DISCUSSION
In this study, we evaluated the effects of roasting and autoclaving on
the major peanut allergens and on peanut allergenicity. We demonstrated
that high-pressure and temperature autoclaving reduces the detection of
the major allergens Ara h 1, Ara h 2, and Ara h 8 as well as
peanut-specific IgE binding when compared to raw or roasted peanuts.
Moreover, the effect of autoclaving was observed even when it was
performed prior to or following roasting of the peanut, further
indicating the strong denaturing effects of autoclaving. Indeed, NMR
analyses showed that while the molecular profiles between raw and
roasted peanuts were similar, those associated with autoclaving
treatment were dramatically different, with evident peak-broadening in
regions corresponding to amino acids associated with proteins. Larger
molecules in solution move or “tumble” more slowly, resulting in a
range of molecular orientations and thus broader peaks in the NMR
spectrum32. Since these experiments were performed
with peanut-soaked solutions using 1H NMR, the results
suggest that the autoclaved peanut sample contained a larger number and
a wider range of sizes of soluble molecules than the raw and roasted
solutions. Overall, the data indicate that of the processing methods
evaluated, autoclaving produces conditions for the most significant
denaturation, resulting in complete degradation of Ara h 8 and partial
degradation of Ara h 1 and Ara h 2. In contrast, under raw and roasted
conditions, these allergens remained largely intact.
Consistent with these findings, autoclaving was associated with the
weakest IgE binding using serum samples of peanut-allergic patients. The
decrease in IgE binding may be partly caused by the complete
denaturation of Ara h 8 or major structural changes affecting the
accessibility of its epitope regions. Likewise, the partial degradation
of Ara h 2 may also affect epitope accessibility. However, due to its
rigid structure maintained by a number of disulfide bonds, Ara h 2 is
more resistant to denaturation14. Nevertheless, we
cannot rule out the possibility that the results observed are the
outcome of a dose-related phenomenon. It is known that Ara h 2 exists in
high proportions relative to others (5.9%-9.3% of total protein
content)33, and Ara h 8 in much lower abundance,
indicating that the remaining levels of Ara h 2 and absence of Ara h 8
could perhaps be proportional to their initial concentrations in the
peanut.
Autoclaving is a condition where temperature, pressure and moisture play
a significant role. There are currently few studies in the literature
addressing the effect of autoclaving on peanut proteins. One major study
to date has investigated the effect of heat and pressure treatments on
peanut allergenicity25. Cabanillas et al. (2012)
demonstrated that peanut-specific IgE binding, as well as the detection
of major allergens Ara h 1, Ara h 2, and Ara h 3, can be reduced by
autoclaving roasted peanuts25. This was explained by
the observation that autoclaving resulted in a decrease of α-helix
content and an increase in random coils and/or loops as a function of
autoclave pressure and duration as shown by circular dichroism
experiments25. Similar decreases in specific IgE
binding have been observed when autoclaving other legumes such as lupine
allergens34 and green pea35. While
our results are in agreement with these findings, it is important to
note that our work is the first report on the complete absence of
detection of Ara h 8 from autoclaved peanut extract. This is in line
with the fact that Ara h 8 is an allergen deprived of disulfide bonds,
thereby leaving its α-helices as the major barrier to denaturation under
autoclaving conditions.
The literature is more extensive on the comparison between the
allergenicity of raw versus roasted peanuts. Maleki et al. (2000) found
that roasted peanut proteins bound to IgE from patients with peanut
allergy at approximately 90-fold higher levels than the raw
proteins15. The proposed explanation for this
enhancement of IgE binding is the glycation of major allergens to form
advanced glycation end-products (AGE) via the Maillard
Reaction17. More recently, Rao et al. (2016) found
that roasting the peanut at temperatures greater than 130°C resulted in
a reduction of IgE binding to Ara h 1 and Ara h 3, but an increase in
binding to Ara h 2 and Ara h 6, two major peanut
allergens36. However, Blanc et al. (2011) found no
difference in IgE binding between raw and roasted Ara h 1
protein37. In this study, our findings are more in
agreement with this work as we did not observe a significant difference
between the allergen detection and IgE binding responses of raw versus
roasted peanut. We believe this may be due to the restriction of our
analysis to only the soluble fractions of the peanut extracts.
Our discovery of complete and partial degradation of Ara h 8 and Ara h
2, respectively, under autoclaving may have significant clinical
implications. In a preliminary study, we observed a striking decrease in
wheal size in a group of patients that experiences oral symptoms to
peanut upon exposure to the autoclaved extract when compared to raw and
roasted extracts. These differential levels of detection of Ara h 2 and
Ara h 8 may be part of the explanation of the results observed from the
SPT. Currently, whole protein extracts created from raw or roasted
peanuts are used routinely in SPTs for the diagnosis of peanut allergy
in the clinic. Ara h 2 has proven to be one of the best predictors of
anaphylaxis in allergic patients38, while isolated Ara
h 8 sensitization indicates only oral symptoms or tolerance to peanut in
almost all cases39. Our results indicate that the use
of an autoclaved peanut extract, in addition to the current whole
protein extract (non-autoclaved), has the potential to serve as an
improved diagnostic technique (patent applied40)
distinguishing between two subsets of peanut-allergic patients: those at
risk for anaphylaxis, and primarily have Ara h 2-specific IgE, versus
those who will only experience oral symptoms to peanut, and
predominantly have Ara h 8-specific IgE. As depicted in Figure 5,
patients with a positive SPT result using both the whole peanut extract
(raw or roasted) and the autoclaved peanut extract will be classified as
at risk for anaphylaxis. Importantly, patients with a positive SPT
result using the whole peanut extract, but a negative SPT result using
the autoclaved extract, will experience only oral symptoms upon peanut
consumption. Those who experience a negative SPT result using both
extracts will be classified as tolerant to peanut.
Altogether, the data reported in this study suggest that high-pressure
and temperature autoclaving lead to a significant denaturation of Ara h
8 and other allergenic proteins. This discovery is being further
developed into an improved diagnostic test for peanut-allergic patient
stratification. Further studies are required to optimize a degree of
complete reduction of intact allergens by autoclaving.
Word Count: 3,498 words
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Tables:
Table 1 Skin Prick Test (SPT) results displaying the mean wheal
diameter in millimetres.