1
1. Introduction
2
The enhanced incorporation of polyunsaturated fatty acids (PUFA) has become an important topic
3
for the food industry due to their wide range of nutritional and health benefits for the end consumer
4
(Gobert et al., 2010; Sorensen et al., 2012). These positive effects have been described mainly for -3
5
and -6 PUFAs (Jacobsen, Let, Nielsen, & Meyer, 2008). Numerous epidemiological, clinical, animal
6
and in situ experiments have shown health benefits due to an increased intake of -3 fatty acids,
7
such as EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). Studies revealed a including
8
decreased risk of coronary heart disease, immune response disorders and mental illness, as well as
9
benefits to infants and pregnant women (Hu, McClements, & Decker, 2004; Dawczynski, Martin,
10
Wagner, & Jahreis, 2010; Dawczynski et al., 2013). Sources containing high levels of these
11
unsaturated fatty acids are nuts, vegetable oils, fish and soybeans. In the last years, increasing
12
attention is given to new, sustainable sources of these PUFAs, such as microalgae or extracts of
13
microalgae that can be integrated in a variety of foodstuffs (Draaisma et al., 2013; Van Durme, Goiris,
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De Winne, De Cooman, & Muylaert, 2013).
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16
Despite the many advantages of increasing the PUFA content in food matrices, a major issue is their
17
high susceptibility to lipid oxidation. This oxidative phenomenon inevitably leads to loss of shelf-life,
18
consumer acceptability, functionality, nutritional value, organoleptic properties and safety (Arab-
19
Tehrany et al., 2012). The intensity of lipid oxidative deterioration of PUFA enriched foodstuffs
20
depends on different factors; particularly the degree of unsaturation of fatty acids and the presence
21
of external factors promoting oxidation, e.g. exposure to oxygen and light, metallic ions or high
22
temperatures (Roman, Heyd, Broyart, Castillo, & Maillard, 2013). The oxidative stability of each of
23
these PUFAs is inversely proportional to the number of bis-allylic hydrogens in the molecule;
24
therefore, EPA and DHA are even more easily oxidized compared to oleic acid, linoleic acid and
25
linolenic acid (Delgado-Pando, Cofrades, Ruiz-Capillas, Triki, & Jimenez-Colmenero, 2012).
26
27
There are few reports on accurate shelf-life tests for the evaluation of lipid oxidation in PUFA
28
enriched food products that specifically focus on the organoleptic changes developing during
29
storage. For food manufacturers it is of high importance to safeguard the initial nutritional and
30
organoleptic characteristics during the shelf-life. In line with the abovementioned trend, the
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development and improvement of methods to evaluate the oxidative stability of food products have
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received growing attention in the last years. Due to practical reasons, researchers have been
33
especially focusing on accelerated shelf-life tests. Such techniques have great application possibilities
1
34
in the study of lipid oxidation, oil stability, off-flavor formation chemistry, the prediction of possible
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intermediate formation and the impact of oxidation on the nutritional properties of food in a faster
36
manner (Van Durme et al., 2014). Moreover, these techniques can also be used for the assessment of
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the functionality of synthetic and natural antioxidants in PUFA-enriched food products (Erkan,
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Ayranci, & Ayranci, 2008; Ojeda-Sana, van Baren, Elechosa, Juarez, & Moreno, 2013).
39
In practice, most of the accelerated oxidation techniques are based on increased temperatures (e.g.
40
Swift test, Rancimat (Garcia-Moreno, Perez-Galvez, Guadix, & Guadix, 2013)). Rancimat is the most
41
widely used test for accelerated lipid oxidation. An oil sample is heated to the desired temperature
42
while air is bubbled through at a constant flow rate. Next the air, loaded with the formed oxidation
43
volatiles, is sent through a water sample in which the volatiles of the oil sample are transferred. After
44
the experiment an oil matrix is left of which all formed oxidation products have been stripped. In this
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way a sensory evaluation of this accelerated ‘aged’ product is not possible. Secondly, outcomes of
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thermally-based techniques poorly correlate with realistic storage tests. This can be explained by the
47
fact that the mechanism of lipid oxidation changes when temperatures exceed 60 °C (Mancebo-
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Campos, Fregapane, & Salvador, 2008). No marked success has ever been achieved in realistically
49
predictioning organoleptic changes and/or shelf-life of edible fats and oils by such thermally based
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stability tests (Farhoosh & Hoseini-Yazdi, 2013). Some studies in literature revealed that most
51
accelerated tests are performed at temperatures of at least 100 °C (Farhoosh & Hoseini-Yazdi, 2013;
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Garcia-Moreno, Perez-Galvez, Guadix, & Guadix, 2013). Next to deviating lipid oxidation kinetics,
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other reactions such as polymerization, thermal degradation, cyclization, Maillard reactions, Strecker
54
degradation, denaturation or oxygen depletion could occur at such high temperatures (Van Durme,
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Nikiforov, Vandamme, Leys, & De Winne, 2014). Secondly, these thermally based techniques remain
56
relatively time-consuming (up to several days). Moreover, some antioxidants are thermally unstable,
57
which leads to an under –or overestimation of their effect.
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Abovementioned factors indicate that innovative accelerated oxidation techniques are required
59
which operate at ambient temperatures and which are able to accelerate lipid oxidation processes in
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both a fast and reliable manner. Moreover, the development of an accelerated oxidation test
61
enabling the user to perform a sensory analysis on the treated sample would be of great value for
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the food industry. In this paper, the applicability of Non-Thermal Plasma (NTP) will be investigated as
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a new innovative accelerated lipid oxidation test using fish oil as a case. NTP is generally described as
64
the fourth state of matter and consists of reactive species (atoms, ions, radicals), formed by
65
dissociative electron attachment processes (Wan, Coventry, Swiergon, Sanguansri, & Versteeg,
66
2009). Several applications of NTP have already been described in literature, such as removal of
2
67
pollutants in water (Magureanu et al., 2011; T. Zhang et al., 2013), medical applications (Bundscherer
68
et al., 2013; Y. Zhang, Yu, & Wang, 2014) surface treatments (Choi et al., 2013; Li et al., 2013;
69
Sohbatzadeh, Mirzanejhad, Ghasemi, & Talebzadeh, 2013) and gas emission treatments (Van Durme,
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Dewulf, Sysmans, Leys, & Van Langenhove, 2007). However, besides sanitation of food products
71
(Baier et al., 2013; Baier et al., 2014) and first experiments on a commercial blend of vegetable oil
72
(Van Durme, Nikiforov, Vandamme, Leys, & De Winne, 2014), no applications of NTP for the
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accelerated oxidation of lipids in food have been reported. The primary goal of this work is to
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investigate whether NTP treatment induces realistic lipid oxidation reactions in fish oil, and to what
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degree they correlate with natural lipid auto-oxidation. This was assessed by measuring and
76
comparing the secondary volatile lipid oxidation products as markers for food ageing. Experiments
77
were performed using Ar/O2 plasma on fish oil as a reference material. These results are compared to
78
thermally oxidized and naturally aged fish oil samples.
79
80
2. Materials and methods
81
2.1 Fish oil samples
82
Menhaden fish oil (Sigma Aldrich, Diegem (Belgium)) was purchased and stored at -80°C to prevent
83
further oxidation. For each test, fish oil samples were used, either pure or enriched with an
84
antioxidant (100 µg/g and/or 1000 µg/g -Tocopherol (Sigma Aldrich)). The fatty acid composition of
85
the Menhaden fish oil was provided by Sigma Aldrich and is expressed in percentage. For the used
86
fish oil, the following initial typical fatty acid composition is applicable; 30.4 % saturated fatty acids
87
(7.94 % C14:0, 15.1 % C16:0, 3.8 % C18:0), 26.7 % mono-unsaturated fatty acids (10.5 % C16:1, 14.5
88
% C18:1, 1.3 % C20:1, 0.4 % C22:1) and 34.2 % poly-unsaturated fatty acids (2.2 % C18:2, 1.5 % C18:3,
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2.8 % C18:4, 1.1 % C20:4, 13.2 % C20:5, 4.9 % C22:5, 8.6 % C22:6). The fish oil already contained a
90
limited amount of lipid oxidation products, as will be further discussed in §3.1.
91
92
2.2 Oxidation tests
93
2.2.1 Natural aging
94
For natural aging (reference) 100 grams of pure fish oil and 100 grams of enriched (1000 µg/g -
95
tocopherol) fish oil was put in an Erlenmeyer and kept in the dark at ambient conditions for 11
96
weeks. Every week 3 g of oil was sampled and stored at -80°C to prevent further oxidation.
3
97
2.2.2 Thermal accelerated oxidation test
98
Thermal treatment of the fish oil was performed at 100 °C for 6 hours, based on the widely used
99
Rancimat test (Lutterodt, Slavin, Whent, Turner, & Yu, 2011; Roman, Heyd, Broyart, Castillo, &
100
Maillard, 2013). In each experiment 50 g of fish oil was put in a glass flask and heated to the desired
101
temperature by placing it in a temperature controlled oven. Air was bubbled for 6 hours through the
102
sample (using a sintered glass disk for maximum contact with the oil) at a flow rate of 1.0 L/min. The
103
oil was continuously stirred by the air stream passing through the sample, creating an optimum
104
transfer of oxygen to the heated oil. After passing through the oil, the air bubbled through an ice-
105
cooled water sample of 100 g in order to capture secondary volatile lipid oxidation compounds. After
106
thermal treatment, 0.5 g of the water sample was transferred into a 20 mL headspace vial and sealed
107
using an inert Teflon septum. Afterwards, the same treatment was applied to oil containing 1000 µg/g
108
-tocopherol .
109
2.2.2 Accelerated oxidation by DBD-plasma treatment
110
DBD plasma operating with Ar/O2 mixture as a feed gas in ambient air can be considered as a source
111
of a broad range of active species. The species generated in the active zone of the discharge located
112
in between electrodes can be divided in (listed according to increasing reactivity): charged particles
113
(electrons, positive and negative ions); neutral excited states of Ar (metastables, resonance states
114
and electron excited states); UV and VUV photons (appearing due to excimer radiation, OH and NO
115
bands emission); oxygenated species including O3, O2 singlet, and O. The production mechanisms of
116
different excited species have been intensively studied in the last decade worldwide. In the research
117
of van Gils, Hofmann, Boekema, Brandenburg, and Bruggeman (2013) and Reuter et al. (2012)
118
production of VUV and UV radiation in plasma of Ar using a slightly higher power of 20 W has been
119
studied and absolute VUV radiance has been estimated around 2-3 mWmm−2sr−1. Such low amount
120
of VUV/UV photons cannot explain observed chemical changes during oil treatments. Therefore the
121
effect of UV radiation can be excluded (van Gils et al., 2013). Considering the low ionization degree of
122
our plasma with an electron density of about 1.5x1013 cm−3 (Sarani, Nikiforov, & Leys, 2010) and
123
taking into account dissociative electron–ion recombination which has a typical rate of 10−13 m3 s−1
124
(van Gils et al., 2013), the actual density of charged particles that reaches the treated surface in the
125
far afterglow is 2–3 orders of magnitude lower than the density of the charged particles in the active
126
zone. The charged particles concentration of about 10-10 cm−3 cannot considerably affect chemical
127
reactions in the liquid phase during our experiments. Active species of Ar, especially those with long
128
lifetime as metastable and resonance states, can reach the surface of the treated oil. Ar excited
4
129
states cannot directly oxidize the oil but can initiate formation of free radicals in the liquid. This
130
process has been checked in an independent experiment of Van Durme et al. (2014) in which Ar
131
plasma jet has been used for olive oil treatment. It was shown that the formation of oxidative
132
products in oil under action of a pure Ar plasma jet is very low, even after 60 minutes of plasma
133
treatment. Considering the above mentioned results, the effect of plasma treatment of liquid
134
samples can be solely attributed to oxygenated species including mainly O3, O2 singlet, and atomic O.
135
25 grams of fish oil was put in a glass container. The oil was pumped through a sintered glass disk,
136
which prevented the oil from being blown away during the NTP-treatment and increased the contact
137
of the plasma jet and the oil. Sample losses were determined by weighing the sample before and
138
after treatment. Less than 3% of sample was lost during 60 minutes of NTP treatment. Previous tests
139
indicated that a direct treatment of the oil surface without a sintered glass disk leaded to an
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insufficient contact of the plasma with the oil. Secondly the oil would gush, leading to contamination
141
of the quartz tube and eventually inhibiting the formation of a stable plasma jet. The plasma jet
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(figure 1) was placed above the sintered glass disk, spreading over the oil surface. The distance
143
between the capillary quartz tube and the sintered glass disk was 5 mm. Exposure times of 60
144
minutes were applied for plasma treatment. The plasma jet consists of a tungsten rod (energetic
145
electrode) with a sharp tip, inserted in a quartz capillary with 1.3 mm inner diameter. The tungsten
146
rod and quartz capillary together are centered inside a grounded aluminum tube (ground electrode).
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Alternating peak to peak voltage of 6 kV is applied to the tungsten rod by a 50 kHz power supply
148
(Bayerle, Germany). Gas is fed into the plasma jet through two separated lines each controlled by a
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mass flow controller (Bronckhorst, Belgium). For the experimental configuration used in this study, a
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stable discharge was obtained when the voltage input was fixed at 6.00 kV (peak to peak) and
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current of 128 mA while maintaining an Argon gas flow rate of 2.00 slm (standard liters per minute).
152
The Argon stream was doped with oxygen gas (0.6 %) in order to create the abovementioned
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oxidative species and eventually induce lipid oxidation, while maintaining the treated oil sample at
154
ambient temperatures. Atomic oxygen concentration was measured using spectroscopy, based on
155
the method described by Hong, Lu, Pan, Li, and Wu (2013). More specific, an Ocean Optics s2000
156
spectrometer with resolution of 1.5 nm has been used for emission spectrometry of the plasma jet.
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Sensitivity of the spectrometer has been corrected with the use of a NIST calibrated Oriel model
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65355 spectral lamp. Adding 0.6% of oxygen led to a total atomic oxygen concentration of
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7.21*1017cm-3.
160
It has to be noted that the measurement of singlet delta oxygen (SDO) molecules in the plasma jet is
161
a technically challenging task due to the small size of the jet and a correspondingly low absorption
5
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signal. Among available results, most of the experiential studies of the singlet oxygen production
163
have been carried out in conditions similar to those of our plasma jet but for He/O2 mixtures by
164
means of IR absorption. In the study of Sarani et al. (2010) the SDO absolute density was estimated
165
to be around 6 × 1015 cm−3 for RF and DBD jets in an optimal He/O2 mixture. Similar values in the
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order of 1015 cm-3 were obtained in the study of Lu and Wu (2013) for a low power plasma jet
167
operating in ambient air. A density of 1.7 × 1015 cm−3 of O2 (a1g) was found in a microplasma jet
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operating in He+2% O2 (J.S. Sousa, 2013). These experimental results have also been confirmed by
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numerical simulations where the SDO density was estimated at 1015 cm-3 in the He plasma jet (He &
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Zhang, 2012; Zhang, Chi, & He, 2014). In recent work SDO densities were also estimated in an Ar
171
plasma jet by a numerical study (Van Gaens & Bogaerts, 2014). The authors have found that up to 1
172
cm away from the nozzle the O2 (a1g) concentration is about 0.7 × 1015 cm−3 and comparable with
173
the density of atomic oxygen. They found that O2 (a1g) initiated chemistry starts to be important
174
only in the very far effluent, as its internal energy is rather low (0.98eV) compared with OH, Ar
175
excited states and atomic O.
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2.3 Chemical analysis of volatile lipid oxidation products
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Isolation of the volatiles originated from lipid oxidation, was performed with an autosampler
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(MultiPurpose Sampler® or MPS®, GERSTEL®, Mülheim am der Rur, Germany), equipped with a
179
headspace-solid phase microextraction unit. Solid-phase microextraction combined with one
180
dimensional gas chromatography-mass spectrometry has been applied in many food related
181
researches and already proved to be a sensitive and reliable methodology for the evaluation of
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volatile lipid oxidation products (Ryckebosch et al., 2013 , Van Durme et al., 2013;(Van Durme et al.,
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2014). Based on experiments (§3.1) the following sample preparation conditions were selected: 0.5
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g of fish oil sample or water sample (§2.2.1) was hermetically sealed in brown 20 mL vials to be
185
incubated 30 min. Next, the headspace was extracted at 60°C on a well-conditioned CAR/PDMS
186
SPME fiber for another 30 minutes by means of a thermostatic agitator.
187
A fully automated sample preparation unit (MultiPurpose Sampler® or MPS®, GERSTEL®, Mülheim an
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der Rur, Germany), combined with a 6890/5973 GC–MS system (Agilent Technologies®, Palo Alto, CA)
189
was used for compound separation and identification. Helium was used as a carrier gas (1 mL/min).
190
Injector and transfer lines were maintained at 250 °C and 280 °C, respectively. The total ion current
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(70 eV) was recorded in the mass range from 40 to 230 amu (scan mode) using a solvent delay of
192
2 min and a run time of 5 min. For GC–MS profiling, both a cross-linked methyl silicone column (HP-
193
PONA), 50 m × 0.20 mm I.D., 0.5 μm film thickness (Agilent Technology®) and a ZB-WAX column, 30
6
194
m x 0.25 mm I.D., 0.25 µm film thickness (Phenomenex®) were used and programmed: 40 °C (5 min)
195
to 160 °C at 3 °C/min, from 160 °C to 220 °C at 5 °C/min, held for 3 min. Identification of volatile
196
organic compounds in the fish oil headspace was performed by comparison with the mass spectra of
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the Wiley® 275 library. Additionally, confirmation of identified compounds was done by
198
determination of Kovats indices, determined after injection of a series of n-alkane homologues using
199
the analytical configuration as described above. Thirdly, some authentic reference standards were
200
injected to confirm the identity of some important volatiles. Concentration of identified oxidation
201
products were expressed semi-quantitatively, using an internal standard, 4-Hydroxyl-4-methyl-2-
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pentanone (10 µL, 0.309 µg/µL). All samples were measured in triplicate (n=3).
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3. Results and discussion
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3.1 Naturally aged fish oil
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3.1.1 Identification of odor active volatile oxidation markers
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208
In the following section, the naturally aged fish oil was evaluated over a period of 11 weeks by
209
identifying and quantifying volatile organic compounds in the headspace of the matrix. The goal is to
210
profile the aroma compounds in function of storage time and to identify a number of volatiles that
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are clear markers for lipid oxidative phenomena in fish oil. Although this approach, using secondary
212
volatiles to evaluate the lipid oxidation progress, is most realistic, today most researchers still focus
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on measuring primary oxidation products by means of peroxide value (PV) (Ahn, Kim, & Kim, 2012).
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Secondary oxidation products are often evaluated by the thiobarbituric acid reactive substances
215
(TBARS) (Beltran, Pla, Yuste, & Mor-Mur, 2003) or the p-anisidine value (AV) (Guillen & Cabo, 2002).
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Research papers in which volatiles are measured typically select hexanal as a typical lipid oxidation
217
marker (Panseri, Soncin, Chiesa, & Biondi, 2011; Sanches-Silva, de Quiros, Lopez-Hernandez, &
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Paseiro-Losada, 2004). In fish oil however, hexanal is not a typical lipid oxidation marker. Other
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oxidation products such as 1-penten-3-one (pungent green odor), Z-4-heptenal (fishy odor), (E,E)-2,4-
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heptadienal (fatty, rancid odor), (E,Z)-2,6-nonadienal (cucumber odor) and 1-octen-3-ol (mushroom
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odor) have been characterized as very potent odorants, contributing to the unpleasant rancid and
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fishy off-flavor (Iglesias, Lois, & Medina, 2007; (Venkateshwarlu, Let, Meyer, & Jacobsen, 2004). For
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this study, different solid-phase microextraction (SPME) fibers were compared (CAR/PDMS, PDMS,
224
CAR/DVB/PDMS) at 60 °C and an extraction time of 30 min. The most effective fiber type proved to
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225
be CAR/PDMS. Using the selected fiber type (CAR/PDMS), extractions were performed at 40, 60, 80°C
226
for 15, 30, 45 min. .It was observed that a 30 minute extraction time was optimal, when preceded by
227
incubating the sample for 30 min at 60 °C. Naturally aged fish oil was used for this optimization.
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Table 1 represents semi-quantitatively determined concentrations of volatile compounds present in
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fresh and naturally aged fish oil samples. In total 55 volatiles were identified of which the aldehydes
230
proved to be the most dominant, followed by hydrocarbons and ketones. While in fresh fish oil a
231
total volatile organic compound (VOC) concentration of 1.64*103 µg/g was already measured, a
232
significant increase in VOC variety and concentration was observed after 11 weeks of storage in
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ambient and dark conditions (3.82*103 µg/g). It is generally known that in this matrix practically no
234
enzymatic lipid oxidation or other microbial or fermentative processes can occur. Since enzymes are
235
present in the watery phase of a biological system, amounts of enzymes in the extracted oil are
236
considered negligible. Therefore, these observations can only be explained by lipid auto-oxidation,
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typically resulting in volatiles such as aldehydes (2-propenal, propanal, pentanal, heptanal, (E,E)-2,4-
238
heptadienal and (E,E)-2,4-octadienal), ketones (1-octen-3-one, 3,5-octadien-2-one, 2-nonanone) and
239
several hydrocarbons (tridecane, pentadecane). The lipid oxidation mechanism is initiated by free
240
radicals which abstract a hydrogen atom at carbon atoms adjacent to a double bond. Triplet oxygen
241
reacts with these lipid radicals leading to lipid peroxides formation. Further propagation reactions
242
include hydroperoxide formation and -scissions eventually resulting in the formation of secondary
243
lipid oxidation volatiles. Reaction mechanism pathways of these oxidation volatiles are well
244
described in literature (Frankel, 1987, 1991). Above mentioned results illustrate that HS-SPME-GC-
245
MS is a sensitive, reproducible and relevant analytical technique to study oxidation phenomena in
246
fish oil, hence this approach will be also used when studying lipid oxidation chemistry in both thermal
247
and non-thermal plasma based lipid oxidation (§3.3). From Table 1 it can be derived that formic acid,
248
1-penten-3-ol, propenal, (E)-2-pentenal, heptanal, (E)-2-heptenal, (E,E)-2,4-heptadienal, (E,E)-2,4-
249
octadienal, (E)-2-nonenal and (E)-2-decenal strongly increased during natural storage, making them
250
important lipid oxidation products. Since it is well described that oxidized fish oil develops important
251
off-aromas, it is of high importance to consider Odor Activity Values (OAV) when studying lipid
252
oxidation. OAV values are calculated by dividing the specific headspace concentration by the
253
corresponding odor threshold value. For the naturally aged oil most odor active lipid oxidation
254
compounds proved to be 1-octen-3-one (14.40 µg/g, OAV = 2.9*106), (E,Z)-2,6-nonadienal (24.99
255
µg/g, OAV = 2.5*106), (E)-2-nonenal (51.74 µg/g, OAV = 5.2*105), (E,E)-2,4-decadienal (17.87 µg/g,
256
OAV = 2.6*105), (E)-2-decenal (31.95 µg/g, OAV = 1.1*105), 3,5-octadien-2-one (85.68 µg/g, OAV =
257
7.1*104), 1-penten-3-one (43.13 µg/g, OAV = 4.3*104), (E,E)-2,4-heptadienal (517.9 µg/g, OAV =
258
3.5*104), 1-octen-3-ol (33.28 µg/g, OAV = 3.3*104), (E)-2-octenal (55.23 µg/g, OAV = 1.9*104),
8
259
nonanal (16.76 µg/g, OAV = 1.7*104), pentanal (25.28 µg/g, OAV = 2.1*10³) and propanal (38.99
260
µg/g, OAV = 1.1*10³). Using this approach, completed by literature study, enabled to select a list of
261
the most important Lipid Oxidation Markers (LOMs) as summarized in Table 2. These LOMs were
262
used in this paper to evaluate and compare both the thermal (§ 3.2) and non-thermal plasma (§ 3.3)
263
based accelerated lipid oxidation methods.
264
265
3.1.2 Lipid oxidation marker assessment for evaluation of antioxidant effectiveness during the natural
266
aging test
267
Figures 2 illustrates changes in headspace concentrations above fish oil samples for a number of the
268
selected LOMs summarized in Table 2, more specific 2-propenal, (E)-2-pentenal, (E)-2-decenal, 1-
269
octen-3-ol and (E,E)-2,4-octadienal. In agreement with other studies (Horn, Nielsen, & Jacobsen,
270
2009; Zuta, Simpson, Zhao, & Leclerc, 2007) a clear anti-oxidative effect of adding 1000 µg/g -
271
tocopherol is visualized in Figure 1, showing a reduced formation after 11 weeks for 2-propenal, (E)-
272
2-pentenal, (E)-2-decenal, 1-octen-3-ol and (E,E)-2,4-octadienal. This result indicates that -
273
tocopherol and -tocopherol both have antioxidant properties when used in the conditions described
274
earlier. Horn et al. (2009) determined a prooxidative effect of -tocopherol addition below 200 µg/g.
275
This experiment has not been repeated in this work since this effect has already been well described.
276
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3.2 Thermal Treatment
278
Based on VOC measurements of thermally treated fish oil, it could be concluded that some
279
compounds identified in naturally aged fish oil could not be detected after the thermal treatment.
280
This was for example the case for ethanol, acetaldehyde, 2-methyl-2-butenal, (E,E,E)-2,4,6-
281
octatrienal, (E,E)-2,4-decadienal, 1-hydroxy-2-butanone and 5-ethyl-2(5H)-furanone.
282
Secondly the relative VOC composition after thermal treatment proved to be completely different
283
compared to that measured in naturally aged fish oil. For example the relative class importance of
284
aldehydes for naturally aged fish oil was 33%, while this was 82% for thermally oxidized fish oil.
285
Figure 2 illustrates the formation of the earlier identified volatile lipid oxidation markers. Thirdly, the
286
overall concentration range of the VOCs seems to be much higher after the thermal treatment
287
compared to the natural aging process. Since 11 weeks of natural aging resulted in concentrations up
288
to 35 µg/g for 1-octen-3-ol and 55 µg/g for (E)-2-pentenal, a thermal treatment of 6 hours resulted in
289
concentrations for these compounds of respectively 550 µg/g and 1100 µg/g. Formation of 2-
9
290
propenal, (E)-2-pentenal, (E)-2-decenal, 1-octen-3-ol and (E,E)-2,4-octadienal are presented in figure
291
3.
292
Furthermore, in contrary to the results as measured during ambient storage test, the addition of
293
1000 µg/g -tocopherol clearly resulted in a prooxidative effect during thermal exposure, leading to
294
increased lipid oxidation products. Instead of working as a chain-breaking antioxidant preventing
295
propagation of free radicals (Brigelius-Flohe & Traber, 1999), the high temperature inverted these
296
antioxidative properties of -tocopherol into prooxidative effects.
297
Based on these results it can be concluded that the thermal accelerated lipid oxidation test
298
insufficiently correlates with natural oxidation of fish oil. Besides the different composition and
299
higher concentration of oxidation products, the addition of -tocopherol (1000 µg/g) results in a
300
prooxidative effect during the thermal treatment, while an antioxidative effect was observed at
301
ambient temperature. Since the adverse effects of elevated temperatures have already clearly been
302
proven during this study and in other research papers (Mancebo-Campos, Salvador, & Fregapane,
303
2014; Rubén H. Olmedo, 2015; Van Durme et al., 2014), experiments with -tocopherol enriched fish
304
oil at 100 µg/g were not performed.
305
306
3.3 Non-Thermal Plasma treatment
307
Fish oil samples, either fresh or containing -tocopherol at 1000 µg/g, based on Horn et al. (2009)
308
were both treated with the plasma jet over a period of maximum 60 minutes. The temperature of
309
each sample was measured directly after treatment, using a calibrated infrared thermometer
310
(Voltcraft, IR900-30S).
311
treatment revealed that no increase in temperature occurred. As described earlier NTP experiments
312
were performed at a constant voltage input of 6.00 kV, while maintaining an argon gas flow rate of 2
313
slm (standard liters per minute). The same analytical approach, using HS-SPME-GCMS, as described in
314
Materials and Methods was used to evaluate the performance of the NTP. Moreover, addition of -
315
tocopherol should indicate if this new technique accelerates the lipid oxidation, with a more realistic
316
prediction of the antioxidant properties of -tocopherol.
Several temperature measurements of the sample during the plasma
317
318
Following NTP-treatment, a significant increase of several lipid oxidation products was detected
319
which were also found in the naturally aged fish oil. 2-propenal, 1-penten-3-one, pentanal, 2-
320
undecanone, (E)-2-pentenal, (E)-3-hexenal, nonanal, hexanoic acid, butanoic acid and heptanal were
10
321
the compounds that increased in concentration following the NTP-treatment. These oxidation
322
products are formed as a result of the reactive species present in the plasma jet, in particular atomic
323
oxygen and singlet oxygen.
324
The compounds that increased in function of treatment time are displayed in figure 4. Contrary, for a
325
number of volatile lipid oxidation markers (e.g. (E,E)-2,4-heptadienal, (E)-2-decenal and 1-octen-3-
326
ol)no significant increase was observed after NTP-exposure. This result could be explained by the fact
327
that the plasma jet was sustained by an argon gasflow of 2 slm, which creates a very turbulent
328
atmosphere near the contact zone, causing a partial stripping of volatile compounds. Diffusion of
329
volatiles from the oil matrix to the headspace is a well-known physical phenomenon which depends
330
on various parameters, e.g. specific VOC/oil partitioning coefficient, temperature, turbulence… When
331
the stripping effect is more dominant than the formation of specific oxidation products, a decrease in
332
concentration is observed. This might result in an underestimation of the formation rate of some
333
volatile oxidation markers. Since the scope of this study is to evaluate to what extent the NTP treated
334
sample correlates with a naturally aged sample, no further measurements have been performed on
335
the stripped volatiles.
336
As mentioned in §2.3 the addition of oxygen in the plasma results in the formation of several active
337
species of which atomic oxygen and singlet oxygen are considered the most reactive. Singlet oxygen
338
is an excited state of triplet oxygen (ground state) and highly reactive. This highly reactive oxygen
339
species (ROS) can be formed in nature under influence of UV-light, and is responsible for photo-
340
oxidation of lipids. Singlet oxygen directly reacts with an unsaturated fatty acid, without the prior
341
formation of a radical, as is the case in the reaction mechanism with triplet oxygen. As discussed
342
earlier, during the initiation step hydroperoxides are formed on the carbon atoms adjacent to a
343
double bond, which in its turn leads to the formation of various secondary oxidation compounds
344
through a various range of reaction mechanisms (Frankel, 1991). Pentanal can be formed from a 13-
345
hydroperoxide of linoleic acid and the -scission mechanism. (E)-2-Propanal in its turn can be formed
346
from a 3-hydroperoxide of any omega-3 fatty acid including linoleic acid, DHA and EPA . Atomic
347
oxygen is also a short-lived highly reactive species which also initiates the lipid oxidation mechanism
348
by immediate reaction with the fatty acid, eventually leading to the formation of secondary oxidation
349
products.
350
351
Based on the NTP oxidation experiment it could be concluded that the addition of -tocopherol
352
resulted in an antioxidant effect when added at 1000 µg/g for most LOMs (in some cases no
353
significant difference was observed). An additional NTP-treatment was performed on fish oil,
11
354
enriched with 100 µg/g -tocopherol. In agreement with literature data, the antioxidative properties
355
after adding 1000 µg/g a-tocopherol were not observed when the same compound was added at 100
356
µg/g concentration. This effect is clearly visible in case of pentanal, 2-undecanone, (E)-3-hexenal and
357
nonanal. In case of 2-propenal and 1-penten-3-one, an antioxidative effect was observed. Heptanal
358
on the other hand was formed much more rapidly when 100 µg/g -tocopherol was added, while an
359
addition of 1000 µg/g did not have a significant effect. Similar conclusions were made by Horn et al.
360
(2009) who added different concentrations of -tocopherol to fish oil in order to evaluate the
361
antioxidant effect. Addition of -tocopherol at concentrations above 440 µg/g fish oil proved to result
362
in a clear antioxidant effect, while addition at concentrations below 220 µg/g resulted in a
363
prooxidative effect. Prooxidative effects have been shown to rely on the ability of tocopherols to
364
participate in side reactions in some food systems (Huang, Frankel, & German, 1994; Yanishlieva,
365
Kamal-Eldin, Marinova, & Toneva, 2002). It has been described that -tocopherol reacts not only
366
with peroxyl radicals (ROO•), but also with alkoxyl radical intermediates (RO•) to form hydroxy
367
compounds. Such side reactions may, to some extent, explain the present findings (Horn, Nielsen, &
368
Jacobsen, 2009). Another explanation could be interactions between -tocopherol and plasma-
369
immanent species leading to the formation of oxidative compounds which in turn lead to the pro-
370
oxidative effect. Future research is needed to unravel these mechanisms. Based on these results, it
371
can be concluded that the NTP-technique approaches the natural oxidation process more closely
372
than the thermal oxidation test, considering the effects of -tocopherol addition at different
373
concentrations.
374
375
4. Conclusions
376
Measurements of secondary oxidation volatiles during a natural storage test resulted in an accurate
377
evaluation of the lipid oxidation process, thermal accelerated test and NTP-treatments. A natural
378
aging test of 11 weeks resulted in the formation of many lipid oxidation volatiles, of which aldehydes
379
proved to be the most important group. Compounds such as (E,E)-2,4-Heptadienal, (E,Z)-2,6-
380
Nonadienal, 1-octen-3-ol, (E)-2-decenal and others proved to be important oxidation compounds.
381
Based on this natural oxidation test a list of lipid oxidation markers was chosen. Addition of 1000
382
µg/g -tocopherol clearly resulted in an antioxidative effect in accordance with results found in
383
another study of Horn et al. (2009).
384
The thermal accelerated lipid oxidation test, based on the well-known Rancimat test, proved to be
385
insufficiently correlated with the natural aging of fish oil. Next to the formation of deviating types
12
386
and concentrations of products and a different ratio of molecular groups, also a prooxidative effect
387
was observed when 1000 µg/g of -tocopherol was added.
388
The NTP-treatment resulted in the formation of several lipid oxidation products, which were also all
389
found in the naturally aged fish oil, such as 2-propenal, (E)-2-pentenal, heptanal and 1-penten-3-one.
390
However, other lipid oxidation markers found in the naturally aged fish oil, such as (E,E)-2,4-
391
heptadienal and (E,E)-2,4-decadienal, did not seem to be formed during the NTP-treatment. This
392
result could be explained by the highly turbulent atmosphere near the reaction zone, causing many
393
volatiles to be stripped from the oil sample. In this way, an underestimation is made about the
394
formation of oxidation products. Secondly, the addition of 1000 µg/g -tocopherol resulted in a clear
395
antioxidative effect, in accordance with the natural aging test. When 100 µg/g -tocopherol was
396
added however, prooxidative properties were correctly predicted. Non-thermal plasma proved to be
397
able to accelerate the oxidation process in the fish oil, with a more accurate prediction of the
398
antioxidative properties of -tocopherol. In this way, the use of NTP as a non-thermal accelerated
399
oxidation technique has more potential to evaluate additions of antioxidants than the thermal
400
accelerated oxidation test.
401
The results from this work have provided some interesting insights into the use of NTP for
402
accelerated lipid oxidation in fish oil. However, further research is required on this highly innovative
403
and challenging plasma-technique. One important advantage of this plasma-technique is the high
404
steerability. Many parameters, such as voltage, treatment time, oxygen concentration, configuration,
405
water concentration and carrier gas can be altered, resulting in other plasma characteristics. When
406
water is doped in the argon jet for example, a high concentration of hydroxyl radicals can be
407
expected in the plasma. Since these highly reactive species are also responsible for natural oxidation
408
processes, it could move the oxidation chemistry closer to natural oxidation. An important
409
bottleneck of the used NTP configuration technique is the stripping of volatiles during the treatment.
410
This could be overcome by treating the oil in a reaction chamber, and for example capturing the
411
stripped volatiles in a solvent or sorbent tube, or measuring their concentrations by means of an
412
electronic nose. Further experiments with this promising Non-Thermal Plasma for accelerated lipid
413
oxidation in complex food matrices will determine to what extent it can correlate to the natural aging
414
process.
415
416
13
417
5. References
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531
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534
535
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540
541
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545
546
547
548
549
550
551
552
553
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Zhang, Y. T., Chi, Y. Y., & He, J. (2014). Numerical Simulation on the Production of Reactive Oxygen
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16
566
a)
567
568
b)
c)
569
570
571
572
573
574
575
576
577
578
Figure 1: NTP-treatment of fish oil: a) overall NTP-configuration; b) NTP-treatment of oil sample (detail); c)
NTP-contact with fish oil sample
579
a)
b)
c)
d)
580
581
17
582
583
e)
584
585
586
587
Figure 2. Evaluation of average headspace concentrations of typical fish oil oxidation products during 11 weeks of natural
aging with and without addition of -tocopherol antioxidant (AO) (1000 µg/g): a) 2-propenal, b) E-2-pentenal, c) 2decenal, d) 1-octen-3-ol and e) (E,E)-2,4-octadienal (n=3).
588
a)
b)
c)
d)
589
590
591
18
592
e)
593
594
595
596
Figure 3. Evaluation of average headspace concentrations of typical fish oil oxidation products after 6 hours of thermal
treatment with and without addition of -tocopherol antioxidant (AO) (1000 µg/g): a) 2-propenal, b) E-2-pentenal, c) 2decenal, d) 1-octen-3-ol and e) (E,E)-2,4-octadienal (n=3).
597
598
a)
b)
c)
d)
e)
f)
599
600
601
602
19
603
604
g)
h)
605
606
607
I)
608
609
610
611
Figure 4. Average headspace concentrations of fish oil oxidation products after 60 minutes of NTP treatment with
additional restults of 100 µg/g and 1000 µg/g -tocopherol antioxidant (AO) addition: a) propanal, b) propenal, c)
pentanal, d) 2-undecanone, e) 3-hexenal, f) E-2-pentenal, g) nonanal, h) heptanal, i) 1-penten-3-one (n=3).
20
612
613
Table 1: Average headspace concentration of volatile organic compounds found in naturally aged fish oil (11
weeks) with their Odor Activity Values (n=3).
Compounds
Alcohols
ethanol
1-penten-3-ol
2-penten-1-ol
1-octen-3-ol
conc w0
conc w11
(μg/g) stdev (μg/g)
stdev OAV (-) KIexp KIlit Idm
0.00
49.76
34.40
8.45
0.00
3.36
2.53
0.70
8.67
123.9
24.01
33.28
0.25
6.24
1.03
1.27
0.09
310
60
33278
440
685
757
959
440
665
746
968
B
A
B
A
Aldehydes
acetaldehyde
2-propenal
propanal
butanal
2-butenal
pentanal
2-butenal, 2-methylE-2-pentenal
trans,trans-2,4-heptadienal
2-octenal
E,E-2,4-octadienal
Z,Z-2,4-octadienal
nonanal
2,6-nonadienal (E,Z)
2-Nonenal
2,4,6-octatrienal
2-decenal
2,4-decadienal
0.00
0.00
8.10
14.12
49.96
0.00
0.00
0.00
367.4
64.10
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.16
0.99
1.06
0.00
0.00
0.00
16.7
4.09
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
15.59
110.3
38.99
9.93
92.59
25.28
43.19
53.99
517.9
55.23
25.69
41.08
16.76
24.99
51.74
15.91
31.95
17.87
0.28
130 560 427 C
1.40
572
C
0.42
1054 574 506 C
0.45
268 601 596 B
1.51
637 623 B
1.37
2106 677 690 A
3.04
696 611 A
1.23
36 718 731 B
17.10
34527 1000 996 B
3.80
18411 1052 1056 B
1.67
1088 1084 B
1.75
C
0.19
16757 1102 1083 A
1.13 2499223 1144 1154 B
1.65 517429 1152 1147 B
0.66
1161
C
3.03 106510 1246 1250 B
2.22 255313 1273 1297 A
Aromatics
benzene
3,4-dihydropyran
2-Ethylfuran
2-methylfuran
2-Methoxyfuran
phenol
2-(2-propenyl)-furan
34.30
0.00
144.2
11.05
0.00
10.84
0.00
1.26
0.00
11.0
0.56
0.00
1.36
0.00
4.05
7.74
94.75
27.41
33.03
10.98
24.12
0.21
0.27
2.45
0.86
0.63
0.11
0.67
680
502
715
810
1.9 974
1008
C
705 B
698 C
633 C
C
981 B
C
Carbon acids
formic acid
acetic acid
0.30
26.59
0.08
11.7
243.4
242.8
3.73
7.41
0.54 510
2.4 572
543 B
600 B
ester
isopropyl dodecanoate
0.00
0.00
10.79
0.97
C
hydrocarbons
2-pentene, 4-methyl2,5-octadiene
octatriene,1,3-trans-5-trans2,4,6-octatriene
1-acetyl-1-cyclohexene
1-tridecene
tridecane
tetradecane
hexadecane, 2,6,10,14-tetramethyl
1-pentadecene
pentadecane
hexadecane
heptadec-8-ene
1-heptadecene
heptadecane
pentadecane,2,6,10,14-tetramethylisoprene
0.00
264.9
39.61
33.59
0.00
7.83
12.72
18.25
0.00
0.00
287.9
27.57
2.17
6.44
36.37
0.00
0.00
0.00
7.65
4.43
3.20
0.00
1.22
0.68
1.23
0.00
0.00
59.0
4.19
1.14
0.22
10.5
0.00
0.00
4.28
19.13
43.37
6.86
25.40
9.00
16.81
17.83
11.32
23.40
655.7
31.08
26.53
85.31
260.9
71.48
6.42
0.11
0.97
2.36
0.25
0.37
0.68
1.17
0.10
0.81
3.36
70.74
7.90
2.90
4.62
22.44
5.04
0.41
C
812
C
878
C
935
C
943
C
1286 1289 B
1299 1300 A
1419 1400 A
C
1538 1540 B
1518 1500 A
1600 1600 A
C
C
1674 1700 A
C
C
ketones
ethyl vinyl ketone
1-hydroxy-2-butanone
5-ethyl-2(5H)-furanone
ethanone,1-(1-cyclohexen-1-yl)1-octen-3-one
3,5-octadiene-2-one
2-nonanone
2-undecanone
26.66
0.00
0.00
0.00
0.00
25.33
23.27
4.48
1.77
0.00
0.00
0.00
0.00
2.19
0.90
1.14
43.13
15.56
152.9
80.01
14.40
85.68
18.53
11.61
2.31
43133
C
1.24
0.31 694 674 A
11.12
925 954 B
6.03
943
C
0.00 2879850 972 1040 B
4.82
71403 1063 1040 B
0.79
93 1090 1093 B
0.05
1659 1277 1296 B
614
615
616
617
618
619
KI = Kovats index. Idm = identification methods: A, identification based on MS database, retention index values from the literature when available
(ascertained from authentic reference compounds), and spiking with authentic reference compound; B, tentative identification based on the MS
database and retention index values from the literature (ascertained from authentic reference compounds); C, when only MS or retention index
values were available (ascertained from authentic reference compound), it must be considered as a tentative identification. Sources for literature
KI values are summarized in Van Durme et al. (2013).
21
620
621
622
623
624
Table 2: List of LOMs for fish oil with concentrations after different oxidation tests
Compound
1-penten-3-ol
2-propenal
propanal
E-2-pentenal
heptanal
(E,E)-2,4-heptadienal
2-nonenal
2,6-nonadienal
2-decenal
3,5-octadien-2-one
1-penten-3-one
3-hexenal
nonanal
2-undecanone
(E,E)-2,4-octadienal
Odor/aroma
mushroom
burnt cooking grease
solvent, pungent
strawberry, fruit, tomato
soap, fat, almond
nut, fat
orris, fat, cucumber
cucumber, wax, green
tallow
fruity, green, grassy
Earthy, Green, Pungent
grassy, green
floral, waxy, green
fruity-rosy, orange-like
fatty
OTV (µg/g) 11 w NA (µg/g) 6h 100°C (µg/g) 60 min O2/Ar (µg/g)
0.4
123.9
290.5
110.3
168.5
11.9
0.0
39.0
45.5
5.7
1.5
54.0
1093.2
6.3
0.0
716.3
22.6
0.0
517.9
6520.0
0.0
51.7
667.8
0.0
25.0
334.1
0.0
32.0
130.9
0.0
85.7
186.0
1.3
43.1
320.4
5.9
0.3
6.1
1.0
16.8
381.9
28.1
7.0
11.6
59.9
19.3
25.7
220.0
-
OTV = OdorThreshold Value, (11 w NA) = 11 weeks of Natural Aging, (6h 100°C) = thermal oxidation test of 6 hours at 100 °C, (60 min O2/Ar) =
Oxygen/ Argon Non-Thermal Plasma treatment for 60 minutes.
625
626
22