1 Introduction
Marine oil products are valued for their high eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA) content. These fatty acids have
been shown to exert positive health effects, such as reducing
inflammation (Allaire et al., 2016) and lowering blood triglycerides
(Zulyniak et al., 2016). However, the highly unsaturated structure of
these fatty acids makes them susceptible to lipid oxidation, a
deteriorative process that leads to off-flavors and off-odors. If
consumed, the oxidized products may have negative health consequences
(Esterbauer, 1993). For regulatory and quality control purposes, it is
common practice to monitor the level of oxidation in marine oils. This
is typically achieved by measuring multiple oxidation markers, such as
peroxides and carbonyls. Peroxide value, as a primary oxidation
indicator, can be measured using the American Oil Chemists’ Society
(AOCS) titration-based method (Cd 8b-90). Carbonyls, generated during
hydroperoxide decomposition, are often used as secondary oxidation
markers. Volatile carbonyls, such as hexanal, are commonly quantified
using gas chromatography methods. Non-volatile carbonyls can be measured
using the 2,4-Dinitrophenylhydrazine (DNPH) assay, thep -Anisidine Value (pAV), or the thiobarbituric acid reactive
substances (TBARS) test.
Concentrations of non-volatile carbonyls, primarily unsaturated
aldehydes such as 2-alkenals and 2,4-alkadienals, are often assessed
using AOCS Official Method for pAV (Cd 18-90). This is a
spectrophotometric method based on the absorbance of light by an imine
chromophore that forms from the reaction of aldehydes with thep -anisidine reagent. Flavoring agents added to marine oils
interfere with the test, as aldehydes in flavors also react with thep -ansidine reagent and form additional imine chromophores,
leading to inaccurate results (The Global Organization for EPA and DHA
(GOED), 2018; Jackowski et al., 2015; Semb, 2012) and incorrect
conclusions about the shelf-life and quality of the oil products.
Unexpectedly high pAV have been reported in previous studies in flavored
marine oil products (Albert et al., 2015; Jackowski et al., 2015).
To compensate for the increased pAV caused by flavors, GOED (2018)
recommends a protocol to assess the pAV of flavored marine oils. There
are four major steps: 1) measure pAV of an unflavored base oil (pAV); 2)
determine the maximum allowable pAV increase in this oil (ΔpAV); 3) add
flavor to the base oil and measure the pAV of this flavored oil (pAV*);
4) determine the maximum allowable pAV of the flavored oil
(pAV*max), which constitutes a useful reference value
for the shelf-life testing. ΔpAV is calculated by subtracting the pAV of
the fresh unflavored base oil from the maximum acceptable pAV suggested
by GOED. pAV*max is calculated by adding ΔpAV to pAV*.
This recommendation is based on the assumption that the flavors
themselves do not degrade or in any way change influence on the measured
pAV over time (GOED, 2018). If this assumption is violated, estimates of
the oxidation level will be inaccurate. However, this is very likely to
occur; several common flavor compounds, such as citral (Djordjevic et
al., 2008; Liang et al., 2004; Schieberle & Grosch, 1988) and vanillin
(Mourtzinos et al., 2009), have been shown to degrade under a variety of
conditions.
In this study, we tested the null hypothesis that flavors do not change
their contribution to the measured pAV over the course of oxidation. To
do this, we first evaluated 14 flavors to identify those with the
greatest contribution to the measured pAV and would thus be most likely
to change in observable ways over the course of oxidation. Following
this, we performed a series of accelerated stability studies to compare
oil samples to which flavor had been added either before or after
oxidation. The pAV of all samples was evaluated at several sampling
points during oxidation. We anticipated finding differences between the
measured pAV of the two sample types, which would suggest the GOED
recommendation may not apply to these flavored marine oils, and that an
alternative protocol is needed to evaluate the aldehydes that result
from lipid oxidation.