4 Discussion
The AOCS pAV test (Cd 18-90), as a simple colorimetric method to measure
non-volatile carbonyls, primarily aldehydes, has limitations due to its
non-specificity. The imine chromophore that forms between an aldehyde
and the p -anisidine reagent differs considerably among aldehydes
(Szabó et al., 2010), with the method being more sensitive to
unsaturated than saturated aldehydes (Gordon, 2004). So, pAV results
provide only relative aldehyde concentrations. Although pAV is expected
to reflect the amount of aldehydes arising from hydroperoxide
decomposition during oxidation, it is known to respond to extraneous
aldehydes as well, such as those from flavors. The interference by
flavors is dose and type-dependent. Jackowski et al. (2015) found that
marine oil products with citrus flavors, as well as products marketed
towards children (e.g., bubblegum-flavored products) often had higher
pAV than other flavored products. This was also confirmed in this study;
the pAV increase caused by 14 different flavors varied from 1.1 to 46.5,
with chocolate-vanilla, citrus, and bubblegum flavors having the largest
impact (Fig. 1). Despite these limitations, pAV is still one of the most
common methods to measure marine oil oxidation, due to its simple
procedure and low cost compared to sensory evaluation or volatile
analysis using GCMS.
GOED recognized these limitations and provided advice for those who want
to use the AOCS pAV method for fish oil testing (GOED, 2018). In the
guidance document, GOED recommended a protocol for flavored fish oils,
based on an assumption that flavors have a constant contribution to the
measured pAV throughout shelf life studies. However, in this study we
found chocolate vanilla and lemon flavors had a diminishing effect on
the pAV (Fig. 2), indicating that the current GOED recommendation may
not apply to all the flavored oils. For both flavors, the significant
difference in interfering components (A ) in FBO and FAO compared
to UFO was expected since the flavors introduced a large amount of
stable interfering aldehydes. Based on the observed differences in pAV
of FBO and FAO, we had also expected to find a significant difference inB (changing aldehydes) and k (rate constant for aldehyde
formation) for both flavors. However, the uncertainty introduced by the
4-day sampling frequency limited our ability to identify statistical
differences. A more frequent sampling plan would have given us a greater
confidence in estimating the model parameters. Despite this, we did find
that chocolate-vanilla FBO samples had a significantly lower rate
constant (k ) than UFO. This might be caused by flavor components
with antioxidant properties or by flavor aldehydes that degraded over
time.
With the current experimental design, it is difficult to determine if
this diminishing effect was due to antioxidant activity, degradation of
flavor aldehydes, or both. It is not concerning if the diminishing
effect on measured pAV was solely due to any antioxidant activity
because in this case, the lower pAV truly reflected the slower oxidation
in the flavored oil. It is concerning if the decreasing contribution to
the pAV was caused by flavor loss. In this case, more oxidation would
have occurred before the maximum pAV allowed by the GOED recommendation
is reached. In other words, following the GOED recommended protocol, we
would underestimate the amount of oxidation that was occurring in the
oil. This underestimation of the extent of oxidation may lead to overly
optimistic inferences about the quality and shelf-life of the flavored
oil in question.
In the chocolate-vanilla flavor used in this study, vanillin had the
highest ARIS (90.7%). Antioxidant activity of vanillin has been
demonstrated in antioxidant capacity assays (Tai et al., 2011) and in
food systems containing polyunsaturated fatty acids (Burri et al.,
1989). Vanillin may be oxidized to vanillic acid, which is 3.3 times
more effective as an antioxidant than vanillin in bulk oils (Mourtzinos
et al., 2009). Vanillic acid has also been shown to exert antioxidant
effects in corn oil subjected to deep-frying conditions (Naz et al.,
2005) and in fish oil at temperatures between 35 to 55°C (Farhoosh et
al., 2016). Thus, vanillin is very likely to have a real influence on
the rate constant for aldehyde formation (k ) in the FBO samples,
through an antioxidant effect.
In the lemon flavored oils, although pAV of FBO and FAO samples were
significantly different on days 12, 16, and 20, no significant
differences were observed in the rate constant (k ) between FBO
and other samples. In the lemon flavor, citral had the highest ARIS
(total of α- and β-citral at 48.8 %). Citral does not exert strong
antioxidant effects in edible fruit coatings (Guerreiro et al., 2015,
2016) and in essential lemon oils (Misharina et al., 2011). In fact, an
antioxidant is often added to inhibit citral degradation. GC analysis of
cold-pressed lemon oil stored in the dark for two months at 30°C showed
significant losses of citral (Nguyen et al., 2009). This effect is
exacerbated at increased temperatures (Djordjevic et al., 2008; Nguyen
et al., 2009). Compounding this, citral is quite volatile. In the open
vials in this study, citral might have been evaporating throughout the
study. Thus, it is very likely that loss of citral caused the lower pAV
in FBO compared to FAO samples, without significantly changing the rate
constant (k ).
As a small study to estimate the influence of some common flavors on pAV
testing in fish oil products, the current results do not allow us to
calculate a pAV that solely reflects the aldehydes generated through
lipid oxidation. Future studies should include a systematic experimental
design that tracks multiple complementary oxidation products, such as
peroxides and volatile aldehydes, and monitors matrix changes, such as
tocopherol and flavor loss. Such a study would demonstrate the overall
oxidative status, elucidate the correlation between various oxidation
indicators, and clarify the source of the diminishing pAV inflation.
This would also allow us to define the maximum pAV through its
correlation with other oxidation markers and their respective limits.
When pAV is the only applicable method for routine analysis for quality
control purpose, this predefined pAV can be used as the control limit.
For example, if we are confident about the correlation between
2,4-heptadienal and pAV after systematic studies on a flavored fish oil
product under normal storage conditions, and if we have established a
standard for 2,4-heptadienal levels, we can predefine our maximum
allowable pAV for this product. For daily quality control, pAV can be
measured and compared with this maximum pAV.