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.

A rapid immunomagnetic beads-based sELISA method for the detection of bovine αs1-casein based on specific epitopes.

Although αs1-casein poses significant health risks to individuals with milk allergies, the availability of quantification methods for this allergen remains limited. In this study, we developed an immunomagnetic beads-based immunoassay (IMBs-ELISA) for the precise quantitative detection of bovine αs1-CN, specifically targeting epitope AA173-194. No cross-reactivity was observed with the other 7 food allergens including milk allergen. The linear detection range of the established IMBs-ELISA method was 0.125 µg/mL-2.000 µg/mL, with a limit of detection of 0.099 µg/mL. The accuracy of this method was 1.048 %, and the intra-plate and inter-plate precision achieved 4.100 % and 6.777 %, respectively. Notably, the entire IMBs-ELISA process could be completed within 75 min, representing a substantial time-saving advantage over traditional ELISA methods. These results proved the reliability and rapidity of the IMBs-ELISA method for detecting αs1-CN in real food.

Glutamine and lysine as common residues from epitopes on α-lactalbumin and ß-lactoglobulin from cow milk identified by phage display technology.

Cow milk is an important source of food protein for children; however, it could lead to allergy, especially for infants. α-Lactalbumin (α-LA) and ß-lactoglobulin (ß-LG) from whey protein make up a relatively high proportion of milk proteins and have received widespread attention as major allergens in milk. However, few studies have identified the epitopes of both proteins simultaneously. In this study, ImmunoCAP and indirect ELISA were first used for detection of sIgE to screen sera from allergic patients with high binding capacity for α-LA and ß-LG. Subsequently, the mimotopes was biopanned by phage display technology and bioinformatics and 17 mimic peptide sequences were obtained. Aligned with the sequences of α-LA or ß-LG, we identified one linear epitope on α-LA at AA 11-26 and 5 linear epitopes on ß-LG at AA 9-29, AA 45-57, AA 77-80, AA 98-101, and AA 121-135, respectively. Meanwhile, the 8 conformational epitopes and their distributions of α-LA and ß-LG were located using the Pepitope Server. Finally, glutamine and lysine were determined as common AA residues for the conformational epitopes both on α-LA and ß-LG. Moreover, we found the addition of mouse anti-human IgE during the biopanning process did not significantly affect the identification of the epitopes.

Allergenicity reduction of cow milk treated by alkaline protease combined with Lactobacillus Plantarum and Lactobacillus helveticus based on epitopes.

This paper has investigated the residual allergenicity of cow's milk treated by enzymatic hydrolysis combined with Lactobacillus fermentation (Lb. Plantarum and Lb. helveticus). The treated products were comprehensively evaluated by SDS-PAGE, RP-HPLC, ELISA, and Caco-2 models. And the allergenic changes of residual allergenic peptides were explored by DC-T co-culture. The results showed that alkaline protease was the most suitable protease that targeted to destroy epitopes of milk major allergen than trypsin, pepsin, and papain by prediction. And the residual epitopes were reduced to four which was treated by alkaline protease combined with Lb. helveticus. The transport absorption capacity of treated products was almost twice than milk. Meanwhile, the seven residual allergenic peptides were obtained from treated products. Among them, αs1-casein (AA84-90) can be used as an immune tolerance peptide for further study. Lb. helveticus combined with alkaline protease treatment may be considered promising strategy of protect from cow's milk allergy.

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.