (5) were previously shown
to contribute to flavor production in oxidized lipid/amino acid
and oxidized lipid/protein reaction mixtures, although Strecker
aldehydes were not detected in those reactions because those
studies were carried out with lysine (14).
1H and 13C NMR. 1H and 13C NMR spectra at 300 and 75.4 MHz,
respectively, were determined in a Bruker AC-300P (Karlsruhe,
Germany), with Me4Si as internal standard. Two-dimensional NMR
was used to assign the 13C NMR spectra.
Browning. Samples incubated in buffer (200 µL) were extracted
with 200 µL of chloroform, and 180-µL aliquots of the resulting organic
and aqueous extracts were diluted to 1 mL with chloroform or water,
respectively. Their colors were determined spectrophotometrically on
a Shimadzu UV-2401 PC UV-vis spectrophotometer. Color differences
(∆E) at the different periods of time were calculated from the
determined Cielab L*, a*, and b* values according to Hunter (17):
EXPERIMENTAL PROCEDURES
Materials. 4,5(E)-Epoxy-2(E)-heptenal (5a) and 4,5(E)-epoxy-2(E)-
decenal (5b) were prepared from heptadienal and decadienal, respec-
tively, as described previously (15). L-Phenylalanine (6), l-phenylalanine
methyl ester hydrochloride (6M), phenylacetaldehyde (14), 2-ethyl-
pyridine (15a), and 2-pentylpyridine (15b) were obtained from Aldrich
Chemical Co. (Milwaukee, WI). All other chemicals were purchased
from reliable commercial sources.
∆E ) [(L* - 100)2 + (a*)2 + (b*)2]1/2
by referring the determined values to an ideal colorless solution of L*
) 100, and a* ) b* ) 0 (18).
Epoxyalkenal/Amino Acid Reaction Mixtures. Mixtures of 0.1
mmol of the epoxyalkenal [4,5(E)-epoxy-2(E)-heptenal (5a) or 4,5-
(E)-epoxy-2(E)-decenal (5b)] and 0.1 mmol of the amino acid [phen-
ylalanine (6) or phenylalanine methyl ester (6M)] were incubated in 1
mL of acetonitrile-water (2:1) or in 1 mL of buffer at 37 °C and
analyzed by GC-MS at different incubation times. The following buffers
were employed: 0.3 M sodium citrate at pH 4, 5, 5.5, and 6; 0.3 M
sodium phosphate at pH 6, 6.5, 7, 7.4, and 8; and 0.3 M sodium borate
at pH 8, 9, and 10. Reaction mixtures also included 50 µL of 2 N
KOH in methanol for phenylalanine methyl ester incubated in aceto-
nitrile-water (2:1). Additionally, major compounds of the reaction were
isolated by column chromatography and their structures were deter-
RESULTS
Isolation and Characterization of the Major Products of
4,5(E)-Epoxy-2(E)-heptenal/Phenylalanine Reaction. To iden-
tify by GC-MS the different compounds produced in the reaction
between epoxyalkenals and phenylalanine, the major products
of reaction between 4,5(E)-epoxy-2(E)-heptenal (5a) and phen-
ylalanine (6) were isolated by column chromatography and
1
characterized by H and 13C NMR and MS.
Analogously to previous studies of the reaction between
epoxyalkenals and amines and amino acids (19-21), this
reaction produced the corresponding N-substituted 2-(1-hy-
droxyalkyl)pyrrole (7) and the N-substituted pyrrole (10) as main
reaction products. The reaction scheme, including the different
compounds produced, is shown in Scheme 2. This reaction
scheme implies in a first step the transformation of the imine
trans carbon-carbon double bond into a cis double bond, which
can occur by conjugate addition-elimination of amine to the
CdC, well-known for R,â-unsaturated carbonyl compounds.
This cis isomer of imine would then convert to the indicated
cyclic intermediate, the transformation of which into products
7 and 10 is likely to occur by electronic arrangement. This
electronic arrangement implies the exit of either an aldehyde
(11), when compound 10 is produced, or the proton at position
5 of the ring of the intermediate, producing compound 7. In
addition, the close presence of the carboxylic group and the
hydroxyl group in 7 may facilitate the formation of lactone 8
by intramolecular dehydration under favorable conditions.
Finally, the thermal treatment of compounds 7 and 10 may
produce their decarboxylation to compounds 9 and 12, respec-
tively. This heating may occur, for example, in the injection
port of the chromatograph.
1
mined by H and 13C NMR and MS.
Samples for GC-MS analyses were prepared differently if reactions
were done in acetonitrile-water (2:1) or in buffer. Samples incubated
in acetonitrile-water (2:1) (100 µL) were diluted with 100 µL of ether-
2-propanol (1:1) and 25 µL of the internal standard solution [1.35 mg
of 3(Z)-nonenol in 1 mL of methanol] and injected in the chromato-
graph. Samples incubated in buffer (200 µL) were treated with 25 µL
of the internal standard solution and extracted with 200 µL of
chloroform. Phases were separated by centrifugation at 2000g for 10
min and the organic extracts were injected in the chromatograph.
Compounds isolated by column chromatography and characterized
by 1H and 13C NMR and MS were obtained from overnight incubations
in 0.3 M sodium phosphate buffer, pH 7, which were taken to pH 3
with HCl and extracted with chloroform. This extract was fractionated
on silica gel with hexane-acetone (2:1) as solvent.
Thiazolidine Derivatives of Aldehydes Produced in Epoxyalkenal/
Amino Acid Reaction Mixtures. In addition to their direct determi-
nation by GC-MS, aldehydes produced in epoxyalkenal/amino acid
reaction mixtures were derivatized with cysteamine according to the
method of Yasuhara et al. (16), which was slightly modified, and the
thiazolidines produced were analyzed by GC-MS. Briefly, the incubated
epoxyalkenal/amino acid reaction (400 µL) was diluted with 200 µL
of methanol-water (1:1), treated with 45 mg of cysteamine hydro-
chloride, and the pH of the solution was immediately adjusted to 8
with 2 N NaOH. The resulting solution was then stirred for 3 h at
room temperature. After that time, samples were diluted with 1 mL of
water and extracted with 1 mL of ethyl acetate. The resulting organic
layer was dried over sodium sulfate and injected in the gas chromato-
graph under the conditions described below. Thiazolidine derived from
propanal (11aT): Rt 8.54 min; mass spectrum m/z (%) 117 (18), 88
(100), 71 (12), 70 (24), 61 (11), and 56 (16). Thiazolidine derived from
hexanal (11bT): Rt 17.85 min; mass spectrum m/z (%) 159 (12), 88
(100), 70 (11), 61 (10), and 56 (25). Thiazolidine derived from
phenylacetaldehyde (14T): Rt 25.24 min; mass spectrum m/z (%) 179
(1), 150 (1), 132 (5), 117 (3), 103 (3), 92 (4), 91 (20), 90 (5), 89 (7),
88 (100), 77 (4), 65 (6), and 61 (8).
When the reaction was carried out between 4,5(E)-epoxy-
2(E)-heptenal (5a) and phenylalanine (6), the major isolated
compounds were the lactone 4-benzyl-1-ethyl-1H-pyrrolo[2,1-
c][1,4]oxazin-3-one (8a) and the N-substituted pyrrole 3-phenyl-
2-pyrrol-1-ylpropionic acid (10), which were characterized by
1H and 13C NMR and MS.
1
4-Benzyl-1-ethyl-1H-pyrrolo[2,1-c][1,4]oxazin-3-one (8a): H
NMR (CDCl3) δ 0.96 t (3H, J ) 7.3 Hz, CH3CH2), 1.90 m
(2H, CH3CH2CH), 3.35 m (2H, PhCH2), 4.10 m (1H, CH3-
CH2CH), 5.07 t (1H, J ) 5.2 Hz, PhCH2CH), 5.88 m (1H, H3
of pyrrole), 6.20 dd (1H, J ) 2.7 and 3.5 Hz, H4 of pyrrole),
6.51 m (1H, H5 of pyrrole), 6.86 m (2H, H2 and H6 of phenyl),
and 7.23 m (3H, H3, H4, and H5 of phenyl); 13C NMR (CDCl3)
δ 8.86 q (CH3), 25.99 t (CH3CH2), 40.86 t (PhCH2), 59.65 d
(PhCH2CH), 76.80 d (CH3CH2CH), 103.10 d (C3 of pyrrole),
109.73 d (C4 of pyrrole), 118.37 d (C5 of pyrrole), 125.83 s
GC-MS Analyses. GC-MS analyses were conducted with a Hewlett-
Packard 6890 GC Plus coupled with an Agilent 5973 MSD (mass-
selective detector, quadrupole type). A fused-silica HP5-MS capillary
column (30 × 0.25 mm i.d., coating thickness 0.25 µm) was used.
Working conditions were as follows: carrier gas, helium (1 mL/min
at constant flow); injector, 250 °C; oven temperature, from 70 (1 min)

7128 J. Agric. Food Chem., Vol. 52, No. 23, 2004
Hidalgo and Zamora
Scheme 2. Formation of Pyrrole Derivatives between 4,5-Epoxy-2-alkenals and Phenylalaninea
a For 5a, 7a, 8a, 9a, and 11a, R1
)
CH3CH2; for 5b, 7b, 8b, 9b, and 11b, R1
)
CH3(CH2)4. M is employed for the corresponding methyl esters; T is employed
for the thiazolidines of the corresponding aldehydes.
(C2 of pyrrole), 127.76 d (C4 of phenyl), 128.69 d (C3 and C5
of phenyl), 129.59 d (C2 and C6 of phenyl), 134.64 s (C1 of
phenyl), and 168.85 s (CO); MS m/z (%, ion structure) 255 (36,
M+), 226 (17, M+ - ethyl), 198 (100, M+ - C2H5CO), 164
(28, M+ - PhCH2), 136 (23), 105 (24), 91 (76, PhCH2+), 77
(24), and 65 (23).
1
3-Phenyl-2-pyrrol-1-ylpropionic acid (10): H NMR (CDCl3)
δ 3.30 m (2H, CH2CH), 4.60 m (1H, CH2CH), 6.02 m (2H, H3
and H4 of pyrrole), 6.60 m (2H, H2 and H5 of pyrrole), 6.96 m
(2H, H2 and H6 of phenyl), and 7.15 m (3H, H3, H4, and H5
of phenyl); 13C NMR (CDCl3) δ 39.20 t (CH2CH), 65.57 d
(CH2CH), 107.93 d (C3 and C4 of pyrrole), 120.34 d (C2 and
C5 of pyrrole), 126.58 d (C4 of phenyl), 128.34 d (C3 and C5
of phenyl), 128.65 d (C2 and C6 of phenyl), 137.67 s (C1 of
phenyl), and 177.92 s (COOH); MS m/z (%, ion structure): 215
(61, M+), 170 (34, M+ - COOH), 147 (11), 124 (88, M+
-
PhCH2), 103 (11), 91 (100, PhCH2+), 78 (21), 65 (16), and 51
(16).
In addition, the decarboxylated products 1-(1-phenethyl-1H-
pyrrol-2-yl)propan-1-ol (9a) and 1-phenethyl-1H-pyrrole (12)
were characterized by GC-MS.
1-(1-Phenethyl-1H-pyrrol-2-yl)propan-1-ol (9a): MS m/z (%,
ion structure) 229 (22, M+), 211 (23, M+ - H2O), 200 (46,
M+ - ethyl), 182 (10), 172 (38), 120 (57, M+ - H2O -
PhCH2), 105 (100, PhCH2CH2+), 104 (36), 103 (24), 91 (48,
PhCH2+), 80 (21), 77 (28), and 65 (20).
1-Phenethyl-1H-pyrrole (12): MS m/z (%, ion structure) 171
(45, M+), 104 (17), 91 (24, PhCH2+), 80 (100, M+ - PhCH2),
77 (10), 65 (10), 53 (19), and 51 (14).
Figure 1. Total ion chromatograms of GC-MS analysis for the reactions
of (A) 4,5(E)-epoxy-2(E)-heptenal (5a) with phenylalanine (6), (B) 4,5-
(E)-epoxy-2(E)-heptenal (5a) with phenylalanine methyl ester (6M), (C)
4,5(E)-epoxy-2(E)-decenal (5b) with phenylalanine (6), and (D) 4,5(E)-
epoxy-2(E)-decenal (5b) with phenylalanine methyl ester (6M) after
overnight incubation at 37 °C in acetonitrile−water (2:1). Structure for
GC-MS Analysis of Epoxyalkenal/Phenylalanine Reaction
Mixtures. Figure 1A shows the total ion chromatogram of GC-
MS analysis for the reaction mixture of 4,5(E)-epoxy-2(E)-
heptenal (5a) and phenylalanine (6) after overnight incubation
in acetonitrile-water (2:1) at 37 °C. In addition to the original
aldehyde, the corresponding pyrrole derivatives 8a, 9a, and 12
were among the major reaction products and could be easily
the identified compounds are given either in Schemes 2 and 3 or in the
text. The internal standard [3(Z)-nonenol] is marked IS.
identified by their mass spectra and retention times. Other major
products were a pyrrole derivative, which has been tentatively
identified as 2-ethyl-1-phenethyl-1H-pyrrole (EP), the Strecker

Strecker-type Degradation Produced by Epoxyalkenals
J. Agric. Food Chem., Vol. 52, No. 23, 2004 7129
Figure 3. Effect of pH on phenylacetaldehyde (14) formation in the reaction
between 4,5(E)-epoxy-2(E)-heptenal (5a) and phenylalanine (6). Samples
were incubated overnight in 0.3 M sodium citrate (pH 4
−6), sodium
Figure 2. Total ion chromatogram of GC-MS analysis for the reactions
of (A) 4,5(E)-epoxy-2(E)-heptenal (5a) with phenylalanine (6) and (B) 4,5-
(E)-epoxy-2(E)-decenal (5b) with phenylalanine (6) after overnight incuba-
phosphate (pH 6 8), or sodium borate (pH 8 10) buffer and then extracted
−
−
with chloroform, and phenylacetaldehyde (14) was determined by GC-
MS. Phenylacetaldehyde/internal standard (14/IS) area ratios are given.
The IS was 3(Z)-nonenol.
tion at 37 °C in acetonitrile−water (2:1) and derivatization with cysteamine.
Thiazolidines of propanal, hexanal, and phenylalanine are marked 11aT,
11bT, and 14T, respectively. The internal standard [3(Z)-nonenol] is marked
IS. Structures for other identified compounds are given either in Schemes
2 and 3 or in the text.
(24), 131 (29), 121 (23), 118 (45), 108 (32), 104 (33), 91 (100,
Ph CH2+), 80 (26), 77 (35), and 65 (26).
3-Phenyl-2-pyrrol-1-ylpropionic acid methyl ester (10M): MS
m/z (%, ion structure) 229 (67, M+), 170 (60, M+ - CO2CH3),
168 (29), 162 (18), 138 (100, M+ - PhCH2), 131 (13), 110
(12), 103 (14), 91 (59, PhCH2+), 78 (15), 77 (14), 65 (14), 59
(16), and 51 (12).
aldehyde phenylacetaldehyde (14), and 2,4-heptadienal (HD).
These last two compounds were identified by comparison of
mass spectra and retention times with authentic standards.
Analogous products were also formed in 4,5(E)-epoxy-2(E)-
decenal/phenylalanine reaction mixtures (Figure 1C). Thus,
compound 12 was identical to the one obtained in the 4,5(E)-
epoxy-2(E)-heptenal/phenylalanine reaction mixtures, and com-
pound 9b could be identified by analogy of its mass spectrum
to that of 9a. The mass spectrum of 1-(1-phenethyl-1H-pyrrol-
2-yl)hexan-1-ol (9b) was m/z (%, ion structure) 271 (13, M+),
253 (31, M+ - H2O), 210 (56, M+ - H2O - propyl), 200 (56,
M+ - pentyl), 172 (26), 162 (14, M+ - H2O - PhCH2), 118
(20), 106 (17), 105 (100, PhCH2CH2+), 104 (27), 103 (16), 91
(31, PhCH2+), 80 (19), and 77 (21). In addition, the formation
of hexanal (11b), 2-pentylfuran (PF), the Strecker aldehyde
phenylacetaldehyde (14), 2-octenal (OA), 2-pentylpyridine
(15b), 2,4-decadienal (DD), and a pyrrole derivative, which has
been tentatively identified as 2-pentyl-1-phenethyl-1H-pyrrole
(PP), could be identified.
The identification of compounds 8, 9, 11, and 12 in both
reactions confirmed that the reaction scheme indicated in
Scheme 2 was being produced. However, the presence of
Strecker aldehyde phenylacetaldehyde (14) suggested that
pyrrole formation was competing with other reaction(s) that
produced this aldehyde.
GC-MS Analysis of Epoxyalkenal/Phenylalanine Methyl
Ester Reaction Mixtures. To confirm that formation of
compound 14 was not an artifact, the reactions of epoxyalkenals
5a and 5b with phenylalanine methyl ester (6M) were studied.
Figure 1B shows the total ion chromatogram of GC-MS analysis
for the reaction mixture of 4,5(E)-epoxy-2(E)-heptenal (5a) and
phenylalanine methyl ester (6M) after overnight incubation in
acetonitrile-water (2:1) at 37 °C. Because methyl esters are
not easily decarboxylated, the corresponding 9a and 12 were
absent. However, the expected N-substituted 2-(1-hydroxyalkyl)-
pyrrole (7) and N-substituted pyrrole (10) were present, and they
could be easily identified by their mass spectra.
In addition, the pyrrole derivative EP and its analogous ester
(EPM) were also present. However, the phenylacetaldehyde (14)
was absent, therefore suggesting that the acid group is necessary
for its formation.
Analogous results were also obtained when the reaction of
4,5(E)-epoxy-2(E)-decenal (5b) with phenylacetaldehyde methyl
ester (6) was studied. Figure 1D shows the total ion chromato-
gram of GC-MS analysis obtained for this reaction in aceto-
nitrile-water (2:1) after overnight incubation at 37 °C. The
corresponding N-substituted 2-(1-hydroxyalkyl)pyrrole (7) and
N-substituted pyrrole (10) were also major products of the
reaction and could be easily identified by their mass spectra.
The mass spectrum of 2-[2-(1-hydroxyhexyl)pyrrol-1-yl]-3-
phenylpropionic acid methyl ester (7bM) was m/z (%, ion
structure) 329 (17, M+), 311 (41, M+ - H2O), 268 (51, M+ -
H2O - propyl), 258 (33, M+ - pentyl), 242 (17), 232 (17),
230 (100, M+ - C6H11O), 226 (48), 203 (22), 198 (64, 230 -
methanol), 170 (58), 162 (31), 151 (29), 131 (35), 121 (43),
104 (29), 103 (23), 91 (43, PhCH2+), 80 (29), and 68 (35).
Other compounds formed were hexanal (11b), pentylfuran
(PF), and the pyrrole (PP) and its analogous ester (PPM).
However, neither phenylacetaldehyde (14) nor 2-ethylpyridine
(15) was present, therefore confirming that the acid group is
necessary for the formation of these compounds.
Thiazolidine Derivatives of Aldehydes Produced in
Epoxyalkenal/Amino Acid Reaction Mixtures. To confirm
that phenylacetaldehyde (14) was produced at 37 °C and not as
a consequence of the heating at the injection port of the
chromatograph, aldehydes produced in epoxyalkenal/phenyla-
lanine reaction mixtures were derivatized with cysteamine, and
the produced thiazolidines were determined by GC-MS. Figure
2A shows the total ion chromatogram of GC-MS analysis for
the reaction mixture of 4,5(E)-epoxy-2(E)-heptenal (5a) and
phenylalanine (6) after overnight incubation in acetonitrile-
water (2:1) at 37 °C and derivatization with cysteamine. Two
aldehydes were detected: propanal (11aT) and phenylacet-
aldehyde (14T). The former is produced as a byproduct of
N-substituted pyrrole 10 formation (Scheme 2), and the ap-
2-[2-(1-Hydroxypropyl)pyrrol-1-yl]-3-phenylpropionic acid
methyl ester (7aM): MS m/z (%, ion structure) 287 (M+, 29),
269 (49, M+ - H2O), 258 (24, M+ - ethyl), 230 (75, M+
-
C3H5O), 210 (22), 198 (75, 230 - methanol), 170 (79), 168

7130 J. Agric. Food Chem., Vol. 52, No. 23, 2004
Hidalgo and Zamora
Effect of pH on Phenylacetaldehyde Formation and
Browning Development in Epoxyalkenal/Phenylalanine Re-
action Mixtures. Phenylacetaldehyde (14) formation was pH-
dependent and was mainly produced at pH 6-7. Figure 3 shows
the formation of phenylacetaldehyde as a function of pH. It
followed a Gaussian curve (r2 ) 0.92) with the center at pH
6.9. Its formation was not related to color development in these
reactions, which is believed to be a consequence of the
polymerization of N-substituted 2-(1-hydroxyalkyl)pyrrole (7)
and favorably produced under basic conditions (19, 22).
However, when epoxyalkenal/phenylalanine reaction mixtures
were extracted with chloroform, the effect of pH on reaction
browning was different for both aqueous and organic extracts
(Figure 4). Thus, although the main browning was observed in
the aqueous extract, which increased linearly (r ) 0.994, p <
0.0001) between pH 5 and 10, the browning in the organic
extract followed a Gaussian curve (r2 ) 0.96) with the center
at pH 6.4. The similarity of this last curve with the curve of
phenylacetaldehyde formation (Figure 3), and their apparent
correlation (r ) 0.92, p ) 0.000 13), suggested a relationship
between the formation of phenylacetaldehyde and the browned
pigments extractable with chloroform.
Figure 4. Effect of pH on browning development in the reaction between
4,5(E)-epoxy-2(E)-heptenal (5a) and phenylalanine (6). Samples were
incubated overnight in 0.3 M sodium citrate (pH 4−6), sodium phosphate
(pH 6 8), or sodium borate (pH 8 10) buffer and then extracted with
−
−
chloroform. Colors in (A) aqueous and (B) organic extracts were
determined spectrophotometically.
DISCUSSION
Fats and oils are notorious for their role in the development
of off-flavors through autoxidation, and their mechanisms of
formation have been broadly studied (23-25). Thus, aldehydes
and ketones are the main volatiles from autoxidation, and these
compounds can cause painty, fatty, metallic, papery, and
candlelike flavors in foods when their concentrations are
sufficiently high. On the other hand, the pleasant flavors that
contribute to general nutty, meaty, roasted, burnt, floral, plant,
or caramel odors in processed foods have been traditionally
believed to be produced as a consequence of Maillard reactions
(25, 26).
The results obtained in this study suggest that this difference
among flavors produced as a consequence of either lipid
oxidation or Maillard reaction may be not so clear, and flavors
traditionally assigned to Maillard reactions may also be produced
as a consequence of lipid oxidation. This is the case of the
Strecker-type degradation of amino acids that have been found
to be produced by the lipid oxidation products 4,5-epoxy-2-
alkenals.
pearance of the latter confirmed its production at 37 °C. In
addition, 2-ethylpyridine (15a) was also identified in this
reaction.
Analogous results were obtained when 4,5(E)-epoxy-2(E)-
decenal (5b) was employed. Figure 2B shows the total the total
ion chromatogram of GC-MS analysis for the reaction mixture
of 4,5(E)-epoxy-2(E)-decenal (5b) and phenylalanine (6) after
overnight incubation in acetonitrile-water (2:1) at 37 °C and
derivatization with cysteamine. This reaction produced the
corresponding aldehyde 11bT (hexanal, which is the aldehyde
produced from n - 6 fatty acid oxidation) and the Strecker
aldehyde phenylacetaldehyde (14T), therefore confirming the
formation of this last aldehyde at 37 °C from lipid oxidation
products. In addition, 2-pentylfuran (PF) and 2-pentylpyridine
(15b) were also identified, analogously to the above-described
direct injection of 4,5(E)-epoxy-2(E)-decenal/phenylalanine
reaction mixture.
Scheme 3. Strecker-type Degradation of Phenylalanine Produced by 4,5-Epoxy-2-alkenalsa
a For 5a, 13a, and 15a, R1
) ) CH3(CH2)4. T is employed for the thiazolidines of the corresponding aldehydes.
CH3CH2; for 5b, 13b, and 15b, R1

Strecker-type Degradation Produced by Epoxyalkenals
J. Agric. Food Chem., Vol. 52, No. 23, 2004 7131
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The mechanism of the reaction between epoxyalkenals and
amino acids leading to the formation of Stecker aldehydes is
suggested in Scheme 3. Analogously to the Strecker degradation
produced by R-dicarbonyl compounds, the imine is produced
in a first step. However, and in contrast to the evolution to the
pyrrole derivatives indicated in Scheme 2, the imine decar-
boxylation and its later hydrolysis must produce the Strecker
aldehyde 14. This mechanism is supported by the hydroxyl
amino derivative produced (13). This derivative should produce
2-ethylpyridine (15a) or 2-pentylpyridine (15b) when starting
from 4,5(E)-epoxy-2(E)-heptenal (5a) or 4,5(E)-epoxy-2(E)-
decenal (5b), respectively. Both 2-ethylpyridine (15a) and
2-pentylpyridine (15b) have been detected in these reactions,
and this may be one of the origins of the appearance of these
compounds in foods (27-29). In addition, the hydroxyl amino
derivative produced (13) might also contribute, by polymeri-
zation, to the browning color that is extracted by chloroform.
All these data indicate that Strecker-type degradation of amino
acids is produced at 37 °C by some lipid oxidation products.
Although additional studies are needed to know if this reaction
is also produced by other lipid oxidation products and to fully
elucidate the real importance of these reactions in final food
flavors produced during food processing and storage, the results
obtained in this study constitute a new proof of the interrelations
between lipid oxidation and Maillard reaction, which are able
to produce common products, both volatilesas described
abovesand nonvolatile (30), by analogous mechanisms and
starting from different reactants.
(19) Hidalgo, F. J.; Zamora, R. Fluorescent pyrrole products from
carbonyl-amine reactions. J. Biol. Chem. 1993, 268, 16190-
16197.
ACKNOWLEDGMENT
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Lipids 1994, 29, 243-249.
(21) Zamora, R.; Hidalgo, F. J. Phosphatidylethanolamine modifica-
tion by oxidative stress product 4,5(E)-epoxy-2(E)-heptenal.
Chem. Res. Toxicol. 2003, 16, 1632-1641.
(22) Hidalgo, F. J.; Zamora, R. Nonenzymatic browning and fluo-
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model system. J. Food Sci. 1993, 58, 667-670.
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We are indebted to Dr. Jorge Ruiz, University of Extremadura,
for valuable discussions and to Jose´ L. Navarro for technical
assistance.
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Received for review July 7, 2004. Revised manuscript received August
20, 2004. Accepted September 5, 2004. This study was supported in
part by the European Union and the Plan Nacional de I + D of the
Ministerio de Educacio´n y Ciencia of Spain (Project AGL2003-02280).
JF048883R