Allergy is an adverse or augmented immunological reaction against certain substances that are basically supposed to be harmless. Peanuts are notoriously known as allergenic legumes. Peanut allergy (PA) can be topical (cutaneous exposure or inhalation) or systemic. It garners massive attention since it is a nut that presents a prevalent, lifelong-persistent, and severe allergic reaction. Figure 1 illustrates the mechanism of allergic reaction. Allergenicity and IgE reactivity are determined by the interplay of a protein’s digestibility, solubility, stability, structural features, heat resistance, and enzyme activity, in addition to post-translational and chemical modifications ^[1][2]. On the other hand, several risk factors contribute to the increased prevalence of peanut allergenicity by amplifying its intensity and its spread. These factors may include genetics ^[3][4], epigenetics ^[5], and some processing methods such as roasting or dry heating, but also unintended exposure. This fact represents one of the main motivations for research on how to mitigate peanut allergenicity, since measures taken are not always fully adequate. Inconsistent food labeling standards (precautionary allergen labels) or inadequate safety measures in public settings (e.g., restaurants, airlines, and schools) create an environment conducive to accidental ingestion of peanuts.

2. Peanut Allergens and Their Mechanisms of Action

The Allergen Nomenclature Subcommittee of the World Health Organization/International Union of Immunological Societies (WHO/IUIS) reported 17 protein allergens contributing to PA and named after the species ^[6][7]. Upon revealing the peanut genome sequence, various proteomic studies were carried out to determine the allergens’ profile, their levels and modifications, such as reverse-phase high-pressure liquid chromatography ^[8], and more recently liquid chromatography–mass spectrometry ^[9]. Peanut allergens are classified as major and minor allergens, depending on their contribution to the initiation and propagation of an allergic response in sensitive hosts. A major peanut allergen binds specific IgE in more than 50% of allergic patients; otherwise, it would be classified as a minor peanut allergen. Among these proteins, scientists define Ara h 1, Ara h 2, and Ara h 3 as being the major protein allergens. In addition to their strong IgE-binding capacity, they represent more than 80% of the total protein pool contained in peanuts ^[10][11]. Recent studies stated that Ara h 6 displays a great recognition by immunoglobulins in the serum of allergenic patients, and that its biochemical structure and properties show a great similarity to those of Ara h 2 ^[6]. Peanut allergens, as reported in Table 1, are categorized into different superfamilies and families based on their biochemical, structural, and functional characteristics ^{[1][6][12][13][14][15][16][17][18]}.

Table 1. Physicochemical properties of peanut allergens

3. Thermomechanical Processing of Peanuts

The allergenicity of peanuts is closely related to the linear and conformational allergic epitopes of the indigenous proteins. Altering the structure of peanut allergens will result in downstream modifications in their physicochemical properties, thus modifying their immunoreactivity. The degree of alteration of the allergic potential of immunoreactive proteins in peanuts is strictly dependent on the processing conditions. Physical methods encompass not only traditional cooking processes but also novel techniques that are heat-based, wave-based, high-pressure-based, or any possible combination. Thermo-pressure-based treatments can range from simple cooking under mild pressure to autoclaving and high-pressure processing. Presented in Figure 2, such treatments include, among others, boiling, frying (shallow and deep), roasting/baking, microwaving, and ultrasonication.

Thermomechanical treatments of peanuts

3.1. Boiling

Boiling is the immersion of food in water at a temperature approaching 100 °C under atmospheric pressure. While it is conducted for cooking purposes, boiling can be considered an efficient way to partially decrease sensitivity towards peanuts. Ingestion of boiled peanuts by sensitized mice led to a subtle etiology (e.g., weight loss, itching, and diarrhea) compared to the severe allergic reaction caused by raw peanuts ^[19]. Changes in solubility were assessed by measuring the changes in protein content after boiling treatment under mimicked physiological conditions by regulating the addition of gastrointestinal juice. Bicinchoninic acid (BCA) assay and SDS–PAGE detected a change in protein content ranging from 30% to 55% after treatment ^[20]. The total extractable allergen content out of the total peanut protein decreased from 71% in the untreated peanuts to 29% in the boiled peanuts ^[21]. Total Ara h 2 concentrations gradually decreased as the duration of boiling increased due to leaching into cooking water ^[22]. Protein digestion is a commonly used factor to evaluate allergenicity. In a simulated gastric fluid (SGF) experiment, boiled peanut allergens manifested low stability upon digestion ^[23]. Several experiments in mice and humans confirmed the efficiency of boiling in decreasing elicitation through IgE and IgG titers ^[23][24]. The IgE-binding capacity of Ara h 1 in boiled peanuts was significantly lower than in crude peanuts ^[25]. It was also noted that the degranulation i.e., β-hexosaminidase activity based on the RBL-2H3 cells model, was decreased. This might be due to the uncomplimentary fitting of allergens after structure modification to IgE, or decreased IgE activation by the treated allergens ^[26].

3.2. Roasting/Baking

Roasting and baking, used interchangeably in the literature, are carried out through the exposure of peanuts to dry heat: convection, conduction, radiation, or a combination. Roasting has gained a lot of attention over the last few years since it was discovered to be having hyper-allergic rather than hypo-allergic effects. Etiologically, scratching behavior and diarrhea were prominent in mice fed roasted or raw peanuts ^[19]. The weight of mice fed with roasted peanuts was the lowest among treatment groups. In contrast to the boiled peanut group, jejunal breakage and splenomegaly were more prominent using roasted peanuts than raw ^[19][23]. Severe modifications in the structure of peanut allergens were observed after roasting. Circular dichroism spectra implied the modification of the original secondary structure of the allergens. It shows new, previously hidden amino acids in crude peanuts, on the surface: neoepitope development ^[23][27]. UV spectra analysis comparison revealed that the absorbance at 280 nm was higher after roasting. This explains the massive denaturation and alteration of secondary and tertiary structures of proteins by roasting. In another experiment, Western blots of roasted and raw peanut extracts showed high intensities of Ara h 2 and Ara h 8 bands ^[28]. The total extractable allergen content decreased from 71% in raw peanuts to 21% in roasted peanuts ^[21]. Recently, Ðukić and collaborators investigated the post-translational modifications (PTMs) of peanut proteins as affected by roasting ^[29]. The most common PTMs observed were oxidation (met), formylation (Arg/Lys), hydroxylation (Trp), and oxidation or hydroxylation (Asn). Importantly, this study shed light on the structural alteration, and hence the digestibility, of peanut proteins occurring following roasting, as revealed by the proteomic profiling. Not only was the structure of the protein modified, but also the concentration of allergens was multiplied after roasting. It was recorded that Ara h 1 concentration increased after roasting, ultimately in the presence of reducing sugars, resulting in a phenomenon called Advanced Glycosylation End Products (AGE). In several simulated gastric fluid experiments, roasted peanut proteins manifested stronger resistance to digestion than both raw and boiled proteins ^[23]. Mass spectrometry showed that even when intestinal fluid was added to the simulated gastro-intestinal/duodenal fluid experiment, Ara h 1 persisted and appeared as a 65 kDa on the gel in both reducing and non-reducing conditions ^[20]. Ara h 2 obtained an anti-trypsin digestibility ^[21] and lasted a longer time on the SDS–PAGE than the raw peanuts when exposed to SGF ^[19] and even simulated gastric digestion, regardless of the electrophoretic conditions ^[20]. This idea was endorsed by other experiments in which some of the peanut allergens, and more prominently Ara h 2, resisted simulated oral–gastroduodenal digestion in raw ^[30] and roasted peanuts ^[31]. The ability of allergens’ penetration into the body and absorbance through the GI, specifically the intestines, was highly improved after roasting.

3.3. Microwaving

Microwaving is the processing of food materials by means of electromagnetic waves, creating multiple changes at the sample–wave interface. The process is characterized by an uneven heat distribution due to the positional effect of the sample in the microwave oven. It can be used for many purposes in food processing, such as baking, defrosting, pasteurization, and heating. Recent studies assess the role of this electromagnetic treatment in mitigating the allergenicity of several foods, including peanuts. Microwaved peanuts showed a 54% decrease in their total protein content ^[21]. The molecular analysis of the extracted proteins exhibited an insoluble aggregate of high-molecular-weight proteins, whose formation is proportional to the processing time. However, the IgE-binding capacity of proteins obtained from microwaved peanuts remained high, especially in the case of Ara h 2. The reactivity of the 19 kDa and 17 kDa isoforms of Ara h 2 was retained by 71% and 59%, respectively, taking raw peanuts as a reference ^[21]. This may be due to irregularities of the heating process or an insufficient duration of exposure to the treatment.

3.4. Ultrasonication

Ultrasonication is classified under the novel processing methods applied to food materials. It consists of irradiating food samples with high-energy ultrasonic waves, resulting in physical and chemical modifications. The interaction between the waves and the food samples results in the formation/collapse of bubbles within the medium. The sudden decompression of these bubbles creates pressure and temperature gradients and generates high shear energy waves in treated materials, lealteringding to the alteration of their internal composition ^[32]. The impact of this constraint on food matrices is proportional to the treatment power and frequency. These events impose several structural changes on the resident macromolecules, including proteins. In fact, ultrasonication-induced matrix decompression is shown to affect hydrogen bond formation, increasing the susceptibility of proteins to unfolding and cleavage. It also impacts protein-to-protein interactions due to the alteration of protein conformation ^[6]. The treatment of roasted peanuts by ultrasound waves resulted in increased protein solubility and induced peptide bond cleavage. Moreover, ultrasound treatment resulted in a significant drop in Ara h 1 levels of treated samples with respect to those of untreated roasted peanuts, although the levels of Ara h 2 were not markedly lowered after the treatment ^[33].

3.5. Frying

It is essential to differentiate between shallow and deep frying. Shallow frying is the introducingtion of a food into a thin hot oil layer, whereas deep frying is when the food is entirely submerged in the hot oil. It has been noted that shallow frying slightly decreased the immunogenic potential of some major allergens by causing extensive modifications to their structure. A 21% and 2% decrease in α-helices and β-sheets content in Ara h 1, respectively, were detected in the secondary structure, taking raw peanuts as a reference ^[25]. In the tertiary and quaternary structures, a remarkable change was observed in the occurrence of irregular coils and in the leap of approximately 13-fold in the hydrophobic index. The inner hydrophobic residues that surfaced indicated the release of polar amino acids and cross-linking of the aromatic amino acids. This implies the aggregation, or, in other words, the decrease in solubility of the allergens that collectively affect the function of those proteins. This thermal treatment still needs further investigation, since there is no definitive conclusion as to whether it slightly decreases or increases the immunogenic potential of the allergens. Although a decrease of approximately 9.4% in the IgE-binding capacity was noted, high aggregation and rearrangement of Ara h 1 might protect the epitopes from losing their abilities or even form neoepitopes that can interact with the immune system, increasing the elicitation. Regarding the deep frying, smearing of low-molecular-mass (10 kDa) fragments revealed that they gradually became darker as frying time increased, with the 8-minute result (about 1.72 g/100 g peanut) being the highest (1, 3, 6, and 8 min) ^[34][35]. At the maximum interval, the peanuts’ color changed to brown, and they were at risk of burning. Similarly, their analysis referred to this intensity increase as a decrease in the solubility of the allergens. With all these factors combined, a decrease in allergen solubility of 66.5% after deep frying was observed.

3.6. High-Pressure Steaming/Autoclaving

The use of steam under high pressure is one of the most common processing methods applied ton peanuts. High-pressure steaming and dry autoclaving are two sides of the same coin, used interchangeably in the literature. Nonetheless, wet autoclaving is steaming following the hhydration of peanuts by presoaking them in ultrapure water. In a study investigating the effect of various thermal processing methods, the highest aggregation state and fading speed were recorded for pre-soaked high-pressure-steamed peanuts ^[21]. Interpretation of this observation might be correlated to overnight soaking, which increased water activity and contributed to the higher structural change. Unlike most studies, only a few were skeptical about this conclusion, claiming that it could not be valid because of the presence of two variables, high pressure, and heat treatment, along with the hydration factor ^[34]. It causes extensive fragmentation of allergens, as LC–MS/MS analysis detected a vast increase in the number of peptides. Like other treatments, more efficient wet autoclaving alters secondary and tertiary structures. It also increases the percentage of extended-sheet structures along the moiety ^[21][36]. This promotes the aggregation of large protein complexes along with a series of unfolding, crosslinking, and chemical fluctuation (such as glycosylation and targeted oxidation) episodes. Wet autoclaving induces modifications in lysine, cysteine (sulfide bridges), and arginine residues, thus altering solubility. The total extractable proteins decreased by 30–40% after wet autoclaving. A 91%, 61%, 55%, and 60% decrease in peanut content was measured for Ara h 1, Ara h 2, Ara h 3, and Ara h 6, respectively, with Western blot and SDS–PAGE. Subsequently, IgE-binding capacity was remarkably diminished ^{[35][37][38][39]}. Others, however, claim that the protein content is unchanged, or slightly altered ^[38]. All aforementioned experiments agree that the number of peptides associated with allergens increased with much lower molecular mass and concentration, specifically Ara h 2 and Ara h 7 on SDS–PAGE ^[35][39].  Wet autoclaving ceased the anti-proliferative effect of the allergens in Caco-2 cells. The results of cell viability showed improvement after treatment. Presumably, the release of small bioactive peptides that boost growth and reduce the penetration of allergens into the cells justifies increased cell viability. The decreased stimulation of the immune cells (T cell activation and inflammatory mediators), the feeble and massively reduced IgE capacity, and the managed chemokines, cytokines, and growth factors reflect the decrease in the polarization of the immune cells resulting in a milder immune response ^[35][38]. Without presoaking, etiology in humans was detected through SPT for raw and autoclaved peanuts. The effectiveness of autoclaving was revealed by the noticeably reduced wheal diameter (SPT) in patients suffering from mild oral symptoms ^[28][34]. This was not, however, the case for patients who had previously suffered from anaphylaxis. The same applies to autoclaved roasted peanuts. Structure change in autoclaved and autoclaved roasted peanuts was consistent with this conclusion. It was a result of oligomerization, degradation due to free radicals attack on the side chains and peptide fragments, and even reassociation and aggregation due to cross-linking and hydrophobic interaction of peanut allergens ^{[21][27][28][34][35]}. Although complete degradation of Ara h 2, Ara h 7, and Ara h 8 and reduction in Ara h 1 were observed, the resistance of some fragments of Ara h 3 was noted after 20 min of autoclaving, suggesting that some peanut proteins might not be sufficiently susceptible to this treatment ^[21][28][35]. As for the protein content, SDS–PAGE and Western blot assessments showed that Ara h 1 monomer band faded proportionally to the processing time. The total extractable allergen content in raw peanuts decreased from 72% to 25% in steaming ^[21][34]. As we explained for previous treatments, these changes collectively cause a decrease in solubility as supported by the Bradford method measurements. Ara h 1 and Ara h 2 content were nearly null, less than 0.1 g and 0.2 g per 100 g, respectively ^[34]. On the other hand, at a short cooking time (1 min), the three-dimensional structure of the Ara h 1 and Ara h 2 that is rich in disulfide bonds was slightly and insufficiently altered, and hence incapable of causing a change in resistance, solubility, or function ^[28].

4. Conclusions

During the last decade, peanut consumption has risen to unprecedented values per capita. Mitigation of peanut allergenicity has the potential to be a game changer for allergic individuals. The dDevelopment of trivial and novel processing methods has become a necessity not only for scientists, but also for food industrialists, to impose structural and/or chemical modifications on allergens. Although none of the existing methods is qualified as fully effective in eliminating the allergenic potency of peanut proteins, the methods combined with a hydration step proved their higher efficacy. From this viewpoint, consuming wet autoclaved peanuts could be a promising alternative for susceptible individuals. Water penetration within the peanut matrix under pressure may result in a softer texture associated with a higher efficiency in allergenicity mitigation. Likewise, boiling showed a decreased level of peanut allergens. However, boiled peanuts retained some of their allergenic properties due to the formation of neoallergens during the treatment. Lastly, deep frying of peanuts forduring 6 min resulted in reducing their allergenicity. The limitation of this process resides in the color and taste alterations following longer treatment times. To conclude, it is fundamental to unveil the mechanistic details of primary peanut sensitization in order to fully understand peanut allergenicity ^[2]. Further studies should be directed towards the optimization of all of these techniques in order to ensure a sustainable level of hypoallergenicity in the treated peanuts, while preferably preserving their structural integrity and nutritional value. More importantly, the digestibility of modified allergens must be investigated to fully assess their ultimate allergic potential once consumed by peanut-allergic individuals.