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Cow milk protein allergy (CMPA) is the most common food allergy in infants. Currently, hypoallergenic infant formulas on the market are mainly divided into extensively hydrolyzed whey protein formulas and extensively hydrolyzed casein formulas. There are few extensively hydrolyzed infant formulas (EHFs) with a similar protein composition to breast milk. Therefore, we developed a hypoallergenic infant formula based on extensively hydrolyzed cow milk protein (whey protein-to-casein ratio of 6 : 4) and evaluated its allergenicity in vitro and in vivo . The results showed that the antigenicity of EHF was significantly decreased. The levels of Treg and Th1 cells were increased, while the levels of Th2 cells, IgE and IgG1, plasma histamine and serum mast cell enzymes were significantly decreased. At the same time, the allergic symptoms of the jejunum and lungs of mice were relieved. This study provides a solution for the development of cow milk protein based hypoallergenic infant formulas.

Human clinical trials have shown that a specific partially hydrolyzed 100% whey-based infant formula (pHF-W) reduces AD risk in the first yeast of life. Meta-analyses with a specific pHF-W (pHF-W1) confirm a protective effect while other meta-analyses pooling different pHF-W show conflicting results. Here we investigated the molecular composition and functional properties of the specific pHF-W1 as well as the stability of its manufacturing process over time. This specific pHF-W1 was compared with other pHF-Ws. We used size exclusion chromatography to characterize the peptide molecular weight (MW), a rat basophil degranulation assay to assess the relative level of beta-lactoglobulin (BLG) allergenicity and a preclinical model of oral tolerance induction to test prevention of allergic sensitization. To analyze the exact peptide sequences before and after an HLA binding assay, a mass cytometry approach was used. Peptide size allergenicity and oral tolerance induction were conserved across pHF-W1 batches of production and time. The median MW of the 37 samples of pHF-W1 tested was 800 ± 400 Da. Further oral tolerance induction was observed using 10 different batches of the pHF-W1 with a mean reduction of BLG-specific IgE levels of 0.76 log (95% CI = -0.95; -0.57). When comparing pHF-W1 with three other formulas (pHF-W2 3 and 4), peptide size was not necessarily associated with allergenicity reduction in vitro nor oral tolerance induction in vivo as measured by specific IgE level ( p < 0.05 for pHF-W1 and 2 and p = 0.271 and p = 0.189 for pHF-W3 and 4 respectively). Peptide composition showed a limited overlap between the formulas tested ranging from 11.7% to 24.2%. Furthermore nine regions in the BLG sequence were identified as binding HLA-DR. In conclusion, not all pHF-Ws tested have the same peptide size distribution decreased allergenicity and ability to induce oral tolerance. Specific peptides are released during the different processes used by different infant formula producers.

Thermal treatments of milk induce changes in the properties of milk whey proteins. The aim of this study was to investigate the specific changes related to nutrients in the whey proteins of dairy cow milk after pasteurization at 85 °C for 15 s or ultra-high temperature (UHT) at 135 °C for 15 s. A total of 223 whey proteins were confidently identified and quantified by TMT-based global discovery proteomics in this study. We found that UHT thermal treatment resulted in an increased abundance of 17 proteins, which appeared to show heat insensitivity. In contrast, 15 heat-sensitive proteins were decreased in abundance after UHT thermal treatment. Some of the heat-sensitive proteins were connected with the biological immune functionality, suggesting that UHT thermal treatment results in a partial loss of immune function in the whey proteins of dairy cow milk. The information reported here will considerably expand our knowledge about the degree of heat sensitivity in the whey proteins of dairy cow milk in response to different thermal treatments and offer a knowledge-based reference to aid in choosing dairy products. It is worth noting that the whey proteins (lactoperoxidase and lactoperoxidase) in milk that were significantly decreased by high heat treatment in a previous study (142 °C) showed no significant difference in the present study (135 °C). These results may imply that an appropriately reduced heating intensity of UHT retains the immunoactive proteins to the maximum extent possible.

Cow's milk allergy is a common food allergy in infants, and is associated with an increased risk of developing other allergic diseases. Dietary selenium (Se), one of the essential micronutrients for humans and animals, is an important bioelement which can influence both innate and adaptive immune responses. However, the effects of Se on food allergy are still largely unknown. In the current study it was investigated whether dietary Se supplementation can inhibit whey-induced food allergy in an animal research model. Three-week-old female C3H/HeOuJ mice were intragastrically sensitized with whey protein and cholera toxin and randomly assigned to receive a control, low, medium or high Se diet. Acute allergic symptoms, allergen specific immunoglobulin (Ig) E levels and mast cell degranulation were determined upon whey challenge. Body temperature was significantly higher in mice that received the medium Se diet 60 min after the oral challenge with whey compared to the positive control group, which is indicative of impaired anaphylaxis. This was accompanied by reductions in antigen-specific immunoglobulins and reduced levels of mouse mast cell protease-1 (mMCP-1). This study demonstrates that oral Se supplementation may modulate allergic responses to whey by decreasing specific antibody responses and mMCP-1 release.

Due to the prevalence and severity of cow milk (CM) allergy (CMA), an ideal substitute is urgently needed to develop hypoallergenic infant formula for infants who experience anaphylaxis to typical whey-based CM formula. Goat milk (GM) and horse milk (HM) are considered appropriate substitutes; however, whether GM and HM are less allergenic than CM is unclear. In the present study, the difference in allergenicity among CM, GM, and HM was investigated using the Balb/c mouse model. The number of mice with severe respiratory symptoms was significantly lower in the GM- and HM-sensitised groups than in the CM-sensitised group. Furthermore, histologic examination of intestinal and lung tissues revealed a thinner lamina propria of the small intestine and obvious inflammation and congestion in lungs in the CM-sensitised group than in the GM- and HM-sensitised groups. CM-specific immunoglobulin (Ig) E, serum IgG1, and plasma histamine levels were also higher in CM-sensitised mice than in GM- or HM-sensitised mice. In addition, higher interleukin (IL) 4 and IL-17A levels and lower interferon-γ (IFN-γ) and IL-10 levels were observed in CM-sensitised mice compared with GM- and HM-sensitised mice, according to qPCR, indicating Th1/Th2 and Treg/Th17 imbalances. The CM-sensitised group had a higher proportion of IL-4- and IL-17A-producing CD3+ T cells but a lower proportion of IFN-γ- and IL-10-producing CD3+ T cells compared with the GM- and HM-sensitised groups, confirming the Th1/Th2 and Treg/Th17 imbalances. In conclusion, GM and HM were less allergenic than CM in mice as a result of a shift in the Th1/Th2 and Treg/Th17 imbalances; however, HM was less allergenic than GM and can be used as an alternative milk to develop infant formulas for children with CMA.

Casein, β-lactoglobulin and α-lactalbumin are major milk protein allergens. In the present study, the structural modifications and antigenic response of these bovine milk allergens as induced by non-thermal treatment by atmospheric cold plasma were investigated. Spark discharge (SD) and glow discharge (GD), as previously characterized cold plasma systems, were used for protein treatments. Casein, β-lactoglobulin and α-lactalbumin were analyzed before and after plasma treatment using SDS-PAGE, FTIR, UPLC-MS/MS and ELISA. SDS-PAGE results revealed a reduction in the casein and α-lactalbumin intensity bands after SD or GD treatments; however, the β-lactoglobulin intensity band remained unchanged. FTIR studies revealed alterations in protein secondary structure induced by plasma, particularly contents of β-sheet and β-turn. The UPLC-MS/MS results showed that the amino acid compositions decreased after plasma treatments. ELISA of casein and α-lactalbumin showed a decrease in antigenicity post plasma treatment, whereas ELISA of β-lactoglobulin showed an increase in antigenicity. The study indicates that atmospheric cold plasma can be tailored to mitigate the risk of bovine milk allergens in the dairy processing and ingredients sectors., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Several formulas are available for the dietary treatment of cow's milk allergy (CMA). Clinical data suggest potentially different effect on immune tolerance elicited by these formulas. We aimed to comparatively evaluate the tolerogenic effect elicited by the protein fraction of different formulas available for the dietary treatment of CMA. Five formulas were compared: extensively hydrolyzed whey formula (EHWF), extensively hydrolyzed casein formula (EHCF), hydrolyzed rice formula (HRF), soy formula (SF), and amino acid-based formula (AAF). The formulas were reconstituted in water according to the manufacturer's instructions and subjected to an in vitro infant gut simulated digestion using a sequential gastric and duodenal static model. Protein fraction was then purified and used for the experiments on non-immune and immune components of tolerance network in human enterocytes and in peripheral mononuclear blood cells (PBMCs). We assessed epithelial layer permeability and tight junction proteins (occludin and zonula occludens-1, ZO-1), mucin 5AC, IL-33, and thymic stromal lymphopoietin (TSLP) in human enterocytes. In addition, Th1/Th2 cytokine response and Tregs activation were investigated in PBMCs from IgE-mediated CMA infants. EHCF-derived protein fraction positively modulated the expression of gut barrier components (mucin 5AC, occludin and ZO-1) in human enterocytes, while SF was able to stimulate the expression of occludin only. EHWF and HRF protein fractions elicited a significant increase in TSLP production, while IL-33 release was significantly increased by HRF and SF protein fractions in human enterocytes. Only EHCF-derived protein fraction elicited an increase of the tolerogenic cytokines production (IL-10, IFN- γ ) and of activated CD4+FoxP3+ Treg number, through NFAT , AP1 , and Nf-Kb1 pathway. The effect paralleled with an up-regulation of FoxP3 demethylation rate. Protein fraction from all the study formulas was unable to induce Th2 cytokines production. The results suggest a different regulatory action on tolerogenic mechanisms elicited by protein fraction from different formulas commonly used for CMA management. EHCF-derived protein fraction was able to elicit tolerogenic effect through at least in part an epigenetic modulation of FoxP3 gene. These results could explain the different clinical effects observed on immune tolerance acquisition in CMA patients and on allergy prevention in children at risk for atopy observed using EHCF., Competing Interests: The Department of Translational Medical Science received research grants from Nestlè, Vevey, Switzerland; Kraft Heinz Chicago, Illinois, USA; Danone, Paris, France; Mead Johnson, Evansville, IN, USA; and Novalac, Paris, France. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Paparo, Picariello, Bruno, Pisapia, Canale, Sarracino, Nocerino, Carucci, Cosenza, Cozzolino and Berni Canani.)

There is no effective therapy for milk allergy. The role of lactic acid bacteria (LAB) and probiotics in protection against allergy-related outcomes is still under investigation. The aim of the study was to evaluate the immunomodulative and therapeutic potential of yogurt drinks in cow's milk allergy (CMA) management. We compared immunoreactivity of α-casein (α-CN), β-casein (β-CN), κ-casein (κ-CN), α-lactalbumin (α-LA), and β-lactoglobulin (β-LG) in 27 yogurt drinks fermented with different basic yogurt cultures, or yogurt cultures enriched with Lactobacillus plantarum and/or Bifidobacterium lactis strains, by competitive ELISA assay. Drinks with the lowest antigenic potential were used as allergoids for CMA therapy. BALB/c mice were sensitized via intraperitoneal injection of α-CN + β-LG mixture with aluminum adjuvant, and gavaged with increasing doses of selected low-immunogenic drinks (YM-basic, or YM-LB-enriched with L. plantarum and B. lactis ) to induce tolerance. Milk- or phosphate-buffered saline (PBS)-dosed mice served as controls. Compared to milk, the immunoreactivity of proteins in drinks increased or decreased, depending on the bacterial sets applied for fermentation. Only a few sets acted synergistically in reducing immunoreactivity. The selected low-immunogenic drinks stimulated allergic mice for profiling Th2 to Th1 response and acquire tolerance, and the effect was greater with YM-LB drink, which during long-lasting interventional feeding strongly increased the secretion of regulatory cytokines, i.e., IL-10 and TGF-β, and IgA and decreased IL-4, IgE, and anti-(α-CN + β-LG) IgG 1 . The studies revealed variations in the potency of yogurt bacteria to change allergenicity of milk proteins and the need for their strict selection to obtain a safe product for allergy sufferers. The YM-LB drink with reduced antigenic potential may be a source of allergoids used in the immunotherapy of IgE mediated CMA, but further clinical or volunteer studies are required.

This research communication relates to the hypothesis that the consumption of raw or unprocessed cow's milk contributes to lowered prevalence of allergies. Thermal pasteurization of bovine milk can result in denaturation of minor whey proteins and loss of their bioactivity. Denaturation of bovine serum albumin (BSA), immunoglobulin G (IgG) and lactoferrin (LF) in skim milk was studied under different temperature (72, 75 or 78°C) and time (0-300 s) combinations. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that denaturation of all 3 proteins occurred at 72°C and progressed with increase in temperature and holding time. About 59% of LF and 12% of IgG denatured under high-temperature short-time (72°C/ 15 s) pasteurization, while BSA was least impacted. To assess modulation of milk immunogenicity, secretion of selected T helper (Th)-type cytokines by human peripheral blood mononuclear cells (PBMCs) was studied in vitro in response to different concentrations of BSA (0.4-1.0 mg/ml) and IgG (0.8-1.6 mg/ml) in unheated skim milk. Addition of IgG at 1.6 mg/ml induced a prominent Th1-skewed cytokine profile that may not trigger a Th2-skewed allergic reaction. BSA did not appear to modulate milk immunogenicity to any significant extent.

Introduction: This 4-center study is part of a project to validate a food allergy murine model for safety testing of hydrolyzed infant formulas., Aim: The aim of the current multi-center experiment was to evaluate the residual allergenicity of three partial hydrolyzed whey proteins (pWH) in a multiple-parameter cow's milk allergy murine model and to compare to the classically used guinea pig model. Previous work showed differences in the magnitude of the allergic response to whey between centers. To get a first insight in the effect of housing on the robustness of the mouse model, microbiota composition of non-sensitized mice was analyzed and compared between centers., Methods: Mice were sensitized intragastrically (i.g.) with whey, pWH or eWH using cholera toxin as an adjuvant. In mice, whey-IgE/IgG1, acute allergic symptoms were determined upon whey challenge. Guinea pigs were orally sensitized ad libitum via the drinking water (day 0-37) and challenged intravenously with whey on day 49. The microbial composition in fecal samples was determined in non-sensitized mice in all 4 research centers before and after conduct of the study., Results: Elevated levels of whey-IgG1 were detected in whey-sensitized mice in all centers. Except for pWH-A in center 4, we observed elevated levels of whey-IgE in whey-sensitized mice and mice sensitized with pWH-A, -B, -C. Center 2 was excluded from further analysis because of non-significant IgE levels in the positive control. In contrast to whey-mice, pWH-A treated mice showed no acute skin response, mMCP-1 release or change in body temperature upon whey challenge in all centers, which corresponds with the absence of anaphylactic shock symptoms in both the mouse and guinea pig model. pWH-B and -C induced anaphylactic shock symptoms in the guinea-pig and mice whereas results on the remaining allergic outcomes in mice were inconclusive. No differences in microbiota composition were measured in response to the challenge and Microbiota composition depended on the location of the centers., Conclusions: Both animal models showed comparable results on the residual allergenicity of partial hydrolyzed whey proteins, but none of the centers was able to differentiate between the residual sensitizing capacities of the pWH-B and -C based on a single elicitation parameter in the murine model. Differences in microbiota composition might contribute to the robustness of the food allergy murine model. For a well-balanced prediction on the potential allergenicity of hydrolyzed infant formulas a multiple murine parameter model is suggested to decrease the risk of false positive or false negative results. A future challenge is to develop an overall scoring system for proper risk assessment, taking all parameters into account., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Food processing, including heat-treatment, can affect protein structure and stability, and consequently affect protein immunogenicity and allergenicity. A few studies have shown that structural changes induced by heat-treatment impact the intestinal protein uptake and suggest this as a contributing factor for altered allergenicity., Objective: To investigate the impact of heat-treatment of a whey-based protein product on allergenicity and tolerogenicity as well as on intestinal uptake in various animal models., Methods: Immunogenicity and sensitizing capacity of the heat-treated whey product were compared to that of the unmodified product by intraperitoneal and oral exposure studies, while tolerogenic properties were assessed by oral primary prevention and desensitization studies in high-IgE responder Brown Norway rats., Results: Heat-treatment of whey induced partial protein denaturation and aggregation, which reduced the intraperitoneal sensitizing capacity but not immunogenicity. In contrast, heat-treatment did not influence the oral sensitizing capacity, but the heat-treated whey showed a significantly reduced eliciting capacity compared to unmodified whey upon oral challenge. Heat-treatment did not reduce the tolerogenic properties of whey, as both products were equally good at preventing sensitization in naïve rats as well as desensitizing already sensitized rats. Results from inhibitory ELISA and immunoblots with sera from sensitized rats demonstrated that heat-treatment caused an altered protein and epitope reactivity. Protein uptake studies showed that heat-treatment changed the route of uptake with less whey being absorbed through the epithelium but more into the Peyer's patches., Conclusion and Clinical Relevance: These results support the notion that the physicochemical features of proteins affect their route of uptake and that the route of uptake may affect the protein allergenicity. Furthermore, the study highlights the potential for heat-treatment in the production of efficient and safe cow's milk protein-based products for prevention and treatment of cow's milk allergy., (© 2020 John Wiley & Sons Ltd.)

Milk from schistosomotic mothers can modulate the immune response of their offspring. However, its characterization and potential of modulating immunity has not yet been fully elucidated. Thus, the aim of this study was to evaluate whey proteins from the milk of Schistosoma mansoni-infected mice in order to identify the fractions which can act as potential immunomodulatory tools. For this, we did a mass spectrometry (nanoUPLC-MSE) analysis to characterize the proteomic profile of milk from infected (MIM) and non-infected mice (MNIM). It was possible to identify 29 differentially expressed proteins: 15 were only found in MIM, 10 only found in MNIM, and 4 were downregulated in MIM group. Gene Ontology (GO), pathway enrichment analysis, and protein-protein interaction (PPI) analyses indicated differentially expressed proteins linked to biological processes and pathways in MIM group such as the following: fructose 1,6-biphosphate metabolic and glycolytic processes, glucose metabolism, and neutrophil degranulation pathways. The downregulated and unique proteins identified in MNIM group were involved in the positive regulation of B cell activation and receptor signaling pathway, in the innate immune response, complement activation, and phagocytosis. The present findings revealed a protein profile that may be involved in the activation and deactivation of the offspring's immune system in the long term, conferring a protective character due to the previous contact with milk from infected mothers.

This work investigated the reduction of bovine whey proteins antigenicity by ultrasonic pretreatment and microbial fermentation. Firstly, bovine whey proteins was pretreated by ultrasonic techniques, and its secondary structure was detected by circular dichroism. The pretreated whey proteins was used as the fermentation substrate by Kluyveromyces marxianus for microbial transformation. The single factor design and Box-Behnken Design (BBD) were carried out with the aim to optimize culture temperature, initial pH, inoculum volume and rotation speed. After optimization process, culture temperature, initial pH, inoculum volume and rotation speed were determined. Under culture temperature 35 °C, pH 7.25, inoculum level 10% and shaking speed 150 rpm, the α-LA and β-LG antigenicity in bovine whey proteins were reduced by 29% and 53%, respectively. The findings showed that combined with microbial fermentation for hydrolysis of whey proteins, ultrasonic pretreatment can be used in order to produce hypoallergenic bovine whey proteins., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

With the aim to reduce the antigenicity of whey protein hydrolysate in milk, the pretreatment method of coupling ultrasonic and ionic liquid (US-IL) and further enzymatic treatments were studied. Papain and alcalase were found to be suitable for ultrasonic-ionic liquid pretreatment. After ultrasound-ionic liquid treatment, the antigenic decline rates of ALA and BLG upon alcalase hydrolysis were 82.82% and 88.01%, and that of the papain hydrolysis was 81.87% and 88.46%, respectively. Upon ultrasonic-ionic liquid pretreatment, the molecular weight of whey protein did not change significantly, but the small molecular weight proportion of components in the enzymatic hydrolysate obviously increased. The findings showed that combining with US-IL pretreatment for further protease hydrolysis of whey proteins, the hydrolysate can be used in order to produce hypoallergenic bovine whey proteins., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Hydrolyzed cow's milk protein formulas are widely used for infants with a history or risk of cow's milk allergy. Based on the current theory that food allergen sensitization occurs via the skin, we investigated the epicutaneous immunogenicity of partially hydrolyzed whey proteins, which are ingredients in infant formulas., Methods: BALB/c mice were exposed epicutaneously to whey protein concentrate (WPC) or partial whey protein hydrolysates (PWH1 or PWH2) on tape-stripped skin. Sensitization was assessed by evaluating serum β-lactoglobulin (β-LG)-specific antibodies, basophil activation, and cytokine production from β-LG-stimulated lymphoid cells. The anaphylaxis reaction was evaluated by measuring the rectal temperature and plasma level of mouse mast cell protease-1 after oral β-LG challenge. Immune cell accumulation in the skin was also analyzed., Results: Substantive sensitization and β-LG-induced anaphylaxis reaction were observed in WPC-exposed mice, whereas no significant changes were observed in PWH1- or PWH2-exposed mice. The basophil and eosinophil counts increased in WPC-exposed murine skin, not but in PWH1- or PWH2-exposed mice., Conclusion: The epicutaneous immunogenicity of PWH1 and PWH2 is markedly decreased, which may reduce the risk of allergen sensitization. Further studies are required to investigate the clinical value of these partial hydrolysates for high-risk infants., (© 2020 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

The high temperatures used in the production of milk may induce modifications in proteins structure. Due to occurrence of the Maillard reaction, lactose binds lysine residues in proteins, affecting the nutritional value. Milk is also an important source of allergenic proteins (i.e., caseins, β-lactoglobulin and α-lactalbumin). Thus, this modification may also affect the allergenicity of these proteins. Focusing on milk whey proteins, a screening on different Ultra High Temperatures (UHT) and pasteurized milk samples was performed to identify lactosylation sites, in particular in protein known epitopes, and to verify the correlation between lactosylation and the harshness of the treatment. Whey proteins were extracted from milk samples after caseins precipitations at pH 4.6 and, after chymotryptic and tryptic in solution digestion, peptides were analysed by UPLC-MS and LTQ-Orbitrap. Results show the presence of lactosylated lysine residues in several known epitopes. Then, a β-lactoglobulin epitope was selected and synthesized by solid phase synthesis followed by in solution lactosylation, obtaining high reaction yields and purities. The synthesis of lactosylated allergenic epitopes, described here for the first time, is a useful tool for further studies on the technological impacts on food allergenicity.

Background: Amino acid-based formulas (AAFs) are used for the dietary management of cow's milk allergy (CMA). Whether AAFs have the potential to prevent the development and/or symptoms of CMA is not known., Objective: The present study evaluated the preventive effects of an amino acid (AA)-based diet on allergic sensitization and symptoms of CMA in mice and aimed to provide insight into the underlying mechanism., Methods: C3H/HeOuJ mice were sensitized with whey protein or with phosphate-buffered saline as sham-sensitized control. Starting 2 weeks before sensitization, mice were fed with either a protein-based diet or an AA-based diet with an AA composition based on that of the AAF Neocate, a commercially available AAF prescribed for the dietary management of CMA. Upon challenge, allergic symptoms, mast cell degranulation, whey-specific immunoglobulin levels, and FoxP3 + cell counts in jejunum sections were assessed., Results: Compared to mice fed with the protein-based diet, AA-fed mice had significantly lower acute allergic skin responses. Moreover, the AA-based diet prevented the whey-induced symptoms of anaphylaxis and drop in body temperature. Whereas the AA-based diet had no effect on the levels of serum IgE and mucosal mast cell protease-1 (mMCP-1), AA-fed mice had significantly lower serum IgG2a levels and tended to have lower IgG1 levels (P = .076). In addition, the AA-based diet prevented the whey-induced decrease in FoxP3 + cells. In sham-sensitized mice, no differences between the two diets were observed in any of the tested parameters., Conclusion: This study demonstrates that an AA-based diet can at least partially prevent allergic symptoms of CMA in mice. Differences in FoxP3 + cell counts and serum levels of IgG2a and IgG1 may suggest enhanced anti-inflammatory and tolerizing capacities in AA-fed mice. This, combined with the absence of effects in sham-sensitized mice indicates that AAFs for the prevention of food allergies may be an interesting concept that warrants further research., (© 2020 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Food protein allergies are a major global concern. Hydrolysis of food proteins reduces their allergenicity, but another novel approach is the covalent attachment of polysaccharides to proteins via the Maillard reaction (i.e., glycation), which blocks some IgE binding epitopes on the protein allergen. We wanted to examine whether enzymatic hydrolysis, combined with glycation, could further reduce IgE binding for people with a cow milk protein allergy. Whey protein isolate (WPI) was hydrolyzed by immobilized trypsin and chymotrypsin to degree of hydrolysis (DH) values of 17 to 27%. Immobilized enzymes were used to avoid heat-treating the hydrolysate (to inactivate the enzymes, because heating could also affect the IgE binding ability of the protein). The resultant whey protein isolate hydrolysates (WPIH) were then glycated with 10-kDa dextran (DX) in aqueous solutions held at 62°C for 24 h. We analyzed the molar mass (M W ) of WPIH samples and their corresponding glycates (WPIH-DX) using size-exclusion chromatography with multi-angle laser light scattering. We obtained blood sera from 8 patients who had been diagnosed with a cow milk protein allergy, and we used a composite serum for IgE binding analysis. The average M W values of samples WPIH-1 to WPIH-3 decreased from 11.15, 9.46, and 7.57 kDa with increasing DH values of 18.7, 22.5, and 27.1%. Glycation significantly reduced the high bitterness of the WPIH samples, as assessed by a trained sensory panel. The WPIH-DX glycates had significantly reduced WPI-specific IgE binding capacity compared to WPI or unglycated WPIH; we found an almost 99% reduction in IgE binding for the WPIH-DX glycate made from WPIH with a DH value of 27.1%. Hydrolysis of WPI followed by glycation with DX via the Maillard reaction significantly decreased the allergenicity of whey proteins., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The aim of the study was to investigate the use of serine protease from Yarrowia lipolytica yeast for reduction of milk proteins allergenicity. Whey protein concentrate (WPC-80), αs-casein and their hydrolysates were analyzed for the capacity to bind IgE and IgG antibodies present in sera from patients with cow milk protein allergy using a competitive ELISA. The hydrolysis of αs-casein and whey protein concentrate contributed to a significant reduction of their immunoreactive epitopes. In case of IgE antibodies, the lowest binding capacity was detected in the 24 h hydrolysates of both proteins in which the inhibition of the reaction was ≤20 and ≤68% for αs-casein and whey protein concentrate respectively. One hour hydrolysis of WPC-80 reduced the protein antigenicity, while the longer time (5 h) might lead to the exposure of new IgE - reactive epitopes., (Copyright © 2019. Published by Elsevier Ltd.)

We investigated the allergenicity, digestibility and functional properties of whey protein isolate (WPI) after covalent conjugation with chlorogenic acid (CHA). The covalent conjugation of CHA may cause an unfolded protein structure. The WPI-CHA conjugate showed lower IgE binding capacity but higher intestinal digestibility than unmodified WPI. Furthermore, after digestion, the IgE binding capacity of β-lactoglobulin and α-lactoalbumin was lower in the digested WPI-CHA conjugate than digested WPI. Moreover, the solubility, emulsifying activity, foaming properties and antioxidant capacity of WPI were enhanced by covalent conjugation of CHA. Covalent conjugation with CHA might reduce the allergenicity in vitro of WPI by improving the functional properties of the protein., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Several 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., Objective: To 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., Methods: C3H/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., Results: Mice 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 Relevance: This 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., (© 2019 The Authors. Clinical & Experimental Allergy Published by John Wiley & Sons Ltd.)

The present study evaluated four laticifer fluids as a novel source of peptidases capable of hydrolyzing proteins in cow's milk. The latex peptidases from Calotropis procera (CpLP), Cryptostegia grandiflora (CgLP), and Carica papaya (CapLP) were able to perform total hydrolysis of caseins after 30 min at pH 6.5, as confirmed by a significant reduction in the residual antigenicity. Casein hydrolysis by Plumeria rubra latex peptidases (PrLP) was negligible. Moreover, whey proteins were more resistant to proteolysis by latex peptidases; however, heat pretreatment of the whey proteins enhanced the degree of hydrolysis and reduced the residual antigenicity of the hydrolysates. The in vivo assays show that the cow's milk proteins hydrolysed by CgLP and CapLP exhibited no immune reactions in mice allergic to cow's milk, similar to a commercial partially hydrolysed formula. Thus, these peptidases are promising enzymes for the development of novel hypoallergenic formulas for children with a milk allergy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: We sought to determine whether an extensively hydrolyzed formula (EHF) supplemented with two human milk oligosaccharides (HMO) was tolerated by infants with cow's milk protein allergy (CMPA)., Methods: A whey-based EHF (Test formula) containing 2'fucosyl-lactose (2'FL) and lacto-N-neotetraose (LNnT) was assessed for clinical hypoallergenicity and safety. The Control formula was a currently marketed EHF without HMO. Children with CMPA, aged 2 months to 4 years, were assessed by double-blind, placebo-controlled food challenges (DBPCFC) to both formulas, in randomized order. If both DBPCFC were negative, subjects participated in a one-week, open food challenge (OFC) with the Test formula. Symptoms and adverse events were recorded. Hypoallergenicity was accepted if at least 90% (with 95% confidence intervals) of subjects tolerated the Test formula., Results: Of the 82 children with CMPA that were screened, 67 (intention-to-treat [ITT] cohort-mean age 24.5 ± 13.6 months; range 2-57; 45 [67.2%] male) were randomized to receive either the Test or the Control formula during the first DBPCFC. Of these, 64 children completed at least one DBPCFC (modified intention-to-treat [mITT] cohort). Three children were excluded due to protocol deviations (per protocol [PP] cohort; n = 61). There was one allergic reaction to the Test, and one to the Control formula. On the mITT analysis, 63 out of 64 (98.4%; 95% CI lower bound 92.8%), and on the PP analysis 60 out of 61 (98.4%; 95% CI lower bound 92.5%) participants tolerated the Test formula, confirming hypoallergenicity., Conclusion: The whey-based EHF supplemented with 2'FL and LNnT met the clinical hypoallergenicity criteria and can be recommended for the management of CMPA in infants and young children., Competing Interests: The authors declare the following potential conflicts of interest: A.N.-W. received research support from DBV Technologies, Astellas Pharma, Nutricia, Nestlé Health Science; Royalties from Up-To-Date; Advisory Board honoraria from Gerber Institute, Merck, ALK, Abello, and Sanofi Aventis. L.C. is an employee of Nestlé Nutrition, USA. B.C. is an employee of Clinipace. K.R. and R.G.H. are employees of Nestlé Health Science.

Background: Failure to induce oral tolerance may result in food allergy. Hydrolysed cow's milk-based infant formulas are recommended in subjects with a high risk of developing allergic disease. Presentation of T cell epitopes is a prerequisite to generate regulatory T cells that could contribute to oral tolerance., Objective: To investigate whether a specific hydrolysed whey-based infant formula contains peptides that function as T cell epitopes to support the development of oral tolerance to whey., Methods: First, a novel liquid chromatography-mass spectrometry (LC-MS) method was developed to characterize β-lactoglobulin-derived peptides present in a specific infant formula with a focus on region AA#13-48 of β-lactoglobulin, which has previously been described to contain T cell epitopes with tolerogenic potential. Second, the formula was subjected to the ProImmune ProPresent ® antigen presentation assay and MHC class II binding algorithm to identify relevant HLA-DRB1-restricted peptides. Third, identified peptides were tested on human cow's milk protein-specific T cell lines to determine T cell recognition., Results: Thirteen peptides of minimal 9AAs long that overlap with AA#13-48 of β-lactoglobulin were identified. Six of them were found across all batches analysed. It was further confirmed that these peptides were processed and presented by human dendritic cells. The identified HLA-DRB1-restricted peptides were correlated to AA#11-30 and AA#23-39 of β-lactoglobulin. Importantly, the proliferation assay showed that the synthetic peptides were recognized by cow's milk protein-specific T cell lines and induced T cell proliferation., Conclusion and Clinical Relevance: This study demonstrates that the tested hydrolysed infant formula contains functional HLA-DRB1-restricted T cell epitopes, which can potentially support the development of oral tolerance to whey., (© 2018 Nutricia Research B.V. Clinical & Experimental Allergy Published by John Wiley & Sons Ltd.)

The conditionally essential amino acid glycine functions as inhibitory neurotransmitter in the mammalian central nervous system. Moreover, it has been shown to act as an anti-inflammatory compound in animal models of ischemic perfusion, post-operative inflammation, periodontal disease, arthritis and obesity. Glycine acts by binding to a glycine-gated chloride channel, which has been demonstrated on neurons and immune cells, including macrophages, polymorphonuclear neutrophils and lymphocytes. The present study aims to evaluate the effect of glycine on allergy development in a cow's milk allergy model. To this end, C3H/HeOuJ female mice were supplemented with glycine by oral gavage (50 or 100 mg/mouse) 4 hours prior to sensitization with cow's milk whey protein, using cholera toxin as adjuvant. Acute allergic skin responses and anaphylaxis were assessed after intradermal allergen challenge in the ears. Mouse mast cell protease-1 (mMCP-1) and whey specific IgE levels were detected in blood collected 30 minutes after an oral allergen challenge. Jejunum was dissected and evaluated for the presence of mMCP-1-positive cells by immunohistochemistry. Intake of glycine significantly inhibited allergy development in a concentration dependent manner as indicated by a reduction in; acute allergic skin response, anaphylaxis, serum mMCP-1 and serum levels of whey specific IgE. In addition, in-vitro experiments using rat basophilic leukemia cells (RBL), showed that free glycine inhibited cytokine release but not cellular degranulation. These findings support the hypothesis that the onset of cow's milk allergy is prevented by the oral intake of the amino acid glycine. An adequate intake of glycine might be important in the improvement of tolerance against whey allergy or protection against (whey-induced) allergy development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A growing concern around the world is the number of people who are suffering from food protein allergies. One potential approach to decrease protein allergenicity is to block IgE-binding epitopes of the protein allergen by attachment of polysaccharides via the Maillard reaction (i.e., glycation). Protein glycation has been extensively studied to modify various functional properties. We wanted to examine whether glycates could reduce IgE binding in patients with cow milk protein allergy and to explore how the size (molar mass; M W ) of the polysaccharide affects this IgE-binding capacity. Glycation was performed using the initial step of the Maillard reaction performed in aqueous solutions. The specific goal of this study was to reduce the IgE-binding capacity of whey protein isolate (WPI) through glycation with dextran (DX). Blood sera were obtained from 8 patients who had been diagnosed with cow milk protein allergy, and a composite sera sample was used for IgE-binding analysis by the ImmunoCap (Phadia, Uppsala, Sweden) method. The WPI was glycated with DX of M W ranging from 1 to 2,000 kDa, and the M W of purified glycates was determined using size-exclusion chromatography coupled with multiangle laser light scattering. The WPI to DX molar ratios in the glycates made from DX that had M W values of 1, 3.5, 10 (G10), 150, 500, and 2,000 kDa were 1:4, 1:3, 1:2, 1:1.5, 1:1, and 1:1, respectively. With the increase in the M W of DX, there was an increase in the M W values of the corresponding glycates but a decrease in the number of bound DX. The WPI-DX glycates had lower whey protein IgE-binding capacity than native WPI, with the lowest IgE-binding capacity obtained in the G10 glycate. The DX binding ratios and morphology results from atomic force microscopy images suggested that glycation of WPI with small-M W DX resulted in extensive protein surface coverage, probably due to the attachment of up to 4 DX molecules per whey protein. The lower IgE binding of the G10 glycate was likely due to greater steric hindrance (or a physical barrier) at the surface of the protein. In summary, our results demonstrate that glycating WPI with DX via Maillard reaction can potentially be used to decrease the allergenicity of whey protein., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Transitory allergies to cow milk proteins in infants or adults have become a public health problem. Although extensively or partially hydrolyzed cow milk protein formulas are available, these products are costly. Therefore, studies into innovative enzymes to digest cow milk proteins are needed. Danaus plexippus gut peptidases were purified and examined with regard to cow milk protein hydrolysis. The peptidases hydrolyzed caseins and whey proteins. However, after heat treatment, there was a significant improvement in β-lactoglobulin hydrolysis. The hydrolyzed cow milk proteins were not recognized by anti-casein antibodies and only reacted slightly with antibodies against whey proteins. This performance was better than that of partially hydrolyzed formulas and similar to that of an extensively hydrolyzed formula. These results suggest that D. plexippus gut peptidases are suitable and innovative enzymes to produce hypoallergenic cow milk protein formulas., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Epicutaneous sensitization to food allergens can occur through defective skin barriers. However, the relationship between oral tolerance and epicutaneous sensitization remains to be elucidated. We aimed to determine whether prior oral exposure to whey proteins or their hydrolysates prevents epicutaneous sensitization and subsequent food-allergic reaction to the whey protein, β-lactoglobulin (β-LG), and investigated the underlying mechanisms., Methods: BALB/c mice were given whey protein concentrate (WPC), two kinds of partial whey protein hydrolysate (PWH1 or PWH2), or extensive whey protein hydrolysate (EWH) in drinking water for 21 days. The mice were then epicutaneously sensitized with β-LG on tape-stripped skin. Sensitization was assessed by basophil activation tests and by measuring the level of serum β-LG-specific antibodies and cytokines secreted from β-LG-restimulated spleen and mesenteric lymph node (MLN) cells. Development of an allergic reaction was assessed by monitoring body temperature and by measuring mast cell protease-1 level in plasma after the β-LG oral challenge. Activated T-cell population among β-LG-restimulated MLN cells was also analyzed., Results: In mice fed with WPC, PWH1, or PWH2, sensitization and the development of an allergic reaction were totally reduced. The acceleration of cytokine release from the spleen and MLN cells or T-cell activation was not evident after β-LG restimulation. In EWH-fed mice, a suppressive effect, though milder than that in WPC-, PWH1-, or PWH2-fed mice, was observed during the development of the allergic reaction., Conclusions: Prior oral exposure to partially hydrolyzed whey protein prevents epicutaneous sensitization and subsequent allergic response to β-LG in mice., (© 2018 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Background: Growing evidence has strengthened the association of food allergy with neuropsychiatric symptoms such as depression, anxiety, and autism. However, underlying mechanisms by which peripheral allergic responses lead to behavioral dysfunction are yet to be determined. Allergen-activated mast cells may serve as mediators by releasing histamine and other inflammatory factors that could adversely affect brain function. We hypothesized that eliciting food allergy in experimental animals would result in behavioral changes accompanied by mast cell accumulation in the brain. Our hypothesis was tested in a mouse model of milk allergy using bovine milk whey proteins (WP) as the allergen., Methods: Male and female C57BL/6 mice at 4 weeks (young) and 10 months (old) of age underwent 5-week WP sensitization with weekly intragastric administration of 20 mg WP and 10 μg cholera toxin as an adjuvant. Age-matched sham animals were given the vehicle containing only the adjuvant. All animals were orally challenged with 50 mg WP in week 6 and their intrinsic digging behavior was assessed the next day. Animals were sacrificed 3 days after the challenge, and WP-specific serum IgE, intestinal and brain mast cells, glial activation, and epigenetic DNA modification in the brain were examined., Results: WP-sensitized males showed significantly less digging activity than the sham males in both age groups while no apparent difference was observed in females. Mast cells and their activities were evident in the intestines in an age- and sex-dependent manner. Brain mast cells were predominantly located in the region between the lateral midbrain and medial hippocampus, and their number increased in the WP-sensitized young, but not old, male brains. Noticeable differences in for 5-hydroxymethylcytosine immunoreactivity were observed in WP mice of both age groups in the amygdala, suggesting epigenetic regulation. Increased microglial Iba1 immunoreactivity and perivascular astrocytes hypertrophy were also observed in the WP-sensitized old male mice., Conclusions: Our results demonstrated that food allergy induced behavioral abnormality, increases in the number of mast cells, epigenetic DNA modification in the brain, microgliosis, and astrocyte hypertrophy in a sex- and age-dependent manner, providing a potential mechanism by which peripheral allergic responses evoke behavioral dysfunction.

Hydrolysates, which are used in hypoallergenic infant formulas, have been found to possess immune modulating effects. For an optimal utilization of hydrolysates, the working mechanisms and responsible proteins underlying the effects should be elucidated. In this study, the immunomodulating activity of whey and soy hydrolysates was studied by quantifying TLR activation and assessing cytokine production in hydrolysate stimulated dendritic cells. The responsible protein fraction was identified and characterized by gel electrophoresis. The immune effects under gastrointestinal conditions were studied by digesting the hydrolysates in an in vitro infant digestion model, after which the digests were analyzed. In both soy and whey hydrolysates, TLR activation and cytokine production in dendritic cells were induced by a fraction containing protein aggregates larger than 1000 kDa, which were formed by electrostatic interactions and disulfide bonds. Only soy aggregates remained intact during duodenal digestion, and maintained the TLR activating capacity. Soy and whey protein aggregates larger than 1000 kDa possess immunomodulatory properties, but only soy aggregates remain under intestinal digestion conditions. This knowledge is important for a better understanding of the effects of hydrolysates.

Beta-lactoglobulin (BLG)-derived peptides may facilitate oral tolerance to whey and prevent cow's milk allergy (CMA). Loading of BLG-peptides in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Pep-NP) may improve this. Here we studied the uptake of NP and the capacity of NP and Pep-NP to activate bone marrow dendritic cells (BMDC). Furthermore, CMA prevention was evaluated by orally exposing three-week-old female C3H/HeOuJ mice to Pep-NP, NP or free peptides (PepMix) for 6 days before oral sensitization with whole whey protein and effects on the spleen and small intestine lamina propria (SI-LP) were studied. In BMDC, NP and Pep-NP enhanced CD40 expression and IL-6 and TNF-α secretion, while tended to decrease CD80 expression and prevented PepMix-induced IL-12 secretion. In vivo, oral exposure to Pep-NP, but not NP or PepMix, prior to whey sensitization tended to partially prevent the acute allergic skin response to whole whey protein. Splenocytes of NP-pre-exposed mice secreted increased levels of whey-specific IL-6, but this was silenced in Pep-NP-pre-exposed mice which also showed reduced TNF-α and IFN-γ secretion. In the SI-LP, Pep-NP pre-exposure reduced the CD4 + T cell frequency in CMA mice compared to PBS pre-exposure. In addition, while NP increased whey-specific IL-6 secretion in the SI-LP, Pep-NP did not and maintained regulatory TGF-β secretion. This study presents a proof-of-concept that PLGA nanoparticles facilitate the capacity of BLG peptides to suppress the allergic response to whole whey protein. Hence, PLGA nanoparticles may be further developed as an adjunct strategy for BLG-peptide-based oral tolerance induction and CMA prevention., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Staphylococcus aureus is the most common contagious pathogen associated with bovine subclinical mastitis. Current diagnosis of S. aureus mastitis is based on bacteriological culture of milk samples and somatic cell counts, which lack either sensitivity or specificity. Identification of milk proteins that contribute to host defense and their variable responses to pathogenic stimuli would enable the characterization of putative biomarkers of subclinical mastitis. To accomplish this, milk whey samples from healthy and mastitic dairy cows were analyzed using a label-free quantitative proteomics approach. In total, 90 proteins were identified, of which 25 showed significant differential abundance between healthy and mastitic samples. In silico functional analyses indicated the involvement of the differentially abundant proteins in biological mechanisms and signaling pathways related to host defense including pathogen-recognition, direct antimicrobial function, and the acute-phase response. This proteomics and bioinformatics analysis not only facilitates the identification of putative biomarkers of S. aureus subclinical mastitis but also recapitulates previous findings demonstrating the abundance of host defense proteins in intramammary infection. All mass spectrometry data are available via ProteomeXchange with identifier PXD007516 ., Competing Interests: The authors declare no conflict of interest.

Healthy foods like polyphenol-rich berries and high quality edible proteins are in demand in today's functional food marketplace, but it can be difficult to formulate convenient food products with physiologically-relevant amounts of these ingredients and still maintain product quality. In part, this is because proteins can interact with other food ingredients and precipitate destabilizing events, which can disrupt food structure and diminish shelf life. Proteins in foods can also interact with human receptors to provoke adverse consequences such as allergies. When proteins and polyphenols were pre-aggregated into stable colloidal particles prior to use as ingredients, highly palatable food formulations (with reduced astringency of polyphenols) could be prepared, and the overall structural properties of food formulations were significantly improved. All of the nutritive and phytoactive benefits of the proteins and concentrated polyphenols remained highly bioavailable, but the protein molecules in the particle matrix did not self-aggregate into networks or react with other food ingredients. Both the drainage half-life (a marker of structural stability) and the yield stress (resistance to flow) of model foams made with the protein-polyphenol particles were increased in a dose-dependent manner. Of high significance in this complexation process, the reactive allergenic epitopes of certain proteins were effectively blunted by binding with polyphenols, attenuating the allergenicity of the food proteins. Porcine macrophages produced TNF-α proinflammatory cytokine when provoked with whey protein, but, this response was blocked completely when the cells were stimulated with particles that complexed whey protein with cinnamon-derived polyphenols. Cytokine and chemokine production characteristic of allergic reactions were blocked by the polyphenols, allowing for the potential creation of hypoallergenic protein-berry polyphenol enriched foods.

Homologous and heterologous cross-linked polymers of whey protein isolate (WPI), soy protein isolate (SPI) and casein (CN) and their binary mixtures, viz., WPI+SPI, WPI+CN and SPI+CN, were produced using transglutaminase, and their in vitro IgE reactivity and digestibility under simulated gastro-intestinal conditions were studied. The results showed that the IgE reactivity of protein components in heterologous polymers was significantly lower than that in homologous polymers, suggesting that each protein component masked the IgE-reactive epitopes in the other protein component more effectively in heterologous polymers than in homologous polymers. In vitro digestion under simulated gastro-intestinal conditions revealed that both homologous and heterologous polymers were less digestible than untreated proteins, but the peptides released during the time course of digestion were less IgE-reactive. The results of this study indicate that hypoallergenic protein products could be produced by transglutaminase-mediated heterologous polymerization of protein mixtures., (Copyright © 2016. Published by Elsevier Ltd.)

Background: Cow milk allergy is the most common food allergy in children. So far, no effective treatment is available to prevent or cure food allergy. This study investigated whether orally administrated probiotics could suppress sensitisation in whey proteins (WP)-induced allergy mouse model. Two types of probiotic Dahi were prepared by co-culturing Dahi bacteria (Lactococcus lactis ssp. cremoris NCDC-86 and Lactococcus lactis ssp. lactis biovar diacetylactis NCDC-60) along with selected strain of Lactobacillus acidophilus LaVK2 and Bifidobacterium bifidum BbVK3. Mice were fed with probiotic Dahi (La-Dahi and LaBb-Dahi) from 7 days before sensitisation with WP, respectively, in addition to milk protein-free basal diet, and control group received no supplements., Results: Feeding of probiotic Dahi suppressed the elevation of whey proteins-specific IgE and IgG response of WP-sensitised mice. In addition, sIgA levels were significantly (P < 0.001) increased in intestinal fluid collected from mice fed with La-Dahi. Production of T helper (Th)-1 cell-specific cytokines, i.e. interferon-γ (IFN-γ), interleukin (IL)-12, and IL-10 increased, while Th2-specific cytokines, i.e. IL-4 decreased in the supernatant of cultured splenocytes collected from mice fed with probiotic Dahi as compared to the other groups. Moreover, the splenic mRNA levels of IFN-γ, interleukin-10 were found to be significantly increased, while that of IL-4 decreased significantly in La-Dahi groups, as compared to control groups., Conclusion: Results of the present study indicate that probiotic Dahi skewed Th2-specific immune response towards Th1-specific response and suppressed IgE in serum. Collectively, this study shows the potential use of probiotics intervention in reducing the allergic response to whey proteins in mice. © 2015 Society of Chemical Industry., (© 2015 Society of Chemical Industry.)

Whey protein concentrate (WPC) is often subjected to heat treatment during industrial processing, resulting in protein denaturation and loss of protein bioactivity. We hypothesized that WPC samples subjected to different degrees of thermal processing are associated with different levels of bioactive proteins and effects on proliferation and immune response in intestinal epithelial cells (IEC). The results showed that low-heat-treated WPC had elevated levels of lactoferrin and transforming growth factor-β2 compared with that of standard WPC. The level of aggregates depended on the source of whey, with the lowest level being found in WPC derived from acid whey. Following acid activation, WPC from acid whey enhanced IEC proliferation compared with WPC from sweet whey or nonactivated WPC. Low-heat-treated WPC from acid whey induced greater secretion of IL-8 in IEC than either standard WPC from acid whey or low-heat-treated WPC from sweet whey. Following acid activation (to activate growth factors), low-heat-treated WPC from sweet whey induced higher IL-8 levels in IEC compared with standard WPC from sweet whey. In conclusion, higher levels of bioactive proteins in low-heat-treated WPC, especially from acid whey, may enhance proliferation and cytokine responses of IEC. These considerations could be important to maintain optimal bioactivity of infant formulas, including their maturational and immunological effects on the developing intestine., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Food allergy is an adverse immune response to dietary proteins. Hydrolysates are frequently used for children with milk allergy. However, hydrolysates effects afterwards are poorly studied. The aim of this study was to investigate the immunological consequences of hydrolyzed whey protein in allergic mice. For that, we developed a novel model of food allergy in BALB/c mice sensitized with alum-adsorbed β-lactoglobulin. These mice were orally challenged with either whey protein or whey hydrolysate. Whey-challenged mice had elevated levels of specific IgE and lost weight. They also presented gut inflammation, enhanced levels of SIgA and IL-5 as well as decreased production of IL-4 and IL-10 in the intestinal mucosa. Conversely, mice challenged with hydrolyzate maintained normal levels of IgE, IL-4 and IL-5 and showed no sign of gut inflammation probably due to increased IL-12 production in the gut. Thus, consumption of hydrolysate prevented the development of clinical signs of food allergy in mice., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Cow's milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow's milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays., Objective: An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays., Methods: Mice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains., Results: After sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5-10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow's milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow's milk allergic children., Conclusion: Usage of our 'unlimited' source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow's milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.