Combination of Structural Analysis and Proteomics Strategy Revealed the Mechanism of Ultrasound-Assisted Cold Plasma Regulating Shrimp Allergy.

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.

Coconut milk allergenicity: Insight into reducing the affinity of coconut globulin to immunoglobulin E by atmospheric cold plasma.

Atmospheric cold plasma (ACP) presents a promising method for the sterilization of coconut milk and exhibits a modifying effect on coconut globulin (CG), the primary allergen in coconut milk. This study investigated the potential role of ACP treatment in mitigating the allergenic properties of coconut milk by examining changes in protein structure. ACP treatment induced structural alterations in CG, disrupting binding sites with immunoglobulin E (IgE). Consequently, this led to a reduction in the affinity between CG and IgE, evidenced by a decrease in Ka from 2.17 × 104/M to 0.64 × 104/M, thereby diminishing IgE-mediated allergic reactions. The findings from allergenic and cellular models further corroborated that ACP treatment decreased the allergenicity of CG by 55.18%, while inhibiting degranulation and the release of allergic mediators. This study presents an innovative methodology for producing hypoallergenic coconut milk, thereby expanding the applicability of ACP technology within the food industry.

Modulating allergenicity of prawn tropomyosin (penaeus chinensis) via pulsed electric field-induced conformational changes.

The effect of electric field intensities (EFIs, 5-20 kV/cm) and treatment times (0.5-2 h) on allergenicity and spatial conformation of prawn tropomyosin was evaluated. The results demonstrated that the IgG and IgE binding capacity of tropomyosin maximally increased by 24.34 % and 29.16 % respectively, followed by a subsequent decrease after 20 kV/cm treatment for 1 h. Interestingly, 5-10 kV/cm treatments significantly decreased the α-helix content (P < 0.05) and fluorescence intensity, while 20 kV/cm treatment promoted extensive spiralization, resulting in a tightly packed structure. The increased flexibility further exposed the hydrolysis sites and strengthened the gastrointestinal digestibility of tropomyosin. Additionally, molecular dynamic simulation indicated that extended EFIs increased structural flexibility and depolymerized the tropomyosin dimers through destroying intermolecular hydrogen bonds (formed within arginine and glutamate), which allowed tropomyosin to be easily recognized by IgG/IgE. Whereas, decrease of solvent-accessibility surface area (SASA), hydrophobic surface area induced conformation folded and caused epitopes masked.

High aerospora levels and associated atmospheric circulation patterns: Pretoria, South Africa.

At high exposure levels, airborne pollen grains and fungal spores (termed aerospora hereafter), can trigger severe allergic respiratory diseases. For South Africa's administrative capital Pretoria, which boasts dense vegetation within a large urban forest, it is valuable from a health perspective to understand daily atmospheric circulation patterns associated with high aerospora levels. Therefore, we utilised a daily aerospora grain count dataset collected in Pretoria from 08/2019-02/2023 to investigate atmospheric circulation patterns (derived from ERA5 reanalysis sea level pressure [SLP] and 500 hPa geopotential height [zg500] fields) associated with high-risk aerospora levels (aerospora grain count > 90th percentile). Concentrated during October-May, there were 128 high-risk days, with 69.6% of days occurring in November, February and April. Although generally above-average mid-tropospheric subsidence levels prevailed over Pretoria during high-risk days, no single distinct atmospheric circulation pattern was associated with these high-risk days. Therefore, using Principal Component Analysis, we classified 14 Circulation Weather Types (CWTs) for October-May months between 08/2019-02/2023 to assess which CWTs most frequently occurred during high-risk days. Three CWTs had a statistically significant proportion of high-risk days - collectively they occurred during 37.1% of days studied, yet accounted for 45.3% of high-risk days. Among these CWTs, two CWTs were similarly associated with surface and mid-tropospheric high-pressure conditions, while the third was associated with a surface and mid-tropospheric trough. By comparing our CWT classification to daily synoptic charts (from the South African Weather Service), our classification can be used to identify days with potentially high allergenicity risk over Pretoria.

Molecular Cloning, Prokaryotic Expression, and Immunological Characterization of ß-Enolase from Grass Carp (Ctenopharyngodon idella).

ß-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 ß-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 ß-enolase was obtained by prokaryotic expression, and its allergenicity was assessed by ß-enolase-sensitized mice, which confirmed the ability of ß-enolase to trigger an allergic response and cause a rise in Th1 and Th2 immune responses in mice. These results suggest that ß-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.

Effect of Thermal Processing on Food Allergenicity: Mechanisms, Application, Influence Factor, and Future Perspective.

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.

The changes induced by hydrodynamic cavitation treatment in wheat gliadin and celiac-toxic peptides.

Hydrodynamic cavitation (HC) is thought weaken the allergenicity of beer gluten proteins. However, the mechanism of action has not been thoroughly studied. In this study, an HC device was used to treat wheat gliadin and two specific celiac-toxic peptides, P1 and P2. FT-IR, MFS, HPLC, and CD were used to monitor the structural characteristics of gliadin and the two peptides. HC reduced the abundance of the coeliac toxic peptides P1 and P2 in solution and the contents of secondary structure ß-turns and PPII, which are related to reduced allergen immunoreactivity. This meant that both the primary and secondary structures of P1 and P2 were affected by HC, leading to fewer allergic reactions. This study was focused on the impact of HC on the secondary structures of allergens produced from gluten raw materials, and it has positive implications for reducing product allergenicity. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-024-05973-7.

Can Physicochemical Properties Alter the Potency of Aeroallergens? Part 1 - Aeroallergen Protein Families.

PURPOSE OF REVIEW: Respiratory allergies are non-communicable diseases caused by the hypersensitivity of the immune system to environmental aeroallergens. The culprits are aero-transported proteins eliciting respiratory symptoms in sensitized/allergic individuals. This review intends to provide a holistic overview on the categorization of aeroallergens into protein families (Part 1) and to exploit the impact of physicochemical properties on inhalant protein allergenicity (Part 2). This first part will focus particularly on aeroallergen organization into families and how this classification fits their physicochemical properties. RECENT FINDINGS: Aeroallergen classification into protein families facilitates the identification of common physicochemical properties, thus aiding a better comprehension of known allergens, while predicting the behavior of novel ones. The available online databases gathering important features of aeroallergens are currently scarce. Information on distinct aeroallergen classification is still lacking, as data is dispersed and often outdated, hampering an efficient evaluation of new aeroallergens.

Can Physicochemical Properties Alter the Potency of Aeroallergens? Part 2 - Impact of Physicochemical Properties.

PURPOSE OF REVIEW: A holistic perspective on how physicochemical properties modulate the allergenicity of proteins has recently been performed for food allergens, launching the challenge of a similar analysis for aeroallergens. After a first review on aeroallergen classification into protein families (Part 1), this second part (Part 2) will exploit the impact of physicochemical properties (abundance/biological function, protein structure/presence of post-translational modifications, ligand/cofactor/lipid-binding) on inhalant protein allergenicity. RECENT FINDINGS: The abundance linked to biological function is correlated with increased allergenic risk for most protein families, while the loss of structural integrity with consequent destruction of conformational epitopes is well linked with decreased allergenicity. Ligand-binding effect totally depends on the ligand type being highly variable among aeroallergens. Knowledge about the physicochemical properties of aeroallergens is still scarce, which highlights the need for research using integrated approaches (in silico and experimental) to generate and analyze new data on known/new aeroallergens.

Research progress in ultrasound and its assistance treatment to reduce food allergenicity: Mechanisms, influence factor, application and prospect.

Food allergy is a serious public health problem, which is mainly induced by food allergens (mainly allergenic proteins). Ultrasound can change protein structure, suggesting its potential to decrease food allergenicity. The review concluded the mechanism and influence factors of ultrasound to reduce food allergenicity. The effects of ultrasound alone on some major allergenic foods such as tree nuts, shellfish, fish, egg, soy, milk, and wheat were also discussed. Moreover, ultrasound pre- and post-treatments were combined with heating, glycation, germination, hydrolysis, fermentation, irradiation and polyphenol treatment for reducing food allergenicity were also evaluated. It was found that ultrasound induced structural changes even degradation of protein to reduce the allergenicity mainly due to cavitation effects. The reduction of allergenicity through ultrasound alone was affected by ultrasound power, time, frequency and food types, while, apart from these factors, it was affected by ultrasound order and the assisted technologies conditions during ultrasound-assisted technologies. Compared to ultrasound alone treatment, the ultrasound-assisted technology exhibited high efficiency of allergenicity reduction because ultrasound treatment caused protein unfolding to accelerate allergen modification of the assisted technologies for masking and disrupting more epitopes. Thus, ultrasound treatment, especially ultrasound-assisted technologies under appropriate conditions, was promising for producing hypoallergenic foods.

A novel sustainable photo/chemical magnetic nanomaterial effectively mitigates the allergenicity of phospholipase A2.

Reducing the allergenicity of edible insects is crucial for the comprehensive utilization of insect resources. Phospholipase A2 (PLA2) exists in various edible insects and mammalian tissues, which can cause serious allergic reactions. Herein, we constructed a magnetic nanocomposite with photo/chemical synergistic capability to mitigate the allergenicity of PLA2. The formation of prepared nanocomposite was systematically confirmed using various techniques. The nanocomposite exhibited uniform diameters, abundant functional groups, excellent magnetic capabilities. An effective photo/chemical method was established to reduce the allergenicity of PLA2 in vitro. The feasibility of the method was demonstrated through circular dichroism, fluorescence spectrum and IgE-binding analysis. The allergenicity and IgE-binding effect of PLA2 were significantly reduced due to conformational changes after nanomaterial treatment. These results demonstrate the sensitivity and effectiveness a strategy for reducing PLA2 allergenicity, providing a basis for development of nanomaterials to reduce the risk of novel food allergies in response to edible insect products.

Evaluation of the potential food allergy risks of human lactoferrin expressed in Komagataella phaffii.

Introduction: Prior to the introduction of novel food ingredients into the food supply, safety risk assessments are required, and numerous prediction models have been developed and validated to evaluate safety. Methods: The allergenic risk potential of Helaina recombinant human lactoferrin (rhLF, Effera™), produced in Komagataella phaffii (K. phaffii) was assessed by literature search, bioinformatics sequence comparisons to known allergens, glycan allergenicity assessment, and a simulated pepsin digestion model. Results: The literature search identified no allergenic risk for Helaina rhLF, K. phaffii, or its glycans. Bioinformatics search strategies showed no significant risk for cross-reactivity or allergenicity between rhLF or the 36 residual host proteins and known human allergens. Helaina rhLF was also rapidly digested in simulated gastric fluid and its digestibility profile was comparable to human milk lactoferrin (hmLF), further demonstrating a low allergenic risk and similarity to the hmLF protein. Conclusion: Collectively, these results demonstrate a low allergenic risk potential of Helaina rhLF and do not indicate the need for further clinical testing or serum IgE binding to evaluate Helaina rhLF for risk of food allergy prior to introduction into the food supply.

Effects of Glycosylation Combined with Phosphate Treatment on the Allergenicity and Structure of Tropomyosin in Litopenaeus vannamei.

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 (K30, K38, S39, K48, K66, K74, K128, K161, S210, and K251) 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.

Effects of physical processing on food protein allergenicity: A focus on differences between animal and alternative proteins.

In recent years, physical technologies have been widely employed to reduce food protein allergenicity due to their simplicity and stability. This paper summarizes recent research advances in these technologies, focusing on differences in their effects on allergenicity between animal and alternative proteins. The mechanisms of allergenicity reduction and the advantages and disadvantages of these technologies were compared. It was found that heating, although affording better allergenicity reduction than non-thermal treatment technologies, affects other properties of the food. Because of their higher molecular weights and more complex structures, animal proteins are less affected by physical technologies than alternative proteins. It is worth noting that there is a scarcity of existing technology to reduce the allergenicity of food proteins, and more technologies should be explored for this purpose. In addition, better allergenicity-reducing processing technologies should be designed from the perspectives of processing conditions, technological innovations, and combined processing technologies in the future.

The differences of characteristics and allergenicity between natural and recombinant tropomyosin of Macrobrachium nipponense.

Tropomyosin (TM) is the main allergen of Macrobrachium nipponense. Recombinant allergens have great prospects in the detection, diagnosis, and treatment of food allergens. The purpose of this study was to compare the differences in structure and allergenicity between natural TM and recombinant TM. Recombinant TM of M. nipponense with a molecular weight of 38 kDa was successfully expressed in the Escherichia coli system. The amino acid sequence as well as secondary structure between natural and recombinant TM were similar, which were verified by mass and CD spectrometry, respectively. Studies showed that both natural TM and recombinant TM had strong allergenicity, and recombinant TM was more allergenic, which could be used as a substitute for natural TM in the diagnosis and treatment of shrimp allergy. This study provided stable and reliable allergen components for the detection of crustacean allergens and the diagnosis and treatment of food allergies caused by crustacean allergens.

Allergenicity assessment of ß-lactoglobulin ferulic acid-glucose conjugates.

We prepared the ß-lactoglobulin (BLG)-ferulic acid (FA)-glucose (Glu) conjugates by alkaline method and Maillard reaction to assess the allergenicity. FA and Glu can form a ternary covalent conjugate with BLG, as evidenced by the shortening of SEC retention time, upward migration of SDS-PAGE protein bands, considerable decrease in free amino and sulfhydryl content, and changes in multistructure. BLG-Glu-FA conjugates weakly bound to immunoglobulin E in allergic sera was weak, reduced interleukin 4 and tumor necrosis factor α levels in RBL-2H3 cells and histamin and interleukin 6 secretion levels in KU812 cells, and inhibited the nuclear factor-κB signaling pathway. In vivo experiments showed that the conjugates regulated T-cell homeostasis in mouse splenic and mesenteric lymphocytes and attenuated splenic and duodenal immune injury. Therefore, the conjugates of BLG with FA combined with Glu altered the epitope structure and exhibited low allergenicity.

A composite enzyme derived from papain and chymotrypsin reduces the Allergenicity of Cow's Milk allergen casein by targeting T and B cell epitopes.

Casein, the major allergen in cow's milk, presents a significant challenge in providing nutritional support for children with allergies. To address this issue, we investigated a composite enzyme, comprising papain and chymotrypsin, to reduce the allergenicity of casein. Enzymatic hydrolysis induced substantial structural changes in casein, diminishing its affinity for specific IgE and IgG antibodies. Additionally, in a BALB/c mouse model, casein hydrolysate alleviated allergic symptoms, evidenced by lower serum IgE and IgG levels, reduced plasma histamine, and decreased Th2 cytokine release during cell co-culture. Peptidomic analysis revealed a 52.38% and 60% reduction in peptides containing IgE epitopes in casein hydrolyzed by the composite enzyme compared to papain and chymotrypsin, respectively, along with a notable absence of previously reported T cell epitopes. These results demonstrate the potential of enzyme combinations to enhance the efficiency of epitope destruction in allergenic proteins, providing valuable insights into the development of hypoallergenic dairy products.

Multi-epitope peptide vaccines targeting dengue virus serotype 2 created via immunoinformatic analysis.

The Middle East has witnessed a greater spread of infectious Dengue viruses, with serotype 2 (DENV-2) being the most prevalent form. Through this work, multi-epitope peptide vaccines against DENV-2 that target E and nonstructural (NS1) proteins were generated through an immunoinformatic approach. MHC class I and II and LBL epitopes among NS1 and envelope E proteins sequences were predicted and their antigenicity, toxicity, and allergenicity were investigated. Studies of the population coverage denoted the high prevalence of NS1 and envelope-E epitopes among different countries where DENV-2 endemic. Further, both the CTL and HTL epitopes retrieved from NS1 epitopes exhibited high conservancies' percentages with other DENV serotypes (1, 3, and 4). Three vaccine constructs were created and the expected immune responses for the constructs were estimated using C-IMMSIM and HADDOCK (against TLR 2,3,4,5, and 7). Molecular dynamics simulation for vaccine construct 2 with TLR4 denoted high binding affinity and stability of the construct with the receptor which might foretell favorable in vivo interaction and immune responses.

Four amino acids play an important role in the allergenicity of hemocyanin allergen.

To identify the key amino acids (AAs) affecting the allergenicity of hemocyanin (HC) allergens from Chinese mitten crabs, in this study, two epitopes, P1-SHFTGSKSNPEQR and P2-LSPGANTITR were employed and four potential key AAs (P1: F3 and N9 and P2: N6 and R10) were predicted. Mast cell and mouse models revealed that four mutants induced lower levels of immunoglobulin E (IgE) and Th2 type cytokines (15.47-49.89 %), proving that F3, N9, N6, and R10 were the key AAs of two epitopes. Mutants reduce allergic responses via the Th2 pathway. However, the roles of every key AA affecting allergenicity were different (P1-F3 > N9 and P2-N6 > R10). In addition, lower transport and higher efflux were observed in the mutants during transport absorption by Caco-2 cells. The allergenicity of HC was stronger when the transport absorption efficiency of epitopes and mutants was higher and their efflux was lower. Our study provides a novel method for revealing the allergenic molecular mechanisms of food allergens.

Reducing the Allergenicity of ß-Lactoglobulin by Covalent Modification with Different Contents of Epigallocatechin Gallate (EGCG): In Vitro and In Vivo Studies.

ß-Lactoglobulin (ßLG) is a major allergen in bovine milk protein. This study was designed to investigate changes in ßLG structure, digestibility, and allergenicity induced by covalent binding modification with different contents of (-)-epigallocatechin 3-gallate (EGCG). The reaction of EGCG conjugation with ßLG reached saturation at a molar ratio of 1:60 ßLG:EGCG. Conjugation with EGCG altered the ßLG structure, decreased IgE-binding capacity, and increased digestibility in a dose-dependent manner. In vivo studies showed that covalent conjugation with EGCG can reduce ßLG-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 ßLG-EGCG conjugates compared to semisaturated conjugates. Observed changes in IFN-γ, IL-4, IL-5, IL-10, and TGF-ß levels suggested that ßLG-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.