A Review of Thermo-Oxidative Degradation of
Food Lipids Studied by 1H NMR Spectroscopy:
Influence of Degradative Conditions and Food
Lipid Nature
Andrea Martı́nez-Yusta, Encarnación Goicoechea, and Marı́a D. Guillén
Abstract: This review summarizes present-day knowledge provided by proton nuclear magnetic resonance (1 H NMR)
concerning food lipid thermo-oxidative degradation. The food lipids considered include edible oils and fats of animal
and vegetable origin. The thermo-oxidation processes of food lipids of very different composition, occurring at low,
intermediate, or high temperatures, with different food lipid surfaces exposed to oxygen, are reviewed. Mention is
made of the influence of both food lipid nature and degradative conditions on the thermo-oxidation process. Interest is
focused not only on the evolution of the compounds that degrade, but also on the intermediate or primary oxidation
compounds formed, as well as on the secondary ones, from both qualitative and quantitative points of view. Very valuable
qualitative and quantitative information is provided by 1 H NMR, which can be useful for metabolomic and lipidomic
studies. The chemical shift assignments of spectral signals of protons of primary (hydroperoxides and hydroxides associated
with conjugated dienes) and secondary, or further (aldehydes, epoxides, among which 9,10-epoxy-12-octadecenoate
[leukotoxin] can be cited, alcohols, ketones) oxidation compounds is summarized. It is worth noting the ability of 1 H
NMR to detect toxic oxygenated α,β-unsaturated aldehydes, like 4-hydroperoxy-, 4,5-epoxy-, and 4-hydroxy-2-alkenals,
which can be generated in the degradation of food lipids having omega-3 and omega-6 polyunsaturated groups in both
biological systems and foodstuffs. They are considered as genotoxic and cytotoxic, and are potential causative agents of
cancer, atherosclerosis, and Parkinson’s and Alzheimer’s diseases.
Keywords: characterization and quantification, food lipids, oxidation compounds, proton nuclear magnetic resonance
(1 H NMR), thermo-oxidation
Introduction
can occur simultaneously with the formation of conjugated double
bonds. The termination stage takes place when the collision of free
radicals provokes the formation of stable molecules, thus making
the free radicals disappear. The variety of compounds which may
form in these reactions is great and their formation occurs sequentially; for this reason, some of them are called primary oxidation
compounds, and those derived from them are called secondary
oxidation compounds, and there may also be tertiary, or further,
oxidation compounds formed.
Pioneer studies carried out with standard compounds made a
very important contribution to our knowledge of these processes.
In these studies, extraction and separation techniques, and in many
cases proton nuclear magnetic resonance (1 H NMR) spectroscopy,
were used to identify and quantify some of the compounds formed
(Perkins and Anfinsen 1971 [using a Varian A-60 spectrometer]; Gardner and Weisleder 1972 [using a 100-MHz spectromMS 20140255 Submitted 16/2/2014, Accepted 25/4/2014. Authors Martinez- eter]; Wineburg and Swern 1974 [using a Varian XL-100 specYusta, Goicoechea, and Guillen are with Dept. of Food Technology, Lascaray Research trometer]; Neff and others 1981, 1982 [using a Bruker WH-90
Center, Faculty of Pharmacy, Univ. of the Basque Country (UPV/EHU), Vitoria, spectrometer]; Neff and others 1983, 1988, 1990; Neff and Frankel
Spain. Direct inquiries to author Guillén (E-mail: [email protected]). 1984 [using a Bruker WM-300 spectrometer]; Frankel and others
Lipid oxidation involves a large group of complex reactions induced by oxygen in the presence of initiators, among which are
heat, free radicals, light, photosensitizing pigments, and metal ions.
It has been described that this process takes place through a sequential free radical chain reaction mechanism, which implies the
well-known 3 stages of initiation, propagation, and termination
(Frankel 2005).
In short, the initiation stage involves the homolytic breakdown
of hydrogen in the α-position in relation to the double bond in an
unsaturated acyl group, forming unstable free radicals that stabilize by abstracting a hydrogen free radical from another chemical
species. It has been postulated that hydroperoxides and hydroxides
are formed at the propagation stage, and that they in turn generate free radicals; the formation of hydroperoxides or hydroxides
838 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
C 2014 Institute of Food Technologists®
doi: 10.1111/1541-4337.12090
Food lipid thermo-oxidation studied by 1 H NMR . . .
1982 [using a Bruker WH-90 spectrometer]; Frankel and others
1990 [using a Bruker WM-300 spectrometer]; Yamauchi and others 1983 [using a Hitachi R-22 90 MHz spectrometer]; Coxon and
others 1984 [using a 300-MHz spectrometer]; Porter and others
1994 [using a 300-MHz spectrometer]; Butovich 2006; Butovich
and others 2006 [using a Varian 400-MHz spectrometer]; Dobson
and others 2013 [using a Bruker Ascend 500 MHz or a JEOL
Eclipse 400-MHz spectrometer]). In recent years, Hämäläinen and
Kamal-Eldin (2005) have reviewed the 1 H and 13 C NMR spectral data of some of these standards of lipid oxidation compounds.
Gas chromatography (GC) has also been essential in the study of
the volatile compounds formed in thermo-oxidation processes of
standard lipids (Thompson and others 1978; Frankel and others
1981, 1982, 1988), as has high-performance liquid chromatography (HPLC) in the study of the polymers, oligomers, and dimers
formed in thermo-degradation processes (Dobarganes and PérezCamino 1987; Márquez-Ruiz and others 1995; Márquez-Ruiz
and Dobarganes 2005; Dobarganes and Márquez-Ruiz 2007).
As it is known that certain compounds or functional groups can
be formed in food lipid thermo-oxidation processes, many studies
have been devoted to the determination of their concentrations.
Thus, parameters such as peroxide value and conjugated dienes
have been employed to evaluate the occurrence of primary oxidation compounds; however, it has been recently shown that certain
secondary oxidation compounds can also support hydroperoxy
groups and conjugated dienic systems (Guillén and Ruiz 2004b,
2005a, 2005b, 2005c). Similarly, anisidine value and thiobarbituric
acid reactive substances (TBARS) tests have been used to evaluate
the occurrence of secondary oxidation products; however, the usefulness of both methods has also been subject to criticism (GuillénSans and Guzman-Chozas 1998; Laguerre and others 2007). These
classical methods give concentration values of certain compounds
or functional groups in a generic way, but do not provide information about the specific nature of the compounds involved in
each determination, which sometimes could give rise to inaccurate conclusions (Frankel 2005). Thus, these classical methods do
not shed further light on the thermo-oxidative mechanisms of oils
and caution is required in their interpretation.
Techniques that study food lipids as a whole have hardly ever
been used in the analysis of food lipid thermo-oxidation. Two of
the most important ones are Fourier transform infrared (FTIR)
spectroscopy (Guillén and Cabo 1997, 1999, 2000) and NMR
spectroscopy. However, as they are fast and do not require modification or special preparation of the sample, studies aimed at
evaluating their usefulness in this field have recently been carried
out.
1
H NMR spectroscopy has been gaining increasing interest because it is used in metabolomic and lipidomic approaches. However, in many of these studies the use of 1 H NMR spectra is
limited to the introduction of spectral profile data in statistical
analysis in order to extract information on processes or on samples
under the light shed by the statistics. Nevertheless, to extract the
relevant information contained in the 1 H NMR spectra a correct
assignment of the signals to the corresponding protons is essential.
This assignment allows a correct identification of molecules or
functional groups involved, and also the development of adequate
quantitative approaches that permit one to obtain not only qualitative but also the quantitative information which is essential to
draw sound conclusions.
In this context, a review of the use of 1 H NMR spectroscopy in
the study of food lipid thermo-oxidation is presented here. This
review covers not only the influence of the food lipid nature and
C 2014 Institute of Food Technologists®
of the degradation conditions on the evolution of lipid thermooxidation processes, but also the nature and concentration of the
compounds formed which may be detected by this technique.
The aim of this review is to help in providing valuable tools
to improve the conclusions in studies in which lipids and their
thermo-oxidation processes are involved.
1
H NMR as a Tool for the Study of Food Lipid ThermoOxidation Processes
As mentioned, this spectroscopic technique is able to study the
food lipid sample as a whole, before and after submission to any
degradative process. For this reason, if the information provided is
sound, it will be a very useful technique.
Characterization of food lipids before submission to oxidative conditions
Several studies have shown the usefulness of this technique in
the characterization of nonoxidized food lipids. Since the pioneer
studies of Hopkins and Bernstein (1959) and also of Johnson and
Shoolery (1962) using equipment of 40 MHz and of 60 MHz,
respectively, many advances have been made. In spite of the limited resolution of the spectra obtained with this equipment, the
above-mentioned authors indicated that this technique could be
useful not only for identification, but also for quantification of oil
components.
Concerning food lipid main components. Nowadays, due to several well-received contributions, the assignment of the signals of
1
H NMR spectra to the protons of the main nonoxidized food
lipid components, namely of triglycerides, and also of some minor
food lipid components, is well established (Hopkins and Bernstein
1959; Johnson and Shoolery 1962; Gunstone 1990; Wollenberg
1991; Aursand and others 1993; Sacchi and others 1993, 1998;
Segre and Mannina 1997; Igarashi and others 2000; Guillén and
Ruiz 2001, 2003a; Siddiqui and others 2003; Guillén and others
2008; Mannina and others 2008; Vidal and others 2012). As an example, Figure 1 shows a region of the 1 H NMR spectra, recorded
with 400 MHz equipment, of lipids of animal origin such as pork
adipose tissue (PAT), farmed sea bass (Dicentrarchus labrax; SB), and
of 3 vegetable oils, namely virgin linseed (VL), extra-virgin olive
(EVO), and sunflower (SF), dissolved in deuterated chloroform.
All signals observable in this figure are due to protons of several
triglycerides of food lipids. Table 1 gives their assignments to the
corresponding protons, letters in Figure 1 and in Table 1 being in
agreement. A thorough explanation of each of these signals and
its assignment to the different kinds of protons of triglycerides
has been carried out previously (Guillén and Ruiz 2001, 2003a;
Guillén and others 2008; Vidal and others 2012).
All spectra given in Figure 1 have been represented on the same
scale. The different intensity of the common signals in all spectra given, and the presence or absence of others, allows an initial
characterization of these food lipids by simple and direct observation; that is to say, without further enlargement of the spectra.
This task is even easier when some of the signals are enlarged,
as shown in Figure 2. Thus, the difference between PAT lipids
and vegetable oils is clear, due to the high content of saturated
(S) groups in the former, which is observable by the intensity of
the signal at 1.259 ppm. It is also evident that SF oil does not
contain linolenic (Ln) acyl groups (absence of signals at 0.972,
2.093, 2.111, and 2.801 ppm), but has a very important concentration of linoleic (L) acyl groups (intensity of signals at 0.889,
2.036, 2.056, and 2.765 ppm). Similarly, it can be seen without further enlargement that PAT and EVO oil have very small
Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 839
Food lipid thermo-oxidation studied by 1 H NMR . . .
Figure 1–1 H NMR spectra of 2 lipid samples extracted from animal matrixes, pork adipose tissue (PAT) and sea bass (D. labrax; SB), together with 3
vegetable oils, which are virgin linseed (VL), extra-virgin olive (EVO), and sunflower (SF). These spectra were acquired in a Bruker Avance 400
spectrometer operating at 400 MHz. Signal letters agree with those in Table 1.
concentrations of Ln and L acyl groups, but important ones of oleic
(O; intensity of signals at 0.879, 1.270, 2.002, and 2.020 ppm)
, this latter acyl group being higher in EVO oil than in the lipids
of PAT. Finally, the abundance of ω-3 acyl groups in linseed oil
and the lipids of sea bass is shown by the intensity of the signals at 0.972, 2.093, and 2.111 ppm and between 2.773 and
2.895 ppm. In addition, the high proportion of Ln acyl groups
in linseed oil is seen by the intensity of the signal at 2.801 ppm,
whereas in sea bass lipids that of eicosapentaenoic (EPA) plus
arachidonic (ARA) and of docosahexaenoic (DHA) acyl groups is
evidenced by the signals at 1.661 to 1.743 and at 2.364 to 2.421
ppm, respectively. It must be pointed out that farmed fish usually
contains lower concentrations of ARA acyl groups than do wild
specimens.
A much more accurate characterization can be carried out by
the determination of the molar percentages of the different kinds
of acyl groups. Taking into account that the area of the abovementioned signals is proportional to the number of protons that
generate them, and that the proportionality constant is the same in
all the cases, several approaches have been developed to determine
the molar percentage of several kinds of acyl groups in food lipids.
These have been thoroughly explained in previous papers (Aursand and others 1993; Igarashi and others 2000; Guillén and Ruiz
2003b, 2004a, 2004b, 2005a, 2005b, 2005c, 2006, 2008; Guillén
and Goicoechea 2007, 2009; Guillén and others 2008; Guillén and
Uriarte 2009, 2012a, 2012b, 2012c; Goicoechea and Guillén
2010; Vidal and others 2012; Sopelana and others 2013; Martı́nezYusta and Guillén 2014a, 2014b). One of them, which is useful for
vegetable oils and food lipids of animal origin, except fish lipids,
involves the following equations:
Linolenic groups Ln (%) = 100(AJ /3AK )
(1)
Linoleic groups L (%) = 100(2AI /3AK )
(2)
Oleic (or monounsaturated) groups O (or MU) (%)
= 100[(AF − 2AI − AJ )/3AK ]
(3)
Saturated groups S (%) = 100[1 − (AF /3AK )]
(4)
In these equations AJ , AI , and AF are the area of the signal J
(bis-allylic protons of Ln groups), signal I (bis-allylic protons of L
groups), and signal F (mono-allylic protons of all unsaturated acyl
groups), respectively, indicated in Table 1 and Figure 1, and AK
is the area of signal K due to the protons of carbons 1 and 3
of the glycerol backbone of triglycerides. Using these equations,
the molar percentages of the above-mentioned acyl groups in the
several lipids were determined and these are given in Table 2;
the results are in agreement with those expected for oils and fats
of this nature. The accuracy of these determinations made from
1
H NMR spectral data has been demonstrated by using standard
mixtures of triglycerides, giving satisfactory results (Guillén and
Ruiz 2003b).
Similarly, the molar percentage of certain acyl groups of fish
lipids can also be determined from 1 H NMR spectra using different approaches, which have been very thoroughly explained
in previous papers (Guillén and Ruiz 2004a; Guillén and others
2008; Vidal and others 2012); one of these possible approaches
involves these equations:
Total ω−3 groups ω−3(%) = 100(4AB )/3(AH +2AG )
(5)
Docosahexaenoic groups DHA (%) = 100AH /(AH +2AG ) (6)
840 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
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Food lipid thermo-oxidation studied by 1 H NMR . . .
Table 1–Chemical shift assignments and multiplicities of the 1 H NMR signals in CDCl3 of the main acyl groups and minor compounds of food lipids. The
signal letters agree with those given in Figures 1 to 4.
Signal
Main acyl groups
A
A
B
C
D
E
F
G
H
I
J
J’
K
L
M
Minor compounds
N
O
P
Q
R
S
T
U
V
Chemical
shift (ppm)
Multiplicity
0.879
0.889
0.972
1.221 to 1.419
1.522 to 1.700
1.661 to 1.743
1.941 to 2.139
t
t
t
ma
m
m
mb
−CH3
−CH3
−CH3
−(CH2 )n −
−OCO−CH2 −CH2 −
−OCO−CH2 −CH2 −
−CH2 −CH=CH−
2.305
2.364 to 2.421
2.765
2.801
2.773 to 2.895
4.139, 4.303
5.225 to 5.296
5.296 to 5.470
dt
m
t
t
m
dd,dd
m
m
−OCO−CH2 −
−OCO−CH2 −CH2 −
=HC−CH2 −CH=
=HC−CH2 −CH=
=HC−CH2 −CH=
−CH2 OCOR
>CHOCOR
−CH=CH−
0.540
0.681
0.684
0.704
1.670
3.350
3.725
4.949 to 5.072
5.749 to 5.858
s
s
s
s
s
s
tc
dq,dq
m
−CH3 (C−18)
−CH3 (C−18)
−CH3 (C−18)
−CH3 (C−18)
−CH3
−N(CH3 )3
−CH2 OH
−CH=CH2
−CH=CH2
Functional group
Saturated, monounsaturated ω-9 and/or ω-7 acyl groups
Unsaturated ω-6 acyl groups
Unsaturated ω-3 acyl groups
Acyl groups
Acyl groups except for DHA, EPA and ARA acyl groups
EPA and ARA acyl groups
Acyl groups except for -CH2 - of DHA acyl group in β-position
in relation to carbonyl group
Acyl groups except for DHA acyl groups
DHA acyl groups
Diunsaturated ω-6 acyl groups
Triunsaturated ω-3 acyl groups
DHA, EPA, ARA and other polyunsaturated ω-3 acyl groups
Glyceryl groups
Glyceryl groups
Acyl groups
7-avenasterol
Cholesterol
β-sitosterol and 5-campesterol
5-stigmasterol and brassicasterol
Squalene
Phosphatidylcholine
1,2-diglycerides
Unsaturated ω-1 acyl groups
Unsaturated ω-1 acyl groups
a Overlapping of multiplets of methylenic protons in the different acyl groups either in β-position, or further, in relation to double bonds, or in γ -position, or further, in relation to the carbonyl group.
b Overlapping of multiplets of the α-methylenic protons in relation to a single double bond of the different unsaturated acyl groups.
c Standard compounds (dioleoin and dipalmitin) give a triplet centered at 3.73 ppm, and in complex mixtures the signal generated is a doublet centered at 3.72 ppm instead of a triplet (Sopelana
and others
2013).
t, triplet; m, multiplet; DHA, docosahexaenoic acyl groups; EPA, eicosapentaenoic acyl groups; ARA, arachidonic acyl groups; d, doublet; s, singlet; q, quartet.
Eicosapentaenoic plus arachidonic groups EPA + ARA (%)
= 100(2AE )/(AH +2AG )
(7)
Diunsaturated ω−6 (mainly linoleic) groups DUω − 6 (%)
(8)
= 100(2AI )/(AH +2AG )
as 7-avenasterol (signal N) in SF oil (Kurata and others 2005;
Zhang and others 2006a; Sopelana and others 2013). The contents of sterols in vegetable oils can be very varied, even in those
of the same vegetable origin; concentrations ranging from 0 to
206 mg/100 g of individual sterols have been found in different
vegetable oils (Schwartz and others 2008). However, it must be
taken into account that when operating at 400 MHz there is an
overlap between the signal of 5-stigmasterol and brassicasterol
and the side band of the methylic protons of linoleic, oleic, and
saturated acyl groups.
Other very interesting minor components are squalene
(signal R), present mainly in EVO oil (Mannina and others 2003,
2008), and phosphatidylcholine (signal S), present in many lipids
of animal origin like sea bass lipids (Siddiqui and others 2003).
Furthermore, some lipids, such as VL, EVO, and SF oils, as well
as PAT, also contain 1,2-diglycerides (signal T; Sacchi and others
1996; Segre and Mannina 1997). In addition, it is worth mentioning the occurrence in sea bass lipids of ω-1 acyl groups which
give typical signals U and V. Although these had been already
observed in previous studies on salmon lipids (Guillén and Ruiz
2004a), they have recently been identified (Shimizu and others
1991; Fiori and others 2012; Vidal and others 2012).
The concentration of these compounds in the corresponding food lipids can be determined from the area of the abovementioned signals. It is possible to do this determination either
in an absolute way, using a standard compound, or in relation to
other food lipid components present in the same sample.
Total unsaturated groups U (%) = 100(2AF +AH )/(2AH +4AG )
(9)
In these equations AB is the area of signal B due to methylic
protons of ω-3 acyl groups, AH is the area of signal H due to
methylenic protons in α- and β-positions in relation to the carbonyl group of DHA groups, AG is the area of the signal G related
to the methylenic protons in the α-position in relation to carbonyl
groups, except those of DHA groups, and finally AI and AF were
as defined. All these signals are indicated in Figure 1 and in Table 1.
Concerning food lipid minor components. In addition to main
components, nonoxidized food lipids also contain minor components, which are also important because some of them can show
antioxidant ability or interesting nutritional or health-related properties. If these compounds are present in high enough concentrations for them to be detected by 1 HNMR, and if the signals of
their protons do not overlap with those of the main lipid components, then this technique provides information about them. Some
of these detected in the above-mentioned food lipids are shown in
Figure 3. The assignment of these signals is also given in Table 1. It
has been found that food lipids of animal origin, like those of PAT
and sea bass, contain cholesterol (signal O) containing approximate
concentrations near 93 and 734 mg/100 g, respectively (Chiz- Characterization of food lipids after submission to thermozolini and others 1999; Orban and others 2003). Vegetable oils oxidative conditions
contain sterols, with β-sitosterol plus 5-campesterol (signal P),
During thermo-oxidative processes changes are produced in
5-stigmasterol plus brassicasterol (signal Q) being present, as well the original food lipid components and, as mentioned before,
C 2014 Institute of Food Technologists®
Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 841
Food lipid thermo-oxidation studied by 1 H NMR . . .
Figure 2–Enlargement of 1 H NMR spectral signals corresponding to the main acyl groups of the triglycerides of pork adipose tissue lipids (PAT),
farmed sea bass lipids (SB), virgin linseed oil (VL), extra-virgin olive oil (EVO), and sunflower oil (SF). These spectra were acquired in a Bruker Avance
400 spectrometer operating at 400 MHz. Signal letters agree with those in Table 1.
new compounds are generated. Those formed in the 1st step
are named primary oxidation compounds and the ones derived
from them are called secondary or further oxidation compounds.
First, the evolution of the original food lipid components will be
commented on, followed by the formation and evolution of the
different oxidation compounds.
Evolution of main and minor original lipid components. Under
thermo-oxidative conditions both main and minor components
can evolve to give new compounds, and this is reflected in the
intensity of the signals of their protons, which undergo changes,
which are observable in the region of the spectra in which these
signals appear. Figure 4(a), as an example, shows the spectral regions of the original SF oil and of the same oil after submission,
for different heating times, to 2 different temperatures (70 °C and
190 °C) under different processing conditions (10 g of oil in Petri
dishes and 4 L of oil in a deep-fryer, respectively; Goicoechea and
Guillén 2010; Guillén and Uriarte 2012c). It can be observed in
the spectrum of SF oil submitted to 70 °C for 264 h (11 d), under certain oxidative conditions, that the intensity of the signals of
linoleic (L) protons (signals at 0.889, 2.036, 2.056, and 2.765 ppm)
have drastically decreased; however, in the spectrum of SF oil submitted to 190 °C for 40 h under other thermo-oxidative conditions, although the intensity of the same signals due to linoleic (L)
groups has been reduced in relation to its original intensity, they
have not disappeared from the spectra. As Figure 4(a) shows, the
evolution of the different acyl groups throughout the processing
time, under any processing conditions, can be observed directly
in the 1 H NMR spectra. Furthermore, this evolution can also
be quantified using the appropriate equations, developed for each
specific case on the basis of the same principles as mentioned for
nonoxidized lipids (Guillén and Uriarte 2012a, 2012b, 2012c).
Figure 4(b) shows the quantification of the evolution throughout
processing time of the molar percentage of the several kinds of
acyl groups in SF oil submitted to the above-mentioned thermooxidative conditions. It can be observed that great differences are
found, depending on the thermo-oxidation conditions.
Similarly, if minor lipid components are affected during the
thermo-oxidative degradation, the intensity of their proton
842 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
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Food lipid thermo-oxidation studied by 1 H NMR . . .
Table 2–Composition of food lipid samples shown in Figures 1 to 3, obtained from 1 H NMR data and expressed as molar percentage of the different
kinds of acyl groups (data taken from Guillén and Uriarte 2012a, 2012b, 2012c; Vidal and others 2012; Martı́nez-Yusta and Guillén 2014a, 2014b).
Food lipid
Pork adipose tissue lipids (PAT)
Sea bass lipids (SB)
Virgin linseed oil (VL)
Extra-virgin olive oil (EVO)
Sunflower oil (SF)
Ln or ω-3 (%)
1.67 ±
22.10 ±
(DHA 8.09 ±
(EPA 9.49 ±
50.37 ±
0.49 ±
-
0.08
1.60
1.45)
1.59)
0.50
0.02
L (%)
O or MU (%)
S (%)
10.21 ± 0.02
46.83 ± 0.31
41.29 ± 0.41
14.28 ± 2.92
36.36 ± 2.97
26.46 ± 2.16
17.85 ± 0.03
4.49 ± 0.28
55.05 ± 4.31
22.56 ± 0.37
82.06 ± 0.98
34.80 ± 2.83
9.20 ± 0.19
12.93 ± 0.65
10.03 ± 0.95
Acyl groups: Ln, linolenic; ω-3, omega-3; L, linoleic; O or MU, oleic or monounsaturated; S, saturated; DHA, docosahexaenoic; EPA, eicosapentaenoic.
Figure 3–Enlargement of 1 H NMR spectral signals related to minor compounds present in pork adipose tissue lipids (PAT), farmed sea bass lipids (SB),
virgin linseed oil (VL), extra-virgin olive oil (EVO), and sunflower oil (SF). These spectra were acquired in a Bruker Avance 400 spectrometer operating
at 400 MHz. Signal letters agree with those in Table 1.
signals in the spectra decreases or increases with the processing
time, and the evolution of their concentration can also be determined, if the signals of their protons do not overlap with other
signals.
Primary oxidation compounds and their evolution throughout
the thermo-oxidation process. The degradation of the acyl groups
of food lipids provokes the formation of new compounds, the ones
formed at the 1st step being named primary oxidation compounds.
Their natures can vary, and among them some acyl chains supporting hydroperoxy or hydroxy groups have been found, as well as
conjugated dienic systems, which can also have either Z,E or E,E
isomerism. The possibility that some of the hydroperoxy groups
can be supported on saturated and unsaturated acyl chains without conjugated dienic systems exists, as well as the possibility of
2 hydroperoxy groups being supported in the same acyl chain,
that is to say the occurrence of di-hydroperoxides in the same acyl
chain (Coxon and others 1984; Neff and others 1988; Schneider
C 2014 Institute of Food Technologists®
and others 2004, 2005, 2008; Sun and Salomon 2004; Zhang and
others 2006b). The formation of each of these functional groups
depends on both the food lipid nature and the thermo-oxidation
conditions. In fact, under certain degradative conditions the
occurrence of these primary oxidation compounds is not detected
by this technique, which indicates either that they are not formed
or that, if they are formed, their degradation rate is too high for
them to be detected by 1 H NMR (Guillén and Uriarte 2009,
2012a, 2012b, 2012c).
Some of the protons of these compounds give signals in the 1 H
NMR spectra that do not overlap with those of acyl groups, thus
making their identification and also their quantification possible.
The identification of these signals has been based on previous
studies carried out with standard compounds (Neff and others
1990; Frankel and others, 1990; Porter and others 1994; Neff
and El-Agaimy 1996; Kuklev and others 1997; Jie and Lam 2004;
Lin and others 2007; Pajunen and others 2008). Table 3 gives the
Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 843
Food lipid thermo-oxidation studied by 1 H NMR . . .
Figure 4–(a) 1 H NMR spectra of sunflower oil (SF) submitted to 70 °C (10 g oil, Petri dish) and 190 °C (4 L oil, deep-fryer) for different periods. These
spectra were acquired in a Bruker Avance 400 spectrometer operating at 400 MHz. (b) Evolution in both processes of the molar percentages of
linoleic (L), monounsaturated (MU), and saturated + modified (S+M) acyl groups along the heating time (Goicoechea and Guillén 2010; Guillén and
Uriarte 2012c).
chemical shifts of the signals of the protons of the above-mentioned
primary oxidation compounds. In addition, Figure 5(a) gives the
1
H NMR spectral region of nonoxidized corn oil and of this same
oil after storage at room temperature in a closed receptacle for 121
mo (Guillén and Goicoechea 2009), of SF oil submitted to 70 °C
for 72 h and to 100 °C for 9 h (Goicoechea and Guillén 2010).
It can be observed that, depending on the degradative conditions,
different kinds of primary oxidation compounds or functional
groups can be formed or not.
The evolution of these primary oxidation compounds or functional groups throughout a thermo-oxidative process can be observed directly in the spectra and quantified, as mentioned before.
This quantification can be carried out either in an absolute way
by using a standard compound or in relation to main functional
groups present in the food lipids, such as triglycerides. As will be
seen in subsequent sections, great differences have been found in
relation to the formation, or not, of primary oxidation compounds
in high enough concentrations for detection by this technique, in
relation to their nature and also to their formation and degradation
rates. These differences between primary oxidation compounds
are functions of both the food lipid nature and of the conditions
under which the thermo-oxidation occurs.
Secondary or further oxidation compounds and evolution
throughout the thermo-oxidation process. Primary oxidation
compounds are unstable and degrade to generate secondary oxidation compounds, which can have very varied structures. Some
of them have protons whose signals in the 1 H NMR spectra
do not overlap with any other, making it possible to identify
and quantify them. Table 3 gives the chemical shifts of some
of the protons of different secondary oxidation compounds or
functional groups which can be present in oxidized food lipids.
Among them, there are different kinds of aldehydes, alcohols,
epoxides, and ketones. The formation of these compounds also
depends on both the original food lipid composition and the
conditions of the thermo-oxidation process. Thus, aldehydes including n-alkanals, (E)-2-alkenals, (E,E)-2,4-alkadienals, (Z,E)-2,
4-alkadienals, 4,5-epoxy-2-alkenals, 4-hydroxy-(E)-alkenals, 4hydroperoxy-(E)-alkenals, as well as 4-oxo-alkanals can be found
(Guillén and Ruiz 2004a, 2004b, 2005a, 2005b, 2005c, 2006,
2008; Guillén and Goicoechea 2007, 2009; Guillén and Uriarte
2009, 2012a, 2012b, 2012c; Goicoechea and Guillén 2010;
Martı́nez-Yusta and Guillén 2014a,2014b). It should be noticed
that although aldehydes are generally considered as secondary oxidation compounds, all α,β-unsaturated aldehydes have conjugated
844 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
C 2014 Institute of Food Technologists®
Food lipid thermo-oxidation studied by 1 H NMR . . .
Table 3–Chemical shifts and multiplicities of the 1 H NMR signals in CDCl3 of some protons of primary and secondary (or further) oxidation compounds
generated in edible oils submitted to different degradative conditions (data taken from Porter and others 1994; Kuklev and others 1997; Jie and Lam
2004; Guillén and Ruiz 2005a, 2005b, 2005c; Guillén and Goicoechea 2007; Lin and others 2007; Guillén and Uriarte 2009, 2012a, 2012b, 2012c;
Goicoechea and Guillén 2010; Martı́nez-Yusta and Guillén 2014a, 2014b). The signal letters agree with those given in Figures 5 to 9 and 11.
Signal
a
a
Chemical
shift
(ppm)
d
6.58
6.00
5.56
5.51
6.45
5.94
5.64
5.40
6.27
6.06
5.76
5.47
5.72
e
8.3 to 8.9
b
c
c
c
Multiplicity
Functional group
Primary oxidation compounds
dddd
−CH=CH−CH=CH−
ddtd
ddm
dtm
ddd
−CH=CH−CH=CH−
dd
dd
ddt
ddm
−CH=CH−CH=CH−
ddtd
dtm
ddm
m
−CHOOH−CH = CH−
—
−OOH
Secondary or further oxidation compounds
Aldehydes
f
g
h
i
j
k
l
m
n
9.49
9.52
9.55
9.57
9.58
9.60
9.75
9.78
9.79
d
d
d
d
d
d
t
t
t
−CHO
−CHO
−CHO
−CHO
−CHO
−CHO
−CHO
−CHO
−CHO
Alcohols
o
3.43
m
−CHOH−CHOH−
3.54–3.59
3.62
m
t
−CHOH−
−CH2 OH−
2.63
2.88
2.90
m
m
m
−CHOHC−
−CHOHC−
−CHOHC−
u
2.90
m
−CHOHC−CHOHC−
v
3.10
m
−CHOHC−CH2 −CHOHC−
6.08
dt
O=C<CH=CH−
6.82
7.50
8.10
m
??
??
??
??
p
q
Epoxides
r
s
t
Ketones and unidentified
w
w
x
y
(Z,E)-conjugated double
bonds associated with
hydroperoxides (OOH)
(Z,E)-conjugated double
bonds associated with
hydroxides (OH)
(E,E)-conjugated double
bonds associated with
hydroperoxides (OOH)
Double bond associated with
hydroperoxides (OOH)
Hydroperoxide group
(E)-2-alkenals
(E,E)-2,4-alkadienals
4,5-epoxy-2-alkenals
4-hydroxy-(E)-2-alkenals
4-hydroperoxy-(E)-2-alkenals
(Z,E)-2,4-alkadienals
n-alkanals
4-oxo-alkanals
n-alkanals of low molecular
weight (ethanal and
propanal)
9,10-dihydroxy-12octadecenoate
(leukotoxindiol)
secondary alcohols
primary alcohols
(E)-9,10-epoxystearate
(Z)-9,10-epoxystearate
9,10-epoxy-octadecanoate;
9,10-epoxy-12octadecenoate
(leukotoxin); 12,13-epoxy9-octadecenoate
(isoleukotoxin)
9,10–12,13-diepoxyoctadecanoate
9,10–12,13-diepoxyoctadecanoate
Double bond conjugated with
a keto
group
Unidentified
Unidentified
d, doublet; t, triplet; m, multiplet.
dienic systems and some of them also contain hydroperoxy groups.
This fact indicates that neither the hydroperoxy group nor conjugated dienic systems are exclusively present in primary oxidation
compounds, as mentioned before.
Concerning alcohols, primary and secondary ones, together
with dialcohols, have been found (Guillén and Uriarte 2009,
2012a, 2012b, 2012c; Goicoechea and Guillén 2010; Martı́nezYusta and Guillén 2014a, 2014b). With regard to epoxides, those
derived from oleic and from linoleic acyl groups have been detected. The latter may be in the form of not only mono- but also
of diepoxides (Goicoechea and Guillén 2010; Guillén and Uriarte
C 2014 Institute of Food Technologists®
2012a; Martı́nez-Yusta and Guillén 2014a). And finally, ketones
having a conjugated dienic system (keto group conjugated with a
double bond) derived from oleic acyl groups have also been found.
Although signals of these latter ketones were observed previously
(Guillén and Ruiz 2005a, 2005c), this review is where they have
been assigned for the first time on the basis of data for pure compounds provided by other authors (Kuklev and others 1997; Jie
and Lam 2004; Lin and others 2007). However, it should be remembered that none of these compounds can be formed in the
oxidation of every food lipid, or under every thermo-oxidative
condition. Figure 5(b) shows the spectral regions in which some
Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 845
Food lipid thermo-oxidation studied by 1 H NMR . . .
Figure 5–(a) 1 H NMR spectra of primary oxidation compounds formed in corn oil (CO), nonoxidized (0 h), and stored at room temperature in closed
receptacles for 121 mo, sunflower oil (SF) submitted (10 g oil, Petri dish) to 70 °C for 72 h and to 100 °C for 9 h. (b) 1 H NMR spectra of secondary or
further oxidation compounds formed in fish lipids extracted from salmon (SA) submitted to 50 °C for 768 h, sunflower oil (SF), and extra-virgin olive oil
(EVO) submitted (10 g oil, Petri dish) to 70 °C for 216 h and 1560 h, respectively, and to 190 °C (4 L oil, deep-fryer) for 32.5 h (Guillén and Ruiz
2004a, 2005a; Guillén and Goicoechea 2009; Goicoechea and Guillén 2010; Guillén and Uriarte 2012a, 2012c). These spectra were acquired in a
Bruker Avance 400 spectrometer operating at 400 MHz. Signal letters agree with those in Table 3.
of these compounds appear in salmon lipids from fillets submitted
to 50 °C for 768 h (extraction using CS2 as solvent and assisted by
an ultrasound bath), SF oil and EVO oil submitted to 70 °C, and
to deep-frying conditions at 190 °C for different periods of time
(Guillén and Ruiz 2004a, 2005a; Goicoechea and Guillén 2010;
Guillén and Uriarte 2012a, 2012c).
The formation of all these compounds and the evolution of
their concentration throughout the thermo-oxidative degradation
process can be observed directly in the 1 H NMR spectra and
can be quantified either in an absolute way, in relation to a standard compound or in relation to a main functional group such
as triglycerides, this latter method being much easier and faster
(Guillén and Goicoechea 2007, 2009; Guillén and Uriarte 2009,
2012a, 2012b, 2012c; Goicoechea and Guillén 2010; Martı́nezYusta and Guillén 2014a, 2014b).
It is worth noting the capacity for 1 H NMR spectroscopy to
detect and quantify oxygenated α,β-unsaturated aldehydes, such
as 4,5-epoxy-, 4-hydroperoxy-, and 4-hydroxy-(E)-2-alkenals,
which are considered as genotoxic and cytotoxic, and potential causal agents of cancer, atherosclerosis, Parkinson’s disease,
or Alzheimer’s disease, among others (Esterbauer and others 1991;
Guillén and Goicoechea 2008). They are generated in the degradation of ω-3 and ω-6 polyunsaturated acyl groups, fatty acids,
and esters, both in biological systems and foodstuffs, and can be
absorbed through the diet. Similarly, it should be noticed that this
technique permits detection and quantification of 9,10-epoxy12-octadecenoate (leukotoxin) and 12,13-epoxy-9-octadecenoate
(isoleukotoxin) groups, derived from linoleic acyl groups and
bonded to triglyceride backbones (Goicoechea and Guillén 2010).
The former is thus named as it can be formed endogenously, causes
degeneration and necrosis of leukocytes, and has been associated
with multiple organ failure, breast cancer, cell proliferation in vitro,
and disruption of reproductive functions in rats (Markaverich and
others 2005; Thompson and Hammock 2007).
In the thermo-oxidation of food lipids of a specific composition, the types and concentrations of primary and also of secondary
oxidation compounds formed, and also the timing of their formation, depend on the conditions under which the process takes
place. It is also true that under the same thermo-oxidative conditions the types and concentrations of primary and also of secondary
846 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
C 2014 Institute of Food Technologists®
Food lipid thermo-oxidation studied by 1 H NMR . . .
oxidation compounds formed and also the timing of their formation depend on the food lipid nature. Both food lipid composition and conditions of the thermo-oxidation process govern the
physico-chemical rules under which the process evolves. It is obvious that not all intermediate or all final oxidation compounds
mentioned are formed in any particular thermo-oxidation process
or from any food lipid.
Evolution of food lipid oxidation under various degradative
conditions as seen in terms of 1 H NMR
The first studies by 1 H NMR (400 and 600 MHz) on the oxidation process of food lipids, and not on pure standard compounds,
were carried out by Grootveld and coworkers, who submitted
10 to 25 g of oil samples to 180 °C for 90 min, 20 to 60 mL
of oil samples to pan-frying conditions with food (180 °C, 30
min), and also analyzed several repeatedly used culinary frying oils,
among them lard, beef fat, ghee, corn, SF, soybean, grape seed, coconut, rapeseed, peanut, and EVO oils (Claxson and others 1994;
Haywood and others 1995; Sheerin and others 1997; Silwood and
Grootveld 1999). In short, they concluded that thermal stressing
of culinary oils rich in polyunsaturated acyl groups generated high
levels of n-alkanals, (E)-2-alkenals, and (E,E)-2,4-alkadienals, and
the tentatively identified 4-hydroxy-(E)-2-alkenals, via decomposition of their precursor hydroperoxides associated with conjugated
dienes. When oils with a low unsaturation degree and lard were
subjected to the above-mentioned heat treatments, only low concentrations of these aldehydes were produced. The authors also
discussed the dietary, physiological, and toxicological significance
of these results regarding frying practices. Furthermore, using
1,3,5-trichlorobenzene as an external standard, the concentration
(mol/kg oil) of total unsaturated and saturated aldehydes generated
in corn oil, submitted to 180 °C for 90 min in vessels of different size, was determined. It was concluded that, as expected, the
longer the heating time and the bigger the vessel size (and therefore the exposure to oxygen), the higher the concentration of both
saturated and unsaturated aldehydes (Haywood and others 1995).
In this section the information obtained by 1 H NMR on the
mechanisms of lipid oxidation that takes place under different
conditions is reviewed, with regard to degradation of main components, namely the acyl groups of triglycerides, and also to the
formation of oxidation compounds. Special attention is paid to the
influence of both degradative conditions and food lipid nature.
Table 4 summarizes the different food lipid oxidation processes
studied by 1 H NMR and reviewed in the following sections.
Evolution of the oxidation of refined SF and corn oils at room
temperature in closed receptacles. As for the experimental con-
ditions of these studies, different amounts of refined SF and corn
oils, both rich in linoleic acyl groups, were stored separately in
closed receptacles, in the presence of different air volumes and
having different air/oil contact surfaces. These oil samples were
maintained at room temperature for different periods of time up
to 10 y (Guillén and Goicoechea 2007, 2009). Under these conditions the oils underwent oxidation very slowly.
A study by 1 H NMR of the different samples evidenced that
some of them were nonoxidized, others were at incipient or intermediate oxidation stages, and some others at very advanced
oxidation stages. The 1 H NMR spectra showed that under these
conditions the oxidation process of the above-mentioned oils has
a prolonged period of initiation (induction period), after which
the formation of primary oxidation compounds begins as a consequence of the degradation of the acyl groups, in this case
mainly linoleic. Figure 6(a) shows the enlarged regions of the
C 2014 Institute of Food Technologists®
1
H NMR spectra of several SF oil samples in different oxidation
stages (samples S6, S7, S17, S18, and S23), in which the evolution of the primary oxidation compounds is shown. It can be
observed that this oxidation process evolves, forming primary oxidation compounds having initially hydroperoxy groups as well
as (Z,E)-conjugated dienic systems. At more advanced oxidation
stages, hydroperoxy groups associated with (E,E)-conjugated dienic systems and hydroxy groups supported on chains also having
(Z,E)-conjugated dienic systems appear, both in smaller concentrations than hydroperoxy-(Z,E)-conjugated dienic systems. According to other authors, hydroperoxy-(Z,E)-conjugated dienic
systems are considered to be formed under kinetic control at low
temperatures, because they are formed earlier (Schneider 2009).
However, with prolonged autoxidation or at higher temperatures,
the double-bond configuration of the conjugated hydroperoxides
rearranges from (Z,E) to (E,E). The apparent driving force for
this transformation is the greater thermodynamic stability of the
(E,E)-conjugated double bond in comparison with the (Z,E).
Hydroperoxy-(E,E)-conjugated dienic systems could be formed
at the expense of the (Z,E), and therefore be considered to form
under thermodynamic control, because they are formed later and
are more stable.
Many different molecular structures can be associated with the
above-mentioned functional groups. Figure 6(b) shows some of
the structures, which are present in oxidation compounds, for example, 13-hydroperoxyoctadeca-9,11-dienoate, 9-hydroperoxy-,
and 9-hydroxyoctadeca-10,12-dienoates, all derived from linoleic
acyl groups and able to be formed in this process.
It is noteworthy that, of all the oxidation processes analyzed,
only in those that occur under these conditions (room temperature and closed receptacles) has the formation of hydroxy groups
supported on long acyl chains having also a conjugated dienic
system been observed; this could be related to the limited concentration of oxygen in the closed receptacles. As far as we know,
only 1 previous paper has reported the formation, in SF oil after
prolonged storage, of (Z,E)-conjugated diene primary oxidation
products having hydroxy groups (Mikolajczak and others 1968).
The formation of this kind of compounds is known to occur in
in vivo oxidation processes, and, in fact, they are considered as
clinical markers of lipid peroxidation and oxidative stress, usually
detected in urine and serum (Spindler and others 1997; Browne
and Armstrong 2000). Similarly, the formation of either (Z,E)- or
(E,E)-conjugated dienic systems related to hydroperoxy groups is
highly dependent on the conditions to which the oil is submitted.
Very different situations are possible, as the following sections will
show.
All the above-mentioned compounds are usually called primary
oxidation products; however, as Figure 6(a) shows (see S17 and S18
samples), the formation of the hydroxy-(Z,E)-conjugated dienic
systems, and of hydroperoxy-(E,E)-conjugated dienic systems, occurs simultaneously with that of aldehydes (signals between 9.4 and
9.8 ppm). It can be observed in Figure 6(a) that the appearance of
these 3 kinds of groups is incipient in S17 sample and that their
signals are clearly observable in the S18 sample and onwards.
The conditions under which these oils are stored affect not only
the nature and concentration of the hydroxy and hydroperoxy
compounds mentioned above, but also those derived from them,
like aldehydes and epoxides. As Figure 7(a) shows, the main aldehydes formed were n-alkanals, (E)-2-alkenals, and 4-hydroxy-(E)
-2-alkenals. In smaller concentrations (E,E)-2,4-alkadienals and
4-hydroperoxy-(E)-2-alkenals and, in very small proportions, 4,5epoxy-2-alkenals were generated. Molecular structures of some of
Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 847
848 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
Virgin olive, olive,
hazelnut, corn,
sunflower, soybean,
and linseed
Virgin olive, corn, and
linseed
Sunflower
Extra-virgin olive
Virgin linseed
Sunflower
Rapeseed, walnut, and
linseed
Sunflower
70
Virgin olive, hazelnut,
and peanut
Corn, and sunflower
Up to 4 h
Up to 32 h
Up to 40 h
Up to 20 h
Up to 40 h
190
190
190
190
Up to 10 y
Up to 10 y
Up to 32 d
Until polymerization
(20 d)
Until polymerization
(72, 20, and 51 d)
Until polymerization
(11 and 12 d)
Until polymerization
(13, 5, and 4 d)
Until polymerization
(55 h)
Until polymerization
(linseed 7 h and
the others 12 h)
190
190
100
70
70
Temperature (°C)
Room temperature
Room temperature
50
70
Food lipids
Sunflower
Corn
Fish lipids (Salmo salar)
Sesame
Time under
oxidative
conditions
Domestic fryer (20.5 × 23.5 × 13.5 cm)
Industrial fryer (12 × 30 × 17 cm)
Industrial fryer (12 × 30 × 17 cm)
Industrial fryer (12 × 30 × 17 cm)
Open Petri dishes
Open Petri dishes
Open Petri dishes
Open Petri dishes
Open Petri dishes
Open Petri dishes
Closed receptacles
Closed receptacles
Salmon tissue
Open Petri dishes
Receptacle
Table 4–Summary of studies on food lipid oxidation processes carried out by 1 H NMR.
2L
4L
4L
4L
10 g
10 g
10 g
10 g
10 g
10 g
Varied
Varied
—
10 g
Oil volume
Heated air
Heated air
Heated air
Microwave
50.3 cm2
50.3 cm2
50.3 cm2
50.3 cm2
Electric resistance
Electric resistance
Electric resistance
Electric resistance
Heated air
50.3 cm2
481.7 cm2
360 cm2
360 cm2
360 cm2
Heated air
50.3 cm2
Heating source
—
—
Heated air
Heated air
Undetermined
Undetermined
—
50.3 cm2
Air/oil
contact
surface
Aeration
Natural
Natural
Natural
Natural
Natural
Forced
Forced
Forced
Forced
Forced
Not
Not
Forced
Forced
Reference
Guillén and Uriarte (2009)
Guillén and Uriarte (2012a)
Guillén and Uriarte (2012b)
Guillén and Uriarte (2012c)
Guillén and Ruiz (2006)
Guillén and Ruiz (2008)
Goicoechea and Guillén (2010)
Guillén and Ruiz (2005c)
Guillén and Ruiz (2005b)
Guillén and Ruiz (2005a)
Guillén and Goicoechea (2007)
Guillén and Goicoechea (2009)
Guillén and Ruiz (2004a)
Guillén and Ruiz (2004b)
Food lipid thermo-oxidation studied by 1 H NMR . . .
C 2014 Institute of Food Technologists®
Food lipid thermo-oxidation studied by 1 H NMR . . .
Figure 6–(a) Some expanded regions of the 1 H NMR spectra of sunflower oil (SF) stored at room temperature in closed receptacles for different
periods (Guillén and Goicoechea 2007). These spectra were acquired in a Bruker Avance 400 spectrometer operating at 400 MHz. Signal letters agree
with those in Table 3. (b) Some of the primary oxidation compounds derived from linoleic acyl groups that can be detected by 1 H NMR.
these compounds are given in Figure 7(b). It should be commented
on that both the nature and concentration of aldehydes formed
from the same oil are very different depending on the conditions
under which the oxidation process takes place. It is noticeable that
under the conditions of this study (room temperature and limited amount of air in closed receptacles) 4-hydroxy-(E)-2-alkenals
are formed in significant concentration and that 4-hydroperoxy(E)-2-alkenals are also formed, but in much smaller concentrations than the former, both known to be genotoxic and cytotoxic
compounds. The fact that the concentration of 4-hydroxy-(E)-2alkenals is higher than that of 4-hydroperoxy-(E)-2-alkenals could
be due to either the limited concentration of oxygen in this oxidation process, which could favor the formation of hydroxy over that
of hydroperoxy aldehydes, or to the presence of hydroxy-(Z,E)conjugated diene systems among the intermediate oxidation products, which can evolve directly to give 4-hydroxy-(E)-2-alkenals,
as has been described by Schneider and others (2004).
It is also worth mentioning that together with the formation of
aldehydes and of the other compounds mentioned, the generation
of epoxy groups supported on acyl chains also occurs. Some theoretical mechanisms proposed for the formation of epoxides involve
the participation of hydroperoxy groups and of double bonds and
entail the disappearance of both groups (Lercker and others 2003).
The protons of the carbon atoms bonded to the epoxy group
give signals in the spectral region between 2.8 and 3.2 ppm, being
clearly observable in increasing concentration in S17, S18, and S23
samples (see Figure 7a). They are derived from linoleic groups and
are composed not only of monoepoxides (signals at 2.9 ppm) but
also of diepoxides (signals at 2.9 and 3.1 ppm), these latter being in
the samples at the most advanced oxidation stages. A wide variety
of structures can be found among epoxides derived from linoleic
C 2014 Institute of Food Technologists®
acyl groups; it is worth noting the monoepoxide 9,10-epoxy-12octadecenoate, whose hydrolysis gives leukotoxin, and its isomer,
the monoepoxide 12,13-epoxy-9-octadecenoate whose hydrolysis
gives isoleukotoxin. As mentioned before, in recent years a great
deal of attention has been paid to these 2 compounds as a result of
their biological activity (Markaverich and others 2005; Thompson
and Hammock 2007). According to 1 H NMR data provided by
other authors, the 2 protons corresponding to the carbon atoms
in positions 9 and 10 of leukotoxin diol give a multiplet signal at
3.43 ppm (Aerts and Jacobs 2004); in S23 spectra a broad signal
can be found there (see Figure 7a) that appears simultaneously
with that of epoxy groups, and that could be assigned to the dihydroxyleukotoxin derivative, as Table 3 shows. Like aldehydes, the
formation of epoxides, their nature, and concentration are highly
dependent on the oil nature and degradation conditions, as will
be seen later.
It is also remarkable that in these polyunsaturated oils (corn
and SF) at room temperature all kinds of oxidation-derived compounds are present simultaneously for a very prolonged period,
except at the early stages, when only hydroperoxy groups and
(Z,E)-conjugated dienic systems are present (see Figure 6a and
7a). This may be because at low temperatures the hydroperoxides
have longer average lives than at higher temperatures.
Evolution of the thermo-oxidation of fish lipids contained in drysalted and unsalted salmon (Salmo salar) fillets submitted to 50 °C
with aeration in a convection oven. This study was carried out
on lipids contained in fillets of dry-salted and unsalted salmon
(Salmon salar) submitted to thermo-oxidative conditions at 50 °C
with aeration in a convection oven (Guillén and Ruiz 2004a).
The lipids were extracted periodically after submitting fillets to
the above-mentioned degradative conditions for different periods
Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 849
Food lipid thermo-oxidation studied by 1 H NMR . . .
Figure 7–(a) Some expanded regions of the 1 H NMR spectra of sunflower oil (SF) stored at room temperature in closed receptacles for different
periods (Guillén and Goicoechea 2007). These spectra were acquired in a Bruker Avance 400 spectrometer operating at 400 MHz. Signal letters agree
with those in Table 3. (b) Some of the secondary or further oxidation compounds derived from linoleic acyl groups that can be detected by 1 H NMR.
and were studied by 1 H NMR; the extraction was carried out at
low temperature in order to avoid additional oxidation. Regarding
salmon lipid nature, their important content in ω-3 acyl groups
(around 25% molar percentage), mainly DHA and EPA groups,
and their low oxidative stability are well known. Nevertheless,
their high carotenoid content, which is able to exhibit antioxidant
ability, is also well known.
In this case the oxidation process was fairly different to that
occurring in the previous room temperature studies, not only regarding the conditions to which the lipids were submitted, but
also their special composition and the environment in which these
lipids were surrounded by water, proteins, and so on. Figure 8
shows the spectral regions of the unsalted samples in which the
1
H NMR signals of the protons of primary and secondary oxidation compounds appear. It is noteworthy that in spite of the
low temperature, neither the signals of hydroperoxides nor those
of the conjugated double bonds are clearly observed. This could
be due to the high degree of unsaturation of salmon lipids, which
generate very reactive and unstable primary oxidation compounds,
which disappear very quickly. After 17 d under degradative conditions very weak signals of saturated aldehydic protons around 9.75
ppm appear. These have greater intensity in the spectra of more
oxidized samples, signals of saturated aldehydes of small size like
ethanal and propanal at 9.79 ppm also being observable, as well
as other less well-resolved signals between 9.48 and 9.60 ppm.
As mentioned before, the signals of protons of oxygenated and
nonoxygenated α,β-unsaturated aldehydes appear in this latter region. A poor resolution of signals in this region has also been
observed in other lipids rich in ω-3 acyl groups oxidized at intermediate temperatures, as will be commented on later.
In summary, the oxidation process in this case displayed a very
prolonged period of initiation, probably due to the high content
of carotenoids and other compounds with antioxidant ability in
the salmon. In spite of these, it was demonstrated that although
the dry-salting process does not provoke any immediate oxidation
of fish lipids, it reduces fish oxidative stability in an important
way, with the lipids of salted fillets being oxidized more quickly
than those of unsalted fillets. Signals of protons of hydroperoxides or of conjugated dienic system were not observed before
the appearance of signals of aldehydic protons in any of them;
this could be due to the fact that they are very unstable and disappear very quickly. The aldehydes formed were n-alkanals of
various sizes and nonoxygenated and oxygenated α,β-unsaturated
aldehydes; the signals of these latter appeared overlapped. Moreover, the presence of amino groups of proteins or of other compounds of similar nature to the compounds formed in fish lipid
oxidation, either primary or secondary, can provoke the establishment of reactions that reduce their presence in the system,
such as the Maillard reaction. However, it should be mentioned
that both facts, the nondetection of hydroperoxides or conjugated dienic systems and the overlapping of oxygenated α,βunsaturated aldehydes, are also observed in studies of the oxidation
850 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
C 2014 Institute of Food Technologists®
Food lipid thermo-oxidation studied by 1 H NMR . . .
Figure 8–Expanded regions of the 1 H NMR spectra of the lipids extracted from unsalted salmon fillets that were submitted to 50 °C for days (Guillén
and Ruiz 2004a). These spectra were acquired in a Varian Plus spectrometer operating at 300 MHz. Signal letters agree with those in Table 3.
process at low temperature of oils rich in ω-3 groups (Guillén and mation of aldehydes, epoxides, and alcohols. Nevertheless, some
differences may be observed. Thus, in relation to aldehydes it
Ruiz 2005c).
Evolution of the thermo-oxidation of edible oils of very differ- is particularly worth mentioning that the main aldehydes inient composition submitted to 70 °C with aeration in a convection tially formed are 4-hydroperoxy-(E)-2-alkenals, with n-alkanals
oven. In these studies vegetable oils rich in oleic (olive, hazel- and (E)-2-alkenals in smaller concentrations. As the process adnut, peanut), linoleic (sesame, SF, corn), and linolenic (rapeseed, vances (E,E)-2,4-alkadienals and 4-hydroxy-(E)-alkenals appear:
walnut, linseed) acyl groups were submitted to accelerated storage the concentration of (E,E)-2,4-alkadienals remains small throughconditions at 70 °C with aeration (10 g of the oil in Petri dishes; out the entire degradation process, whereas that of 4-hydroxyGuillén and Ruiz 2004b, 2005a, 2005b, 2005c; Goicoechea and (E)-alkenals increases significantly and becomes higher than that
Guillén 2010). In these studies the thermo-oxidative conditions of 4-hydroperoxy-(E)-alkenals at the end of the process; it seems
were the same for all the oils; the only difference was the oil com- that the formation of the former involves the disappearance of the
position. The processes were studied until the samples were totally latter. The concentration of 4,5-epoxy-2-alkenals is even smaller
polymerized and it was not possible to take samples to acquire the than that of (E,E)-2,4-alkadienals and they are only observable at
1
H NMR spectra. In all the oils studied the signals of the protons advanced oxidation stages.
As mentioned, epoxides are also formed in the oxidation of SF
of polyunsaturated (linoleic and linolenic) groups disappeared at
oil at 70 °C. A comparison of Figure 9(a) and 7(a) indicates that
the end of the process.
Figure 9 shows some enlarged regions of the 1 H NMR spectra of at 70 °C the same epoxides and dialcohols, as at room tempera3 oils (SF, EVO, and VL), selected as representatives of different acyl ture in closed receptacles, are formed but their concentrations are
group compositions, in which the signals of protons of oxidation different. It is clear that the formation of diepoxides and also of
dialcohols is more favored at 70 °C, probably due to the greater
compounds appear.
Oils rich in linoleic acyl groups: Under these degradative presence of oxygen in the system. The same can be said of alcohols,
conditions the evolution of oils rich in linoleic groups, such as which give proton signals at 3.59 and 3.62 ppm.
In summary, in comparison with room temperature the efrefined SF, is fairly different from that occurring at room temperature in closed receptacles, although the nature of the compounds fect of the temperature (70 °C) in this process provokes the forformed is relatively similar, with certain exceptions. After 11 d mation of greater concentrations of hydroperoxides in chains of
under these degradative conditions this oil is totally polymerized. (E,E)-conjugated dienic systems and the higher availability of oxyOn day 1 signals of hydroperoxy groups associated with (Z,E)- gen gives rise to the formation of greater concentrations of the
conjugated dienic systems are observable in the 1 H NMR spectra, most oxygenated final compounds in the groups of aldehydes (the
and their formation is favored in contrast to those associated with toxic and reactive 4-hydroperoxy-(E)-2-alkenals, 4-hydroxy-(E)(E,E)-conjugated dienic systems until day 5; afterwards, the latter 2-alkenals), epoxides (diepoxides), and alcohols. This higher temare in much higher concentrations than the former (Goicoechea perature also significantly affects the rate of the formation of all of
and Guillén 2010). In agreement with observations made at room these compounds.
Oils rich in oleic acyl groups: The evolution of oils rich
temperature, the hydroperoxide signal near 8.46 ppm is associated
with (E,E)-dienes and those at 8.50 ppm and at 8.53 are asso- in oleic acyl groups, such as EVO oil, under these same degradaciated with (Z,E)-dienes. Furthermore, the formation, at room tive conditions, shares some features with the above-mentioned
temperature in closed receptacles, of hydroxy groups associated linoleic-rich oils, but great differences also exist. This is summarized in Figure 9(b). First, the induction period is much more
with conjugated dienic systems, is not observed here.
As Figure 9(a) shows, the concentration of these primary oxi- prolonged (day 26) than in SF oil, and after 72 d under these
dation compounds, especially the hydroperoxy-(E,E)-conjugated degradative conditions, EVO oil polymerizes totally, and the main
dienic systems, increases as the process advances, reaching a max- compounds formed, even though some of them are the same,
imum value after 7 d under degradative conditions; afterwards are somewhat different. As can be observed in Figure 9(b), the
their sharp degradation occurs. Like at room temperature, the formation of hydroperoxy groups begins after a prolonged period
degradation of primary oxidation compounds provokes the for- under degradative conditions. The antioxidant compounds present
C 2014 Institute of Food Technologists®
Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 851
Food lipid thermo-oxidation studied by 1 H NMR . . .
Figure 9–Expanded regions of the 1 H NMR spectra of oils submitted to accelerated storage conditions in Petri dishes (10 g) in a convection oven at
70 °C with aeration until polymerization: (a) refined sunflower (SF), (b) extra-virgin olive (EVO), and (c) virgin linseed (VL; Guillén and Ruiz 2005a,
2005b; Goicoechea and Guillén 2010). These spectra were acquired in a Bruker Avance 400 spectrometer operating at 400 MHz. Signal letters agree
with those in Table 3.
in the oil and the lower unsaturation degree of the acyl groups
are the reason for this. Among the hydroperoxy groups formed,
some are derived from linoleic groups, which are the same as those
generated in SF oil; however, in this case their concentration is
very small. In addition, there are other ones in higher concentrations derived from oleic, some of whose structures are given in
Figure 10; they can be distinguished from the ones derived from
linoleic by the signal of their olefinic protons at 5.72 ppm (Porter
and others 1994). In any case, under the same degradative conditions the formation of hydroperoxides in EVO oil is much smaller
than in SF oil.
The difference between the compounds formed from olive oil
and SF oil is even more noticeable in the groups of aldehydes,
epoxides, and ketones. Thus, regarding aldehydes, at 70 °C with
aeration in EVO oil practically only n-alkanals and (E)-2-alkenals
are formed, with 4-hydroperoxy- and 4-hydroxy-(E)-2-alkenals
in very small concentrations, although these were the main ones
formed in SF oil. As can be observed in Figure 9(b), at advanced
oxidation stages these oxygenated aldehydes are almost unappreciable in the spectra.
From these results it is evident that the determination of the
concentration of the aldehydic functional group, in a global way,
in different oils submitted to degradation is a very crude way to
define or to establish oil safety.
As in SF oil, epoxy groups are also formed in EVO oil; nevertheless, in this latter case the main ones are those derived
from oleic groups, namely (E)-9,10-epoxystearate groups (signal at 2.63 ppm) and (Z)-9,10-epoxystearate groups (signal at
2.88 ppm; see Figure 9b and 10). By contrast, in SF oil leukotoxin
(9,10-epoxy-12-octadecenoate) and/or isoleukotoxin (12,13epoxy-9-octadecenoate) were formed, in addition to diepoxides,
all of them derived from linoleic acyl groups.
Another distinctive characteristic of oils rich in oleic groups
oxidized at intermediate temperatures with aeration is the formation, in important concentrations, of keto groups conjugated with
a double bond (Gibson 1948), this latter giving the characteristic
signals at 6.08 and 6.82 ppm in the spectral region of protons
supported on conjugated systems (see Figure 9b and 10). These
signals, which had been observed in previous studies (Guillén and
Ruiz 2005a, 2005c) and were unidentified at the time, can now
852 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
C 2014 Institute of Food Technologists®
Food lipid thermo-oxidation studied by 1 H NMR . . .
Figure 10–Some of the primary oxidation compounds that can be detected by 1 H NMR derived from: (a) oleic and (b) linolenic acyl groups.
be identified. This identification has been carried out by comparison with data on similar oxidation compounds provided by other
authors (Kuklev and others 1997; Jie and Lam 2004; Lin and others 2007); these keto groups, like epoxides, are derived from the
hydroperoxides of oleic groups.
Again, this shows that the determination, in a global way, of
conjugated dienic systems is a very crude way of establishing the
oxidation degree, because not only primary but also secondary
compounds, these of very different natures, have conjugated dienic
systems.
In summary, under the same conditions of accelerated storage, the different composition of EVO oil and SF oil determine
that: the rate of oxidation in the former is much smaller than
in the latter; the main hydroperoxides and epoxides formed in
the former are those derived from oleic acyl groups, whereas in
the latter they are those derived from linoleic, with leukotoxin
(9,10-epoxy-12-octadecenoate), isoleukotoxin (12,13-epoxy-9octadecenoate), and diepoxides in important concentrations; the
main aldehydes formed in EVO oil are n-alkanals and (E)-2alkenals, with very small amounts of the genotoxic and cytotoxic 4hydroperoxy-(E)-2-alkenals and 4-hydroxy-(E)-2-alkenals, which
are the main ones in oxidized SF oil; and finally that in the oxidized EVO oil, by contrast with oxidized SF oil, chains having keto
groups conjugated with a double bond are present in significant
concentrations.
Oils rich in linolenic acyl groups: The evolution of the
thermo-oxidative process of oils rich in linolenic groups, such as
VL oil submitted to 70 °C with aeration, is also very different from
that of the previously mentioned oils, as may be expected. The
rate of degradation is very high and in 4 d under these degradative
conditions linseed oil is totally polymerized.
C 2014 Institute of Food Technologists®
As Figure 9(c) shows, the spectra of linseed oil do not show wellresolved signals either of protons of hydroperoxides or of those of
conjugated dienic systems related to them, these signals being
of low intensity in comparison with those observed in SF oil (see
Figure 9a). Their low concentration could be due to the instability
of these hydroperoxides, which do not accumulate and evolve very
quickly to give other compounds. It must be remembered that in
the thermo-oxidation at 50 °C of fish lipids, which are also rich
in ω-3 groups, hydroperoxides, and the dienic conjugated systems
associated with them, were not clearly observed (see Figure 8;
Guillén and Ruiz 2004a).
The low resolution of these signals could be due to the overlapping of those of several different structures of hydroperoxides and
conjugated dienic systems, supported by octadecadienoates and
octadecatrienoates, derived from linoleic and linolenic acyl groups,
respectively. Frankel and others (1990) submitted pure trilinolenin
to 40 °C and studied the primary oxidation products generated by
different techniques, among them 1 H NMR. The main compounds identified were several hydroperoxy-octadecatrienoates
showing 2 of the double bonds conjugated (see Figure 10b). The
characteristic proton signals of these (Z,E)-conjugated dienes of
octadecatrienoates appeared at 6.55, 6.00, 5.56, and 5.42 ppm,
with chemical shifts that were very similar to those of the (Z,E)conjugated dienes present in hydroperoxides derived from linoleic
(see Table 3).
It is noteworthy that many other different structures of oxidation
products derived from pure linolenic acid or ester were identified
by 1 H NMR, such as dihydroperoxides or dihydroxides associated
with conjugated diene–triene systems, hydroxy or hydroperoxyepidioxides (also called hydroxy or hydroperoxy-cyclic peroxides),
and so on (Gardner and Weisleder 1972; Neff and others 1981;
Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 853
Food lipid thermo-oxidation studied by 1 H NMR . . .
Coxon and others 1984; Frankel and others 1990). This great
complexity of structures could explain the above-mentioned lowsignal resolution in certain regions of the spectra.
Regarding secondary oxidation compounds, as in SF oil, 4hydroperoxy-, and 4-hydroxy-(E)-2-alkenals, are formed in important concentrations, in addition to n-alkanals, (E)-2-alkenals,
(Z,E)-2,4-alkadienals, 4,5-epoxy-2-alkenals, and their precursors
(E,E)-2,4-alkadienals. The signals of these aldehydes greatly overlap, as was observed in the oxidation of the fish lipids mentioned
before. It is remarkable that from 2.75 d onwards, when VL oil
started to polymerize, some signals appeared at 9.38 to 9.42 ppm.
Although these signals were previously considered as unknown
(Guillén and Ruiz 2005c), now they can be tentatively attributed to the hydroperoxy proton (OOH) present in the abovementioned hydroperoxy-cyclic peroxides, which are secondary or
further oxidation compounds derived from linolenate (Neff and
others 1981).
As far as epoxides are concerned, mono- and diepoxides can
be tentatively identified, because proton signals near 3 ppm are
highly overlapped, probably due to the great variety of structures
mentioned before. However, they are in lower concentrations than
in SF oil submitted to similar conditions (see Figure 9a and c).
In summary, under the same thermo-oxidative conditions (10 g
of oil at 70 °C with aeration) oil composition decisively influences
not only the rate of the oxidation process, but also the compounds
formed, which are very different from 1 oil to another. The oils
taken as examples in the previous paragraphs show significant
abundances of the 3 kinds of acyl groups: linoleic, oleic, and
linolenic. The performance in the thermo-oxidation processes
of other oils having intermediate acyl group composition is also
intermediate, in comparison with that exhibited by the abovementioned oils.
Evolution of the thermo-oxidation process of refined SF oil submitted to 100 °C with aeration in a convection oven. In this study,
as above, samples of 10 g of SF oil placed in open Petri dishes
were submitted to 100 °C with aeration in a convection oven
and the evolution of their composition was studied by 1 H NMR
(Goicoechea and Guillén 2010). As expected, under these conditions the induction period was shorter than in SF oil submitted
to 70 °C, and the formation of hydroperoxides associated with
(E,E)-conjugated dienic systems predominated from the beginning of the process, attributable to the increase of the temperature
by 30 °C (Schneider 2009).
In relation to the generation of aldehydes, it can be seen that the
same kinds as those previously detected at 70 °C are also formed
at 100 °C; however, the formation of oxygenated α,β-unsaturated
aldehydes is less favored than at 70 °C. It is worth mentioning that
in the process at 100 °C the detection of aldehydes is produced
at lower concentrations of hydroperoxides (77.2 mmol/L oil at
9 h) than in the process at 70 °C (346.8 mmol/L oil at 144 h)
; this proves that the degradation of hydroperoxides at higher temperatures is faster than at lower temperatures. This may be one
of the reasons for discrepancies when classical methods are used
to evaluate the oxidation level of edible oils. Furthermore, the
sequential formation of 4,5-epoxy-2-alkenals and of 4-hydroxy(E)-2-alkenals after (E,E)-2,4-alkadienals and 4-hydroperoxy-(E)2-alkenals, respectively, also observed at 70 °C is maintained, together with the diminution in the concentration of the 2 latter
types of aldehydes as the concentration of the 2 former increases.
Temperature also affects epoxide formation, favoring that
of monoepoxides (leukotoxin [9,10-epoxy-12-octadecenoate]
and isoleukotoxin [12,13-epoxy-9-octadecenoate]) over that of
diepoxides and their diol derivatives, unlike what was seen at
70 °C.
In short, the increase of the temperature from 70 to 100 °C
in the SF oil thermo-oxidative process favors, on the one hand,
the formation of hydroperoxy groups supported by chains having
(E,E)-conjugated dienic systems versus those supported by (Z,E)dienic systems, and, on the other hand, reduces the formation of
oxygenated α,β-unsaturated aldehydes, as well as of diepoxides
versus monoepoxides.
Evolution of the thermo-oxidation of edible oils of very different
compositions submitted at 190 °C with aeration in a convection
oven. In this study, samples of 10 g of oil put in open Petri dishes
were also submitted to 190 °C with aeration in a convection oven
until total polymerization; the oils in this study were rich in oleic
(virgin olive, olive, hazelnut), linoleic (corn, SF), and linolenic acyl
groups (soybean, linseed; Guillén and Ruiz 2008). The decreasing order of resistance to degradation was as follows: virgin-olive
oil > olive oil > hazelnut oil > corn oil > SF oil > soybean
oil > linseed oil, in agreement with the studies carried out at
70 °C with aeration (Guillén and Ruiz 2005a, 2005b, 2005c). In
general, the lower the content in polyunsaturated groups in the
oil, the higher its resistance to degradation; however, other factors,
such as the presence of minor antioxidant components, play a very
important role in the degradation rate of the oils, including when
the oil is submitted to high temperatures. So, the virgin olive oil
studied contains similar proportions of oleic and linoleic groups to
olive oil, but its degradation rate is lower, making it evident that
the differences found in the resistance against degradation among
the different oils studied are not only due to the structure and
composition of the main components.
Regarding the formation of oxidation compounds, neither protons of hydroperoxy groups nor protons of conjugated dienic systems bonded to hydroperoxy or hydroxy derivatives were observed
in the 1 H NMR spectra of the oils kept under these degradative
conditions, while aldehydic signals were the first to be observed.
The signals observed between 6.08 and 7.09 ppm are related to
the conjugated double bonds present in aldehydes (Goicoechea
and Guillén 2010). This fact indicates that, if primary oxidation
compounds are formed, they degrade at very high rates and do
not accumulate in sufficient amounts to be detected by 1 H NMR.
It is noteworthy that oils of different compositions generated
aldehydes of different natures and also in different proportions.
Thus, in virgin olive, olive, and hazelnut oil, only significant
concentrations of (E)-2-alkenals and of n-alkanals are generated.
However, in oils rich in polyunsaturated groups, in addition to nalkanals and (E)-2-alkenals, significant proportions of (E,E)-2,4alkadienals are also formed, as well as 4-hydroxy-(E)-2-alkenals,
those tentatively identified as (Z,E)-2,4-alkadienals, and also small
proportions of 4,5-epoxy-2-alkenals. In all the oils, 4-oxo-alkanals,
a kind of aldehyde typical of frying temperatures, were tentatively
identified (Takeoka and others 1995). In none of the oils were
4-hydroperoxy-(E)-2-alkenals detected. In comparison with the
thermo-oxidation process of these oils at 70 °C with aeration
(Guillén and Ruiz 2005a, 2005b, 2005c), the increase of the temperature to 190 °C provoked a lesser generation of oxygenated
α,β-unsaturated aldehydes. It is also remarkable that oils rich in
oleic acyl groups were not only more resistant to degradation than
oils rich in linoleic and linolenic acyl groups, but also produced
lower proportions of toxic aldehydes.
Regarding other secondary or further oxidation compounds,
small proton signals at 6.08 ppm and 6.82 ppm related to keto
groups conjugated with a double bond were observed in oils rich in
854 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
C 2014 Institute of Food Technologists®
Food lipid thermo-oxidation studied by 1 H NMR . . .
oleic acyl groups; as mentioned before, these signals also appeared
when these oils were submitted to 70 °C with aeration, but in
much greater intensities.
In short, if primary oxidation compounds were formed at
190 °C with aeration, they degraded so fast that they cannot be
observed in the 1 H NMR spectra. As for aldehydes, in comparison
with the thermo-oxidation with aeration at lower temperatures,
smaller concentrations of oxygenated α,β-unsaturated aldehydes
were generated; 4-hydroperoxy-(E)-2-alkenals were not detected,
and 4-oxo-alkanals were.
Evolution of the thermo-oxidation process of virgin olive, refined
corn, and VL oils submitted to microwave action at 190 °C. In
this study, virgin olive, refined corn, and VL oils (10 g of oil in
Petri dishes) were repeatedly submitted to the effect of microwave
heating at 190 °C without exceeding this temperature (Guillén
and Ruiz 2006) for 240 min. In virgin olive oil the highest level
of degradation was undergone by the oleic acyl groups, in corn
oil by the linoleic, and in linseed oil by the linolenic acyl groups.
In contrast with observations made at lower temperatures, none
of these acyl groups was totally degraded in any of the 3 oils, with
a very small remaining molar percentage of linoleic in virgin olive
and in corn oils after 240 min.
The times at which the initiation of the degradation was detected in these 3 oils were very similar in this process and when
submitted to 190 °C with aeration in a convection oven (Guillén
and Ruiz 2008), suggesting that the heat supplied to the oil affects
the rate of its degradation to a higher degree than the means by
which the heat is produced.
Regarding the formation and occurrence of primary oxidation
compounds, only very slight and occasional hydroperoxide proton
signals appear between 8.4 and 8.6 ppm in the 1 H NMR spectra
(in virgin olive oil at minute 70 and in corn oil at minute 40),
which were not accompanied by signals of protons of associated dienic conjugated systems. Signals of aldehydes were detected at the
same time as the appearance of these rare incipient signals of hydroperoxides. In VL oil, hydroperoxides were not clearly detected.
The absence of significant concentrations of hydroperoxides in the
oils submitted to microwave-heating is remarkable, along with the
absence of related dienic systems, and the rapid generation of aldehydes. These results are in agreement with those obtained in oils
submitted to 190 °C with aeration in a convection oven (Guillén
and Ruiz 2008).
The different composition of the 3 oils studied (virgin olive,
corn, and VL), has, as expected, differences in the proportions
of the aldehydes generated. Thus, of the 3 oils, virgin olive oil
produced the smallest amount of (E,E)-2,4-alkadienals, with (E)2-alkenals and n-alkanals the main aldehydes generated from this
oil. As the proportion of polyunsaturated acyl groups in the oil
rises, so does the proportion of (E,E)-2,4-alkadienals, 4-hydroxy(E)-2-alkenals, and 4,5-epoxy-2-alkenals, and that of tentatively
identified (Z,E)-2,4-alkadienals and 4-oxo-alkanals, in such a way
that (E,E)-2,4-alkadienals are produced in higher concentrations
than (E)-2-alkenals in the degradation of corn and linseed oils.
In summary, n-alkanals and (E)-2-alkenals are the main aldehydes
generated from virgin olive oil, whereas those generated from
corn and VL oils are (E,E)-2,4-alkadienals. Furthermore, virgin
olive oil is the only one that does not produce 4-hydroxy-(E)-2alkenals. These facts are very important because of the different
reactivity and toxicity of the several kinds of aldehydes.
In short, the results obtained after submitting the oils to microwave action at 190 °C are in agreement with those obtained
at 190 °C with aeration in a convection oven. The absence of
C 2014 Institute of Food Technologists®
significant concentrations of primary oxidation compounds and
the rapid generation of aldehydes, especially nonoxygenated ones,
is noteworthy.
Evolution of EVO, refined SF, and VL oils under deep-frying conditions at 190 °C in the absence of food. EVO, refined SF, and
VL oils were submitted to deep-frying conditions (4 L oil) without food at 190 °C for a prolonged period of time (Guillén and
Uriarte 2012a, 2012b, 2012c). Regarding the degradation of the
acyl groups, it can be observed that the highest rate occurred in
the main unsaturated acyl group in the oil, independently of its
unsaturation degree. Furthermore, it can also be seen that the molar percentages of the acyl groups, which are less saturated than the
main one, decreased with heating time, whereas those more saturated either increased or remained unchanged. Thus, the highest
decrease per hour in VL oil was produced in the molar percentage of linolenic acyl groups, in SF oil in the molar percentage of
linoleic acyl groups, and in EVO oil in the molar percentage of
oleic acyl groups; these results indicate that the rate of degradation
of the various acyl groups, at frying temperature, depends more on
their concentrations in the oil than on their unsaturation degree,
this latter associated with their facility or tendency to oxidize.
Under these conditions formation of primary oxidation compounds was not observed. As can be observed in Figure 11, proton
signals of hydroperoxy groups, as well as of conjugated dienes,
were not detected, in agreement with what was observed during thermo-oxidation at 190 °C in a convection oven (Guillén
and Ruiz 2008) and at 190 °C under microwave action (Guillén
and Ruiz 2006). This fact indicates that in oils submitted to high
temperatures if hydroperoxides are formed, they decompose very
quickly, independently of the heating method, in such a way that
they are not present in the sample in significant concentrations
at any time. This is in agreement with the absence of significant
changes in the classical peroxide value assay of oils submitted to
frying temperature (Dobarganes and Pérez-Camino 1988), and
with the observations by other authors concerning the formation of aldehydes in oils heated at high temperature without previous induction periods (Silvagni and others 2010). Moreover,
the classical parameter, conjugated dienes, associated with the
concentration of acyl group chains having conjugated dienic systems in the oils, in other words, primary oxidation compounds,
which is determined by absorption at 232 nm in ultraviolet (UV)
spectroscopy, is not useful for this purpose in oils submitted to these
conditions (190 °C). This is so because, as Figure 11 shows, the
only signals present at this temperature, in the spectra of protons
belonging to conjugated dienic systems, are those of unsaturated
aldehydes and of other secondary oxidation compounds.
With regard to the formation of aldehydes under deep-frying
conditions, there are very important differences between oils.
Thus, in EVO oil, as Figure 11(b) shows, the most abundant aldehydes are the less reactive, that is (E)-2-alkenals and n-alkanals, and
the least abundant are the more reactive aldehydes, that is (E,E)
-2,4-alkadienals; neither 4-hydroxy-(E)-2-alkenals nor (Z,E)-2,4alkadienals were detected in this oil (see Figure 11b). However,
as Figure 11(a) shows, the heated SF oil was, of the 3 oils, the
one which had higher concentrations of the most reactive aldehydes such as (E,E)-2,4-alkadienals, (Z,E)-2,4-alkadienals, 4,5epoxy-2-alkenals, and 4-hydroxy-(E)-2-alkenals. And, finally, VL
oil contained higher concentrations of (E,E)-2,4-alkadienals than
of (E)-2-alkenals and n-alkanals; however, the difference in concentration of these kinds of aldehydes was much smaller than in
SF oil (see Figure 11c). This may be because the main (E,E)-2,4alkadienal formed in linseed oil is (E,E)-2,4-heptadienal (Guillén
Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 855
Food lipid thermo-oxidation studied by 1 H NMR . . .
Figure 11–Expanded regions of the 1 H NMR spectra of oils heated in a professional deep-fryer (4 L oil) without food for several hours: (a) refined
sunflower (SF), (b) extra-virgin olive (EVO), and (c) virgin linseed (VL; Guillén and Uriarte 2012a, 2012b, 2012c). These spectra were acquired in a
Bruker Avance 400 spectrometer operating at 400 MHz. Signal letters agree with those in Table 3.
and Uriarte 2012d), which has a lower boiling point (177 °C)
than the frying temperature (190 °C), and for that reason a large
quantity of this aldehyde escapes into the atmosphere. However, in
SF oil the (E,E)-2,4-alkadienals formed were mainly (E,E)-2,4decadienal and (Z,E)-2,4-decadienal, both with higher boiling
points (244 °C) than the frying temperature (190 °C), and for
that reason their ability to escape from the oil matrix into the
atmosphere is greatly diminished, so they remain in the oil liquid
matrix to a large extent. Nevertheless, it has to be noted that the
formation of aldehydes in thermo-oxidation processes at 190 °C
is affected by the air–oil contact surface in relation to the oil volume (Guillén and Ruiz 2006, 2008; Guillén and Uriarte 2009,
2012c).
In short, under frying conditions, once again, EVO oil generates
lower concentrations of the most reactive aldehydes than oils rich
in polyunsaturated groups, such as SF and VL oils.
In addition to aldehydes, other secondary compounds are also
formed under these conditions and they too are different depending on the oil nature. So, the 1 H NMR spectra of SF oil submitted
to deep-frying conditions do not have signals of epoxides or of the
diol derivatives; the only signals that are visible here are the side
bands of the bis-allylic protons (see Figure 11a). However, when
it comes to EVO oil, the formation of 2 epoxides derived from
oleic acyl groups, such as (E)-9,10-epoxystearate (signal at 2.63
ppm) and (Z)-9,10-epoxystearate (signal at 2.88 ppm), is observed
(Du and others 2004; Marmesat and others 2008; Guillén and
Uriarte 2012a; Martı́nez-Yusta and Guillén 2014a). And, finally,
no signals of epoxidic protons were observed in the spectra of VL
oil submitted to the same conditions as above.
Concerning alcohols, only signals attributable to primary alcohols were detected in SF oil spectra, whereas in that of EVO
oil signals attributable to primary and to secondary ones were
observed, the formation of this functional group in the degradation of VL oil under deep-frying conditions being unclear. The
formation of hydroxyl groups in edible oils submitted to frying
temperature has been reported previously (Schwartz and others
1994).
In short, when oils are submitted to deep-frying conditions at
190 °C, in the absence of food, their thermo-oxidation evolves
without the occurrence, in measurable concentrations by this technique, of primary oxidation compounds (hydroperoxy groups and
conjugated dienic systems). In comparison with thermo-oxidation
processes at lower temperatures with aeration, lower concentrations of oxygenated secondary oxidation compounds are generated. Differences between the performance of oils as a function of
their compositions can be observed.
Conclusions
1
H NMR gives very valuable information about the different
types of protons present in food lipids, which can be supported by
856 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014
C 2014 Institute of Food Technologists®
Food lipid thermo-oxidation studied by 1 H NMR . . .
main and minor compounds. This spectroscopic technique is fast
and simple, requires no chemical modification of the sample, and
provides a great deal of information on the lipid sample as a whole,
this latter fact being very important. It is very useful not only for
the study of the nondegraded food lipids, but also for the study of
the degraded ones. For this reason it is very useful to follow, in a
qualitative and quantitative way, the evolution of food lipids when
they are submitted to degradative conditions. It allows not only
the identification of many of the main functional groups formed,
but also to quantify them providing, in this way, information of
the thermodegradative processes.
From the results described here, it can be concluded that the use
of classical methods to determine the concentration of functional
groups in a global way (hydroperoxides, conjugated dienes, aldehydes, and so on) in food lipids of different compositions when
submitted to different degradation conditions, is a very crude way
of analyzing their thermo-oxidation processes. It should be taken
into account that each functional group includes compounds of
very different reactivities and toxicities.
The studies mentioned here have demonstrated that both food
lipid composition and the conditions under which the thermooxidation process takes place govern the mechanisms by which the
process evolves, as well as affecting the nature and concentrations
of intermediate and finally formed compounds.
From the above-mentioned studies it can also be concluded that
polyunsaturated food lipids submitted to oxidative conditions at
low temperatures are able to generate a great number of different kinds of very reactive oxygenated compounds, some of which
are known to be toxic, such as 4-hydroperoxy-, and 4-hydroxy(E)-2-alkenals, or 9,10-epoxy-12-octadecenoate (leukotoxin) and
12,13-epoxy-9-octadecenoate (isoleukotoxin) derivatives. As the
temperature increases, the number of different kinds of compounds
which may be formed from these lipids decreases. At low temperatures the process is slow; however, at high temperatures it takes
place very quickly. Although the number and concentration of the
most reactive compounds which may form from polyunsaturated
lipids at high temperatures is smaller than at low temperatures,
they can accumulate with heating time.
Food lipids with small concentrations of polyunsaturated groups
and significant concentrations of compounds that exhibit antioxidant ability, when submitted to thermo-oxidative conditions at
low temperature, not only generate much lower concentrations
of the most reactive and toxic lipid-derived compounds, but this
formation occurs much more slowly than in the case of the food
lipids, which are rich in polyunsaturated groups. When these lipids
are submitted to high temperatures the rate of their degradation
increases, as does that of those rich in polyunsaturated groups, but
the range of compounds formed is different from those formed in
the latter.
In addition to the influence of both food lipid nature and the
temperature during which the thermo-oxidation evolves, another
important factor is the food lipid surface exposed to the oxygen.
When this surface is high in relation to the oil volume involved,
then higher concentrations of oxygenated derivatives are formed
than when the exposed surface is small. This fact is very important
because some of the oxygenated compounds are very reactive and
toxic.
The results summarized here are worthy of consideration because some of the above-mentioned toxic compounds detected by
this technique during food lipid thermo-oxidation processes have
also been found in human cells and tissues. Finally, it only remains
to be added that the results commented on and discussed here may
C 2014 Institute of Food Technologists®
be helpful in the interpretation of results obtained in metabolomic
and lipidomic studies based on 1 H NMR techniques.
Acknowledgments
This work was supported by the Spanish Ministry of Economy
and Competitiveness (MINECO, AGL2012-36466), the Basque
Government (EJ-GV, GIC10/IT-463–10), and the Univ. of the
Basque Country (UPV/EHU, UFI-11/21). A. Martı́nez-Yusta
thanks the EJ-GV for a predoctoral contract.
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