Thermal processing is the most commonly used technique for preserving food and extending its shelf life. It was initially scientifically defined in 1920 for the sterilization of containers, and since then, numerous forms and applications have been developed. In contemporary society, the need for food items has progressed from basic shelf stability to encompassing food safety and quality [40]. Currently, thermal processing is primarily used to improve food texture, taste, safety against microbes, digestibility, and toxin reduction. Regarding food allergies, heat treatment of proteins through thermal modification can alter their allergenic properties [41, 42]. As the consumption of processed fish increases, it is essential to examine how heating affects different fish allergens and their potential to cause allergic reactions.

As stated earlier, alterations in the structure of native proteins during processing can either diminish or increase allergenicity. In particular, the two primary elements affected by thermal processing of allergenic proteins are digestibility and antigenic traits [42]. Proteins that cause allergic responses must remain undigested or only partially digested (as immunologically active pieces) when they move through the digestive tract and are absorbed by the intestinal lining. Consequently, the resistance of proteins to digestion significantly affects their allergenic potential, which is frequently altered by heat [43].

Thermal processing includes several heat-based methods, such as boiling, roasting, frying, baking, microwave heating, and pressure cooking, all of which can significantly affect the allergenic characteristics of food proteins. These methods are commonly applied in the food sector and household environments, and their impact on allergenicity is complex, encompassing both the alteration and breakdown of protein structures. Heating can cause denaturation, aggregation, or fragmentation of proteins, thereby changing their three-dimensional structure and, consequently, their detection by the immune system [44, 45]. The vulnerability of allergens to digestive enzymes can also be altered, potentially reducing or enhancing their allergenic capacity based on specific proteins and processing conditions [45]. Roasting is one of the most extensively studied thermal processes, particularly for peanut allergens. The Maillard reaction, a non-enzymatic browning process that occurs during roasting, involves alteration of the amino groups of proteins by reducing sugars. This reaction not only enhances flavor and color but also significantly increases the IgE-binding capacity of specific proteins, such as those present in roasted peanuts, as demonstrated primarily by in vitro IgE-binding assays. Structural modifications caused by the Maillard reaction can improve the stability of allergenic epitopes, rendering them more resistant to gastrointestinal digestion and increasing the likelihood of triggering an immune response [44].

Meng et al. [46] suggest that the primary peanut allergens, including Ara h 1, Ara h 2, and Ara h 6, show modified IgE-binding characteristics following thermal processing (assessed by in vitro IgE-binding assays and SDS-PAGE), indicating that heat treatment may either reveal or conceal certain epitopes. Boiling and frying are common heat treatments with different effects on allergenicity. For example, boiling may cause soluble proteins to leach into the cooking water, which may decrease the allergenic properties of the food. Nonetheless, the degree of this reduction is significantly influenced by the type of allergen and the medium in which it is present [44]. Conversely, frying not only exposes proteins to elevated temperatures but also induces interactions with lipids. These lipid-protein interactions can lead to the formation of aggregates that may obstruct or improve the ability of the immune system to extract and identify allergens. The heightened challenge of lipid extraction from peanuts subjected to thermal processing, as noted by Meng et al. [46], indicates that thermal treatment can considerably modify the food matrix, consequently influencing allergen availability.

Microwave heating and pressure cooking are other thermal methods that have been explored for their impact on food allergenicity. These techniques can cause rapid and consistent heating, resulting in protein denaturation and aggregation. However, their effects on allergenicity vary and are influenced by food and processing conditions. Certain allergens, particularly those similar to the primary birch pollen allergen Bet v 1, exhibit lower resistance to heat treatment, leading to a reduction in their IgE-binding capacity in vitro [44]. In contrast, different allergens, such as those in sesame, show significant resistance to heat processing and have a minimal impact on their immunoreactivity, emphasizing the necessity for additional research to clarify the mechanisms underlying these variations [16, 47]. It is crucial to recognize that the modified capacity of food allergens to bind to IgE, assessed using conventional in vitro immunoassays, does not consistently correlate with variations in allergenic activity. Experiments with physiological relevance, such as skin prick tests and mediator release assays, are crucial for evaluating the genuine effects of thermal processing on the ability of allergens to cross-link IgE and induce allergic responses in effector cells [48].

The intricate nature of food matrices and the variety of thermal processing conditions require a case-by-case assessment of changes in allergenicity. Moreover, the incorporation of processed nuts with diminished IgE-binding potential, such as cashews and pistachios, has been suggested as an approach to induce tolerance in people with allergies. Nonetheless, thorough evaluation via clinical oral challenge trials and assessment of sensory attributes is needed to validate the reduction in allergenicity and ensure consumer acceptance [49]. The identification and extraction of allergenic proteins from thermally treated foods pose considerable challenges, as denaturation and aggregation can impede the effectiveness of immunochemical assays. Selecting appropriate antibodies, target analytes, and extraction methods is essential for precise allergen measurements in processed foods [50]. Thus, thermal processing can play a dual role in food allergenicity. While it may lower the IgE-binding capacity of certain proteins by altering their structure or aiding their removal from the food matrix, it can also enhance the allergenic potential of other proteins due to chemical changes and greater resistance to digestion. The interaction between protein structure, food matrix, and processing conditions highlights the necessity for a detailed understanding of the impact of thermal processing, which is vital for creating safer food products and efficient allergy management techniques [44, 45].

As described by Khan et al. [51], thermal processing can modify allergen structure through two pathways: by disrupting epitopes and reducing IgE recognition, or by inducing structural changes that lead to neoepitope formation and increased allergenic potential. The specific outcome depends on allergen identity, processing conditions, and food matrix.

MD simulations have provided direct mechanistic insights into thermally induced structural changes. Studies on Ara h 6 peanut allergen using GROningen MAchine for Chemical Simulations (GROMACS) with the CHARMM36m force field, conducted at temperatures between 300 K and 450 K, demonstrated that thermal stress causes measurable increases in root mean square deviation (RMSD) values, indicating progressive conformational destabilization, with longer simulation times revealing statistically more reliable structural changes [52, 53]. Similarly, microwave processing of shrimp allergens has been shown through MD analysis to induce notable alterations in the secondary structure, including reductions in β-sheet content and turn structures, which correlate with observed shifts in allergenicity at higher temperatures and longer processing times [54]. These simulations confirm that thermal denaturation is not a uniform process; the degree of structural disruption is highly dependent on processing temperature, duration, and the inherent stability of the target protein, underscoring the need for protein-specific processing optimization.

Taken together, the evidence on thermal processing and allergenicity reveals a contradictory picture. While thermal denaturation can disrupt conformational epitopes and improve digestibility, the Maillard reaction simultaneously introduces structural modifications that may stabilize or even enhance allergenic elements, particularly under dry-heat conditions such as roasting. A critical limitation across most thermal processing studies is their reliance on in vitro IgE-binding assays, which do not always predict clinical allergic responses. Furthermore, the vast majority of studies examine single allergens under controlled laboratory conditions, which poorly reflects the complexity of real food matrices. From the authors’ perspective, the field would benefit from standardized processing protocols that allow meaningful cross-study comparisons and greater integration of MD simulation data with scientific outcomes to bridge the gap between structural changes and actual allergenic risk.

Non-thermal processing techniques have emerged as innovative alternatives to traditional thermal treatments, offering the ability to modify food proteins and their allergenic characteristics without significant heat-driven denaturation, which is often associated with conventional methods. These techniques include high hydrostatic pressure (HHP, also referred to as high-pressure processing, HPP), cold plasma treatment, enzymatic treatment, irradiation, and fermentation. Each of these methods produces distinct physicochemical effects on food allergens, frequently resulting in structural changes that may either reduce or, in certain instances, increase allergenicity [44].

For example, cold plasma processing has emerged as an innovative, nonthermal method in the food sector. This technique employs ionized gases to trigger chemical and structural modifications in proteins at room or sub-lethal temperatures, thus preventing significant protein aggregation and solubility reduction observed during thermal processes. Based on available experimental evidence, cold plasma has been shown to alter allergenic proteins by disturbing their tertiary and quaternary structures, potentially decreasing IgE recognition, as assessed primarily through in vitro immunoassay studies [45]. Nonetheless, the decrease in allergenicity is greatly influenced by the specific proteins and processing conditions used.

HHP is another extensively researched nonthermal method. This technique exerts pressure, usually between 100 MPa and 800 MPa, on food matrices, causing conformational alterations in proteins without heat. The authors indicated that HHP can encourage the formation of disulfide bonds via SH/S–S exchange reactions, potentially leading to protein aggregation or unfolding, which is influenced by the pressure and pH levels. These structural changes, including protein unfolding and altered surface hydrophobicity, may modify epitope accessibility, although direct allergenicity outcomes depend on the specific protein system and require dedicated immunological assessment [55]. Nonetheless, the impact of HHP varies among different proteins; certain studies have indicated reduced allergenicity, whereas others have noted negligible or even negative effects, underscoring the significance of the matrix composition and processing conditions [55].

Nonthermal processing techniques can also influence the physicochemical properties of allergens, including their solubility, digestibility, and aggregation state. Villa et al. [47] emphasized that changes in protein structure, such as the formation of disulfide bonds, variations in α-helical content, and shifts in hydrophobic regions, can affect both the stability of allergens and their identification by the immune system. These structural modifications can conceal or reveal IgE-binding sites, thereby influencing allergenicity.

The analytical identification of allergens in foods that are not subjected to thermal processing poses additional challenges. The selection of detection techniques, such as real-time polymerase chain reaction (PCR) or immunoassays, should consider the modified structural and chemical characteristics of the target proteins. The authors also highlighted that no individual analytical method is universally superior, and confirmatory techniques are frequently required to guarantee precise allergen measurements in processed foods. The variety of nonthermal processing methods and their differing impacts on various food allergens highlight the difficulty in forecasting allergenic results. Despite the potential of various nonthermal techniques to decrease the allergenic properties of foods, the interactions among processing conditions, protein structure, and food matrix composition require meticulous optimization and validation. Cabanillas and Novak [44] asserted that understanding these interactions is crucial for creating safer food products and effectively managing food allergies.

Dong et al. [56] examined the allergenic threats posed by alternative protein sources, including insects, algae, and legumes, and assessed current detection techniques, such as ELISA and mass spectrometry. They also explored nonthermal processing methods (e.g., cold plasma, ultrasound, and HPP) for their ability to reduce allergenicity. An important conclusion of this study is the need for additional research to ensure food safety while encouraging sustainable protein options. They assessed and summarized various nonthermal methods to decrease allergenicity, as shown in Table 2 [57, 58, 59–61].

Tsai et al. [12] examined how species, muscle site, food processing, and cold storage impact fish tropomyosin and contrasted it with the primary fish allergen, parvalbumin. They discovered that the location of the muscle influences allergen distribution, as these two allergens are not uniformly spread throughout fish muscle, suggesting that the sampling site is crucial for precise allergen identification. Nonetheless, both allergens remained unchanged and immunoreactive when stored in a refrigerator at 4°C for up to four days. A minor decline in tropomyosin was noted after six days, whereas parvalbumin levels remained consistent during that period.

MD simulations have also shed light on the structural mechanisms underlying non-thermal processing effects. Oscillating electric fields paired with thermal treatments have been shown to unfold secondary structures, specifically α-helices and turns, in the Act d 2 kiwifruit allergen, demonstrating how electromagnetic stress can alter protein conformation in ways that may reduce epitope accessibility [57]. In the context of HHP, MD simulations support experimental findings by showing that elevated pressure increases protein vulnerability to enzymatic degradation, resulting in decreased IgE-binding ability—a key finding for understanding how combined HHP and enzymatic treatments may work synergistically to reduce allergenicity [15]. Together, these computational findings reinforce that nonthermal methods alter allergenicity through specific, protein-dependent structural pathways rather than generalized denaturation, which has direct implications for optimizing processing conditions.

Despite the promise of non-thermal processing technologies, several critical limitations inhibit their widespread application. First, most studies have focused on single proteins or model systems, and the results rarely translate predictably across different food matrices or allergen types. Second, evidence for allergenicity reduction is predominantly in vitro, with very few studies using clinical endpoints or patient trials to validate findings. Third, technologies such as cold plasma and pulsed electric fields remain largely at the laboratory scale, with limited data on industrial feasibility and consumer acceptability. From a critical standpoint, the field lacks standardized protocols for assessing the effects of nonthermal processing on allergenicity, making cross-study comparisons unreliable. Future research should prioritize multi-allergen assessments under realistic food matrix conditions and combine them with clinical validation to move beyond proof-of-concept findings toward actionable processing strategies.

Enzymatic treatment is a targeted approach for the nonthermal modification of food allergens. The use of specific proteases allows the hydrolysis of allergenic proteins into smaller peptides, thereby disrupting the conformational and linear epitopes that bind IgE. This procedure can significantly reduce the IgE-binding capacity of foods, as demonstrated primarily by in vitro immunoassays, because the immune system is less inclined to identify fragmented peptides as allergens. Enzymatic hydrolysis is affected by enzyme specificity, substrate availability, and hydrolysis level [45].

Fermentation, commonly associated with microbial processes, is a nonthermal treatment technique that significantly impacts allergenicity. During fermentation, microbial enzymes break down proteins, including allergens, into smaller fragments. Based on the available in vitro evidence, fermentation has been shown to cause the degradation of allergenic proteins, potentially decreasing their IgE-binding capacity; however, the effectiveness depends on the microbial strains employed and the fermentation conditions [50].

Enzymatic and fermentation methods play crucial roles in altering food proteins and have a major effect on their allergenic capacity. Microbial enzymes facilitate the breakdown of complex food elements, such as proteins, within the gastrointestinal tract through fermentation. This process is mainly aided by the microbiome in the large intestine, which ferments the contents and generates various microbial metabolites. These biochemical changes can modify the structure and immunogenicity of food proteins, which is particularly important in the context of food allergies. The type and quantity of glycans found in gut mucus, which also vary during fermentation, affect the relationship between digested proteins and the immune system, possibly altering allergic responses [44, 45].

The enzymatic breakdown of proteins, whether occurring naturally during fermentation or via a specific enzymatic treatment, can result in breakage of allergenic epitopes. This structural alteration frequently leads to a diminished ability to bind IgE because the epitopes recognized by the immune system are either obscured or eliminated. For instance, glycation, a mechanism by which proteins undergo enzymatic alterations in the presence of sugars, has been demonstrated to reduce the IgE-binding ability of ovalbumin, probably because of conformational modifications that hide allergenic sites. These changes are not restricted to glycation; various enzymatic processes throughout fermentation can modify protein structure and affect allergenicity [55, 62].

The fermentation process varies significantly based on the microbial species present, substrate composition, and environmental factors, such as pH and temperature. Together, these factors influence the degree and characteristics of protein hydrolysis and the subsequent allergenic profile of food. The complexity of these interactions has led to the development of a dynamic field of study aimed at understanding how various fermentation and enzymatic methods can reduce or, in certain instances, increase the allergenic capacity of food proteins. For example, partial hydrolysis or the formation of new peptide fragments during fermentation can reveal new epitopes that may increase allergenicity in susceptible individuals [44].

The relationship between enzymatic and fermentation processes and the host immune system is complicated by the inconsistent IgE groups found in allergic individuals. Each patient’s immune system identifies a distinct set of allergenic components, complicating the prediction of the impact of enzymatic or fermentation treatments on allergenicity. This inter-individual variation highlights the need for tailored food processing and allergy treatment methods, along with the significance of ongoing studies on how enzymatic and fermentation processes affect protein allergenicity.

MD simulations support these experimental findings by demonstrating that pressurized treatments combined with enzymatic hydrolysis increase protein vulnerability to enzymatic degradation at the molecular level, resulting in measurable decreases in IgE-binding ability while preserving the overall nutritional integrity of the food [15]. These computational insights suggest that combined processing approaches, rather than single-method treatments, may offer the most reliable pathway to allergenicity reduction through enzymatic means.

Enzymatic and fermentation approaches hold genuine promise for allergenicity reduction; however, several critical issues limit their current applicability. The effectiveness of enzymatic hydrolysis is highly dependent on enzyme specificity and the degree of hydrolysis; incomplete hydrolysis can generate new peptide fragments that may be allergenic, potentially worsening outcomes for some individuals. Fermentation outcomes vary considerably depending on the microbial strain, substrate composition, and environmental conditions, making reproducibility a persistent challenge. Critically, most studies are conducted under controlled laboratory conditions using purified proteins, which does not reflect the complexity of real food systems. Individual variability in IgE profiles among allergic patients further complicates the prediction of clinical outcomes. Combined processing approaches, such as pressurized enzymatic hydrolysis, show the most computational and experimental promise but require clinical validation before they can be recommended as reliable allergen mitigation strategies.

Pressure-based methods, particularly HHP, have emerged as novel strategies for altering the allergenic characteristics of food proteins. These treatments typically expose foods to pressures between 100 MPa and 800 MPa, resulting in major changes in protein structure without the requirement of high temperatures. Pressure-induced structural changes can alter the exposure and accessibility of amino acid residues, subsequently affecting the immunogenicity and allergenicity of proteins.

Liu et al. [59] investigated largemouth bass subjected to HHP treatment and found that the processing treatment altered the protein structure and composition. Structural modifications were observed under optimal conditions of 400 MPa for 15 min, resulting in the emergence of a new 21 kDa allergenic protein band post-treatment, although allergenicity did not substantially decrease.

Somkuti et al. [60] examined the influence of pressure and temperature on the structure of cod parvalbumin (Gad m 1), a significant fish allergen, and discovered that calcium binding is crucial for the stability of the protein and that it experiences intricate unfolding under different pressure-temperature scenarios. Although partial or complete protein unfolding was achieved via heat and pressure treatments, the allergenicity of the protein remained identical when evaluated using sera from patients allergic to fish. This suggests that traditional pressure-temperature methods may not effectively reduce the allergenic potential of fish proteins.

Liu and Xue [61] investigated the allergenic properties of silver carp protein during HHP processing and found, using in vitro IgE-binding assays and structural analysis, that HHP altered the secondary structure of the allergenic molecule but did not reduce its allergenicity. They recommended different or integrated HHP processing techniques to reduce fish allergenicity.

Experimental findings [55] have shown that high pressure can lead to the unfolding of protein molecules, which in turn may reveal previously hidden residues, such as tyrosine (Tyr) and phenylalanine (Phe), on the protein surface. This exposure was evident from the increased fluorescence intensity of soy β-conglycinin and soy protein isolate (SPI) at 400 MPa for 15 min. The heightened surface visibility of these aromatic residues suggests structural changes that could affect epitope recognition by the immune system of the host. However, when the pressure exceeded this limit, the fluorescence intensity decreased, probably because of the reorganization of the hydrophobic regions and movement of tryptophan (Trp) residues into these regions. These rearrangements can obscure or alter epitopes, possibly decreasing the allergenic potential of the proteins.

The influence of pressure-based methods on allergenicity varies among proteins and is highly dependent on the specific pressure utilized, treatment duration, and inherent characteristics of the protein. Additionally, pressure-based treatments can interact with other elements of the food matrix, potentially affecting allergic responses. For instance, the presence of other proteins or food components during processing can influence the degree of structural change and epitope availability. This interaction emphasizes the difficulty of forecasting allergenicity results after pressure-based processing [63].

Conformational epitopes, which rely on the three-dimensional structure of a protein, are more prone to disruption owing to pressure-induced unfolding. This may result in diminished recognition by immunoglobulin G (IgG) or IgE antibodies, which in turn lowers the likelihood of triggering an allergic reaction [50]. In contrast, linear epitopes, characterized by their primary amino acid sequences, remain mostly unchanged unless significant hydrolysis or fragmentation occurs [63]. Consequently, the net impact of pressure-based therapies on allergenicity involves a trade-off between the degradation of conformational epitopes and the maintenance or unveiling of linear epitopes. It is important to acknowledge that although pressure-based treatments may decrease allergenicity in certain instances, there is a possibility of generating new epitopes or revealing previously concealed epitopes, which could potentially increase the allergenic potential in specific situations [44]. These differences in responses highlight the need for a thorough assessment of every protein system exposed to HPP.

MD simulations offer mechanistic support for these experimental observations. Computational studies have shown that elevated pressure induces the unfolding of protein molecules, exposing previously hidden aromatic residues such as Tyr and Phe on the protein surface, which alters epitope accessibility and potentially affects IgE recognition [55]. Furthermore, MD metrics, such as solvent-accessible surface area (SASA), are particularly useful for quantifying pressure-induced changes in epitope exposure, providing a molecular-level explanation for the variable allergenicity outcomes observed experimentally under different protein systems and pressure conditions.

Pressure-based treatments present a contradiction that is critical to acknowledge: while HHP consistently induces structural changes in allergenic proteins, these changes do not reliably translate into reduced allergenicity. Studies on fish allergens—particularly parvalbumin from cod and silver carp—have demonstrated that even substantial structural disruption, including partial or complete unfolding, may leave the IgE-binding capacity intact. This is a fundamental challenge for HHP as an allergen mitigation strategy, as structural changes and immunological consequences are not equivalent outcomes. Furthermore, the emergence of new allergenic protein bands following HHP treatment, as observed in largemouth bass, raises legitimate safety concerns that have not been adequately addressed in the literature. From the authors’ perspective, HHP shows more promise as a pretreatment to enhance susceptibility to subsequent enzymatic hydrolysis than as a standalone allergen reduction method. Clinical validation of any HHP-based strategy remains an essential and largely unmet requirement.

Chemical alterations in food proteins during processing can significantly influence their allergenic potential. These alterations can occur via different mechanisms, such as glycation, oxidation, and the creation or breaking of covalent bonds within or among protein molecules. The interaction of proteins with reactive carbonyl compounds, such as methylglyoxal (MGO), illustrates a mechanism by which chemical alterations can affect allergenicity. The alteration of β-lactoglobulin by MGO results in variations in its glycation pattern, potentially affecting the functionality and immunogenicity of the protein. Glycation caused by MGO may change the sites of glycation or the total levels of modification, which could either conceal or reveal epitopes that the immune system can recognize [64]. These alterations can either diminish or increase the allergenic characteristics of a protein, depending on the specific structural changes and their effects on immune detection.

Pressure cooking is another method that causes chemical changes in proteins. By exposing food to high temperatures and pressures, pressure-cooking can enhance the denaturation and aggregation of proteins and promote Maillard reactions between amino groups and reducing sugars. These processes lead to the formation of advanced glycation end products (AGEs), which can alter the structure of allergenic proteins, thereby influencing their detection by IgE antibodies [44]. The degree and characteristics of these chemical alterations are affected by variables such as temperature, pressure, and processing time.

Chemical alterations can either conceal epitopes, rendering them unreachable by immune receptors, or reveal new epitopes, possibly forming new allergenic determinants. Computational and experimental methods have improved our knowledge of how specific chemical modifications can help eliminate or diminish allergenic epitopes. Computational predictions specific to antibodies, when used in conjunction with cross-blocking experiments, allow for the precise identification of residues within conformational B-cell epitopes. The combination of computational simulations and experimental validation offers the potential for the precise removal of allergenic epitopes in food proteins, particularly milk allergens [16]. These strategies may facilitate the creation of hypoallergenic food items through targeted chemical alterations. The intricacy of chemical changes in food processing underscores the necessity of comprehensively understanding how these alterations affect protein structure and immune detection. The interactions between glycation, oxidation, and covalent bond rearrangement highlight the complex nature of the modulation of allergenicity in processed foods.

This method was applied in a different study related to HHP, which asserted that it could decrease food allergenicity. Liu et al. [65] applied HHP in conjunction with chlorogenic acid and noted a decrease in crayfish tropomyosin IgE-binding capacity, as assessed by in vitro immunoassays, through covalent or non-covalent interactions, modifying its secondary structure and obscuring tropomyosin’s linear epitopes. Nevertheless, the authors claimed that chlorogenic acid, rather than HHP, decreased allergenicity.

Qiu et al. [66] administered dense-phase carbon dioxide (DPCD) to pompano fish (Trachinotus ovatus) and observed a 39–41% reduction in parvalbumin-triggered allergic reactions, as measured by indirect ELISA and Western blotting (in vitro). This was primarily due to the disruption of the typical conformational epitopes and alteration of the secondary structure from ordered to disordered, accompanied by a reduced presence of α-helical groups, while the internal hydrophobic groups became exposed.

MD simulations have significantly contributed to understanding how chemical modifications alter the structure of allergenic proteins. Glycation-induced changes, such as those caused by MGO on β-lactoglobulin, can be modeled computationally to predict how modified glycation patterns affect epitope accessibility and IgE recognition at the molecular level [64]. Similarly, computational predictions specific to antibody binding, combined with cross-blocking experiments, have enabled the precise identification of residues within the conformational B cell epitopes of milk allergens, demonstrating how targeted chemical modifications can be designed to eliminate specific allergenic determinants [16]. These computational approaches highlight the potential of MD-guided chemical modification strategies as a rational approach for designing hypoallergenic food protein engineering.

Chemical modifications during food processing represent an especially complex and often unpredictable pathway for allergenicity modulation. A critical issue is that the same chemical process, such as glycation, can either reduce or increase allergenicity, depending on the extent of modification, the specific protein involved, and the food matrix context. The chlorogenic acid/HHP study by Liu et al. [65] illustrates this complexity well; the chemical agent, rather than the physical treatment, drove allergenicity reduction, emphasizing that interaction effects between processing methods and food components are frequently overlooked in experimental designs. Additionally, novel processing methods, such as DPCD, remain poorly characterized in terms of their effects across different allergen families and species. From the authors’ perspective, chemical modification strategies show promise primarily when they are targeted and validated; broad application without protein-specific optimization risks unpredictable immunological outcomes, including potential neoallergen formation.

The reduction in the allergenicity of food proteins after different processing treatments is primarily due to the loss of IgE-binding epitopes. The structural stability of epitopes, which are distinct amino acid sequences or conformational patterns identified by IgE antibodies, is vital for triggering allergic reactions. The application of processing methods, such as heating, chemical modification, or enzymatic hydrolysis, can alter, conceal, or eliminate these epitopes, resulting in decreased IgE binding and, consequently, lower allergenic potential.

Thermal treatment, particularly at elevated temperatures and/or for prolonged durations, may cause denaturation and unfolding of allergenic proteins, resulting in the loss of linear IgE-binding epitopes. Meng et al. [46] suggest that in extreme thermal conditions, the Maillard reaction may deteriorate linear epitopes, leading to a reduced antigenic response. Their results showed that purified Ara h 1, a key peanut allergen, exhibited significantly reduced IgE-binding ability following thermal denaturation and unfolding, as assessed by in vitro IgE-binding assays and SDS-PAGE. This occurrence is not exclusive to peanuts; comparable effects have been noted for other allergenic proteins, wherein alterations in epitope structure are linked to diminished IgE reactivity.

Chemical changes, such as those caused by acrolein treatment, may interfere with IgE binding. Lv et al. [67] presented molecular evidence that acrolein adduction affects lysine and histidine residues in the peptide sequence, thereby reinforcing the disruption of essential IgE-binding sites. These chemical alterations emphasize the sensitivity of the epitope structure to minor changes in the protein backbone or its side chains. Enzymatic hydrolysis serves as an additional method for reducing allergenicity by breaking specific peptide bonds within antigenic epitopes. Zeng et al. [68] indicated that the efficient hydrolysis of vital amino acids in antigenic epitopes is crucial for reducing the antigenicity of cow milk proteins. Noorbakhsh et al. [69], using competitive inhibition ELISA and Western blotting (in vitro), noted that various processing and preparation techniques can significantly change protein solubility and IgE-binding characteristics of pistachio extracts, highlighting the necessity of refining processing conditions to attain the intended decrease in allergenicity.

Although processing-induced allergenicity reduction is well documented, a critical limitation is that most evidence comes from in vitro IgE-binding assays, which do not reliably predict clinical outcomes. Residual peptide fragments after processing may retain immunogenic potential, and the sensory and nutritional impacts of the required processing conditions are rarely assessed alongside allergenicity.

The emergence of new epitopes, referred to as neoallergens, is observed during different food-processing techniques, in which alterations in protein structures can lead to the creation of unique antigenic determinants. These newly created epitopes may not exist in the native protein and can trigger immune reactions in sensitized individuals, thereby increasing the allergenic risk of processed foods [44, 49, 50].

As stated by Cuadrado et al. [49], processing can concurrently remove existing epitopes and create new ones, thus emphasizing its dual effect on allergenicity. The fundamental mechanisms frequently include changes in the tertiary and quaternary structures of the protein, resulting in the exposure or formation of amino acid sequences that were previously hidden from the immune system [44, 47, 50]. Heat treatment methods, such as roasting or baking, are particularly effective in inducing these structural changes. Khan et al. [50] suggested that any mechanism that can alter protein conformation may affect the structural properties of epitopes, consequently influencing antigen-antibody interactions. For example, elevated temperatures can lead to protein unfolding, aggregation, or even chemical changes, such as Maillard reactions, all of which may reveal concealed epitopes or create completely new antigenic sites.

The unmasking of allergenicity represents an underappreciated risk in food processing, because standard safety assessments rarely screen for neoallergen formation. A critical concern is that processing conditions optimized to reduce one allergen may simultaneously increase reactivity toward another, particularly through Maillard-induced structural changes. Systematic screening for neoepitope formation should be incorporated into the allergenicity risk assessments of processed foods.

Interactions between food proteins and other components of the food matrix, including carbohydrates, lipids, and proteins, significantly influence allergenicity. The intricate nature of the food matrix suggests that proteins rarely exist independently of each other. Instead, they are integrated into a network of different molecules that can directly or indirectly affect immunogenic characteristics [50]. These interactions may alter epitope accessibility, alter protein shape, and influence the effectiveness of extraction and detection techniques. Carbohydrates, especially reducing sugars, are significant because of their involvement in non-enzymatic glycation processes, such as the Maillard reaction. This procedure entails the condensation of the N-group from lysine residues in proteins with the carbonyl group of reducing sugars, initially yielding glycosamines, which may subsequently rearrange into AGEs [63]. AGE development alters the chemical composition of proteins and can either mask or reveal immunologically significant epitopes, influencing the potential for allergic reactions. Heating accelerates these reactions, and the resulting changes can either improve or reduce the immune system’s recognition of the protein, depending on the context. Interactions between lipids and other proteins complicate the allergic environment. As noted by Villa et al. [47], the food matrix can either stabilize, enhance, or diminish protein allergenicity, depending on the protein type and processing parameters. For example, the attachment of proteins to lipids or other large molecules may limit their accessibility to immune receptors, possibly decreasing their allergenic potential. However, these interactions can also resemble pathogen-associated molecular patterns, thereby improving receptor attachment and increasing the likelihood of allergens.

Therefore, the relationship between protein structure and matrix composition varies significantly depending on the context. Interactions within these matrices also affect the extraction and detection of allergens in food samples. Zhang et al. [70] suggested that thermal treatment influences the extraction of protein elements, as observed in processed peanut extracts, where protein bands shift and blur, indicating changes in molecular weight and possibly modified immunoreactivity. These modifications result not only from direct thermal denaturation but also from interactions with various matrix components, which may mask or cluster proteins, making their identification and quantification more complex. Matrix effects encompass not only chemical interactions but also include physical and structural elements. Food structure may hinder enzymatic access during digestion, consequently influencing the degradation and subsequent absorption of allergenic proteins [47, 63]. This alteration in gastrointestinal digestion can affect the quantity and structure of allergenic peptides that access the immune system, subsequently affecting their allergenicity. Khan et al. [50] noted that the matrix effect refers to the total impact of all sample components, except the analyte, on the measurement of the analyte itself. This definition highlights the significance of considering the overall composition of food when evaluating allergenic risk. The matrix can either disguise allergens, rendering them more difficult to detect and possibly less immunogenic, or enhance their exposure to the immune system, depending on the interaction of its components.

Additionally, processing techniques, such as fermentation, autoclaving, and drying, which intrinsically change the matrix composition and structure, further influence these interactions [45]. The influence of food additives and the selection of extraction techniques in research environments also contribute to the inconsistency of allergenicity evaluations, emphasizing the need for standardized methods that consider matrix effects.

Food matrix effects on allergenicity remain among the least understood variables in the field, largely because most studies use purified proteins rather than whole food systems. This is a critical methodological gap, as matrix interactions can mask or amplify allergenicity in ways that purified protein studies cannot capture, thereby limiting the translational value of current findings.

The oral, gastric, and intestinal stages of digestion are crucial in influencing the allergenic potential of food proteins. In the oral phase, mechanical disruption and enzyme action initiate the breakdown of food matrices; however, the effect on protein allergenicity is usually minimal owing to the brief exposure time and gentle enzymatic environment. The mouth mainly presents proteins to salivary enzymes, such as amylase, which has a limited direct impact on protein allergens. Consequently, most allergenic proteins remain structurally intact as they enter the gastric phase [71].

During the gastric phase, acidic conditions (pH < 2) and pepsin are essential for protein digestion. The upper part of the stomach, comprising the cardia and fundus, plays a role in producing gastric juices and sustaining a low pH, which is crucial for effective pepsin function [45]. Pepsin breaks peptide bonds, particularly those with aromatic amino acids, resulting in the breakdown of protein allergens.

However, the degree of degradation differs significantly for various proteins. Certain allergens, such as the 2S albumins found in sesame seeds, demonstrate significant resilience to acidic pH and pepsin digestion, preserving their structure and allergenic capability even after prolonged exposure to gastric conditions. This stability is attributed to their rigid structure, which is reinforced by several disulfide bonds that shield them from enzymatic hydrolysis [47]. In contrast, certain proteins, such as parvalbumin from fish, may be partially broken down during gastric digestion; however, their durability is influenced by the species and the unique structure of the protein. Koidl et al. [45] suggest that parvalbumin can still be found after 120 minutes of gastric digestion, emphasizing the durability of specific allergenic proteins.

Processing treatments significantly affect the digestibility of food proteins, subsequently altering their potential to cause allergies. Changes in protein structure caused by different processing techniques can either increase or decrease protein vulnerability to digestive enzymes, thus affecting the composition and quantity of digestion products. For example, the ultrasonication of kiwifruit proteins has been shown to enhance peptide levels during simulated gastrointestinal digestion, as assessed by o-phthalaldehyde (OPA) assay. The peptide yield increased with prolonged ultrasound treatment, indicating that this physical method can aid in the decomposition of protein structures, rendering them more accessible to digestive enzymes [57]. This improved digestibility is not exclusive to kiwifruit; comparable effects have been observed in other food matrices that have undergone physical or thermal processing. The vulnerability of food allergens to digestion is significantly influenced by the physicochemical conditions of the digestive process. Elements such as pH, enzyme composition, and digestion duration are crucial in influencing the degree of protein hydrolysis. Koidl et al. [45] emphasize that the level of digestion and subsequent allergenicity are significantly affected by these experimental factors, and that the differences in individual gastrointestinal conditions should be considered when evaluating protein digestibility and the risk of allergies.

The overall findings [16, 45, 46, 57, 58] suggest that alterations in protein structure due to processing, physicochemical conditions of digestion, and inherent characteristics of the food matrix shape the digestibility of food proteins and, therefore, their potential to cause allergies. A thorough understanding of these interactions is vital for the development of processing strategies designed to reduce food allergenicity while preserving nutritional quality.

MD simulations have provided valuable additional insights into digestion dynamics at the molecular level. The simulated gastric fluid conditions modeled computationally have shown how parvalbumins interact with digestive enzymes in acidic environments, yielding information on their structural stability and allergenic persistence during digestion that is difficult to obtain experimentally [43]. Additionally, MD simulations of acidic treatments have revealed that protein cleavage and alterations in surface polarity produce conformational changes that reduce epitope accessibility, offering a mechanistic explanation for the observed reduction in IgE-binding capacity under acidic digestive conditions, though clinical validation of these computationally derived findings remains limited [72]. These findings highlight the value of MD as a tool for probing digestion-allergenicity relationships at a resolution beyond what conventional in vitro models can provide.

A critical limitation of current digestion studies is their reliance on standardized in vitro models that do not reflect the considerable individual variability in gastrointestinal conditions, including differences in pH, enzyme concentrations, and transit time. These variables can substantially alter allergen stability and immunogenic fragment generation; however, they are rarely accounted for in experimental designs. MD simulations offer a promising avenue for modeling these individual differences computationally; however, experimental validation under physiologically diverse conditions remains a priority.