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.
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References:
1. Ben-Shoshan M, Turnbull E, Clarke A. Food Allergy: Temporal Trends and Determinants. Curr Allergy Asthma Rep 2012;12(4):346-372.
2. Ben-Shoshan M, La Vieille S, Eisman H, et al. Anaphylaxis treated in a Canadian pediatric hospital: Incidence, clinical characteristics, triggers, and management. J Allergy Clin Immunol 2013;132(3):739-741.
3. Urisu A, Ando H, Morita Y, et al. Allergenic activity of heated and ovomucoid-depleted egg white. J Allergy Clin Immunol 1997;100(2):171-6.
4. Eigenmann PADoP, the Division of I, Allergy DoMUoGSoMGS. Anaphylactic reactions to raw eggs after negative challenges with cooked eggs. J Allergy Clin Immunol 2000;105(3):587-588.
5. Des Roches A, Nguyen M, Paradis L, Primeau MN, Singer S. Tolerance to cooked egg in an egg allergic population. Allergy 2006;61(7):900-901.
6. Nowak-Wegrzyn A, Bloom KA, Sicherer SH, et al. Tolerance to extensively heated milk in children with cow’s milk allergy. J Allergy Clin Immunol 2008;122(2):342-347.
7. Leonard SA, Sampson HA, Sicherer SH, et al. Dietary baked egg accelerates resolution of egg allergy in children. J Allergy Clin Immunol 2012;130(2):473-480.
8. Goldberg MR, Nachshon L, Appel MY, et al. Efficacy of baked milk oral immunotherapy in baked milk–reactive allergic patients. J Allergy Clin Immunol 2015;136(6):1601-1606.
9. Kim JSMD, Nowak-Węgrzyn AMD, Sicherer SHMD, Noone SRN, Moshier ELMS, Sampson HAMD. Dietary baked milk accelerates the resolution of cow’s milk allergy in children. J Allergy Clin Immunol 2011;128(1):125-131.
10. De Schryver S, Mazer B, Clarke AE, et al. Adverse Events in Oral Immunotherapy for the Desensitization of Cow’s Milk Allergy in Children: A Randomized Controlled Trial. J Allergy Clin Immunol Pract 2019;7(6):1912-1919.
11. Mueller GA, Gosavi RA, Pomés A, et al. Ara h 2: crystal structure and IgE binding distinguish two subpopulations of peanut allergic patients by epitope diversity Structure and IgE binding of Ara h 2. Allergy 2011;66(7):878-885.
12. Chruszcz M, Maleki SJ, Majorek KA, et al. Structural and immunologic characterization of Ara h 1, a major peanut allergen. J Biol Chem 2011;286(51):44294.
13. Hurlburt BK, Offermann LR, McBride JK, Majorek KA, Maleki SJ, Chruszcz M. Structure and Function of the Peanut Panallergen Ara h 8. J Biol Chem 2013;288(52):36890-36901.
14. Koppelman SJ, Hefle SL, Taylor SL, de Jong GAH. Digestion of peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6: A comparative <i>in vitro</i> study and partial characterization of digestion-resistant peptides. Mol Nutr Food Res 2010;54(12):1711-1721.
15. Maleki SJ, Chung S-Y, Champagne ET, Raufman J-P. The effects of roasting on the allergenic properties of peanut proteins. J Allergy Clin Immunol 2000;106(4):763-768.
16. Chung SY, Champagne ET. Allergenicity of Maillard reaction products from peanut proteins. J Agric Food Chem 1999;47(12):5227-5231.
17. Chung SY, Champagne ET. Association of end-product adducts with increased IgE binding of roasted peanuts. J Agric Food Chem 2001;49(8):3911-3916.
18. Mueller GA, Maleki SJ, Johnson K, et al. Identification of Maillard reaction products on peanut allergens that influence binding to the receptor for advanced glycation end products. Allergy 2013;68(12):1546-1554.
19. Vissers YM, Blanc F, Skov PS, et al. Effect of heating and glycation on the allergenicity of 2S albumins (Ara h 2/6) from peanut. PLoS One 2011;6(8):e23998.
20. Kholief TS. Chemical composition and protein properties of peanuts. Z Ernahrungswiss 1987;26(1):56-61.
21. Hoffpauir CL. Peanut Composition, Relation to Processing and Utilization. J Agric Food Chem 1953;1(10):668-671.
22. Verhoeckx KC, Vissers YM, Baumert JL, et al. Food processing and allergenicity. Food Chem Toxicol 2015;80:223-40.
23. Mondoulet L, Paty E, Drumare MF, et al. Influence of Thermal Processing on the Allergenicity of Peanut Proteins. J Agric Food Chem 2005;53(11):4547-4553.
24. Comstock SS, Maleki SJ, Teuber SS. Boiling and Frying Peanuts Decreases Soluble Peanut (Arachis Hypogaea) Allergens Ara h 1 and Ara h 2 But Does Not Generate Hypoallergenic Peanuts. PLoS One 2016;11(6):e0157849.
25. Cabanillas B, Maleki SJ, Rodriguez J, et al. Heat and pressure treatments effects on peanut allergenicity. Food Chem 2012;132(1):360-6.
26. Walczyk NE, Smith PMC, Tovey ER, Roberts TH. Peanut protein extraction conditions strongly influence yield of allergens Ara h 1 and 2 and sensitivity of immunoassays. Food Chem 2017;221:335-344.
27. Bradford MM. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal Biochem 1976;72(1-2):248-254.
28. Koppelman SJ, Wensing M, Ertmann M, Knulst AC, Knol EF. Relevance of Ara h1, Ara h2 and Ara h3 in peanut-allergic patients, as determined by immunoglobulin E Western blotting, basophil-histamine release and intracutaneous testing: Ara h2 is the most important peanut allergen. Clin Exp Allergy 2004;34(4):583-590.
29. Palmer GW, Dibbern DA, Burks AW, et al. Comparative potency of Ara h 1 and Ara h 2 in immunochemical and functional assays of allergenicity. Clin Immunol 2005;115(3):302-312.
30. Hales BJ, Bosco A, Mills KL, et al. Isoforms of the Major Peanut Allergen Ara h 2: IgE Binding in Children with Peanut Allergy. Int Arch Allergy Immunol 2004;135(2):101-107.
31. El Mezayen R, Pons L, Burks AW, et al. Ara H 2.02 Is A More Potent Cross-linker Of Anti- Peanut IgE Than Is Ara H 2.01. J Allergy Clin Immunol 2007;119(1):S193.
32. Foster MP, McElroy CA, Amero CD. Solution NMR of Large Molecules and Assemblies †. Biochemistry 2007;46(2):331-340.
33. Koppelman SJ, Vlooswijk RAA, Knippels LMJ, et al. Quantification of major peanut allergens Ara h 1 and Ara h 2 in the peanut varieties Runner, Spanish, Virginia, and Valencia, bred in different parts of the world. Allergy 2001;56(2):132-137.
34. Álvarez-Álvarez J, Guillamón E, Crespo JsF, et al. Effects of Extrusion, Boiling, Autoclaving, and Microwave Heating on Lupine Allergenicity. J Agric Food Chem 2005;53(4):1294-1298.
35. Malley A, Baecher L, Mackler B, Perlman F. The isolation of allergens from the green pea. J Allergy Clin Immunol 1975;56(4):282-90.
36. Rao H, Tian Y, Tao S, Tang J, Li X, Xue W-T. Key factors affecting the immunoreactivity of roasted and boiled peanuts: Temperature and water. LWT - Food Sci Technol 2016;72:492-500.
37. Blanc F, Vissers YM, Adel-Patient K, et al. Boiling peanut Ara h 1 results in the formation of aggregates with reduced allergenicity. Mol Nutr Food Res 2011;55(12):1887-1894.
38. Kukkonen AK, Pelkonen AS, Mäkinen-Kiljunen S, Voutilainen H, Mäkelä MJ. Ara h 2 and Ara 6 are the best predictors of severe peanut allergy: a double-blind placebo-controlled study. Allergy 2015;70(10):1239-1245.
39. Asarnoj A, Nilsson C, Lidholm J, et al. Peanut component Ara h 8 sensitization and tolerance to peanut. J Allergy Clin Immunol 2012;130(2):468-72.
40. Cohen C, Jean-Claude BJ, Mazer B. Peanut Hypoallergenic Formulations for Determining the Risk of Anaphylaxis. Canada patent application 3,097,204. 2020.
Tables:
Table 1 Skin Prick Test (SPT) results displaying the mean wheal diameter in millimetres.