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The underlying cause of differences in sensitization between bovine and caprine milk β-lactoglobulin (β-LG) remains unclear. In this study, denatured forms of bovine and caprine milk β-LG were obtained through reductive alkylation and evaluated for allergenicity and digestibility in Balb/c mice. Results indicated weaker sensitization to nondenatured caprine milk β-LG compared to nondenatured bovine milk β-LG, with no significant difference in sensitization observed between denatured β-LG from both sources. The nondenatured β-LG of caprine milk and two types of denatured β-LG were degraded more rapidly than nondenatured bovine milk β-LG in the small intestine of mice. In terms of undenatured proteins, mouse intestinal tissues absorbed more bovine milk β-LG than caprine milk β-LG. Overall, structural disparities in β-LG between bovine and caprine milk resulted in varying digestion rates. Moreover, the slower-degraded bovine milk β-LG and its enzymatic fragments facilitated easier absorption by the intestine, disrupting the Th1/Th2 balance and increasing susceptibility to severe allergic reactions in mice.

Allergic incidents of crustacean aquatic products occur frequently, and tropomyosin (TM) is the main allergen. Therefore, it is worthwhile to develop technologies to efficiently reduce the allergenicity of TM. In this study, ultrasound-assisted cold plasma (UCP) treatment was used to regulate shrimp allergy. The remarkable changes in TM structure were substantiated by alteration in secondary structure, reduction in sulfhydryl content, change in surface hydrophobicity, and disparity in surface morphology. The IgE and IgG binding ability of TM significantly decreased by 52.40% and 46.51% due to UCP treatment. In the Balb/c mouse model, mice in the UCP group showed most prominent mitigation of allergic symptoms, proved by lower allergy score, changes in levels of TM-specific antibodies, and restoration of Th1/Th2 cytokine imbalance. Using a proteomics approach, 439 differentially expressed proteins (DEPs) in the TM group (vs phosphate-buffered saline group) and 170 DEPs in the UCP group (vs TM group) were determined. Subsequent analysis demonstrated that Col6a5, Col6a6, and Epx were potential biomarkers of TM allergy. Moreover, Col6a5, Col6a6, Dcn, and Kng1 might be the target proteins of UCP treatment, while PI3K/Akt/mTOR might be the regulated signaling pathway. These findings proved that UCP treatment has great potential in reducing TM allergenicity and provide new insights into the development of hypoallergenic shrimp products.

β-Enolase is a cross-allergen commonly found in fungi, plants, and aquatic products. Although studies on the allergenicity of fish enolase have been reported in recent years, they are still limited to a few species of marine fish. Therefore, the detection of freshwater fish in the food industry requires more studies of the molecular characterization as well as the allergenicity of enolase. In this study, the nucleotide sequence of β-enolase from grass carp was obtained by molecular cloning technology. Structural domain analysis showed that it contained the characteristic structural domains of the enolase superfamily, and homology analysis indicated that enolases are highly conserved evolutionarily. Recombinant β-enolase was obtained by prokaryotic expression, and its allergenicity was assessed by β-enolase-sensitized mice, which confirmed the ability of β-enolase to trigger an allergic response and cause a rise in Th1 and Th2 immune responses in mice. These results suggest that β-enolase could be used as a characterizing substance for the detection of fish allergens in the food industry as well as the preparation of drugs for allergy-related studies.

Thermally processed foods are essential in the human diet, and their induced allergic reactions are also very common, seriously affecting human health. This review covers the effects of thermal processing on food allergenicity, involving boiling, water/oil bath heating, roasting, autoclaving, steaming, frying, microwave heating, ohmic heating, infrared heating, and radio frequency heating. It was found that thermal processing decreased the protein electrophoretic band intensity (except for infrared heating and radio frequency heating) responsible for destruction of linear epitopes and changed the protein structure responsible for the masking of linear/conformational epitopes or the destruction of conformational epitopes, thus decreasing food allergenicity. The outcome was related to thermal processing (e.g., temperature, time) and food (e.g., types, pH) condition. Of note, as for conventional thermal processing, it is necessary to control the generation of the advanced glycation end products in roasting/baking and frying, and the increase of structural flexibility in boiling and water/oil bath heating, autoclaving, and steaming must be controlled; otherwise, it might increase food allergenicity. As for novel thermal processing, the temperature nonuniformity of microwave and radio frequency heating, low penetration of infrared heating, and unwanted metal ion production of ohmic heating must be considered; otherwise, it might be the nonuniformity and low effect of allergenicity reduction and safety problems.

Tropomyosin (TM) is the main allergen in shrimp ( Litopenaeus vannamei ). In this study, the effects of allergenicity and structure of TM by glycosylation (GOS-TM), phosphate treatment (SP-TM), and glycosylation combined with phosphate treatment (GOS-SP-TM) were investigated. Compared to GOS-TM and SP-TM, the IgG/IgE binding capacity of GOS-SP-TM was significantly decreased with 63.9 ± 2.0 and 49.7 ± 2.7%, respectively. Meanwhile, the α-helix content reduced, surface hydrophobicity increased, and 10 specific amino acids (K 30 , K 38 , S 39 , K 48 , K 66 , K 74 , K 128 , K 161 , S 210 , and K 251 ) were modified by glycosylation on six IgE linear epitopes of GOS-SP-TM. In the BALB/c mice allergy model, GOS-SP-TM could significantly reduce the levels of specific IgE, IgG1, and CD4 + IL-4 + , while the levels of IgG2a, CD4 + CD25 + Foxp3 + , and CD4 + IFN-γ + were increased, which equilibrated Th1 and Th2 cells, thus alleviating allergic symptoms. These results indicated that glycosylation combined with phosphate treatment can provide a new insight into developing hypoallergenic shrimp food.

β-Lactoglobulin (βLG) is a major allergen in bovine milk protein. This study was designed to investigate changes in βLG structure, digestibility, and allergenicity induced by covalent binding modification with different contents of (-)-epigallocatechin 3-gallate (EGCG). The reaction of EGCG conjugation with βLG reached saturation at a molar ratio of 1:60 βLG:EGCG. Conjugation with EGCG altered the βLG structure, decreased IgE-binding capacity, and increased digestibility in a dose-dependent manner. In vivo studies showed that covalent conjugation with EGCG can reduce βLG-induced allergic symptoms with reducing levels of IgE, histamine, and mast cell protease-1 (mMCP-1) and the percentage of sensitized mast cells. Allergenicity was reduced more effectively in saturated βLG-EGCG conjugates compared to semisaturated conjugates. Observed changes in IFN-γ, IL-4, IL-5, IL-10, and TGF-β levels suggested that βLG-EGCG conjugates were able to promote Th1/Th2 immune balance. These findings further our understanding of the relationship between the degree of polyphenol conjugation and the allergenicity of food allergens.

Food allergies are a main public health disease in the world. Ultrasound is an environmentally friendly technology that typically leads to protein unfolding and loss of protein structure, which means it has the potential to be combined with other technologies to achieve a great reduction of allergenicity in foods. This review concludes the effects of the combined ultrasound with other technologies on food allergenicity from three combinations: ultrasound before other technologies, ultrasound under other technologies, and ultrasound after other technologies. Each combination affects food allergenicity through different mechanisms: (1) as for ultrasound before other technologies, ultrasound pretreatment can unfold and lose the protein structure to improve the accessibility of other technologies to epitopes; (2) as for ultrasound under other technologies, ultrasound can continuously affect the accessibility of other technologies to epitopes; (3) as for ultrasound after other technologies, ultrasound further induces structural changes to mask and disrupt the epitopes. The reduction of allergenicity is related to the ultrasound/other technologies conditions and food types/cultivars, etc. The comparison of ultrasound before, under, and after other technologies to decrease food allergenicity should be further investigated in the future. The combination of ultrasound with other technologies is promising to produce hypoallergenic foods.

As a key component of cell-cultured fish, fish skin gelatin (FSG)-based cell scaffold provides support structures for cell growth, proliferation, and differentiation. However, there are potential allergenicity risks contained in FSG-based scaffolds. In this study, 3D edible scaffolds were prepared by phase separation method and showed a contact angle of less than 90°, which indicated that the scaffolds were favorable for cell adhesion. Besides, the swelling ratio was greater than 200%, implying a great potential to support cell growth. The sequence homology analysis indicated that FSG was prone to cross-reaction with collagen analogues. Additionally, a food allergic model was constructed and represented that mice gavaged with cod FSG exhibited higher levels of specific antibodies, mast cell degranulation, vascular permeability, and intestinal barrier impairment than those gavaged with pangasius and tilapias FSG. Its higher allergenicity might be attributed to a higher number of digestion-resistant linear epitopes. Moreover, the higher hydrolysis degree linked to the exposure of linear epitopes to promote the combination with IgE, which was also responsible for maintaining the higher allergenicity of cod FSG. This study clarifies allergenic risks in cell-cultured fish and further study will focus on the allergenicity reduction of FSG-based cell scaffolds.

Scy p 8 (triosephosphate isomerase) as a crab allergen in inducing distinct T-helper (Th) cell differentiation and a linear epitope associated with allergenicity remain elusive. In this study, mice sensitized with Scy p 8 exhibited significantly upregulated levels of IgE, IgG1, and IL-4 release, inducing a Th2 immune response. Moreover, the release of IFN-γ (Th1) and the levels of Treg cells were downregulated, while IL-17A (Th17) was upregulated, indicating that Scy p 8 disrupted the Th1/Th2 balance and Th17/Treg balance in mice. Furthermore, bioinformatics prediction and serum samples from crab-allergic patients and mice enabled the discovery of 8 linear epitopes of Scy p 8. Meanwhile, the analysis of peptide similarity and tertiary superposition revealed that 8 epitopes of Scy p 8 exhibited conservation across various species, potentially resulting in cross-reactivity. These findings possess the potential to enhance the comprehension of crab allergens, thereby establishing a foundation for investigating cross-reactivity.

Plant-based peptides (PBPs) benefit functional food development and environmental sustainability. Proteolysis remains the primary method of peptide production because it is a mild and nontoxic technique. However, potential safety concerns still emanate from toxic or allergenic sequences, amino acid racemization, iso-peptide bond formation, Maillard reaction, dose usage, and frequency. The main aim of this review is to investigate the techno-functions of PBPs in food matrices, as well as their safety concerns. The distinctive characteristics of PBPs exhibit their techno-functions for improving food quality and functionality by contributing to several crucial food formulations and processing. The techno-functions of PBPs include solubility, hydrophobicity, bitterness, foaming, oil-binding, and water-holding capacities, which subsequently affect food matrices. The safety and quality of foodstuff containing PBPs depend on the proper source of plant proteins, the selection of processing approaches, and compliance with legal regulations for allergen labeling and safety evaluations. The safety concerns in allergenicity and toxicity were discussed. The conclusion is that food technologists must apply safe limits and consider potential allergenic components generated during the development of food products with PBPs. Therefore, functional food products containing PBPs can be a promising strategy to provide consumers with wholesome health benefits.

This study found that, after microwave treatment at 560 W for 30 s, alkaline protease enzymolysis significantly reduced the allergenicity of ovalbumin (OVA). Furthermore, specific adsorption of allergenic anti-enzyme hydrolyzed peptides in the enzymatic products by immunoglobulin G (IgG) bound to magnetic bead further decreased the allergenicity of OVA. The results indicated that microwave treatment disrupts the structure of OVA, increasing the accessibility of OVA to the alkaline protease. A comparison between 17 IgG-binding epitopes identified through high-performance liquid chromatography-higher energy collisional dissociation-tandem mass spectrometry and previously reported immunoglobulin E (IgE)-binding epitopes revealed a complete overlap in binding epitopes at amino acids (AA)125-135, AA151-158, AA357-366, and AA373-381. Additionally, partial overlap was observed at positions AA41-59, AA243-252, and AA320-340. Consequently, these binding epitopes were likely pivotal in eliciting the allergic reaction to OVA, warranting specific attention in future studies. In conclusion, microwave-assisted enzymolysis synergized with magnetic bead adsorption provides an effective method to reduce the allergenicity of OVA.

Allergenicity of soybean 7S protein (7S) troubles many people around the world. However, many processing methods for lowering allergenicity is invalid. Interaction of 7S with phenolic acids, such as chlorogenic acid (CHA), to structurally modify 7S may lower the allergenicity. Hence, the effects of covalent (C-I, periodate oxidation method) and noncovalent interactions (NC-I) of 7S with CHA in different concentrations (0.3, 0.5, and 1.0 mM) on lowering 7S allergenicity were investigated in this study. The results demonstrated that C-I led to higher binding efficiency (C-0.3:28.51 ± 2.13%) than NC-I (N-0.3:22.66 ± 1.75%). The C-I decreased the α-helix content (C-1:21.06%), while the NC-I increased the random coil content (N-1:24.39%). The covalent 7S-CHA complexes of different concentrations had lower IgE binding capacity (C-0.3:37.38 ± 0.61; C-0.5:34.89 ± 0.80; C-1:35.69 ± 0.61%) compared with that of natural 7S (100%), while the noncovalent 7S-CHA complexes showed concentration-dependent inhibition of IgE binding capacity (N-0.3:57.89 ± 1.23; N-0.5:46.91 ± 1.57; N-1:40.79 ± 0.22%). Both interactions produced binding to known linear epitopes. This study provides the theoretical basis for the CHA application in soybean products to lower soybean allergenicity.

A novel strategy combining ferulic acid and glucose was proposed to reduce β-lactoglobulin (BLG) allergenicity and investigate whether the reduction in allergenicity was associated with gut microbiome and serum metabolism. As a result, the multistructure of BLG changed, and the modified BLG decreased significantly the contents of IgE, IgG, IgG 1 , and mMCP-1 in serum, improved the diversity and structural composition of gut microbiota, and increased the content of short-chain fatty acids (SCFAs) in allergic mice. Meanwhile, allergic mice induced by BLG affected arachidonic acid, tryptophan, and other metabolic pathways in serum, the modified BLG inhibited the production of metabolites in arachidonic acid metabolism pathway and significantly increased tryptophan metabolites, and this contribution helps in reducing BLG allergenicity. Overall, reduced allergenicity of BLG after ferulic acid was combined with glucose modification by regulating gut microbiota, the metabolic pathways of arachidonic acid and tryptophan. The results may offer new thoughts alleviating the allergy risk of allergenic proteins.

Effective reduction of the allergenicity of instant soy milk powder (ISMP) is practically valuable for expanding its applications. This study optimized the enzymolysis technology of ISMP using single-factor experiments and response surface methodology, combined serological analysis, cellular immunological models, bioinformatics tools, and multiple spectroscopy techniques to investigate the effects of alcalase hydrolysis on allergenicity, spatial conformation, and linear epitopes of ISMP. Under the optimal process, special IgE and IgG1 binding abilities and allergenic activity to induce cell degranulation of alcalase-hydrolyzed ISMP were reduced by (64.72 ± 1.76)%, (56.79 ± 3.72)%, and (73.3 ± 1.19)%, respectively ( P < 0.05). Moreover, the spatial conformation of instant soy milk powder hydrolysates (ISMPH) changed, including decreased surface hydrophobicity, a weaker peak of amide II band, lower contents of α-helix and β-sheet, and an enhanced content of random coil. Furthermore, the linear epitopes of major soy allergens, 9 from glycinin and 13 from β-conglycinin, could be directionally disrupted by alcalase hydrolysis. Overall, the structure-activity mechanism of alcalase hydrolysis to reduce ISMP allergenicity in vitro was preliminarily clarified. It provided a new research direction for the breakthrough in the desensitization of ISMP and a theoretical basis for revealing the potential mechanism of alcalase enzymolysis to reduce the allergenicity of ISMP.

Casein (CN) is the primary allergenic protein in cow's milk, contributing to the worldwide escalating prevalence of food allergies. However, there remains limited knowledge regarding the effect of structural modifications on CN allergenicity. Herein, we prepared three modified CNs (mCN), including sodium dodecyl sulfate and dithiothreitol-induced linear CN (LCN), transglutaminase-cross-linked CN (TCN), and glucose-glycated CN (GCN). The electrophoresis results indicated widespread protein aggregation among mCN, causing variations in their molecular weights. The unique internal and external structural characteristics of mCN were substantiated by disparities in surface microstructure, alterations in the secondary structure, variations in free amino acid contents, and modifications in functional molecular groups. Despite the lower digestibility of TCN and GCN compared to LCN, they significantly suppressed IL-8 production in Caco-2 cells without significantly promoting their proliferation. Moreover, GCN showed the weakest capacity to induce LAD2 cell degranulation. Despite the therapeutic effect of TCN, GCN-treated mice displayed the most prominent attenuation of allergic reactions and a remarkably restored Th1/Th2 imbalance, while LCN administration resulted in severe allergic phenotypes and endotypes in both cellular and murine models. This study highlighted the detrimental effect of linear modifications and underscored the significance of glycation in relation to CN allergenicity.

Glycinin is an important allergenic protein. A1a is the acidic chain of the G1 subunit in glycinin (G1A1a), and it has strong allergenicity. In this study, we used phage display technology to express the protein of G1A1a and its overlapping fragments and an indirect enzyme-linked immunosorbent assay (iELISA) to determine the antigenicity and allergenicity of the expressed protein. After three rounds of screening, it was determined that fragment A1a-2-B-I ( 151 SLENQLDQMPRRFYLAGNQEQEFLKYQQEQG 181 ) is the allergenic domain of G1A1a destroyed by thermal processing. In addition, three overlapping peptides were synthesized from fragments A1a-2-B-I, and a linear epitope was found in this domain through methods including dot blot and iELISA. Peptide 2 ( 157 DQMPRRFYLANGNQE 170 ) showed allergenicity, and after replacing it with alanine, it was found that amino acids D 157 , Q 158 , M 159 , and Y 164 were the key amino acids that affected its antigenicity, while Q 158 , M 159 , R 162 , and N 168 affected allergenicity.

The rising prevalence of allergy demands efficient and accurate bioinformatic tools to expedite allergen identification and risk assessment while also reducing wet experiment expenses and time. Recently, pretrained protein language models (pLMs) have successfully predicted protein structure and function. However, to our best knowledge, they have not been used for predicting allergenic proteins/peptides. Therefore, this study aims to develop robust models for allergenic protein/peptide prediction using five pLMs of varying sizes and systematically assess their performance through fine-tuning with a convolutional neural network. The developed pLM4Alg models have achieved state-of-the-art performance with accuracy, Matthews correlation coefficient, and area under the curve scoring 93.4-95.1%, 0.869-0.902, and 0.981-0.990, respectively. Moreover, pLM4Alg is the first model capable of handling prediction tasks involving residue-missed sequences and sequences containing nonstandard amino acid residues. To facilitate easy access, a user-friendly web server (https://f6wxpfd3sh.us-east-1.awsapprunner.com) has been established. pLM4Alg is expected to become the leading machine learning-based prediction model for allergenic peptides and proteins. Its collaboration with other predictors holds great promise for accelerating allergy research.

Soy allergens are susceptible to inducing allergic reactions in infants and young animals, which have an impact on the effective daily utilization of proteins. In this study, we used Alcalase-hydrolyzed instant soybean powder (ISP) to clarify the sensitization changes of instant soybean powder hydrolysates (ISPH), and we explored the assisted memory-enhancing effects. BALB/c mice in the ISPH group showed significant improvement in the allergy symptoms, with their allergy symptom scores decreasing to (1.57 ± 0.53) and their specific serum IgE and IgG1 binding capacity decreasing by 28.00 and 25.73% ( P < 0.05), which suppressed the mast cell degranulation rate. Meanwhile, the plasma HIS and IL-4 levels decreased by 12.59 and 25.32%, and the plasma INF-γ and IL- 10 levels increased by 30.64 and 27.79%, which obviously regulated the imbalance of Th1/Th2 cells and attenuated the tissue damage ( P < 0.05). Furthermore, ISPH improved behavioral characteristics, increased cholinergic system activity, reduced neuronal cell damage or apoptosis, and increased the number of Nissl bodies to help improve memory in Kunming mice ( P < 0.05). In general, alcalase-hydrolyzed ISP had the dual effects of reducing allergenicity and aiding in memory improvement.

Although tropomyosin has been identified as a major allergen in Antarctic krill, the digestive fate of Antarctic krill tropomyosin and its relationship with allergenicity are unknown. In this study, Antarctic krill tropomyosin was administered to BALB/c mice via both gavage and intraperitoneal injection to explore its sensitizing and eliciting capacity, and its digestion products were analyzed for structural changes and digestion-resistant linear epitopes. Mice gavaged with tropomyosin exhibited lower levels of specific IgE and IgG 1 , mast cell degranulation, vascular permeability, and anaphylaxis symptoms than those in the intraperitoneal injection group. This may be due to the destruction of macromolecular aggregates, loose expansion of the tertiary structure, complete disappearance of α-helix, and significant changes in molecular force upon the digestion of tropomyosin. Nevertheless, the intragastric administration of Antarctic krill tropomyosin still triggered strong allergic reactions, which was attributed to the existence of seven digestion-resistant linear epitopes (Glu26-His44, Thr111-Arg125, Glu157-Glu164, Glu177-Gly186, Val209-Ile225, Arg244-Arg255, and Val261-Ile270).

Ovalbumin (OVA) is a major allergen in hen eggs. Enzymolysis has been demonstrated as an efficient method for reducing OVA allergenicity. This study demonstrates that microwave pretreatment (MP) at 400 W for 20 s assisting bromelain enzymolysis further decreases the allergenicity of OVA, which was attributed to the increase in the degree of hydrolysis and promoted the destruction of IgE-binding epitopes. The results showed that MP could promote OVA unfolding, expose hydrophobic domains, and disrupt tightly packed α-helical structures and disulfide bonds, which increased the degree of hydrolysis by 7.28% and the contents of peptides below 1 kDa from 43.55 to 85.06% in hydrolysates compared with that for untreated OVA. Biological mass spectrometry demonstrated that the number of intact IgE-binding epitope peptides in MP-assisted OVA hydrolysates decreased by 533 compared to that in hydrolysis without MP; consequently, their IgG/IgE binding rates decreased more significantly. Therefore, MP-assisted enzymolysis may provide an alternative method for decreasing the OVA allergenicity.

Allergic reactions caused by silkworm pupae greatly limit their utilization, and studies suggest that silkworm pupae proteins of 25-30 kDa may be the principal allergens. To further understand these allergens, we attempted to purify a protein of about 30 kDa by ammonium sulfate salting, pH-graded precipitation, and ion-exchange chromatography. The protein was identified by mass spectrometry and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, enzyme-linked immunosorbent assays, circular dichroism, and fluorescence spectroscopy analyses. We identified the purified protein as Bombyx mori lipoprotein 3 (Bmlp3), which has high IgE reactivity and is a novel uncharacterized allergen that we named Bomb m 6 according to the WHO/IUIS Allergen Nomenclature Sub-Committee. This allergen is stable against heat, acids, bases, and digestion. In conclusion, we successfully purified and characterized a novel silkworm pupa allergen, which may inform the diagnosis and treatment of silkworm pupa allergies.

Egg is one of the eight major food allergens, and ovalbumin (OVA) is the most abundant allergenic protein in eggs. In this study, the effects of pulsed electric field (PEF)-assisted Alcalase hydrolysis on the spatial conformation and potential allergenicity of OVA were studied, and the mechanism of its inhibiting allergic reactions effect was revealed. PEF-assisted Alcalase hydrolysis increased the degree of hydrolysis, surface hydrophobicity, and free sulfhydryl group content. Moreover, the reduction in the α-helix content, fluorescence intensity, and disulfide bond content suggested that PEF promoted the OVA hydrolysis by Alcalase. Additionally, enzyme-linked immunosorbent assay data indicated that PEF-assisted Alcalase hydrolysis hindered OVA binding to immunoglobulins E and G1. Finally, based on bioinformatics combined with mass spectrometry, PEF-assisted Alcalase reduced OVA-induced allergic reactions by destroying epitopes in OVA. Overall, PEF technology further destroyed the epitopes of allergens by targeting the binding sites of substrates and enzymes to improve the affinity of enzymes and substrates, reducing allergic reactions.

Fermentation techniques may induce alterations in fish allergen immunoreactivity. In this study, the influence of fermentation with three different strains of Lactobacillus helveticus ( Lh187926 , Lh191404 , and Lh187926 ) on the immunoreactivity of Atlantic cod allergens was investigated via several methods. Gradually reduced protein composition and band intensity due to the fermentation by strain Lh191404 were found in SDS-PAGE analysis, and decreased immunoreactivity of fish allergens was confirmed by Western blotting and ELISA analysis due to the fermentation of strain Lh191404 . Additionally, results from nLC-MS/MS and immunoinformatics tools analysis demonstrated that the protein polypeptide and allergen composition of Atlantic cod showed evident alterations after fermentation, with the epitopes of the main fish allergens being heavily exposed and destroyed. These results indicated that the fermentation of L. helveticus could destroy the structure and linear epitopes of the allergens from Atlantic cod and may have considerable potential in mitigating the allergenicity of fish.Lh191404 could destroy the structure and linear epitopes of the allergens from Atlantic cod and may have considerable potential in mitigating the allergenicity of fish.

The mechanism by which thermal/pressure processing influences the allergenicity of shrimp ( Penaeus vannamei ) was explored by anaphylaxis in mice, the protein structure, gastrointestinal digestion, and linear epitopes. Roasting induced the unfolding of the structure, which may reduce the allergenicity, but it made more linear epitopes to be exposed, causing mice to exhibit similar systemic anaphylaxis as mice fed with the raw shrimp protein ( p > 0.05). However, the roasted + reverse-pressure-sterilized shrimp can significantly reduce specific antibodies, mast cell degranulation, vascular permeability, and histopathological morphology in mice compared with the raw and roasted shrimp ( p < 0.05) because reverse-pressure sterilization causes protein to aggregate, hiding the heat/digested stable epitopes of arginine kinase (Glu59-Ser63, Asn112-Lys118, Leu131-Phe136, and Ser158-Glu162) and sarcoplasmic calcium-binding protein (Asn57-Phe67, Ser159-Cys165, and Glu126-Ala130) inside a 3D structure, while gastrointestinal digestion can destroy immunodominant, minor epitopes and the epitopes exposed by roasting. Meanwhile, the low binding frequency of IgE to troponin C was also responsible for maintaining the hypoallergenicity of shrimp.

To accurately map the B-cell linear epitopes of lysozyme (LYS) in eggs, five bioinformatics tools were first used to obtain the mimotopes. Afterward, based on the Chinese egg-allergic sera samples screened by the indirect enzyme-linked immunosorbent, the epitopes possessing the capability of binding to IgG/IgE were mapped at both pooled and individual levels by using overlapping peptides covering the complete amino acid sequence of LYS. Six B-cell linear epitopes and two dominant B-cell linear epitopes that could bind to LYS-sIgG were mapped for the first time. Seven IgE-binding epitopes and three dominant IgE-binding epitopes were also obtained. Furthermore, AA31-34 and AA88-91 were the shared dominant epitopes of LYS-sIgG and LYS-sIgE at pooled and individual levels. Overall, the mapped B-cell linear epitopes filled in the gaps in the study of LYS epitopes, and the results may provide theoretical support for the following immunotherapy of egg allergy.

Physically assisted chemical modifications can effectively reduce the allergenicity of ovomucoid (OVM). However, only a few studies have used pulsed electric field (PEF)-assisted alcalase hydrolysis to reduce the allergenicity of OVM. Herein, we investigated the effect of PEF-assisted alcalase treatment on the spatial conformation, allergenicity, and antigenic epitopes of OVM based on multispectroscopic analyses, bioinformatics, and mass spectrometry. The results showed that PEF-assisted alcalase treatment promoted the hydrolysis of OVM; moreover, the α-helix content and surface hydrophobicity of OVM significantly decreased, which disordered its spatial conformation and weakened its intermolecular interactions. Additionally, enzyme-linked immunosorbent assay (ELISA) results showed that the PEF-assisted alcalase treatment significantly reduced the binding levels of IgE and IgG1, which were 47.66 and 36.41%, respectively. Finally, eight epitopes of OVM were obtained by immunoinformatic tools. Nano-high performance liquid chromatography coupled to tandem mass spectrometry (nano-HPLC MS/MS) results showed that the hydrolysate of OVM and alcalase (HOVM) had nine more peptide-containing epitopes than the hydrolysate of PEF-treated OVM and PEF-treated alcalase (HOVM-PP'), indicating that PEF could promote the elimination of linear epitopes in OVM, thereby reducing OVM allergenicity.

Peanut allergy is the leading pediatric food allergy. Many attempts have been made to reduce its allergenicity by processing. After roasting, Ara h 2 and its derivatives in the matrix were isolated by immunoaffinity chromatography (IAC). The structure and allergenicity of Ara h 2 were analyzed by circular dichroism, mass spectrometry (MS), western blotting, the enzyme-linked immunoassay, and cell modeling. Our results showed that a large portion of Ara h 2 was fragmented and cross-linked. Ara h 2 monomers accounted for only 13% of the total proteins after IAC purification. In addition, the structure of Ara h 2 changed after roasting. In addition to methylation and oxidation modification, the disulfide bonds of Ara h 2 were found to be rearranged after roasting. In the conformational structure of Ara h 2, the content of the α-helix decreased from 27.1 to 21.6% after roasting, while the content of the random coil increased from 29.1 to 34.3%. Six cleavage sites of trypsin were exposed, while three were covered. In terms of allergenicity, most of the cross-linking products were not recognized by patients' sera. Only one faint band around 40 kDa was observed in our blotting. For Ara h 2 monomers, roasting enhanced their IgE binding capacity and ability to stimulate the degranulation of basophils. The potential allergenicity increase of Ara h 2 monomers did not reflect the allergenicity change of Ara h 2 in the matrix due to the amount and property of its derivatives after roasting.

Shrimp is a major allergic food that could trigger severe food allergy, with the most significant and potent allergen of shrimp referred to as tropomyosin (TM). Glycation modification (Maillard reaction) could reportedly weaken the allergenicity of TM and generate hypoallergenic TM, while up to now, there is still a lack of investigations on the hypoallergenic glycated tropomyosin (GTM) as a candidate immunotherapy for desensitizing the shrimp TM-induced allergy. This study analyzed the effects of glycation modification on decreasing the allergenicity of TM and generated hypoallergenic GTM and how GTM absorbed to the Al(OH) 3 function as a candidate immunotherapy for desensitizing allergy. As the results, in comparison to TM, the saccharides of smaller molecular sizes could lead to more advanced glycation end products in GTMs than saccharides of greater molecular sizes, and TM glycated by saccharides of different molecular sizes (glucose, maltose, maltotriose, maltopentaose, and maltoheptaose) exhibited lower allergenicity as a hypoallergen upon activating the allergic reactions of the mast cell and mouse model, while TM glycated by maltose had insignificant allergenicity changes upon activating the allergic reactions of the mast cell and mouse model. In addition, the hypoallergenic GTM + Al(OH) 3 was efficient as a candidate immunotherapy; this work intended to offer preclinical data to promote GTM + Al(OH) 3 as a candidate allergen-specific immunotherapy for desensitizing the allergy reactions for patients allergic to shrimp food.

The effects of ultrasound combined with glycation (UCG) on the allergenicity and human microbial community of β-Lg during in vitro digestion were studied by ELISA, cell experiments, and 16S rRNA high-throughput sequencing. UCG modification and subsequent digestion significantly reduced allergenicity. The decrease in the allergenicity of β-Lg depended not only on the low digestibility of glycated β-Lg, which led to the decrease of some peptides with complete immunogenicity, but also the masking effect of glycation on allergen epitopes of β-Lg. Meanwhile, UCG modification and subsequent digestion could alter the structures of intestinal microbiota and the community abundance at phylum, family, and genus levels, such as Bacteroidota, Fusobacteriota, Enterobacteriaceae, Bacteroidaceae, Ruminococcaceae, Bacteroides, and Faecalibacterium . These results show that simulated in vitro digestion of modified β-Lg reduces allergenicity and alters human intestinal microbiota, which could provide a theoretical basis for studying the relationship between intestinal dysbiosis and cow's milk allergy.

The present study aims to investigate the structure of covalent conjugates of bovine β-lactoglobulin (BLG) and flavonoids (luteolin, myricetin, and hyperoside), and their effect on the allergenicity and human intestinal microbiota. Covalent modification of amino acids in BLG by flavonoids was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and o -phthaldialdehyde assay. The secondary and conformational structures of BLG were changed by the covalent modification, which were determined by the circular dichroism, Fourier transform infrared spectroscopy, fluorescence spectroscopy, and UV spectroscopy. The enzyme-linked immunosorbent assay (ELISA) and cell experiments indicated that BLG covalent conjugates could reduce IgE/IgG binding capacities and suppress the allergy reactivity of RBL-2H3 cells, suggesting that the covalent modification modulated the balance of T cells. Meanwhile, covalent modification of BLG with these flavonoids can alter the diversity of human intestinal microbiota and the community abundance at phylum, family, and genus levels. The results revealed that covalent modification of BLG with flavonoids alters human intestinal microbiota, might result in the reduction of allergenicity, which could provide information for confirming the relationship between food allergy and the intestinal microbial ecosystem.

Physical-assisted chemical modification is effective to reduce the allergenicity of α-lactalbumin (ALA). However, there are few in-depth studies on the allergenicity changes of physical-assisted chemical-modified ALA during digestion. The effect of gastroduodenal digestion on the allergenicity changes of ALA treated by sonication-assisted glycation was assessed. Digestion of both ALA and its glycated forms generated peptide fractions, and intact undigested glycated ALA in the hydrolysates still covalently bound to d-galactose. High-resolution mass spectrometry revealed that a higher glycation degree was discovered in sonication-preprocessed ALA compared to native ALA. Enzyme-linked immunosorbent assay and basophil degranulation showed that sonication-assisted glycation could significantly reduce ALA allergenicity. The allergenicity of both gastric and gastroduodenal hydrolysates was further increased, and the hydrolysates of sonication-assisted glycated ALA showed the lowest allergenicity. The reason could be the shielding effect of the linear epitope found to be caused by a higher glycation degree; although linear epitopes were exposed, d-galactose covalently bound to intact undigested glycated ALA in the hydrolysates retained its masking role. These results indicated that sonication-assisted glycation could be a promising method to prepare immunotherapeutic agents for allergen immunotherapy to achieve the purpose of allergy desensitization.

The impact of covalent attachment of (-)-epigallocatechin gallate (EGCG) to lactoferrin (LF) on the structure, morphology, functionality, and allergenicity of the protein was studied. These polyphenol-protein conjugates were formed using various enzymatic (laccase- and tyrosinase-catalyzed oxidation) and nonenzymatic (free radical grafting and alkali treatment) methods. The preparation conditions for forming the enzymatic conjugates were optimized by exploring the influence of order-of-addition effects: protein, polyphenols, and enzymes. The total phenol content of the LF-EGCG conjugates was quantified using the Folin-Ciocalteu method. The nature of the conjugates formed was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) spectroscopy analyses. These studies showed that enzymatic cross-linking was a highly effective means of forming LF-EGCG conjugates. Analysis of these conjugates using various spectroscopic methods showed that conjugation to EGCG changed the molecular structure of LF. Atomic force microscopy showed that the four covalent cross-linking methods affected the size and morphology of these LF-EGCG conjugates formed. The antioxidant activity and emulsifying stability of LF were significantly improved by conjugation to EGCG. Finally, an enzyme-linked immunosorbent assay (ELISA) and a western blot assay indicated that conjugation of EGCG reduced the binding capacity of LF to immunoglobulin E (IgE) and immunoglobulin G (IgG), which is consistent with a decrease in allergenicity. Overall, this study suggests that LF-EGCG conjugates formed using enzymatic or nonenzymatic methods have potential applications as functional ingredients in foods.

This study analyzed the effect of lipid peroxidation using 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) and acrolein on the in vitro and in vivo allergenicity of α-lactalbumin (α-La). The structure of oxidized α-La was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, fluorescence spectroscopy, and circular dichroism, whereas the changes in the allergenic properties were evaluated. Lipid peroxidation induced changes to the structural properties that might destroy and/or mask α-La epitopes. In comparison to native α-La, oxidation complexes caused a decrease in the immunoglobulin E (IgE) binding capacity, as observed via immunoblotting. Moreover, the capacity to release mediators and cytokines from KU812 cells was also greatly reduced. In vivo , oxidation with AAPH and acrolein caused a significant reduction in IgE, IgG, IgG1, mast cell protease 1, and plasma histamine, along with the reduction of mast surface c-Kit + and FcεRI + expression. Therefore, these results indicate that oxidation via AAPH and acrolein can potentially reduce the allergenicity of α-La, which can help with the better understanding of the changes in allergenicity of milk allergen by lipid peroxidation.

A high content of potentially allergenic lectin in Phaseolus vulgaris L. beans is of increasing health concerns; however, understanding of the protein allergenicity mechanism on the molecular basis is scarce. In the present study, low-pH treatments were applied to modify black turtle bean lectin allergen, and a sensitization procedure was performed using the BALB/c mice for the allergenicity evaluation, while the conformational changes were monitored by the spectral analyses and the details were explored by the molecular dynamics simulation. Much milder anaphylactic responses were observed in BALB/c mice experiments. At the molecular level, the protein was unfolded in low acidic environments because of protonation, and α-helix was reduced with the exposure of trypsin cleavage sites, especially the improvement of protease accessibility for Lys121, 134, and 157 in the B cell epitope structural alterations. These results indicate that a low-pH treatment might be an efficient method to improve the safety of legume protein consumption.

A previous study demonstrated decreased allergenicity in vitro of some food allergens after conjugation with polyphenols. However, little is known about how polyphenol conjugation with food allergens affects in vivo allergenicity. We conjugated a well-known food allergen, ovalbumin (OVA), with quercetin (QUE) to assess the potential allergenicity of OVA in vitro and in vivo in a BALB/c mouse model. QUE could covalently conjugate with OVA and changed the protein structure, which might destroy and/or mask OVA epitopes. Conjugation with QUE decreased IgE binding properties and the release capacity of the conjugated OVA. In vivo , as compared with native protein, conjugation with QUE decreased the levels of IgE, IgG1, IgG, plasma histamine, and mast cell protease-1 (mMCP-1) on the surface of sensitized mast cells, along with decreased FcεRI + and c-kit + expression. The levels of Th2-related cytokines (IL-4, IL-5, IL-13) decreased and that of a Th1-related cytokine (IFN-γ) increased slightly, which suggests that conjugation with QUE modulated the imbalance of the Th1/Th2 immune response. Conjugation of OVA with QUE could reduce OVA allergenicity in vitro and in vivo , which could provide information for reducing food allergenicity by conjugation with polyphenols.

Gliadins are major allergens responsible for wheat allergies. Food processing is an effective strategy to reduce the allergenicity of gluten. In the present study, we determined the secondary and tertiary structures of gluten and gliadins treated by chemical, physical, and enzymatic means through FTIR, surface hydrophobicity, intrinsic fluorescence spectra, and UV absorption spectra. The results showed that the three treatments of phosphorylation and alcalase and papain hydrolyses significantly changed the conformational structures of gliadins, especially the secondary structure. Then, the potential allergenicity of the phosphorylated and alcalase and papain hydrolyzed gliadins were further characterized, and we observed a significant decrease in the allergenicity through the results of the index of spleen, serum total IgE, gliadin-specific IgE, histamine, and serum cytokine concentrations. An elevation of Th17 cells, the absence of Treg cells, and an imbalance in Treg/Th17 are associated with allergy. On the basis of the expression levels of related cytokines and key transcription factors, we also confirmed that phosphorylation and alcalase and papain hydrolysis could effectively reduce the allergenicity of gliadins by improving the imbalance of both Th1/Th2 and Treg/Th17 in the spleens of sensitized mice. This study suggested that the changes in conformational structure contribute to gliadin hyposensitization and that phosphorylation and alcalase and papain hydrolysis may be promising strategies for the production of wheat products with low allergenicity.

Ovalbumin (OVA) is a major allergen in avian egg white. Here, we investigated the conjugation of OVA and chlorogenic acid (CHA) to reduce the allergenic capacity of OVA. OVA-CHA conjugate was characterized by SDS-PAGE, MALDI-TOF-MS, differential scanning calorimetry, and multispectroscopic methods. Sites of the OVA-CHA conjugate were identified by LC-MS/MS. CHA possibly conjugated with Lys20 and Lys17 in OVA, which resulted in the unfolding of OVA. ELISA and Western blot assay indicated that the OVA-CHA conjugate reduced the IgE binding capacity of OVA. The results also indicated that the ability of the OVA-CHA conjugate to activate histamine release was reduced. The decreased allergenic capacity of OVA was attributed to changes in the protein structure. Moreover, the CHA binding site in OVA might directly shield the linear IgE epitope, thereby reducing the IgE binding ability. Also, the OVA-CHA conjugate showed high antioxidant activity. OVA conjugated with CHA may be a promising method of OVA hyposensitization.

As a naturally occurring reaction during food processing, glycation, also known as non-enzymatic browning or Maillard reaction, can improve food protein physiochemical properties and functionality. In this perspective, three aspects of glycation (terminology confusion between glycation and glycosylation, its current application, and its impact on immunoreactivity) are elaborated. Overall, the immunoreactivity of glycated proteins may decrease, remain unchanged, or even increase after food glycation. Also, it should be noted that the effect of glycation on the immunoglobulin (Ig)E- or IgG-binding capacity of allergens does not necessarily and correctly predict the allergenicity of the glycated protein in the allergic patient population.

Baker's asthma is a serious airway disease triggered by wheat protein CM3 α-amylase/trypsin inhibitor. The purpose of the present study was to investigate the impact of genotype and crop year on allergen CM3 α-amylase/trypsin inhibitor associated with baker's asthma. A historical series of Tunisian durum wheat (100 accessions), derived from three crop years, was used to compare the amount of CM3 from landraces to advanced cultivars. CM3 protein quantification was assessed after an enzymatic cleavage of the soluble protein extracts on a UPLC/ESI-MS system, using a marker peptide for its quantification. Combined data analysis of variance revealed an important effect of genotype, crop year, and their interaction. The CM3 allergenic proteins were found to significantly vary among studied genotypes, as confirmed by genetic variability, coefficient of variance, heritability, and genetic advance.

Soybean (Glycine max L. Merrill) is one of eight major allergenic foods with endogenous proteins identified as allergens. To better understand the natural variability of five soybean allergens (Gly m 4, Gly m 5, Gly m 6, Gly m Bd 28k, and Gly m Bd 30k), validated enzyme-linked immunosorbent assays (ELISAs) were developed. These ELISAs measured allergens in 604 soybean samples collected from locations in North and South America over five growing seasons (2009-2013/2014) and including 37 conventional varieties. Levels of these five allergens varied 5-19-fold. Multivariate statistical analyses and pairwise comparisons show that environmental factors have a larger effect on allergen levels than genetic factors. Therefore, from year to year, consumers are exposed to highly variable levels of allergens in soy-based foods, bringing into question whether quantitative comparison of endogenous allergen levels of new genetically modified soybean adds meaningful information to their overall safety risk assessment.

Myosin light chain (MLC) plays a vital role in cell and muscle functions and has been identified as an allergen in shrimp. In this study, MLC with a molecular mass of 18 kDa was purified from crayfish (Procambarus clarkii) muscle. Its physicochemical characterization showed that the purified MLC is a glycoprotein with 4.3% carbohydrate, highly stable to heat, acid-alkali, and digestion, and weakly retains IgE-binding activity when its secondary structure was altered. Serological assays suggested that conformational epitopes predominate over linear epitopes in the purified MLC. Two isoforms of the MLC gene (MLC1 and MLC2) were cloned, and the purified MLC was identified as MLC1. Analysis of the secondary and tertiary structures of the MLCs indicated that MLC1 has four conformational epitopes and three linear epitopes, whereas MLC2 had a major conformational epitope and three linear epitopes. These results are significant for understanding hypersensitization of humans to crayfish.