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Modulation of Milk Allergenicity by Baking Milk in Foods: A Proteomic Investigation.

ABSTRACT: Cow's milk is considered the best wholesome supplement for children since it is highly enriched with micro and macro nutrients. Although the protein fraction is composed of more than 25 proteins, only a few of them are capable of triggering allergic reactions in sensitive consumers. The balance in protein composition plays an important role in the sensitization capacity of cow's milk, and its modification can increase the immunological response in allergic patients. In particular, the heating treatments in the presence of a food matrix have demonstrated a decrease in the milk allergenicity and this has also proved to play a pivotal role in developing tolerance towards milk. In this paper we investigated the effect of thermal treatment like baking of cow's milk proteins that were employed as ingredients in the preparation of muffins. A proteomic workflow was applied to the analysis of the protein bands highlighted along the SDS gel followed by western blot analyses with sera of milk allergic children in order to have deeper information on the impact of the heating on the epitopes and consequent IgE recognition. Our results show that incorporating milk in muffins might promote the formation of complex milk-food components and induce a modulation of the immunoreactivity towards milk allergens compared to milk baked in the oven at 180 °C for ten minutes. The interactions between milk proteins and food components during heating proved to play a role in the potential reduction of allergenicity as assessed by in vitro tests. This would help, in perspective, in designing strategies for improving milk tolerance in young patients affected from severe milk allergies.

SUBMITTER: Bavaro SL

PROVIDER: S-EPMC6683078 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Modulation of Milk Allergenicity by Baking Milk in Foods: A Proteomic Investigation.

Bavaro Simona L SL De Angelis Elisabetta E Barni Simona S Pilolli Rosa R Mori Francesca F Novembre Elio M EM Monaci Linda L

Nutrients 20190706 7

Cow's milk is considered the best wholesome supplement for children since it is highly enriched with micro and macro nutrients. Although the protein fraction is composed of more than 25 proteins, only a few of them are capable of triggering allergic reactions in sensitive consumers. The balance in protein composition plays an important role in the sensitization capacity of cow's milk, and its modification can increase the immunological response in allergic patients. In particular, the heating tr ...[more]

PMID: 31284583

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Project description:BackgroundSeveral studies demonstrated the adverse effect of milk processing on the allergy-protective capacity of raw cow's milk. Whether milk processing also affects the allergenicity of raw milk is hardly investigated.ObjectiveTo assess the allergenicity of raw (unprocessed) and processed cow's milk in a murine model for food allergy as well as in cow's milk allergic children.MethodsC3H/HeOuJ mice were either sensitized to whole milk (raw cow's milk, heated raw cow's milk or shop milk [store-bought milk]) and challenged with cow's milk protein or they were sensitized and challenged to whey proteins (native or heated). Acute allergic symptoms, mast cell degranulation, allergen-specific IgE levels and cytokine concentrations were determined upon challenge. Cow's milk allergic children were tested in an oral provocation pilot with organic raw and conventional shop milk.ResultsMice sensitized to raw milk showed fewer acute allergic symptoms upon intradermal challenge than mice sensitized to processed milk. The acute allergic skin response was low (103 ± 8.5 µm vs 195 ± 17.7 µm for heated raw milk, P < 0.0001 and vs 149 ± 13.6 µm for shop milk, P = 0.0316), and there were no anaphylactic shock symptoms and no anaphylactic shock-induced drop in body temperature. Moreover, allergen-specific IgE levels and Th2 cytokines were significantly lower in raw milk sensitized mice. Interestingly, the reduced sensitizing capacity was preserved in the isolated native whey protein fraction of raw milk. Besides, native whey protein challenge diminished allergic symptoms in mice sensitized to heated whey proteins. In an oral provocation pilot, cow's milk allergic children tolerated raw milk up to 50 mL, whereas they only tolerated 8.6 ± 5.3 mL shop milk (P = 0.0078).Conclusion and clinical relevanceThis study demonstrates that raw (unprocessed) cow's milk and native whey proteins have a lower allergenicity than their processed counterparts. The preclinical evidence in combination with the human proof-of-concept provocation pilot provides evidence that milk processing negatively influences the allergenicity of milk.

Project description:BACKGROUND:Novel foods may provide new protein sources for a growing world population but entail risks of unexpected food-allergic reactions. No guidance on allergenicity assessment of novel foods exists, while for genetically modified (GM) crops it includes comparison of sequence identity with known allergens, digestibility tests and IgE serum screening. OBJECTIVE:As a proof of concept, to evaluate non-/allergenic tropomyosins (TMs) regarding their potential as new calibrator proteins in functional biological in vitro assays for the semi-quantitative allergy risk assessment of novel TM-containing animal foods with mealworm TM as an example. METHODS:Purified TMs (shrimp, Penaeus monodon; chicken Gallus gallus; E coli overexpression) were compared by protein sequencing, circular dichroism analysis and in vitro digestion. IgE binding was quantified using shrimp-allergic patients' sera (ELISA). Biological activities were investigated (skin testing; titrated basophil activation tests, BAT), compared to titrated biological mediator release using humanized rat basophil leukaemia (RBL) cells. RESULTS:Shrimp and chicken TMs showed high sequence homology, both alpha-helical structures and thermal stability. Shrimp TM was stable during in vitro gastric digestion, chicken TM degraded quickly. Both TMs bound specific IgE from shrimp-allergic patients (significantly higher for shrimp TM), whereas skin reactivity was mostly positive with only shrimp TM. BAT and RBL cell assays were positive with shrimp and chicken TM, although at up to 100- to 1000-times lower allergen concentrations for shrimp than chicken TM. In RBL cell assays using both TM as calibrators, an activation of effector cells by mealworm TM similar to that by shrimp TM confirmed the already reported high allergenic potency of mealworm TM as a novel protein source. CONCLUSIONS & CLINICAL RELEVANCE:According to current GM crops' allergenicity assessment, non-allergenic chicken TM could falsely be considered an allergen on a weight-of-evidence approach. However, calibrating allergenic potency in functional BAT and RBL cell assays with clinically validated TMs allowed for semi-quantitative discrimination of novel food protein's allergenicity. With TM calibration as a proof of concept, similar systems of homologous protein might be developed to scale on an axis of allergenicity.

Dataset Information

Modulation of Milk Allergenicity by Baking Milk in Foods: A Proteomic Investigation.

Publications

Modulation of Milk Allergenicity by Baking Milk in Foods: A Proteomic Investigation.

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