Short-time ozone treatment promotes protease-mediated destruction of B cell allergen epitopes by altering the structural characteristics of whey protein.

A novel processing method combining short-time ozone pretreatment with hydrolysis has been developed to reduce whey protein allergenicity. The results showed that ozone treatment altered the whey protein spatial structure, initially increasing the surface hydrophobicity index, and then decreasing due to polymer formation as the time increased. Under the optimized conditions of alkaline protease-mediated hydrolysis, a 10-second pre-exposure to ozone significantly promoted the reduction in the IgE binding capacity of whey protein without compromising the hydrolysis efficiency. Compared with whey protein, the degranulation of KU812 cells stimulated by this hydrolysate decreased by 20.54%, 17.99%, and 22.80% for IL-6, ß-hexosaminidase, and histamine, respectively. In vitro simulated gastrointestinal digestion confirmed increased digestibility and reduced allergenicity. Peptidomics identification revealed that short-time ozonation exposed allergen epitopes, allowing alkaline protease to target these epitopes more effectively, particularly those associated with α-lactalbumin. These findings suggest the promising application of this processing method in mitigating the allergenicity of whey protein.

Extracellular adenosine triphosphate skews the T helper cell balance and enhances neutrophil activation in mice with food allergies.

Exposure to food allergens elicits fast changes in the intestinal microenvironment, which guides the development of allergic reactions. Investigating the key information about these changes may help in better understanding food allergies. In this research, we explored the relationship between a food allergy and extracellular adenosine triphosphate (ATP), a danger molecule that has been proved to regulate the onset of allergic asthma and dermatitis but has not been studied in food allergies, by developing a unique animal model through allergen-containing diet feeding. After consuming an allergen-containing diet for 7 days, the allergic mice exhibited severe enteritis with elevated luminal ATP levels. The dysregulated luminal ATP worsened food-induced enteritis by enhancing Th17 cell responses and increasing mucosal neutrophil accumulation. In vitro experiments demonstrated that ATP intervention facilitated Th17 cell differentiation and neutrophil activation. In addition, the diet-induced allergy showed noticeable gut dysbiosis, characterized by decreased microbial diversity and increased diet-specific microbiota signatures. As the first, we show that food-induced enteritis is associated with an elevated concentration of luminal ATP. The dysregulated extracellular ATP exacerbated the enteritis of mice to a food challenge by manipulating intestinal Th17 cells and neutrophils.

Novel Perspective on the Regulation of Offspring Food Allergy by Maternal Diet and Nutrients.

There has been a dramatic surge in the prevalence of food allergy (FA) that cannot be explained solely by genetics, identifying mechanisms of sensitization that are driven by environmental factors has become increasingly important. Diet, gut microbiota, and their metabolites have been shown to play an important role in the development of FA. In this review, we discuss the latest epidemiological evidence on the impact of two major dietary patterns and key nutrients in early life on the risk of offspring developing FA. The Western diet typically includes high sugar and high fat, which may affect the immune system of offspring and increase susceptibility to FA. In contrast, the Mediterranean diet is rich in fiber, which may reduce the risk of FA in offspring. Furthermore, we explore the potential mechanisms by which maternal dietary nutrients during a window of opportunity (pregnancy, birth, and lactation) influences the susceptibility of offspring to FA through multi-interface crosstalk. Finally, we discuss the limitations and gaps in the available evidence regarding the relationship between maternal dietary nutrients and the risk of FA in offspring. This review provides novel perspective on the regulation of offspring FA by maternal diet and nutrients.

Lipid matrix-specific pretreatment method for enhancing the extractability and allergenicity maintenance of bovine milk allergens in ELISA detection.

The presence of various components in the food matrix makes allergen detection difficult and inaccurate, and pretreatment is an innovative breakthrough point. Food matrices were categorised based on their composition. Subsequently, a pretreatment method was established using a combination of ultrasound-assisted n-hexane degreasing and weakly alkaline extraction systems to enhance the detection accuracy of bovine milk allergens. Results showed that more allergens were obtained with less structural destruction, as demonstrated using immunological quantification and spectral analysis. Concurrently, allergenicity preservation was confirmed through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, a KU812 cell degranulation model, and western blotting. The method exhibited good accuracy (bias, 8.47%), repeatability (RSDr, 1.52%), and stability (RSDR, 5.65%). In foods with high lipid content, such as chocolate, the allergen content was 2.29-fold higher than that of commercial kits. Laser confocal scanning microscopy (LCSM) and scanning electron microscopy (SEM) analyses revealed a significant decrease in fat content after post-pretreatment using our method. In addition, colloidal stability surpassed that achieved using commercial kits, as indicated through the PSA and zeta potential results. The results demonstrated the superiority of the extractability and allergenicity maintenance of lipid matrix-specific pretreatment methods for improving the accuracy of ELISA based allergen detection in real food.

Blockade of exosome release alleviates the hypersensitive reaction by influencing the T helper cell population in cow's milk allergic mice.

The aim of this work was to evaluate the ameliorative effects of exosome biogenesis in cow's milk allergy (CMA) response. In this context, BALB/c mice were systemically sensitized with cow's milk proteins plus an aluminum adjuvant to induce CMA. The inhibitor GW4869 of exosome biogenesis was added before sensitization and then the anaphylactic reactions were evaluated both in vivo (clinical score and body temperature) and in vitro (serum histamine, allergen-specific antibodies, cytokines by ELISA and cell analysis by flow cytometry) to explore the role of exosomes in the development of CMA. Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) showed that the size distribution and morphology of CMA-derived exosomes were not changed after GW4869 preconditioning, and the concentration of exosomes was much lower than that of the CMA group. In the GW4869 group, inhibition of release of exosomes modulated the induction of T helper 2 cell (Th2)-related substances, with a decrease in histamine and allergen-specific immunoglobulin (Ig) E, and the expression of Th1, Th2, and Th17 cells all decreased as well. Moreover, the experimental data were integrated by means of principal component analysis (PCA) to give an overview that the percentage of Th cells and concentrations of cytokines were more influenced by GW4869 treatment. These data for the first time demonstrated that exosomes are involved in the development of CMA and the blockade of exosome release with GW4869 suppressed the IgE-mediated immune response in CMA.

Effects of medicine food homologous materials on food allergy-associated factors: intestinal oxidative stress, intestinal inflammation and Th2 immune response.

BACKGROUND: Food allergies could be regulated via Th1/Th2 balance, intestinal oxidative stress and inflammation, which were considered as food allergy-associated factors. Medicine-food homologous materials (MFHM) were considered as a significant factor with respect to preventing human diseases. To evaluate the associations between MFHM and food allergy-associated factors, two types of MFHM with the remarkable function of anti-oxidation and anti-inflammation, Gardeniae fructus (Gar) and Sophorae glos (Sop), were chosen. RESULTS: By constructing an H2O2-induced oxidative stress model of Caco-2 cells and an intestinal inflammatory cell model of Caco-2 cells with tumor necrosis factor-α and interleukin (IL)-13, the contents of anti-oxidative enzymes (SOD and GSH), inflammatory factor (IL-8) and tight junction proteins (zonula occludens-1, occludin and claudin-1) in Caco-2 cells were determined. Moreover, the anti-allergic effects of digestive Sop and Gar were evaluated by measuring the levels of Th1/Th2/Treg cytokines in the spleen cells of sensitized mice. The results showed that the SOD and GSH were obviously increased and the gene and protein expression of IL-8 and claudin-1 were improved with the incubation of digested Sop. Th2 cytokine was reduced and Th1/Th2 balance was promoted on coincubation with ovalbumin (OVA) and digested Sop in the splenocytes. However, the digested Gar had no effect. CONCLUSION: The digested Sop not only had suppressive effects on intestinal oxidative stress and inflammation, but also had regulative effects on Th1/Th2 balance. This finding demonstrated that not all of the MFHM with anti-oxidant and anti-inflammatory effects have anti-allergic activities. The present study may be contributing toward establishing a screening model to identify the anti-allergic MFHM. © 2024 Society of Chemical Industry.

Active polysaccharides: a new roadmap for the prevention and treatment of food allergy.

Active polysaccharides are extensively utilized in the fields of food and medicine because of their rich functional properties and structural plasticity. However, there are still few systematic studies and reviews on active polysaccharides for allergy. Allergy, especially food allergy, occurs frequently around the world and is related to a variety of factors such as age, genetics and dietary habits. Currently in medicine, avoiding allergens and desensitizing can effectively relieve allergy symptoms, but these are difficult to maintain over the long term and come with risks. Based on the supplementation of dietary nutrition to these two treatments, it has been discovered in recent years that the use of active ingredients from natural substances can effectively intervene in allergies. Considering the potential of active polysaccharides in this regard, we systematically characterize the latent patterns of polysaccharides in allergic symptoms and pathogenesis, including the aspects of gut, immunomodulatory, oxidative stress and signaling pathways, as well as the application prospect of them in allergy. It can be found that active polysaccharides have excellent anti-allergic potential, especially from the ocean. We believe that the active polysaccharides are associated with the treatment of allergic diseases, which may provide the benefits to allergy sufferers in the future.

Oral exposure to Staphylococcus aureus enterotoxin B could promote the Ovalbumin-induced food allergy by enhancing the activation of DCs and T cells.

Introduction: Recent work highlighted the importance of environmental contaminants in the development of allergic diseases. Methods: The intestinal mucosal barrier, Th (helper T) cells, DCs (dendritic cells), and intestinal flora were analyzed with flow cytometry, RNA-seq, and 16s sequencing in the present study to demonstrate whether the exposure of enterotoxins like Staphylococcus aureus enterotoxin B (SEB) in allergens could promote the development of food allergy. Results and discussion: We found that co-exposure to SEB and Ovalbumin (OVA) could impair the intestinal barrier, imbalance the intestinal Th immune, and cause the decline of intestinal flora diversity in OVA-sensitized mice. Moreover, with the co-stimulation of SEB, the transport of OVA was enhanced in the Caco-2 cell monolayer, the uptake and presentation of OVA were promoted in the bone marrow dendritic cells (BMDCs), and Th cell differentiation was also enhanced. In summary, co-exposure to SEB in allergens should be considered a food allergy risk factor.

Staphylococcus aureus Enterotoxin B Is a Cofactor of Food Allergy beyond a Superantigen.

Staphylococcus aureus enterotoxin B (SEB), one of the most common bacterial toxins in food contamination, has been poorly understood in relationship to food allergy outcomes. To investigate whether the ingestion of enterotoxins in food allergens could affect the development of food allergy, OVA-sensitized female BALB/c mice were challenged with OVA added with different doses of SEB or LPS. Allergic symptoms, such as diarrhea rate and hypothermia, could be aggravated in mice challenged with OVA and a low dose of SEB. The increased differentiation of Th2 and reduced expression of CD103 in dendritic cells was found in mice coexposed to SEB and OVA. Additionally, there was an increasing differentiation of Th1 induced by a high dose of SEB. The expression of ST2+ in intestinal mast cells was also increased in mice sensitized with a low dose of SEB and OVA. Employing several in vitro cell culture models showed that the secretion of IL-33 from intestinal epithelial cells and IL-4 from group 2 innate lymphoid cells, activation of bone marrow-derived dendritic cells, and differentiation of naive T cells were induced by SEB and OVA. Our work proved that challenge with low-dose SEB and OVA partly aggravated the food allergy, suggesting a (to our knowledge) new finding of the potential cofactor of food allergy and that the contamination of SEB in food allergens deserves attention for allergic and normal individuals.

Development of a Two-Step Hydrolysis Hypoallergenic Cow's Milk Formula and Evaluation of Residue Allergenicity by Peptidomics and Immunoreactivity Analysis.

Cow's milk allergy (CMA) is an abnormal immune response that severely affects the nutritional supplementation of allergic infants. Currently, only a limited number of hypoallergenic formulas are available on the market, and these are only categorized according to their degree of hydrolysis, which still poses an allergy risk and cannot be consumed by CMA patients, especially infants. To address this issue, we developed a two-step hydrolysis hypoallergenic formula targeting destruction of allergen epitope from whey protein. Then, a comprehensive evaluation system was constructed, including peptidomics analysis, in vivo and in vitro allergenicity assessments, revealing allergic changes in the product from the epitope structure level to the immunological level. The results showed that 97.14% of hydrolyzed peptides from α-lactalbumin and ß-lactoglobulin did not contain allergenic epitopes after treatment with trypsin and flavourzyme. In vitro and in vivo allergenicity assessment results confirmed that the two-step hydrolysis method effectively reduced the allergenicity of whey protein. Compared with the common milk powder, the hypoallergenic formula induced lower levels of basophil degranulation and relieved the body's anaphylactic symptoms caused by cow milk. This study provides a promising solution to the limited hypoallergenic formula problem and may benefit allergic infants who require nutritional supplements.

New Perspectives on Food Matrix Modulation of Food Allergies: Immunomodulation and Component Interactions.

Food allergy is a multifactorial interplay process influenced not only by the structure and function of the allergen itself but also by other components of the food matrix. For food, before it is thoroughly digested and absorbed, numerous factors make the food matrix constantly change. This will also lead to changes in the chemistry, biochemical composition, and structure of the various components in the matrix, resulting in multifaceted effects on food allergies. In this review, we reveal the relationship between the food matrix and food allergies and outline the immune role of the components in the food matrix, while highlighting the ways and pathways in which the components in the food matrix interact and their impact on food allergies. The in-depth study of the food matrix will essentially explore the mechanism of food allergies and bring about new ideas and breakthroughs for the prevention and treatment of food allergies.

A Higher Dose of Staphylococcus aureus Enterotoxin B Led to More Th1 and Lower Th2/Th1 Ratio in Th Cells.

Exposure to Staphylococcus aureus enterotoxin B (SEB) is one of the causes of food poisoning and is associated with several immune diseases due to its superantigen capability. This study aimed to characterize the differentiations of naïve Th cells stimulated with different doses of SEB. The expression of T-bet, GATA-3, and Foxp3 or secretion of IFN-γ, IL-4, IL-5, IL-13, and IL-10 were evaluated in wild-type (WT) or DO11.10 CD4 T cells co-cultured with bone marrow dendritic cells (BMDCs). We found that the balance of Th1/Th2 could be dominated by the doses of SEB stimulation. A higher SEB dose could induce more Th1 and a lower Th2/Th1 ratio in Th cells co-cultured with BMDCs. This different tendency of Th cell differentiation induced by the SEB complements the existing knowledge about SEB acting as a superantigen to activate Th cells. Additionally, it is also helpful in managing the colonization of S. aureus and food contamination of SEB.

Goblet cell-associated antigen passage: A gatekeeper of the intestinal immune system.

Effective delivery of luminal antigens to the underlying immune system is the initial step in generating antigen-specific responses in the gut. However, a large body of information regarding the immune response activation process remains unknown. Recently, goblet cells (GCs) have been reported to form goblet cell-associated antigen passages (GAPs). Luminal antigens can be transported inside GAPs and reach subepithelial immune cells to induce antigen-specific immune responses, contributing largely to gut homeostasis and the prevention of some intestinal diseases like allergic enteritis and bacterial translocation. In this article, we summarized recent observations on the formation of intestinal GAPs and their roles in mucosal immunity. We hope that this review can offer a fresh perspective and valuable insights for clinicians and researchers interested in studying the intestinal immune system.

Establishment of Food Allergy Model in Dextran Sulfate Sodium Induced Colitis Mice.

Food allergy (FA) has become a global food safety issue. Evidence suggests that inflammatory bowel disease (IBD) can increase the incidence of FA, but it is mostly based on epidemiological studies. An animal model is pivotal for unraveling the mechanisms involved. However, dextran sulfate sodium (DSS)-induced IBD models may cause substantial animal losses. To better investigate the effect of IBD on FA, this study aimed to establish a murine model to fit both IBD and FA symptoms. Firstly, we compared three DSS-induced colitis models by monitoring survival rate, disease activity index, colon length, and spleen index, and then eliminated the colitis model with a 7-day administration of 4% due to high mortality. Moreover, we evaluated the modeling effects on FA and intestinal histopathology of the two models selected and found the modeling effects were similar in both the colitis model with a 7-day administration of 3% DSS and the colitis model with long-term administration of DSS. However, for animal survival reasons, we recommend the colitis model with long-term administration of DSS.

Changes in structural and functional properties of whey protein cross-linked by polyphenol oxidase.

The natural whey protein is unstable, to achieve more efficient utilization, the functional properties of whey protein were modified by changing its structure, and enzymatic cross-linking is one of the common methods in dairy products to change the functional characterization. This study was conducted with objective to evaluate the structural and functional of whey protein which was cross-linked by polyphenol oxidase from Agaricus bisporus. Whey protein was cross-linked by polyphenol oxidase, and the polymers and dimers were revealed by SDS-PAGE and LC-MS/MS, the structural alterations of the polymers were analyzed by UV-vis, fluorescence spectroscopy and SEM, and the effects of functional properties of whey protein after cross-linked were also explored. Results showed that dimer and high polymer of ß-lactoglobulin were formed, the secondary structure of whey protein was exhibited a significant variation, and the microstructure changed obviously. Moreover, the foaming and antioxidant activity of whey protein was enhanced although the emulsifying was reduced after cross-linked. These findings emphasize the feasible application of enzymatic cross-linking in improving the functional properties of whey protein, and provide a new direction for changing the traditional processing technology of whey protein and developing high-quality products.

Potential Allergenicity Response to Moringa oleifera Leaf Proteins in BALB/c Mice.

The reported association of Moringa oleifera seeds and allergic disease clinically resembling occupational asthma in cosmetic manufacturing workers has resultedin the need to identify such components in the manufacturing process. However, Moringa oleifera leaves from the same plant, an important food ingredient, have limited immunotoxicity data. This study aimed to determine if Moringa oleifera leafproteins (MLP) can elicit allergic responses in BALB/c mice. The BALB/c mice were sensitized twice and challenged 10 times to evaluate the potential allergenicityof MLP in vivo. The results showed increased levels of mast cells, total and specific IgE and IgG, severe signs of systemic anaphylaxis, and reduced body temperature compared with controls. The sensitized mice serum observed enhanced levels of histamine and Th-related cytokine release. Compared with the control group, increased levels of interleukins IL-4, IL-9, and IL-17A and enhanced expression and secretion of normal T cells were found in the culture supernatant of splenocytes treated with MLP.This study suggeststhat MLPcanelicit allergic responses; this providesmore comprehensive guidance for identifying new allergen candidates and developing hypoallergenic MLP products.

Allergenicity evaluation of peptides from milk and yogurt after gastrointestinal digestion based on epitopes.

As we know, milk and yogurt have good nutritional value and it is reported that some peptides can induce tolerance to alleviate or eliminate cow's milk allergy (CMA). However, there is a lack of detailed information on the peptides after digestion which could induce tolerance. In this study, the distribution pattern of digested proteins was detected during gastrointestinal digestion in infants and adults by Tricine-SDS-PAGE and RP-HPLC, and the digestive products were transported by the model of Caco-2 cells and the sequence of peptides was identified by LC-MS/MS. Residual allergenicity was evaluated by indirect ELISA during gastrointestinal digestion and the released peptides were aligned with T cell/IgE epitopes and biological functions by prediction software and previous information. These results indicated that the major allergens in yogurt were more easily digested with stronger transport capacity and had lower IgE-binding capacity. We obtained 113 peptides from the major allergens in the digested products and 38 of these peptides existed in all the digested products, among which 13 peptides had specific biological functions, such as ACE-inhibitory, antimicrobial and DPP-IV inhibitory properties. Although there was no obvious difference in the number of epitopes between fresh milk and yogurt, the difference in the properties and content of specific peptides might be the key factor for the difference in allergenicity. Most importantly, 11 peptides that contained T cell epitopes but not IgE epitopes might induce immune tolerance in CMA, which should be confirmed further.

Potential allergenicity and hydrolysis assessment of bovine casein and ß-casein by treatment with lactic acid bacteria.

Casein is one of the main allergens in cow's milk, accounting for 80% of cow's milk proteins. The ability of hydrolyzing proteins by bacteria is also different. In this study, the capacity of lactic acid bacteria to hydrolyze casein or ß-casein and the IgG/IgE-binding capacity of hydrolysates were evaluated. The intensity of casein and ß-casein degradation was analyzed by SDS-PAGE and RP-HPLC. The hydrolysates were tested for their capacity to inhibit IgG and IgE binding by ELISA. The peptides in the hydrolysate were also analyzed by LC-MS/MS. In these strains, Lactobacillus rhamnosus (CICC No. 22175) had the strongest hydrolysis of casein and ß-casein. The hydrolysate of Lactobacillus rhamnosus (CICC No. 22175) showed the lowest antigenicity and potential allergenicity. It also hydrolyzed major allergen IgE epitopes and preserved T cell epitopes. Thereore Lactobacillus rhamnosus (CICC No. 22175) could be used for developing hypoallergenic dairy products and the development of tolerance. PRACTICAL APPLICATIONS: By the study, it obtained that a strain of Lactobacillus rhamnosus could effectively degrade casein and reduced the potential allergenicity of casein. At the same time, some major allergic epitopes were hydrolyzed and T cell epitopes were preserved. Therefore, it is very valuable for the application and development of lactic acid bacteria. The hydrolysate can also be used in a new hypoallergenic dairy formula with specific health benefits and promoting oral tolerance.

Identification of immunoglobulin E epitopes on major allergens from dairy products after digestion and transportation in vitro.

Dairy processing can alter the digestion stability and bioavailability of cow milk proteins in the gastrointestinal tract. However, analysis of stable linear epitopes on cow milk allergens that could enter into intestinal mucosal is limited. Thus, this study aimed to investigate the digestion and transportation properties and residual allergen epitopes entering into gastrointestinal mucosa of 3 commercial dairy products, including pasteurized milk (PM), ultra-heat-treated milk (UHTM), and dried skim milk (DSM). In this work, the digestive stability of the 3 kinds of dairy products has been performed in a standard multistep static digestion model in vitro and characterized by Tricine-SDS-polyacrylamide gel electrophoresis and reversed-phase HPLC. With respect to gastrointestinal digestion in vitro, the main allergens including ß-lactoglobulin (ß-LG), α-lactalbumin (α-LA), and caseins were degraded gradually, and the resistance peptides remained in the PM with a molecular weight of range from 3.4 to 5.0 kDa. Simultaneously, the potential allergenicity of the cow milk proteins was diminished gradually and is basically consistent after 60 min of gastrointestinal digestion. After gastrointestinal digestion, the remaining peptides were transported via an Ussing chamber and identified by liquid chromatography-MS/MS. By alignment, 10 epitopes peptides were identified from 16 stable peptides, including 5 peptides (AA 92-100, 125-135, 125-138, and 149-162) in ß-LG, 2 peptides in α-LA (AA 80-93 and 63-79), 2 peptides in αS1-casein (AA 84-90 and 125-132), and 1 peptide (AA 25-32) in αS2-casein were identified by dot-blotting mainly exist in UHTM and PM. This study demonstrates dairy processing can affect the digestion and transport characteristics of milk proteins and in turn alter epitope peptides release.

Digestive stability and transport ability changes of ß-lactoglobulin-catechin complexes by M cell model in vitro.

The current research on interaction between catechin and protein has focused on non-covalent crosslinking, however, the mechanism of free radical-induced crosslinking between catechin and ß-lactoglobulin (BLG) is not known. In this study, BLG bound to four catechins [epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG)]. The structure change of complex was investigated by circular dichroism spectroscopy, ultraviolet-visible (UV-vis) spectroscopy and Acid and 8-Anilino-1-naphthalenesulfonic acid (ANS) fluorescence spectroscopy. M cell model was constructed to evaluate the transintestinal epithelial transport capacity of complex digestive products. The results showed that catechins were covalently bound to BLG by C-S and C-N bonds and their binding content was EGCG>EGC>ECG>EC. Moreover, catechins could change the secondary structure of BLG, with the decrease of α-helix and reduction of the irregular coilings, which leads to the loose spatial structure of the protein. Moreover, the catechin could enhance further the digestibility of BLG. Transport capacity of digestive products of M cell model was about twice of that of the Caco-2 cell model, indicating that M cell model had better antigen transport capacity. The difference between groups indicated that the transport efficiency of digestive products was decreased with the presence of catechin, in which BLG-EGCG and BLG-EGC groups were transported more strong than those of BLG-EC and BLG-ECG groups. The transport efficiency of BLG-catechin complexes were lower than that of BLG, indicating that catechin had the protective and repair roles on intestinal barrier permeability.