Introduction

Efforts to develop a sustainable food supply are increasing in the market, as a means of mitigating the environmental damage caused by farming and addressing the challenges currently faced in the industry. Animal-based proteins are considered traditional protein sources, but they have been associated with various environmental, ethical, and health issues due to the conventional farming practices used to produce them. These issues include higher levels of greenhouse gas emissions, significant water and land usage, and ethical concerns related to animal welfare. Sustainable food provides us with more options in food selection, including meat substitutes and alternative proteins. Alternative proteins mainly refer to the proteins that can provide an equal protein supply for daily human consumption but are derived from sources such as plants, microorganisms, algae and even insects [55]. In comparison to traditional protein sources, insects and pulses require less water and land for cultivation, while algae are naturally rich in fresh and sea water [5, 12, 145, 155]. Recently, the potential of various alternative proteins, including pulses, algae, plant-based meat alternatives, and insects has been investigated [59, 148]. Insects, for example, are a rich source of protein, omega-3 fatty acids, vitamins and essential minerals like iron and calcium [65]. Insects are also more efficient at converting feed into protein, making them a more sustainable option. Moreover, pulses, such as lentils, chickpeas, and beans are a nutritious source of protein and fibre, as well as B vitamins, iron, and potassium, which make them a healthy and beneficial alternative source of protein [12, 18, 154]. Algae is rich in protein, fibre, vitamins, fatty acids and minerals, which are essential for maintaining a healthy diet [53, 137]. Algae has also a low environmental impact, as it can help absorb carbon dioxide from the atmosphere and use it for microalgae cultivation [28]. An often overlooked alternative protein source is canola meal, a byproduct of the canola oil extraction process. After extracting the oil from rapeseed, canola meal still retains an impressive protein content and is commonly used worldwide as animal feed, second only to soybean meal [34].

However, alongside the exploration of the potential of alternative protein sources, the critical aspect of food safety emerges as a pivotal area of concern. There are several potential concerns of food safety related to the presence of microbes, toxins, or allergens that might have adverse effects on the health of consumers, and therefore, it is precise their risk assessment, detection and control [143]. In particular, this review focuses on the potential presence of allergens in alternative food proteins.

Allergies are triggered by the immune system in response to a foreign substance. This allergic reaction can induce different symptoms that can range from mild (e.g. cough, rhinitis) to severe (e.g. anaphylaxis), depending on the type and severity of the response [40]. Food allergies are defined as adverse, immune-related reactions that occur in particular individuals and can be immunoglobulin E (IgE) and cell-mediated reactions [103]. A growing body of research indicates that certain food allergens continue to provoke allergic reactions despite significant. alterations to their three-dimensional structures following gastrointestinal digestion [158]. The prevalence of food allergies (FAs) appears to have risen in recent decades [121]. Furthermore, Sicherer and Sampson [130] reported that the incidence of recorded food allergies increased by up to 10% over four years. Thus, food allergies are a global health concern. The introduction of novel alternative proteins in the agri-food sector may lead to a food safety risk and therefore, a proper risk evaluation of the presence of food allergens in these products has to be conducted [56]. Considering the potentially adverse impacts that may be caused by the presence of food allergens in these novel protein sources. It is important to minimise the presence of allergens present in final products. In order to minimise the risk of allergic reactions in patients caused by an abnormal immune response to certain food allergens, a number of approaches are adopted such as detection of the allergens, risk assessment and reduction of the allergen employed [29, 30, 123, 160]. Commonly, food allergens are proteins or peptides that present a specific region or specific regions formed by several amino acids, also known as epitopes. Epitopes, which are segments typically comprising 8–26 amino acids, are categorized into linear and conformational types [172]. Linear epitopes consist of sequential amino acids, whereas conformational epitopes are formed by amino acids that are non-sequential but spatially close [158]. Both linear and conformational epitopes can be recognized by specific IgE. The allergens’ three-dimensional configuration can be significantly changed by digestion in the gastrointestinal tract, affecting the availability and types of epitopes for IgE binding [158]. However, linear epitopes are deemed crucial for food allergens as they are more adept at maintaining their structure throughout the digestive process compared to conformational epitopes, positioning them as the primary epitopes with the potential to initiate allergic responses [87, 142, 158, 172].

In response to the growing demand for sustainable food options, the exploration of alternative proteins has gained significant attention. In this study, we have narrowed our scope to allergen detection and control methods within alternative protein sources, such as pea, soybean, seaweed and other protein sources. This intentional emphasis arises from our examination of the current literature, which has highlighted gaps and limitations in the development of alternative protein sources. This review seeks to enrich the existing body of knowledge by examining the evolution and current landscape of alternative proteins in the European context, with a particular focus on their impact on the food industry. A critical aspect of this exploration is the identification of allergens originating from these novel protein sources. The review also meticulously scrutinizes potential food allergens found in alternative proteins and evaluates current detection assays used to identify and mitigate allergen presence. Different types of assays, such as analysis based on proteomics, immunoblotting assays and DNA-based assays, are presented with their specific limitations and advantages, crucial for effective labelling and ensuring consumer safety. Additionally, in this review, the use of non-thermal technologies to mitigate the reactivity of food allergens in these new products is proposed. Cold plasma (CP), High Pressure Processing (HPP), Pulsed Electricity Field (PEF), Ultrasound (US) and gamma irradiation technologies are described with particular emphasis on describing the effects and mechanisms of each technology on allergen mitigation. It is therefore hoped that this review could provide valuable information to highlight the importance of alternative proteins in the sustainable food landscape and the critical need to address allergens associated with these sources, contributing some insights to both researchers and industry professionals for the development of safer and more sustainable food products for consumers.

Potential Food Allergens of Alternative Food Proteins

Table 1 summarises the potential main food allergens found in alternative protein sources. Research in the field of insect allergens is relatively undeveloped. A few studies have successfully identified certain allergens, which are documented in the recognized database (http://www.allergen.org/). The identified allergens include such as Pro c 2 (arginine kinase) in red swamp crayfish, Der f 4 (α-amylase ) and Der p 4 (α-amylase ) in dust mite, and Per a 17 (α-tublin), Bla g 12 (chitinase) in cockroach [25, 36, 117]. In plant-based proteins, peas, a member of the legume group, have been considered as an alternative protein source. Pis s 1 (vicilin), Pis s 2 (convicilin), and Pis s 3 (non-specific lipid transfer protein-nsLTP) belonging to legumin-like globulin proteins were identified as the allergens of pea, reported by and accepted as food allergens by the International Union of Immunological Sciences [138]. In a comprehensive study on pea allergens, two sulfur-rich albumin proteins, PA1 and PA2, found in peas, were shown to trigger an immune response [138].

Table 1 Main allergens in alternative protein sources
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When discussing food allergens, it is worth mentioning the phenomenon known as cross-reactivity. The mechanism of cross-reactivity among allergens is complex, potentially stemming from the homologous protein structural similarities, identical amino acid sequences, or even shared common epitopes within the proteins [153]. Thus, an individual can experience a similar allergic reaction to another substance that they were not exposed to before [23]. For instance, cross-reactivity between lupines and peanuts or soybeans is a common phenomenon in individuals allergic to peanuts or soybean that were not previously exposed to lupines [3, 153]. Specifically, the study done by Sirtori et al. [131] revealed a sequence homology of 63% between allergen Ara h 1 (vicilin) in peanuts and β-conglutin precursor from lupins when comparing peptides from reactive regions in lupin peptides.

The use of seaweed as a novel alternative protein is well known. Currently, to the best of the authors’ knowledge scarce evidence reported seaweed as a potential source of allergenic proteins. However, a risk associated with seaweed consumption remains in terms of its ability to induce an allergic reaction. Allergic reactions caused by algae species may be caused indirectly (e.g. as feed for organisms such as shrimps, crustaceans, etc.) [169]. Furthermore, shellfish (crustaceans and molluscs) can be found in seaweed and act as parasites in the seaweed’s natural habitat and seaweed cultivation spaces. Therefore, shellfish allergens are one of the major causes of allergic reactions in seaweeds [17]. For instance, a reported case of an allergic reaction to seaweed was when a patient allergic to Crustacean protein (tropomyosin) consumed a seaweed product contaminated with bycatch [60]. Although studies are limited to seaweed, it has been reported that allergenic proteins were found in the green Ulva species [7, 139]. By comparing potentially allergenic proteins extracted from Ulva spp. in the Allergenic Protein Database, Polikovsky et al. [116] found that the protein responsible for the allergenicity of this protein in Ulva spp. was superoxide dismutase. Few studies reporting allergic reactions to certain algae species are described in the literature. As an example, generalized urticaria with facial angioedema was observed in an individual after the consumption of sushi containing red seaweed (Chondrus crispus and Palmaria palmata) [139]. Another alternative protein source, Spirulina, which belongs to the Cyanobacteria family, is shown to induce allergic rhinitis and dermatitis when aerosol particles invade a patient’s respiratory system [89]. The first case of Spirulina-related anaphylaxis was reported in 2010 when the photosynthetic pigment phycocyanin was discovered to be a potentially allergenic protein [111].

Allergen Detection

The most effective and established method of reducing and controlling the risk of allergic reactions is to detect allergens in foods and food substances and label them accordingly [45]. Deoxyribonucleic acid (DNA) and proteins in food are the principal targets for allergen detection [45, 118]. Direct analysis method targets the specific allergenic proteins, while indirect methods detect the DNA (as markers). Some of the classical assays will be introduced in this review, including the mass spectrometry-based assay, SDS-PAGE Western blotting, Enzyme-Linked Immunosorbents assay (ELISAs) and polymerase chain reaction (PCR) techniques. A comparative summary of the various methods mentioned above is shown in Table 2.

Table 2 Summary of allergen detection methods
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Mass Spectrometry-Based Proteomic Methods

Mass spectrometry (MS) is central to the field of proteomics and the utilisation of proteomic techniques to identify food allergens has given rise to what is referred to as ‘allergenomics’ [4]. In the MS method, samples are ionised and broken down into gaseous ions according to mass-to-charge (m/z) ratios in the protein or peptide molecule, which are then identified by their molecular weight and analysed for their isotopic abundance. Following this, the specific spectral information of extracted or digested proteins or peptides is matched to known protein information in protein databases such as the National Centre for Biotechnology Information [93, 144]. Various software and algorithms are utilized in the protein/peptide analysis process, which includes tools such as MASCOT, SEQUEST, and Andromeda, in addition to commonly used software like MaxQuant, PEAKS Studio, and Protein Prospector [14, 88, 93, 122]. MS-based proteomics enables qualitative and quantitative analysis of allergenic proteins. The workflow of this method is illustrated in Fig. 1.

Fig. 1
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The workflow of MS-based proteomics

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Depending on the level of information required, two alternative types of analytical strategies are used in MS-based assays, namely “bottom-up” analysis and “top-down’’. Specifically, the “top-down” technique is a direct analysis of intact proteins and their fragmentation products at the protein level [99]. On the other hand, when proteins are digested into peptides or polypeptides during digestion, the “bottom-up” technique is typically used to analyse the information provided by these molecules at the peptide level [74, 99]. Shotgun proteomics applies a “bottom-up” strategy [37], which is an undirected approach to protein discovery and identification [35]. Shotgun proteomics most commonly starts with the proteins in the digestion mixture, followed by the separation of the resulting peptides by liquid chromatography [61]. Another commonly used proteomic assay assisting MS-based methods is selected reaction monitoring (SRM) also known as multiple reaction monitoring (MRM) proteomics, which is used to quantify known allergenic proteins [93]. A triple quadrupole mass spectrometer is the standard spectrometer for SRM assay, with ionised proteins or protein fragments separated and filtered via three phases [93]. The stringent selection criteria reduce interference from isobaric precursor ions and improve the performance of SRM in terms of peptide quantification rates and measurement sensitivity [35, 114].

According to Monaci and Visconti [99], the MS-based methods offer greater advantages in the sensitive detection of processed foods that have been heat-treated or chemically preserved, even when food processing causes changes in protein structure [149]. In addition, the MS-based approach enables the quantification of allergens in a wide range of protein matrices [93]. Along with the previously discussed strategies and software that support MS techniques for allergen detection. Data independent acquisition (DIA) has emerged as a bioinformatics-based data processing approach. DIA systematically fragments all precursor ions within specific m/z intervals, enabling the effective detection and analysis of even low-abundance ions. This method ensures a comprehensive peptide scan of potential allergenic proteins, minimizing false negatives and improving the detection and analysis of low-abundance allergens. The capacity of DIA to detect both known and unknown allergens in complex food matrices strengthens allergen detection processes, contributing significantly to food safety applications [14]. Uvackova et al. [146] detected and quantified 15 allergenic protein isoforms and early identification of several peptides in four epitopes of two specific allergenic proteins by using the MS-based techniques and alongside the DIA approach. As a developing method, mass spectrometry also can be performed either in a specific allergen protein or in a matrix of allergen proteins. In addition, it is an effective tool to achieve the goal of quantifying allergens in food not only at the protein level but also at the peptide level [74]. However, MS-based proteomics assays have limitations such as the simultaneous elution of large numbers of different proteins or peptides occurring when a significant number of proteins or proteins elute from LC, and ion suppression occurs [61, 74]. The mass spectrum then shows a superposition of many peaks due to the appearance of many different proteins [74] which lead to less sensitivity. In addition, the limitations of this technology are also reflected in the high cost of the equipment and skilled operators are demanded in the analysis process [93].

Immunoassay

Allergens and antibodies have a strong affinity for one another, and the immunoassay detects allergens by binding antibodies to epitopes of the antigen. Figure 2 illustrates the basic principles of the immunoassay in detecting food allergens. Western blot and enzyme-linked immunosorbent assay (ELISA) are two classic immunoassays for the detection of allergens in food.

Fig. 2
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The basic principle of immunoassays in detecting allergens

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Western Blot

The western blot, also known as the immunoblot, is a classic method for identifying proteins. It is typically used following sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) for food allergy testing [120]. Western blot procedure involves three primary steps: protein separation by SDS-PAGE, transfer of separated proteins onto a membrane, and specific detection through antibody probing. Protein separation was completed by using the SDS-PAGE technique, which is based on the interaction between proteins and SDS. During this, proteins are denatured and uniformly negatively charged by binding to SDS [118, 133], ensuring their separation during electrophoresis is based solely on molecular weight, rather than their intrinsic charge. After separation, proteins are transferred to a membrane with a high affinity for proteins (e.g., nitrocellulose or polyvinylidene difluoride (PVDF)) [118]. SDS-PAGE can be one-dimensional (1D) for separating proteins based on their molecular weight [74, 110], or two-dimensional (2D), which includes preceding steps of separation by isoelectric point for more detailed proteome mapping [74].

The detection of target proteins in the western blot assay involves a series of steps culminating in chromatic changes due to enzymatic reaction. Initially, allergenic proteins separated by SDS-PAGE are transferred from the gel into the membrane and immobilized on the membrane. During antibody probing, specific antigens within these proteins are then targeted by primary antibodies, which bind to them with high specificity [16]. The secondary antibody recognizes and binds to the primary antibodies [69, 77]. Secondary antibodies can be labelled with radioisotopes, enzymes, or fluorophores, with the latter two being the preferred methods for signal generation due to common practice [9, 91]. Enzyme-conjugated secondary antibodies are prevalent, with horseradish peroxidase (HRP) and alkaline phosphatase (AP) being commonly employed due to their efficiency in catalyzing chromogenic or fluorogenic reactions with the substrates [9, 91]. Colour development occurs upon substrate addition triggered by the reaction between the enzyme-labelled antibody and the substrate. This signal, which correlates with the presence and quantity of the target antigen, is then detected and analyzed, often through enhanced chemifluorescence (ECF) methods. It has been therein reported that when the substrate is cleaved by the enzyme, a higher fluorescence end product is formed and a more stable signal (absorbance) occurs, along with a greater linear range of protein detection than the traditional one that does not use ECF [91]. Allergen analysis can then be completed by spectrophotometry. In the end, the analysis of allergens, or other proteins of interest, can be quantitatively assessed using spectrophotometry.

There are some limitations of western blot that must be considered. Firstly, this is a time-consuming assay that requires a lot of time for protein migration and sample preparation [74]. Secondly, the western blot is used for allergen detection at a quantitative or semi-quantitative level, so if the allergens need to be identified, other methods such as MS, are required.

ELISA

Another classic immunoassay for the detection of food allergens is the enzyme-linked immunosorbent assay (ELISA), which detects known allergens and involves specific and enzyme-labelled antibodies [45]. Similar to the western blot, the ELISA process involves enzyme-labelled antibodies binding to specific target antigens. The enzymatic activity of these antibodies, upon interaction with a specific substrate, generates a colourimetric or fluorescent signal, indicating the presence of the antigen [74]. The two epitopes in the antigen are specifically recognised and bound by two types of antibodies, where the primary antibody specifically binds to the allergen and the enzyme-labelled antibody binds to the antibody-antigen (Ab-Ag) complex and reacts with the substrate and forms a coloured product.

There are two types of ELISA for the detection of allergens, based on different purposes, namely the “sandwich” ELISA and competitive ELISA. In a “sandwich” ELISA, a capture polyclonal antibody specific to the target allergen is bound to the solid phase (e.g. multi-well plate) and the capture antibody selectively binds to the food antigen present in the sample, forming an Ag-Ab complex. Subsequently, an enzyme-labelled analyte-specific antibody, which recognizes a different epitope on the antigen, binds to the Ag-Ab complex. The enzyme reacts with the substrate showing a colour change, and the absorbance of the coloured product correlates positively with the allergen concentration [45, 161]. Given that the antigen must offer more than one epitope, the “sandwich” ELISA approach is suited for large molecules of antigens (EFSA Panel on Dietetic Products & Allergies [45], Schubert-Ullrich et al [124]. As the other classic ELISA method, competitive ELISA is used for the detection of small molecules of allergens, where the concentration is indirectly detected (EFSA Panel on Dietetic Products & Allergies [45]). In a competitive ELISA, the reference antigen is immobilised on a plate. The reference antigen and the antigen in the food sample compete to bind to the enzyme-labelled analyte-specific antibody and form the Ag-Ab complex. The enzyme-substrate reaction shows a colour change. When the plate is washed, Ag-Ab formed by the food antigen is washed away, and then the remaining Ag-Ab in the plate is the reference antigen-antibody complex. Therefore, the absorbance of the coloured product is negatively correlated with the allergen concentration when performing the analysis (EFSA Panel on Dietetic Products & Allergies [45], Wu et al [161]. However, one limitation of ELISA, as with all other immunoassay tests, is cross-reactivity [74]. The recognition between antigen and antibody is based on the epitopes rather than the entire antigens. Therefore, the uniqueness of the epitope determines the specific recognition of the antibody [74]. When the antibodies do not fully bind to one antigen but bind to other allergens with that epitope, cross-reactivity can occur, resulting in a false positive test result (EFSA Panel on Dietetic Products & Allergies [45]). In addition, the sensitivity is strongly influenced by changes in the conformation of proteins after food processing [32]. Since the epitope plays a critical role in antibody recognition when food processing such as roasting and boiling alters the structure (quaternary) of the protein, the epitope will shift position (fold or hide) and the ELISA test’s sensitivity will be diminished [74].

Other Detection Assays

Other methods that can be used for the detection of allergens include enzyme-allegro sorbent test (EAST), radioallergosorbent test (RAST), lateral flow devices (LFDs), dip sticks, rocket immune electrophoresis (RIE), dot immunoblots, protein microarrays, and protein biosensors. The principle of EAST and RAST is similar to that of ELISA, albeit the RAST test employs a radioisotope-labelled antibody and the colour products are measured using a gamma counter [74]. As a simplified ELISA test, LFDs and dipsticks employ a similar principle where the Ag-Ab complex is coloured during the test. Dot immunoblotting, on the other hand, is similar to western blot, except that it does not require pre-separation of proteins and is much easier to perform (EFSA Panel on Dietetic Products & Allergies [45, 118]. RIE employs the precipitation and migration of Ag-Ab complexes during electrophoresis. In addition, the graphs displayed during electrophoresis often resemble rockets, hence the name RIE [118]. This method is often used for semi-quantitative analysis. Antibodies are fixed as microarrays identifying and qualifying the specific antigens in the protein microarrays method. In addition, a number of assay combinations and innovations are gradually being discovered, such as ELISA inductively coupled plasma MS [22], DNA microarrays [13], Surface Plasmon Resonance [2], Magneto-Chemical Sensor [85], smartphone-based immunoassays [123]. The validity of the immunoassay will be affected by the processing of the food due to changes in protein structure during processing. Specific recognition and binding between antigens and antibodies will be affected by alteration in protein structure, which will adversely affect the effectiveness of the immunoassay.

Deoxyribonucleic Acid-Based Methods

DNA has a stable structure and is resistant to treatment processes such as heating, chemical reactions and hydrolysis. These properties provide an alternative method for quantifying allergens [66]. As a marker, DNA is a powerful tool for detecting food allergens when the concentration level of an allergen is low within a food sample or when the allergens in the processed food may be altered (EFSA Panel on Dietetic Products & Allergies [45]). Polymerase chain reaction (PCR) is a widely DNA-based detection technique and it involves rapidly amplifying the DNA fragment of the allergenic protein of interest, generating many copies and using the genetic code to identify the suspected allergenic protein in the food [74].

There are two types of PCR amplification of DNA, end-point PCR and real-time PCR (EFSA Panel on Dietetic Products & Allergies [45]). End-point PCR is used to identify the presence of the allergenic DNA fragment, which is a qualitative method. While real-time PCR is used for quantitative assay [62]. In addition, some novel methods based on PCR were developed to detect food allergens. The multiplex PCR method allowed the simultaneous identification of several allergenic DNA fragments [78]. Ultrafast PCR system allowed a shorter time thermal cycle in PCR (20 min, 40 cycles) with the microfluidic chips to detect allergens in food [73].

Although PCR is an alternative assay in cases where immunoassays are not applicable, the use of PCR to amplify DNA sequences does not always reveal the presence of allergenic proteins in the food matrix. This technique focuses on specific DNA fragments encoding amino acids within active peptide regions, rather than analyzing the entire protein. Consequently, it only offers gene-level information and does not directly reflect the structural and functional changes of proteins (EFSA Panel on Dietetic Products & Allergies [45], Mulalapele & Xi [102]. The allergenic potential of proteins depends not only on their amino acid sequences but also on factors such as tertiary structures and post-translational modifications (e.g., folding or glycosylation) [1]. However, PCR is unable to directly detect the structural changes in the protein, which are crucial for understanding protein expression levels and modifications. Moreover, certain issues associated with PCR technology such as non-specific sequence amplification and challenges in amplifying GC-rich DNA sequences, can lead to unsatisfactory results [135]. The presence of small amounts of DNA in allergenic foods can sometimes also result in false-negative outcomes (EFSA Panel on Dietetic Products & Allergies [45]). These challenges underscore the need for caution when relying solely on PCR for allergen detection.

Non-Thermal Mitigation of Allergens

Several studies highlighted the use of food processing technologies as a potential allergen mitigation strategy. In terms of reducing food allergenicity, traditional heat treatments are divided into moist heat (e.g., autoclaving, boiling, frying at 120°C) and dry heat (e.g., microwave, baking, roasting at 150°C) [129]. Heat-induced protein denaturation, changes in the conformational structure of the allergen, particularly in secondary and tertiary structures, the formation and breaking of new molecular bonds and intermolecular aggregation, lead to changes in the reactivity of the allergen [100]. Vanga et al. [150] explained the effect of heat treatment on proteins, where the structure of proteins, especially tertiary structures, was lost when temperatures reached 50–60°C. However, consequently, heat can negatively affect the quality of food products. High temperatures could cause chemical reactions of amino acids and certain components of the food (carbohydrates), leading to a change in colour (e.g., browning) or texture of the food [129]. Even protein allergenicity can occur or be maintained after heat treatment. Heat processing may result in the formation of “neo-allergenic” ingredients or epitopes, in other words, heat processing enhances the allergenicity of certain allergenic foods [24, 39, 92]. According to Davis and Williams [39], one patient with a reported allergy had an allergic reaction after consuming fish that had been cooked but did not experience a reaction after consuming raw fish. The heat-treated protein β-lactoglobulin was found to be reactive to the antibody, whereas the native protein was not. It was inferred that the refolding of conformational epitopes remaining in the denatured protein formed new allergenic epitopes or maintained the original allergenicity. The degradation of secondary and tertiary structures and non-covalent interactions that occur in the protein structure caused by thermal treatment, can affect the allergenicity of the protein by either eliminating or exposing conformational epitopes. This can lead to a decrease or increase in allergenicity [29]. Although it is not clear whether a ‘neo-allergic’ ingredient occurs when proteins are treated with nonthermal techniques, there is great interest in developing nonthermal mitigation techniques. To our current understanding, many food processing methods entail a heat effect. However, to counteract the negative impacts on food quality associated with traditional thermal processing, non-thermal technologies strive to process foods with minimal temperature increase. The heating effect becomes significant during prolonged technology application, whereas shorter processing durations typically prevent heat from interfering with the outcomes of non-thermal processing. In addition to this, given that some allergenic proteins, such as TM in shrimp, caseins in milk, and vicilin in peanuts, are heat-resistant [11, 33, 43, 112], pivoting towards non-thermal technologies represents a growing trend in reducing allergens in food. Nonthermal techniques including pulsed light, HPP, CP, gamma irradiation treatment, US, PEF, fermentation treatment and enzyme hydrolysis treatment, ribonucleic acid (RNA) interference techniques, magnetic beads techniques, were discussed thoroughly by Chizoba Ekezie et al. [29], based on the various applications of the techniques and their impact on allergenicity. According to the literature, techniques, such as CP, HPP, PEF, US and gamma irradiation are promising methods that show huge potential in mitigating allergens in traditional food protein. However, there is a lack of research on the effectiveness of these techniques for mitigating allergens in alternative proteins, such as those from algae, peas, lupine and insect, which have not been investigated.

Considering these challenges, the application of nonthermal treatment in the reduction of allergens in food is a promising area of research. Because of the similarities and cross-reactivity between some allergens and traditional allergens, such as tropomyosin in seaweed can trigger allergic reactions similar to those caused by tropomyosin in seafood such as shrimp, lobster and crustacean seafood [17]. Therefore, studies applying non-thermal techniques to treat traditional allergens can yield valuable insights. These similarities provide valuable perspective on the application of similar techniques to address allergens in foods from alternative protein sources. Table 3 summarises several studies that focus on the effect of different nonthermal processing techniques on food samples and allergens.

Table 3 Summary of studies on food samples and allergens processed by different non-thermal techniques
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Physical processing methods-including heat, pressure, radiation, and ultrasound-primarily influence the allergenicity of food proteins by modifying their secondary and tertiary structures, which can lead to the destruction, masking, or revelation of conformational epitopes. In contrast, linear epitopes are predominantly altered by biochemical interventions, such as fermentation and enzymatic hydrolysis [150]. These alterations at the epitopes have the potential to either attenuate or amplify allergenicity [119]. Table 4 gives an overview of the mechanism and limits of those techniques in mitigating allergens of alternative proteins. Figure 3 provides a comprehensive depiction of the hypothesized impact of non-thermal processing technologies, including CP, HPP, US, and Gamma Radiation, on the modification of protein allergenic properties.

Table 4 Summary of nonthermal allergen mitigation techniques
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Fig. 3
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(adapted from Huang et al. [64], Pan et al. [108], Sengar et al. [125], Zhang et al. [167];)

Mechanistic Insights into Allergen Reduction: Comparative Analysis of Non-Thermal Techniques - Cold Plasma, High-Pressure Processing, Ultrasound, and Gamma Radiation.

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Cold Plasma

Plasma is the fourth state of matter and is produced by the ionization of gas at ambient or atmospheric temperature. CP has been employed in the food industry as a decontamination method to target microbes, serving as an innovative approach in food preservation technologies [44, 46]. Due to the capability of CP to alter protein structure, it has been recently investigated for its potential to reduce allergenicity in foods. The exact mechanism of CP is not well understood, one of the most prevalent hypotheses is that the reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated by the plasma gas react with antigens and alter their protein structure or binding sites between antigen and antibodies [29, 38, 132, 141, 152]. The effectiveness of CP treatment usually depends on the duration and temperature of the treatment [104]. When combined with time [26], temperature, and glycation [157], as hurdle technologies in food processing, cold plasma (CP) shows enhanced performance in reducing allergenicity. According to the effect of plasma on mitigating the allergens from alternative protein sources, numerous research studies have speculated on diverse mechanisms. Figure 3 outlines the potential pathways through which plasma acts to mitigate the effects of food allergens.

In a study investigating the application of CP to treat soybean allergens, reduced immunoreactivity of allergens after CP treatment was speculated to be due to the oxidation of amino acids in proteins by ROS, leading to protein fragmentation and disruption of antibody binding sites [96]. Similarly, while studying the effect of CP on dried defatted peanut flour and whole peanut, Venkataratnam et al. [152] also observed decreased antigenicity of Ara h 1 in CP-treated peanuts. According to the further detection of protein structure by using the circular dichroism, it was reported that the secondary structure showed alterations and concluded that the alteration of the antigen was due to the ROS and RNS. As previously mentioned, tropomyosin, an allergenic protein present in seaweed proteins, is exposed in post-harvest environments and adheres to seaweed surfaces. Despite the lack of specific research on treating this allergen in seaweed proteins, insights can be gleaned from studies on CP processing of tropomyosin in other seafood, offering theoretical support for potential applications in reducing the allergenicity of seaweed-derived proteins. For instance, Ekezie et al. [47] investigated the effect of cold argon plasma treatment on the binding capacity of King Prawn tropomyosin, observing a decrease in the recognition capability for both immunoglobulin E (17.6% reduction) and immunoglobulin G (IgG) (26.87% reduction) post- treatment. It is concluded from that study, which encompassed the examination of the primary structure of the protein and the scond and tertiary structure, that the reduced antibody binding to tropomyosin was due to oxidative modifications by ROS impacting both the linear and conformational epitopes.

CP offers the advantages of being ecologically sustainable, and no significant loss and changes in colours and texture [51, 109]. However, there are some limitations regarding this technology. Firstly, the investment in a CP system is a significant cost compared to other heat treatments such as boiling and roasting. Secondly, as the mechanisms of CP allergen reduction are not fully understood, the application of this technology should be carefully studied. Further investigations are required to elucidate this. Finally, according to Boehm et al. [19, 52], possible cytotoxic activity associated with CP-treated fluids should be taken into account when conducting relevant studies. Therefore, more investigations on toxicity will be needed in the future to secure this technology.

High-Pressure Processing

HPP, also known as high hydrostatic processing, is a non-thermal processing technology applied in the food industry. High-Pressure Processing (HPP) has been demonstrated to effectively deactivate spoilage-causing microorganisms and enzymes, thus its potential for reducing allergenicity in food proteins is being rigorously evaluated [8, 27]. HPP treatment is effective in reducing the allergenicity of proteins while maintaining the sensory quality of food samples [72]. High pressure affects the secondary structure of proteins, by damaging the non-covalent bonds such as high-pressure breaks the hydrogen bonds [63, 64, 97]. Studies have shown that high pressure induced the formation of multimers, leading to the burial of immune active sites (conformational epitopes), which is a possible mechanism for reducing allergenicity [107]. It was reported that high pressure was effective in extracting or releasing allergens from the membrane, thus facilitating the enzymatic hydrolysis of these molecules [72].

Many studies have been conducted to investigate the relationship between changes in protein structure and the reduction in protein allergenicity caused by high pressure. One of the prominent areas of research involving the application of HPP in the development of safe food products from alternative protein sources focuses on soybean protein isolation (SPI), particularly in infant milk powder formulations. Li et al. [81] conducted a study demonstrating that HPP significantly reduced the allergenicity of SPI by 45.5% at 300 MPa for 15 min, suggesting its potential incorporation into infant milk formulas. Further mass spectrometry analysis revealed that HPP treatment led to changes in the subunits of 7 S globulin and 11 S globulin, resulting in alterations in allergenicity profiles. In another study, the allergenicity of tropomyosin was effectively reduced after shrimps were treated with high pressure, especially when the pressure was increased from 100 to 500 MPa [89]. Zhang et al. [168] studied the structural changes of squid hemocyanin (Hc) after HPP treatment and the reduction in the content of α-helix indicated that the secondary structure of squid Hc changed after HPP treatment. The comparison of control and treated samples, particularly the amount of α-helix and random coils in the samples confirmed that HPP weakened the binding capacity of IgE and IgG. However, there were no significant differences between the HPP-treated samples and the control in terms of the tertiary structure according to the indicators (surface hydrophobicity index and free sulfhydryl content). Pan et al. [107] found that HPP significantly reduced the immunoreactivity of Ara h 1 in treatments at 400 MPa and 600 MPa. Furthermore, an increase in particle size was also found in the 400 MPa treated Ara h1. It was therein confirmed that the secondary structure changed when 400 MPa acted on Ara h1, suggesting that the effect of HPP treatment on allergenicity was associated with changes in secondary structure. Based on the results of fast protein liquid chromatography or molecular weight analysis and particle size distribution for particle size analysis, it was discovered that when allergens were treated beyond 400 MPa, unfolded proteins aggregated and became multimers, resulting in a reduction in allergenicity. It was elaborated that such alteration in the protein results in the concealment of the immunological active site leading to the allergens not being identified. However, in some studies, the effect of HPP processing does not have an effect on reducing allergenicity. Lavilla et al. [79] reported the effect of high pressure on the peach allergen protein caused it to unfold, exposing the epitope and making it more readily recognisable thus increasing the potential of the major allergen in peach to elicit an allergic reaction. Given the varying performances in reducing the allergenicity of the protein, Zhou et al. [173] hypothesised that the two opposite effects on antigens caused by HPP treatment were related to the linear epitopes of antigens. It has been noted that linear epitope-dominated antigens did not perform well in HPP processing. Moreover, the variability in the effects of HPP in reducing allergenicity may be caused by the detection assays used in the research [80]. Therefore, futher studies are required to investigate the effects and mechanisms of HPP work on allergens to better apply the technique.

As mentioned previously, HPP may not may not consistently reduce the allergenicity of protein antigens, particularly those with linear epitopes. Studies have shown that the allergenicity of specific allergens was reduced through the synergistic application of HPP with other factors, such as temperature and proteolysis [46, 75, 79, 80, 89]. This suggests that the hurdle approach, which combines high pressure with additional methods could potentially offer a more robust solution for reducing allergenicity across a wider spectrum of food allergens.

Pulsed Electric Field (PEF)

PEF applies high voltage pulses (10–80 kV cm− 1) over a very short period of time (millisecond or microsecond) to food for various processing applications, resulting in the disruption of cellular structures [54, 126]. In the food industry, PEF is widely applied in the food industry and has been proven to significantly enhance meat quality, such as improving tenderness and extending the shelf-life of foods [42, 54]. Recent research has discovered that PEF shows great potential to reduce the immune activity of allergens [140, 163]. Although the exact mechanism of PEF on allergens has not been clearly elucidated, there is evidence to suggest that PEF processing may induce changes in protein structure, including alterations in hydrogen bonds and hydrophobic interactions within the secondary and tertiary structures, potentially affecting the concealment or exposure of conformational epitopes [83, 165]. Wei et al. [159] reported that the linear unfolding of the secondary structure of the ovalbumin protein molecule increased with increasing pulse intensity (ranging from 27 to 53 kV cm− 1) and treatment time (ranging from 720 to 4320 µs).

Yang et al. [163] showed a loss of immunity to Ovalbumin antigens (OVA) when OVA was isolated and purified from fresh eggs and treated with PEF at 35 kV cm− 1 and 180 µs. The conformational structure of OVA was altered after treatment with PEF, which was confirmed when analysing the OVA samples using circular dichroism, UV absorption and fluorescence spectroscopy. Moreover, they implied that the binding of IgG to OVA or IgE to OVA was influenced by these structural changes. A similar result was obtained by Li et al. [84] when treating one of the main types of allergens (ovomucin) in egg whites with PEF. PEF was reported to reduce the ability of IgG and IgE to ovomucin at 30 kV cm− 1. Structural changes in the treated proteins were also confirmed with the help of scanning electron microscopy, Fourier transform infrared (FTIR) and CD spectroscopy, SDS-PAGE methods, and analysis of changes in surface hydrophobicity and total sulfhydryl content. Thus a correlation between the spatial conformation and the ability of IgE and IgG to bind to ovalmucin was indicated therein.

Same to HPP, PEF can modify the proteins in food products while offering the advantage of being non-destructive. However, as few studies have been conducted on the treatment of foods with PEF, its mechanisms have not been clearly described. In this way, there is limited scientific guidance on the use of PEF to mitigate allergens in foods for operational purposes, limiting its application in food allergen mitigation. In addition, PEF technology can be effective independently for various food processing applications, such as microbial inactivation and improving textural properties. The efficacy of the PEF in certain areas, like reducing allergenicity or enhancing protein digestibility, can be significantly enhanced when combined with other methods like proteolysis [82]. This synergistic approach, known as hurdle technology, allows for wider applications in food processing but also adds complexity to the implementation process. Therefore, more efforts should be made to carry out research on PEF in food allergen mitigation, especially focusing on understanding the underlying mechanisms.

Ultrasound

Ultrasound applies power ultrasonic waves with 20–100 kHz to a sample, this energy generates a gradient of pressure and temperature within a matrix, inducing physical changes and stimulating chemical reactions [126]. It has been widely used as a non-thermal approach to keep fruits and vegetables fresh by sterilizing the microbial inside [68], extraction [76], oxidation [86] and homogenization [136]. The exploration of US technology in food preservation has prompted a closer examination of its potential for addressing other concerns within the food industry. US has been observed to induce modifications in the structure of proteins, including allergens, while simultaneously extending the shelf life of food products. As shown in Fig. 3, the possible mechanism of US is the cavitation effect that US treatment has on the food sample, altering the structure of the allergen protein [128]. The energy released during the cavitation process propagates through the ultrasonic medium, generating active free radicals, particularly hydroxyl radicals (OH); these factors cause damage to the peptide bonds, leading to changes in protein structure that may reduce allergenicity [29, 31, 126, 170]. Yang et al. [162] investigated the effect of ultrasound on soybean sprouts in which the samples were subjected to different ultrasonic conditions at 100, 200 and 300 W with a holding time of 30 min. According to the study, the IgE binding capacity of the US- treated sample considerably decreased. It was claimed that the degradation of the allergen to a number of peptides and amino acids after US treatment allowed the epitopes of the allergen to be destroyed. Wang et al. [156] studied casein (CN), an allergenic protein in cow’s milk. Treatment of fresh milk and purified CN in the US revealed a decrease in the binding capacity of IgE to CN, indicating that the US mitigated the IgE binding capacity of CN in milk. Despite CN being characterized by its linear peptide chains, which typically indicate the presence of linear epitopes, the study observed only a slight reduction in the IgE-binding capacity of CN. This suggests that while CN may predominantly contain linear epitopes, the ultrasound treatment might also impact the secondary or tertiary structures of protein to some extent, thereby affecting conformational epitopes. The result of SDS-PAGE and transmission Electron Microscopy (TEM) analyses confirmed that the reported reduction in allergenicity was significantly related to the alteration of conformational epitopes in CN. Another study conducted by X. Dong et al. [43] on the effect of US treatment on allergenic proteins in shrimp, showed that ultrasound treatment is a potentially effective method to mitigate allergens in shrimp. In this study, analysis of changes in tropomyosin (TM) content obtained by ELISA and changes in the banding intensity obtained by SDS-PAGE showed that shrimp allergenicity decreased with increasing duration of US treatment. FTIR analysis revealed alterations in secondary structures post-treatment, with a rise in β-sheets and α-helices alongside a declines in turns and unordered structures. Multiple studies have observed a correlative phenomenon where ultrasound treatment is linked to changes in protein secondary and tertiary structures, potentially reducing allergenicity by modifying conformational epitopes.

Compared to conventional treatment of allergens, the US is environmentally friendly, energy-efficient and inexpensive [29]. However, studies by Gallo et al. [50, 166] concluded that there were some negative effects on food quality, particularly on colour, flavour and even nutritional value, when food was treated using US at high power levels for a long time. Therefore, future studies should aim to optimize US parameters, such as intensity, duration, and frequency, to mitigate allergens effectively while preserving the sensory and nutritional attributes of food products.

Gamma Irradiation

Food irradiation has been recognized for its role in pasteurizing microbial (Pi, Yang, et al [113]), and gamma irradiation was found to be an effective non-thermal technology used to decrease the allergenicity of food products [108]. Gamma irradiation is a form of ionising radiation and the process of food irradiation treatment involves the irradiation of food or raw materials by highly energetic and penetrating rays [108, 115, 127]. Gamma irradiation has a dual mechanism in mitigating allergens. The first one involved the direct impact of gamma photons on proteins, with the absorbed energy inducing structure changes by breaking covalent bonds, leading to changes in linear and conformational epitopes [108, 151, 171]. Another mechanism is that after radiation, free radicals produced from water molecules react with amino acids, causing decarboxylation, reduction and oxidation of sulfhydryl groups, further modifying the protein structure [29, 108, 151]. The mechanism of gamma radiation’s effect on allergens is depicted in Fig. 3, including the modification in either linear or conformational epitopes. Van der Spiegel et al. [147] established that the changes in the protein molecular were irreversible in the gamma-irradiated allergens.

The application and mechanism of Gamma irradiation application on food allergens have been investigated for a long time. Zhenxing et al. [171] confirmed that gamma irradiation was potentially beneficial in reducing the allergenicity of shrimps, especially in processing shrimp extract with optimal functionality. It was found that the allergenicity of shrimp extract decreased with increasing irradiation dose, while the allergenicity of shrimp muscle decreased with increasing irradiation up to more than 10 kGy of radiation treatment. The results have been therein interpreted that the different effects of irradiation on the samples might be related to the radiolysis of water. There are other components of shrimp muscle, such as lipids, that protect proteins from being catalysed by free radicals formed by small doses of irradiation. The interpretation was supported by a study conducted by Muanghorn et al. [101], which focused on the effects of gamma irradiation on the allergenicity of freshwater prawns. An increase in protein band density was shown on immunoblot images at lower irradiation levels (5–7 kDa), suggesting that lipids might protect proteins from ROS attack, potentially leading to increased protein allergenicity. Moreover, the study demonstrated the gamma irradiation only at irradiation levels of 15 kGy showed the best effect on the food samples, with a notable decrease in tropomyosin band density. Another study conducted by Meinlschmidt et al. [96] who applied gamma irradiation to soy protein isolates found that gamma irradiation was effective with a complete reduction in Gly m6 when the applied dose exceeded 100 kGy.

Gamma Irradiation offers an option for reducing allergens in food without causing sensory damage to the food. However, some limitations also accompany the advantages of gamma radiation. Firstly, the most effective gamma radiation for handling food allergens is 25 kGy or more, however, the FDA set a safe radiation level for food use of only 10 kGy in 2004 [49]. Secondly, there is a list of food and food ingredients that can be treated with ionising radiation according to the regulation of the European Union (EU, [48]). However, most of the food or food ingredients in the list are traditional foods, like chicken meat, fish, milk, eggs, onions, garlic, dried fruit, and rice. As alternative protein foods including seaweed and insects are novel foods there may be some legislative issues to overcome. Finally, key consumers are sceptical of residues in irradiated food, leading to lower customer acceptance [127]. In addition, there were currently insufficient substantial studies to assess the relationship between irradiation dose and allergenic effects [108, 119]. Therefore, in order to ensure the safe and effective development and dissemination of irradiation treatment in food allergens, and to optimise gamma treatments for food allergenicity reduction, more detailed and targeted radiation studies (e.g., the range of effective radiation doses required for specific food allergens) should be investigated in depth in the future.

Discussion on the Impact of Protein Physicochemical Properties on Allergen Modification

The allergenic potential of a protein, or the ability to elicit allergic reactions, is modulated by multiple attributes such as its solubility, stability to digestion, molecular size, and conformational structure [1, 57, 94]. Research above has shown that food processing can significantly reduce the risk of food allergies by altering these protein attributes, thereby modifying their allergenic properties. Structural changes in protein are critical for both conformational and linear epitopes, which play a crucial role in the exposure or concealment of immune-reactive sites [119]. In addition, the physicochemical properties of proteins, including solubility and digestibility, can affect their interactions with the immune reaction due to modification of proteins during processing [126]. For food allergens, the solubility and stability of the protein to be digested are critical, as these factors can impact the allergenicity of the protein and sometimes the assessment of allergens in food [119]. This section focuses on discussing how changes in solubility and digestibility lead to varying outcomes in protein allergenicity.

Solubility

Numerous studies indicate a potential link between solubility and protein allergenicity [15, 20, 21]. A common hypothesis is that the aggregation and denaturation of protein post-treatment, due to changes such as the masking or unveiling of target epitopes, affect allergenicity. However, the solubility-allergenicity relationship is complicated by the unpredictable alterations in epitopes within the protein structure. Generally, processing leads to protein molecule aggregation, concealing certain conformational epitopes and thus, potentially decreasing allergenicity in tests [95]. For instance, heating processing induces the formation of insoluble aggregates of the allergens Ara h 1 and Ara h 6 in peanuts, which impedes antibody recognition by concealing native epitopes, thus resulting in a diminished allergenicity assessment [15, 90]. Recent research has further confirmed the relationship between protein solubility and its allergenic potential. Nugraha et al. [105] investigated the effects of extraction buffers on the solubility and immunoreactivity of Pacific oyster allergens, finding that buffers with high salt and pH levels enhanced the protein solubility, which subsequently resulted in increased IgE reactivity. In the study by Kasera et al. [71], the effects of thermal processing and gamma irradiation on the allergenicity and solubility of proteins from three leguminous plants (peanut, black gram and kidney bean) were investigated. It was found that thermal processing led to a decrease in the solubility of proteins from all three legumes, accompanied by a reduction in their IgE binding capacity. An interesting result was observed by Kasera et al. [71] when they verified whether the decrease in allergenicity was due to the aggregation of allergenic proteins into insoluble fractions during thermal processing. Immunological analysis was conducted on the insoluble protein fractions of the three legumes. The findings revealed that only in the case of kidney beans, there was no significant decrease in IgE binding capacity. Thermal treatment reduced the solubility of kidney bean proteins, causing them to aggregate and form less detectable insoluble aggregates in immunological assays. However, different result obtained from similar tests on peanuts and black gram which is the IgE binding capacity reduced significantly in the insoluable fraction of the samples.The discrioency between two results indicating that there is no comfirmed correlation between the protein aggregation, solubility and allergenicity. Therefore, a decrease in solubility does not consistently result in lower allergenicity, suggesting that the impact varies among different allergens. There is another research which applied the ultrasound and pressure homogenizer to treat the CN in milk, found that the solubility increased while the allergenicity reduced after the treatment [58]. Further review on aggregation and sensitization within protein families suggests that protein aggregation can enhance the allergenic potential of 2 S albumins. In contrast, for legume proteins and cereal prolamins, similar aggregative processes may lead to a reduction in allergenicity [33]. The observed correlation between solubility and allergenicity post-treatment indicates that solubility might act as a preliminary indicator for allergenic alterations. Nevertheless, a comprehensive understanding emerges only when solubility assessments are integrated with examinations of protein structure and epitope mappings, thereby elucidating the processing-induced modifications in allergenic properties.

Digestibility

In the context of food allergen research, the impact of food processing on protein digestibility and allergenic potential is complex and multifaceted. Digestibility encompasses not only the ability of the protein to be cleaved into smaller molecules, such as small peptides and single amino acids, but also the extent to which these breakdown products are absorbed by the human body. Digestibility is a crucial factor influencing the allergenic potential of proteins. However, when proteins are digested, their epitopes may be exposed, hidden, or even degraded [174]. This change in epitope unpredictability directly affects the protein’s ability to bind to antibodies, a key factor in triggering allergic reactions. The correlation between digestibility and the allergenicity is complex.

Highly digestible proteins generally have their epitopes disintegrated more easily, reducing their potential to cause allergies [134]. Conversely, proteins that resist digestion can maintain their structure, including epitopes, and may cross the intestinal barrier to elicit an immune response, heightening allergy risks [106, 119]. For instance, in the study conducted by Jin et al. [70], high-pressure processing high-pressure processing led to the unfolding of the protein structure of the squid allergen Tod p1. This structural change not only disrupted the conformational epitopes, thereby reducing its capacity to bind IgE antibodies and consequently its allergenicity but also exposed new cleavage sites for digestive enzymes, enhancing its digestibility. However, there are some cases of increased allergy due to increased digestibility. During the digestion of autoclaved peanuts, some proteins are efficiently processed, improving overall digestibility. However, particular allergenic fragments, especially those near 42 kDa, persist or are more evident. This suggests that despite the general increase in digestibility, the allergenic potential of some components persists [6, 10, 41].

The digestibility and allergenic potential of food proteins can undergo simultaneous changes due to modifications in the protein structure. However, the unpredictable digestion patterns of epitopes in allergenic proteins make it challenging to determine the allergenicity level of a processed protein solely based on its digestibility. Future research should focus on elucidating these complex interactions, potentially through in vivo studies, to better predict the allergenicity of processed food proteins [33, 70].

Conclusion

This review highlights advancements in the study of alternative proteins and their allergenic properties, focusing on innovative allergen detection methods such as MS-based proteomics and ELISA, alongside the emerging role of non-thermal processing techniques like CP, US, HPP, PEF and gamma irradiation in allergen mitigation. These innovations hold the potential for diminishing allergenicity, though challenges remain, especially in comprehensively understanding the impact on allergenic epitopes. Current research primarily on allergen processing mechanisms predominantly addresses changes in conformational epitopes. However, linear epitopes, as the most prevalence epitopes in food protein, require more focused research. on allergen mitigation. Integrating hurdle technology could address these challenges, enhancing both allergen modification and microbial safety. Furthermore, the translation of reductions in allergenicity from in vitro studies to real-world outcomes remains uncertain, largely due to variability in protein digestibility. These challenges underscore the necessity for more in-depth research on processing effects on epitope modifications and their implications for allergenicity.

As the field moves forward, the need for in-depth research becomes evident, particularly studies that bridge the gap between in vitro allergenicity assessments and actual clinical outcomes in humans. Future explorations should leverage hurdle technology to devise safer, hypoallergenic food solutions, taking into account both conformational and linear epitope modifications to achieve effective allergen management. This comprehensive strategy is crucial for advancing our understanding and capability to mitigate allergenicity in alternative protein sources.