2 Materials and methods
2.1 Fish Oil and Flavors
Fish oil was manufactured by DSM Nutritional Products (Dartmouth, NS,
Canada) and donated by Nature’s Way of Canada Ltd. The fresh base oil
contained approximately 18% EPA and 12% DHA and was stabilized with 3
mg g-1 natural mixed tocopherols. Flavoring agents
were sourced from FONA International (Geneva, IL, USA). The flavors
consisted of medium-chain triacylglycerols and natural flavor compounds.
They were not stabilized with any additional antioxidants.p -Anisidine crystals and solvents used in this study were
purchased from Sigma Aldrich (Oakville, ON, Canada)
2.2 Screening Flavored Fish Oils
The fourteen flavors screened were apple, blackcurrant, bubblegum,
chocolate-vanilla, citrus punch, coconut, cranberry,
grapefruit-tangerine, lemon, mandarin, mango, meat, orange, and peanut
butter. Fourteen 4 mL screw top vials were loaded with accurately
weighed fish oil (about 2.5 g per vial). As a common industrial
practice, 2% (w/w) flavor was added to each vial. All vials were purged
with nitrogen, capped, and vortexed for 30 seconds. pAV of these
flavored oils and the unflavored base oil were measured in duplicate,
following AOCS Official Method Cd 18-90 (AOCS, 2011).
2.3 Identifying Major Flavor Compounds Using GC-MS
After determining that chocolate-vanilla and lemon flavors had the
largest influence on the measured pAV, GC-MS was used to identify major
flavor components in these flavors. A Thermo Trace 1310 GC was coupled
with a Thermo ISQ 7000 single quadrupole mass spectrometer equipped with
an autosampler. Flavors were diluted 100 times in hexane with 50 ppm
internal standard (tribenzylamine). Half a microliter of each diluted
flavor was injected in split mode with a split ratio of 150:1 at 250 °C.
Separation of compounds was achieved on a Zebron ZB-5 capillary column
(5% phenyl, 95% dimethylpolysiloxane; 5 m GUARDIAN; 30 m length, 0.25
mm I.D., 0.25 µm film thickness). The oven temperature was: 1) held at
60 °C for 2 min, 2) increased to 80 °C at a rate of 7 °C
min-1, 3) increased again to 150 °C at a rate of 3 °C
min-1, 4) further increased to 260 °C at 7 °C
min-1, 5) increased to 300 °C at 3 °C
min-1, and 6) held for 20 min. Total run time was 78
min. This long temperature program ensured large molecules, including
the triacylglycerol carrier oil, had eluted from the column at the end
of each run. The MS was operated in TIC mode, with a scanning range from
60 to 600 amu and dwell time of 0.2 sec. Transfer line and ion source
were both set at 300 °C. MS data were processed by Chromeleon
Chromatography Studio. Peaks with area larger than 1.0 % of internal
standard were tentatively identified through the NIST library search and
confirmed using an MS processing method with three confirmation ions
that had been developed and employed for routine in-house analysis of
flavors. The retention indices of all the identified compounds were
compared to further confirm the peaks’ identities.
2.4 Oxidation of Chocolate-Vanilla and Lemon Flavored Fish Oils
The following experiment and subsequent analyses were performed twice,
once with chocolate-vanilla flavor and once with lemon flavor.
Accurately weighed aliquots of fish oil (~3.5 g) were
added to fifty-one 4 mL screw top vials. To eighteen of these vials, 2%
(w/w) flavor was added. All vials were then purged with nitrogen,
capped, and vortexed for 30 seconds. Following vortexing, three vials
without flavor and three with flavor added were set aside to serve as
unoxidized baseline samples. The remainder of the samples were placed
uncapped in a 40 ± 2°C oven for oxidation. Every four days for a period
of 20 days, nine oil samples (two triplicate sets of unflavored oils,
and one triplicate set of oil to which flavor had been added prior to
incubation) were taken out of the oven and cooled to room temperature.
To one set of triplicate unflavored oil samples, 2% (w/w) flavor was
added, thus creating three streams of oxidized oil: unflavored oil
(UFO), oil to which flavor had been added before oxidation (FBO),
and oil to which flavor was added after oxidation (FAO). All
three triplicate sets of oil samples were then purged with nitrogen,
capped, and vortexed for 30 seconds. pAV of oils was measured following
the protocol described above.
2.5 Statistical Analyses
The following equation was used to model (SPSS, IBM, New York) the pAV
growth data for each treatment: